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Last Updated :2024/10/03

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Master

Affiliation (Master)

  • Research Faculty of Agriculture　Fundamental AgriScience Research　Animal Science

Affiliation (Master)

  • Research Faculty of Agriculture　Fundamental AgriScience Research　Animal Science

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Profile and Settings

Affiliation

  • Hokkaido University, Graduate School of Agriculture Research Faculty of Agriculture Division of Applied Bioscience

Profile and Settings

  • Name (Japanese)

    Kobayashi

  • Name (Kana)

    Ken

  • Name

    201301061596148220

Alternate Names

Affiliation

  • Hokkaido University, Graduate School of Agriculture Research Faculty of Agriculture Division of Applied Bioscience

Achievement

Research Interests

  • milk production   乳産生   claudin   mammary gland   tight junctions   milk   casein   epithelial cells   parturition   paracellular pathway   mastitis   hair   regenerative medicine   

Research Areas

  • Life sciences / Veterinary medicine
  • Life sciences / Clinical pharmacy
  • Life sciences / Physiology

Research Experience

  • 2016/04 - Today Hokkaido University Graduate School of Agriculture Research Faculty of Agriculture
  • 2015/10 - 2016/03 Hokkaido University Graduate School of Agriculture Research Faculty of Agriculture
  • 2010/10 - 2015/09 Hokkaido University Graduate School of Agriculture Research Faculty of Agriculture
  • 2006/10 - 2010/09 Keio University School of Medicine
  • 2005/04 - 2006/09 Fukushima Medical University

Education

  • 1999/04 - 2005/03  Hokkaido University
  • 1995/04 - 1999/03  Hokkaido University  Faculty of Agriculture  Department of Animal Science

Committee Memberships

  • 2021/06 - Today   Journal of Mammary gland and Neoplasia   editor

Awards

  • 2017/10 Japanese Society of Bovine Mastitis Takai Yuriko Award
     
    受賞者: Ken Kobayashi

Published Papers

  • Liang Han, Shan-Ni Lu, Takanori Nishimura, Ken Kobayashi
    Experimental cell research 439 (1) 114090 - 114090 2024/06/01 
    Dopamine D2 receptors (D2Rs) play crucial roles in regulating diverse physiological functions of the central nervous system and peripheral organs. D2Rs are also expressed in mammary glands. However, which cell types express D2Rs and whether they are involved in milk production remains unclear. The present findings revealed that D2Rs are expressed in the apical regions of the lateral membranes of mammary epithelial cells (MECs) in lactating mice. We also investigated the effects of the D2R agonist bromocriptine and/or antagonist domperidone on intracellular cAMP levels, milk protein production, and apoptosis in a lactation culture model of MECs that produce major milk components like lactating MECs in vivo. We found that bromocriptine decreased intracellular cAMP levels, whereas domperidone dose-dependently neutralized this effect. Bromocriptine also inhibited casein and lactoferrin production and suppressed activities of STAT5 and glucocorticoid receptors (GRs). Domperidone neutralized the inhibition of casein production as well as STAT5 and GR inactivation induced by bromocriptine. Furthermore, D2R activation by bromocriptine induced apoptosis and inactivated ERK, a signaling molecule responsible for promoting cell proliferation and survival. Domperidone attenuated ERK inactivation and apoptosis induced by bromocriptine. These findings suggest that D2Rs play regulatory roles in milk protein production and apoptosis in MECs.
  • Emi Uenaka, Koichi Ojima, Takahiro Suzuki, Ken Kobayashi, Susumu Muroya, Takanori Nishimura
    In vitro cellular & developmental biology. Animal 2024/05/17 
    Skeletal muscle tissue increases or decreases its volume by synthesizing or degrading myofibrillar proteins. The ubiquitin-proteasome system plays a pivotal role during muscle atrophy, where muscle ring finger proteins (Murf) function as E3 ubiquitin ligases responsible for identifying and targeting substrates for degradation. Our previous study demonstrated that overexpression of Ozz, an E3 specific to embryonic myosin heavy chain (Myh3), precisely reduced the Myh3 replacement rate in the thick filaments of myotubes (E. Ichimura et al., Physiol Rep. 9:e15003, 2021). These findings strongly suggest that E3 plays a critical role in regulating myosin replacement. Here, we hypothesized that the Murf isoforms, which recognize Myhs as substrates, reduced the myosin replacement rates through the enhanced Myh degradation by Murfs. First, fluorescence recovery after a photobleaching experiment was conducted to assess whether Murf isoforms affected the GFP-Myh3 replacement. In contrast to Murf2 or Murf3 overexpression, Murf1 overexpression selectively facilitated the GFP-Myh3 myosin replacement. Next, to examine the effects of Murf1 overexpression on the replacement of myosin isoforms, Cherry-Murf1 was coexpressed with GFP-Myh1, GFP-Myh4, or GFP-Myh7 in myotubes. Intriguingly, Murf1 overexpression enhanced the myosin replacement of GFP-Myh4 but did not affect those of GFP-Myh1 or GFP-Myh7. Surprisingly, overexpression of Murf1 did not enhance the ubiquitination of proteins. These results indicate that Murf1 selectively regulated myosin replacement in a Myh isoform-dependent fashion, independent of enhanced ubiquitination. This suggests that Murf1 may have a role beyond functioning as a ubiquitin ligase E3 in thick filament myosin replacement.
  • Culture models to investigate lactation and mastitis in bovine mammary epithelial cells
    Tsugami Y, Nagasawa Y, Kobayashi K
    Japan Agricultural Research Quarterly 2024 [Refereed]
  • Yuka Katayama, Osamu Ichii, Teppei Nakamura, Keita Yanase, Masaya Hiraishi, Takashi Namba, Yuki Otani, Teppei Ikeda, Erika Tsuji, Natsuko Tsuzuki, Ken Kobayashi, Yasuhiro Kon, Takanori Nishimura
    Animals : an open access journal from MDPI 13 (23) 2023/11/29 [Refereed]
     
    Foreign body reactions (FBRs) are inadvertently observed in invading or artificially embedded materials, triggering inflammation and subsequent fibrotic processes to occur in situ. Here, we assessed the spatiotemporal formation of connective tissue around implanted materials to establish a technique using connective tissue formed by FBRs as xenografts. An acrylic resin implant, comprising a columnar inner rod and a tubular outer cylinder (OC) with several slits, was embedded in adult dairy cows. Tissues formed in the inner rod and OC groups were histologically analyzed at weeks 2, 4, 8, and 12. Edematous tissues with non-collagenous fibers formed for 2 weeks and showed increased cellularity after 4 weeks. The weight, thickness, amounts of total protein, collagen, DNA, and quantitative scores of α-smooth muscle actin-positive myofibroblasts or elastic fibers notably increased after 8 weeks, with condensed collagen fibers showing orientation. Inflammatory cells were primarily localized in tissues close to the OC, and their numbers increased, with the count of CD204+ cells peaking at 8 weeks and declining at 12 weeks. The count of Ki67+ proliferating cells slightly increased in tissues close to the OC; however, the number and lumen of CD31+ vessels increased. These results may help understand FBR-related tissue remodeling.
  • Ken Kobayashi, Liang Han, Shan-Ni Lu, Kazuki Ninomiya, Naoki Isobe, Takanori Nishimura
    Experimental Cell Research 431 (1) 113762 - 113762 0014-4827 2023/10
  • Takahiro Maeno, Rio Arimatsu, Koichi Ojima, Yuki Yamaya, Hikaru Imakyure, Naruha Watanabe, Yusuke Komiya, Ken Kobayashi, Mako Nakamura, Takanori Nishimura, Ryuichi Tatsumi, Takahiro Suzuki
    Experimental Cell Research 430 (1) 113698 - 113698 0014-4827 2023/09 [Refereed]
  • Yusaku Tsugami, Takahiro Nii, Ken Kobayashi, Naoki Isobe
    Innate Immunity 1753-4259 2023/08/22 [Refereed][Not invited]
     
    In the mammary glands, production of antimicrobial components and formation of less-permeable tight junctions (TJs) are important for safe milk production. Previously, we reported that local heat treatment of udders using disposable heating pad enhances the components of innate immunity in lactating goat mammary glands. Gingerol is a polyphenol present in ginger that can induce heat-like effects. However, oral administration of polyphenols causes a decrease in biological activity through conjugation and metabolic conversion. Here, we investigated the effects of gingerol on antimicrobial components and TJs by topically applying it to lactating goat udders. Gingerol application increased the somatic cell count, cathelicidin-2 concentration, and proportion of polymorphonuclear cells in the milk and interleukin-8 production. Moreover, gingerol treatment enhanced β-defensin-1 production in milk, cultured mammary epithelial cells, and cultured somatic cells. Contrastingly, gingerol treatment did not affect the concentrations of blood-derived components (Na+, albumin, and IgG) in the milk or the TJ barrier function of cultured mammary epithelial cells. These findings suggest that the topical application of gingerol, similar to local heat treatment, to udders enhances the components of innate immunity in mammary glands. These findings may be useful for the prevention of mastitis in milk-producing animals and, hence, safe and stable dairy production.
  • Ken Kobayashi
    Journal of Mammary Gland Biology and Neoplasia 28 (1) 1083-3021 2023/05/01 [Refereed][Invited]
     
    Abstract Mammary epithelial cells (MECs) are the only cell type that produces milk during lactation. MECs also form less-permeable tight junctions (TJs) to prevent the leakage of milk and blood components through the paracellular pathway (blood-milk barrier). Multiple factors that include hormones, cytokines, nutrition, and temperature regulate milk production and TJ formation in MECs. Multiple intracellular signaling pathways that positively and negatively regulate milk production and TJ formation have been reported. However, their regulatory mechanisms have not been fully elucidated. In addition, unidentified components that regulate milk production in MECs likely exist in foods, for example plants. Culture models of functional MECs that recapitulate milk production and TJs are useful tools for their study. Such models enable the elimination of indirect effects via cells other than MECs and allows for more detailed experimental conditions. However, culture models of MECs with inappropriate functionality may result in unphysiological reactions that never occur in lactating mammary glands in vivo. Here, I briefly review the physiological functions of alveolar MECs during lactation in vivo and culture models of MECs that feature milk production and less-permeable TJs, together with a protocol for establishment of MEC culture with functional TJ barrier and milk production capability using cell culture inserts.
  • Ken Kobayashi, Liang Han, Taku Koyama, Shan-Ni Lu, Takanori Nishimura
    Biochimica et biophysica acta. Molecular cell research 1870 (4) 119448 - 119448 2023/04 [Refereed]
     
    During lactation, mammary epithelial cells (MECs) on the apical membrane are in contact with lactose in milk, while MECs on the basolateral membrane are in contact with glucose in blood. Both glucose and lactose are sweeteners that are sensed by a sweet taste receptor. Previously, we have shown that lactose exposure on the basolateral membrane, but not the apical membrane, inhibits casein production and phosphorylation of STAT5 in MECs. However, it remains unclear whether MECs have a sweet taste receptor. In this study, we confirmed that the sweet taste receptor subunit T1R3 existed in both the apical and basolateral membranes of MECs. Subsequently, we investigated the influence of apical and basolateral sucralose as a ligand for the sweet taste receptor using a cell culture model. In this model, upper and lower media were separated by the MEC layer with less-permeable tight junctions. The results showed in the absence of glucose, both apical and basolateral sucralose induced phosphorylation of STAT5, which is a positive transcriptional factor for milk production. In contrast, the T1R3 inhibitor basolateral lactisole reducing phosphorylated STAT5 and secreted caseins in the presence of glucose. Furthermore, exposure of the apical membrane to sucralose in the presence of glucose inhibited the phosphorylation of STAT5. Simultaneously, GLUT1 was partially translocated from the basolateral membrane to the cytoplasm in MECs. These results suggest that T1R3 functions as a sweet receptor and is closely involved in casein production in MECs.
  • Ruri Ohno, Naoki Suzuki, Yusaku Tsugami, Takahiro Nii, Ken Kobayashi, Naoki Isobe
    Animal Science Journal 94 (1) e13832  1344-3941 2023/01 [Refereed]
     
    Mammary glands with mastitis are usually treated with antibiotics in combination with anti-inflammatory drug application on the udder skin. Menthol is an anti-inflammatory drug. The aim of the present study was to investigate the effect of surface application of menthol on goat udders on the production of antimicrobial components in milk. Goats (5 Shiba and 11 Tokara goats) were subjected to menthol application to the udder under both healthy and inflammatory conditions. An intramammary infusion of lipopolysaccharides was carried out to induce inflammatory conditions in the udder. Milk samples were collected to determine somatic cell count (SCC) and sodium ion (Na+ ), antimicrobial component and cytokine concentrations. In healthy udders, menthol application increased the concentration of antimicrobial components (S100A7 and S100A8), but not in the control. In the inflamed udder, antimicrobial component (lactoferrin, S100A7, and S100A8) and inflammatory cytokine (IL-1β) concentrations were higher in the menthol group than in the control group. These results suggest that menthol application on udders augments the antimicrobial component concentration in the mammary gland under both healthy and inflammatory conditions.
  • Michael G Ross, Ken Kobayashi, Guang Han, Mina Desai
    Nutrients 14 (23) 2022/11/22 [Refereed]
     
    Human milk synthesis is impacted by maternal diet, serum composition, and substrate uptake and synthesis by mammary epithelial cells (MECs). The milk of obese/high-fat-diet women has an increased fat content, which promote excess infant weight gain and the risk of childhood/adult obesity. Yet, the knowledge of milk synthesis regulation is limited, and there are no established approaches to modulate human milk composition. We established a 3-dimensional mouse MEC primary culture that recreates the milk production pathway and tested the effects of the major saturated fatty acid in human milk (palmitate) and a lipoprotein lipase inhibitor (orlistat) on triglyceride production. Positive immunostaining confirmed the presence of milk protein and intracellular lipid including milk globules in the cytoplasm and extracellular space. The treatment with palmitate activated "milk" production by MECs (β-casein) and the lipid pathway (as evident by increased protein and mRNA expression). Consistent with these cellular changes, there was increased secretion of milk protein and triglyceride in MEC "milk". The treatment with orlistat suppressed milk triglyceride production. Palmitate increased milk and lipid synthesis, partly via lipoprotein lipase activation. These findings demonstrate the ability to examine MEC pathways of milk production via both protein and mRNA and to modulate select pathways regulating milk composition in MEC culture.
  • Ken Kobayashi, Naoki Omatsu, Liang Han, Lu Shan-Ni, Takanori Nishimura
    Experimental cell research 420 (1) 113352 - 113352 2022/11/01 [Refereed]
     
    Staphylococcus aureus causes subclinical mastitis; lipoteichoic acid (LTA) from S. aureus causes mastitis-like adverse effects on milk production by mammary epithelial cells (MECs). Here, we investigated the early effects of LTA from S. aureus on mouse MECs using a culture model, in which MECs produced milk components and formed less permeable tight junctions (TJs). In MECs of this model, Toll-like receptor 2 (receptor for LTA), was localized on the apical membrane, similar to MECs in lactating mammary glands. LTA weakened the TJ barrier within 1 h, concurrently with localization changes of claudin 4. LTA treatment for 24 h increased αS1-casein and decreased β-casein levels. In MECs exposed to LTA, the activation level of signal transducer and activator of transcription 5 (major transcriptional factor for milk production) was low. LTA activated signaling pathways related to cell survival (extracellular signal-regulated kinase, heat shock protein 27, and Akt) and inflammation (p38, c-Jun N-terminal kinase, and nuclear factor κB). Thus, LTA caused abnormalities in casein production and weakened the TJs by affecting multiple signaling pathways in MECs. LTA-induced changes in signaling pathways were not uniform in all MECs. Such complex and semi-negative actions of LTA may contribute to subclinical mastitis caused by S. aureus.
  • Koichi Ojima, Masahiro Kigaki, Emi Ichimura, Takahiro Suzuki, Ken Kobayashi, Susumu Muroya, Takanori Nishimura
    American journal of physiology. Cell physiology 323 (2) C520-C535  2022/06/27 [Refereed]
     
    Skeletal muscle consists of slow and fast myofibers in which different myosin isoforms are expressed. Approximately 300 myosins form a single thick filament in the myofibrils, where myosin is continuously exchanged. However, endogenous slow and fast myosin dynamics have not been fully understood. To elucidate those dynamics, here we generated mice expressing green fluorescence protein-tagged slow myosin heavy chain (GFP-Myh7) and Kusabira Orange fluorescence protein-tagged fast myosin heavy chain (KuO-Myh1). First, these mice enabled us to distinguish between GFP- and KuO-myofibers under fluorescence microscopy: GFP-Myh7 and KuO-Myh1 were exclusively expressed in slow myofibers and fast myofibers, respectively. Next, to monitor endogenous myosin dynamics, fluorescence recovery after photobleaching (FRAP) was conducted. The mobile fraction (Mf) of GFP-Myh7 and that of KuO-Myh1 were almost constant values independent of the regions of the myofibers and the muscle portions where the myofibers were isolated. Intriguingly, proteasome inhibitor treatment significantly decreased the Mf in GFP-Myh7 but not in KuO-Myh1 myofibers, indicating that the response to a disturbance in protein turnover depended on muscle fiber type. Taken together, the present results indicated that the mice we generated are promising tools not only for distinguishing between GFP- and KuO-myofibers but also for studying the dynamics of endogenous myosin isoforms by live-cell fluorescence imaging.
  • Ken Kobayashi, Haruka Wakasa, Liang Han, Taku Koyama, Yusaku Tsugami, Takanori Nishimura
    Cell and tissue research 389 (3) 501 - 515 2022/06/24 [Refereed]
     
    Mammary epithelial cells (MECs) are the only cells capable of synthesizing lactose. During lactation, alveolar MECs secrete lactose through the apical membrane into the alveolar lumen, whereas alveolar tight junctions (TJs) block the leakage of lactose into the basolateral sides of the MECs. However, lactose leaks from the alveolar lumen into the blood plasma in the mastitis and after weaning. This exposes the basolateral membrane of MECs to lactose. The relationship between lactose in blood plasma and milk production has been suggested. The present study determined whether lactose exposure on the basolateral membrane of mouse MECs adversely affects milk production in vitro. Restricted exposure to lactose on the basolateral side of the MECs was performed using a culture model, in which MECs on the cell culture insert exhibit milk production and less-permeable TJs. The results indicated that lactose exposure on the basolateral side inhibited casein and lipid production in the MECs. Interestingly, lactose exposure on the apical side did not show detectable effects on milk production in the MECs. Basolateral lactose exposure also caused the inactivation of STAT5, a primary transcriptional factor for milk production. Furthermore, p38 and JNK were activated by basolateral lactose exposure. The activation of p38 and JNK following anisomycin treatment reduced phosphorylated STAT5, and inhibitors of p38 blocked the reduction of phosphorylated STAT5 by basolateral lactose exposure. These findings suggest that lactose functions as a partial inhibitor for milk production but only when it directly makes contact with the basolateral membrane of MECs.
  • Haruka Wakasa, Yusaku Tsugami, Taku Koyama, Liang Han, Takanori Nishimura, Naoki Isobe, Ken Kobayashi
    Journal of mammary gland biology and neoplasia 27 (2) 155 - 170 2022/05/17 [Refereed]
     
    In the mammary glands during pregnancy, the alveolar buds are first branched from the mammary ducts after which they form the alveolar luminal structure for milk production postparturition. Body temperature could increase for several reasons, such as infectious disease and heat stress. We have previously reported that high temperature adversely effects on the lactation capacity of mouse mammary epithelial cells (MECs). However, it remains unclear how high temperature influences mammary morophogenesis during pregnancy. In this study, we investigated the effects of high temperature on this mammary alveolar development process using two types of culture models including embedded organoids of MECs in Matrigel; these models reproduced mammary alveolar bud induction and alveolar luminal formation. Results showed that a culture temperature of 41 °C repressed alveolar bud induction and inhibited alveolar luminal formation. In addition, the treatment at 41 °C decreased the number of proliferating mammary epithelial cells but did not affect cell migration. Levels of phosphorylated Akt, -ERK1/2, -HSP90, and -HSP27 were increased in organoids cultured at 41 °C. The specific inhibitors of HSP90 and HSP27 exacerbated the disruption of organoids at 41 °C but not at 37 °C. Furthermore, the organoids precultured at 37 and 41 °C in the alveolar luminal formation model showed differences in the expression levels of caseins and tight junction proteins, which express in MECs in lactating mammary glands, after induction of MEC differentiation by prolactin and dexamethasone treatment in vitro. These results suggest that elevated temperature directly hinders mammary alveolar development; however, heat shock proteins may mitigate the adverse effects of high temperatures.
  • Emi Ichimura, Koichi Ojima, Susumu Muroya, Ken Kobayashi, Takanori Nishimura
    FEBS open bio 12 (4) 852 - 863 2022/04 [Refereed]
     
    Myosin plays a fundamental role in muscle contraction. Approximately 300 myosins form a bipolar thick filament, in which myosin is continuously replaced by protein turnover. However, it is unclear how rapidly this process occurs and whether the myosin exchange rate differs depending on the region of the thick filament. To answer this question, we first measured myosin release and insertion rates over a short period and monitored myotubes expressing a photoconvertible fluorescence protein-tagged myosin, which enabled us to monitor myosin release and insertion simultaneously. About 20% of myosins were replaced within 10 min, while 70% of myosins were exchanged over 10 h with symmetrical and biphasic alteration of myosin release and insertion rates. Next, a fluorescence pulse-chase assay was conducted to investigate whether myosin is incorporated into specific regions in the thick filament. Newly synthesized myosin was located at the tip of the thick filament rather than the center in the first 7 min of pulse-chase labeling and was observed in the remainder of the thick filament by 30 min. These results suggest that the myosin replacement rate differs depending on the regions of the thick filament. We concluded that myosin release and insertion occur concurrently and that myosin is more frequently exchanged at the tip of the thick filament.
  • Yusaku Tsugami, Haruka Wakasa, Manabu Kawahara, Takanori Nishimura, Ken Kobayashi
    Animal Science Journal 93 (1) e13720  1344-3941 2022/01 [Refereed]
     
    Dairy cows feed on isoflavones as physiologically active substances present in legumes. However, the influences of isoflavones (biochanin A, genistein, formononetin, and daidzein) and their metabolites (p-ethylphenol and equol) on milk components production, tight junctions (TJs), and their regulatory pathways are unclear in bovine mammary epithelial cells (BMECs). In this study, we investigated the influences of isoflavones and their metabolites in BMECs using an in vitro culture model. The influences of isoflavones on milk components production, TJ proteins, and STAT5/STAT3 signaling pathways were different in a type-specific manner. Biochanin A decreased the mRNA expression and secretion of both β-casein and lactoferrin while a decrease in activated STAT5 and an increase in activated STAT3. In contrast, equol increased claudin-3, which is the main components for less-permeable TJs in lactation, while an increase in activated STAT5. In addition, a mixture of multiple isoflavones based on the intake of red clover increased secretion of lactoferrin, mRNA expression of β-casein, and amount of claudin-3, but a mixture based on soy did not affect the BMECs. Thus, these results indicate that isoflavones in legumes and the metabolic activity of isoflavones in dairy cows when feeding legumes may affect the milk production ability in BMECs.
  • Ken Kobayashi, Kota Matsunaga, Yusaku Tsugami, Haruka Wakasa, Takanori Nishimura
    Experimental cell research 409 (2) 112938 - 112938 2021/12/15 [Refereed]
     
    In lactating mammary glands, alveolar mammary epithelial cells (MECs) produce milk and form less-permeable tight junctions (TJs). However, alveolar TJs are weakened with a reduction in milk production in mammary glands due to mastitis or weaning in the presence of high levels of IL-1β, IL-6, or TNF-α. In this study, using in vitro cultured model of MECs with milk-producing ability and lactation-specific TJs, we investigated whether the aforementioned cytokines affect MEC TJs. The results showed that TNF-α, IL-1β, and IL-6 affected lactation-specific TJs in different ways. In particular, upon activation of p38 and JNK signalling, IL-1β caused rapid disruption of TJs at tricellular contact points. IL-1β treatment led to decreased CLDN3, CLDN4, and OCLN levels and a weakened TJ barrier. The adverse effects of IL-1β on TJs were mimicked by anisomycin, which is an activator of p38 and JNK signalling, and were blocked by MEC pretreatment with a p38 inhibitor but not a JNK inhibitor. The mislocalization of tricellulin at tricellular contact areas was confirmed in MECs treated with IL-1β or anisomycin. These results indicate that IL-1β is a key cytokine that adversely affects the TJs between MECs by activating p38.
  • Yusaku Tsugami, Yuki Ishiba, Naoki Suzuki, Takahiro Nii, Ken Kobayashi, Naoki Isobe
    Journal of mammary gland biology and neoplasia 26 (4) 387 - 397 2021/12 [Refereed]
     
    Heat stress and mastitis adversely affect milk production in dairy ruminants. Although the udder temperature is elevated in both conditions, the influence of this local temperature rise on milk production and immune function of ruminant mammary glands remains unclear. To address this question, we heated the mammary glands of goats by covering one half of the udder with a disposable heating pad for 24 h, the other uncovered half served as a control. Sixteen Tokara goats (1-5 parity) and three Shiba goats (1-2 parity) at the mid-lactation stage were individually housed, fed 0.6 kg of hay cubes and 0.2 kg of barley per day, and had free access to water and trace-mineralized salt blocks. Milk samples were collected every 6 h for 24 h after covering (n = 16), and deep mammary gland tissue areas were collected after 24 h (n = 5). The concentrations of antimicrobial components [lactoferrin, β-defensin-1, cathelicidin-2, cathelicidin-7, and immunoglobulin A (IgA)] in milk were measured by the enzyme-linked immunosorbent assay (ELISA). The localization of IgA was examined by immunohistochemistry. The mRNA expression and protein concentrations of C-C motif chemokine ligand-28 (CCL-28) and interleukin (IL)-8 in the mammary gland tissue were measured using quantitative polymerase chain reaction and ELISA, respectively. The somatic cell count in milk was significantly higher in the local heat-treatment group than in the control group after 12 h of treatment. The treatment group had significantly higher concentrations of cathelicidin-2 and IgA than the control group after 24 h of treatment. In addition, the number of IgA-positive cells in the mammary stromal region and the concentration of CCL-28 in the mammary glands were increased by local heat treatment. In conclusion, a local rise in udder temperature enhanced the innate immune function in mammary glands by increasing antimicrobial components.
  • Hideki Miwa, Ken Kobayashi, Shinobu Hirai, Mitsuhiko Yamada, Masahiko Watanabe, Haruo Okado, Yuchio Yanagawa
    The Cerebellum 21 (6) 905 - 919 2021/10/21 [Refereed]
  • Yusaku Tsugami, Haruka Wakasa, Takanori Nishimura, Ken Kobayashi
    ACS omega 6 (35) 22765 - 22772 2021/09/07 [Refereed]
     
    Genistein is a soy isoflavone and shows various physiological activities, such as affinities for estrogen receptors (ERs) and inhibitory effects on the epidermal growth factor receptor (EGFR) pathway. A previous study reported that genistein downregulates milk production ability in mammary epithelial cells (MECs) while decreasing the phosphorylation of STAT5. The ER and EGFR pathways indirectly regulate STAT5. In this study, the repressing mechanism of genistein against the phosphorylation of STAT5 was investigated using a culture model of mouse MECs with milk production ability. The results revealed that genistein did not influence the behavior of ERα and ERβ, whereas genistein immediately repressed the phosphorylation of ERK1/2. However, the decrease in phosphorylated STAT5 occurred independent of the phosphorylation of EGFR. Genistein repressed new phosphorylation of STAT5 by prolactin without influencing the phosphorylation of JAK2. In conclusion, this study indicates that genistein directly inhibits the phosphorylation of STAT5 in lactating MECs.
  • Emi Ichimura, Koichi Ojima, Susumu Muroya, Takahiro Suzuki, Ken Kobayashi, Takanori Nishimura
    Physiological reports 9 (17) e15003  2021/09 [Refereed]
     
    Myosin, the most abundant myofibrillar protein in skeletal muscle, functions as a motor protein in muscle contraction. Myosin polymerizes into the thick filaments in the sarcomere where approximately 50% of embryonic myosin (Myh3) are replaced within 3 h (Ojima K, Ichimura E, Yasukawa Y, Wakamatsu J, Nishimura T, Am J Physiol Cell Physiol 309: C669-C679, 2015). The sarcomere structure including the thick filament is maintained by a balance between protein biosynthesis and degradation. However, the involvement of a protein degradation system in the myosin replacement process remains unclear. Here, we show that the muscle-specific ubiquitin ligase Ozz regulates replacement rate of Myh3. To examine the direct effect of Ozz on myosin replacement, eGFP-Myh3 replacement rate was measured in myotubes overexpressing Ozz by fluorescence recovery after photobleaching. Ozz overexpression significantly decreased the replacement rate of eGFP-Myh3 in the myofibrils, whereas it had no effect on other myosin isoforms. It is likely that ectopic Ozz promoted myosin degradation through increment of ubiquitinated myosin, and decreased myosin supply for replacement, thereby reducing myosin replacement rate. Intriguingly, treatment with a proteasome inhibitor MG132 also decreased myosin replacement rate, although MG132 enhanced the accumulation of ubiquitinated myosin in the cytosol where replaceable myosin is pooled, suggesting that ubiquitinated myosin is not replaced by myosin in the myofibril. Collectively, our findings showed that Myh3 replacement rate was reduced in the presence of overexpressed Ozz probably through enhanced ubiquitination and degradation of Myh3 by Ozz.
  • Takahiro Suzuki, Aika Mori, Takahiro Maeno, Rio Arimatsu, Emi Ichimura, Yuriko Nishi, Kouga Hisaeda, Yuki Yamaya, Ken Kobayashi, Mako Nakamura, Ryuichi Tatsumi, Koichi Ojima, Takanori Nishimura
    International journal of molecular sciences 22 (9) 2021/04/26 [Refereed]
     
    Resident myogenic stem cells (satellite cells) are attracting attention for their novel roles in myofiber type regulation. In the myogenic differentiation phase, satellite cells from soleus muscle (slow fiber-abundant) synthesize and secrete higher levels of semaphorin 3A (Sema3A, a multifunctional modulator) than those derived from extensor digitorum longus (EDL; fast fiber-abundant), suggesting the role of Sema3A in forming slow-twitch myofibers. However, the regulatory mechanisms underlying fast-twitch myotube commitment remain unclear. Herein, we focused on netrin family members (netrin-1, -3, and -4) that compete with Sema3A in neurogenesis and osteogenesis. We examined whether netrins affect fast-twitch myotube generation by evaluating their expression in primary satellite cell cultures. Initially, netrins are upregulated during myogenic differentiation. Next, we compared the expression levels of netrins and their cell membrane receptors between soleus- and EDL-derived satellite cells; only netrin-1 showed higher expression in EDL-derived satellite cells than in soleus-derived satellite cells. We also performed netrin-1 knockdown experiments and additional experiments with recombinant netrin-1 in differentiated satellite cell-derived myoblasts. Netrin-1 knockdown in myoblasts substantially reduced fast-type myosin heavy chain (MyHC) expression; exogenous netrin-1 upregulated fast-type MyHC in satellite cells. Thus, netrin-1 synthesized in EDL-derived satellite cells may promote myofiber type commitment of fast muscles.
  • Yusaku Tsugami, Haruka Wakasa, Manabu Kawahara, Takanori Nishimura, Ken Kobayashi
    Experimental cell research 400 (2) 112472 - 112472 2021/03/15 [Refereed]
     
    Lipopolysaccharide (LPS) and lipoteichoic acid (LTA) are cell wall components of Escherichia coli and Staphylococcus aureus, which cause clinical and subclinical mastitis, respectively. However, the reason of the difference in symptoms by pathogen type remains unclear. In this study, the influence of LPS and LTA on early response and milk production in lactating bovine mammary epithelial cells (BMECs) was comparatively investigated. The results showed that LPS decreased the secretion of β-casein, lactose, and triglycerides, whereas LTA decreased the secretion of lactose and triglycerides but increased lactoferrin production without any influence on β-casein secretion. In addition, the influence of milk lipid droplet size in BMECs and gene expression related to milk fat synthesis was different between LPS and LTA. LPS increased the gene expression of interleukin (IL)-1β, tumor necrosis factor-α, and IL-8 through the activation of the nuclear factor-κB (NF-κB), p38, and c-Jun N-terminal kinase pathways, whereas LTA increased IL-1β and CC chemokine ligand 5 expression through the activation of the NF-κB pathway. Moreover, these cytokines and chemokines differently affected the milk production ability of BMECs. These results suggested that the pathogen-specific symptoms may be related to the differences in the early response of BMECs to bacterial toxins.
  • Yusaku Tsugami, Haruka Wakasa, Manabu Kawahara, Atsushi Watanabe, Takahiro Suzuki, Takanori Nishimura, Ken Kobayashi
    Cell and tissue research 384 (2) 435 - 448 2021/01/12 [Refereed]
     
    Mastitis causes a decrease in milk yield and abnormalities in milk components from dairy cows. Escherichia coli and the E. coli lipopolysaccharide (LPS) cell wall component directly downregulate milk production in bovine mammary epithelial cells (BMECs). However, the detailed mechanism by which this occurs in BMECs remains unclear. Various membrane proteins, such as immune sensors (Toll-like receptors, TLR), nutrient transporters (glucose transporter and aquaporin), and tight junction proteins (claudin and occludin) are involved in the onset of mastitis or milk production in BMECs. In this study, we investigated the influence of LPS on membrane proteins using an in vitro culture model. This mastitis model demonstrated a loss of glucose transporter-1 and aquaporin-3 at lateral membranes and a decrease in milk production in response to LPS treatment. LPS disrupted the tight junction barrier and caused compositional changes in localization of claudin-3 and claudin-4, although tight junctions were maintained to separate the apical membrane domains and the basolateral membrane domains. LPS did not significantly affect the expression level and subcellular localization of epidermal growth factor receptor in lactating BMECs with no detectable changes in MEK1/2-ERK1/2 signaling. In contrast, NFκB was concurrently activated with temporal translocation of TLR-4 in the apical membranes, whereas TLR-2 was not significantly influenced by LPS treatment. These findings indicate the importance of investigating the subcellular localization of membrane proteins to understand the molecular mechanism of LPS in milk production in mastitis.
  • Ken Kobayashi, Yusaku Tsugami, Norihiro Suzuki, Takahiro Suzuki, Takanori Nishimura
    Phytomedicine : international journal of phytotherapy and phytopharmacology 80 153360 - 153360 2021/01 [Refereed][Not invited]
     
    BACKGROUND: Curcumin is a naturally occurring polyphenol found in Curcuma longa with multiple therapeutic properties, such as anti-inflammatory, wound healing and anti-cancer effects. Curcuma longa is also used as a galactagogue to improve milk production during lactation. PURPOSE: To assess curcumin could have therapeutic potential for breastfeeding mothers, we investigated whether and how curcumin influences milk production in lactating mammary epithelial cells (MECs) at the cellular and molecular levels. METHODS: We prepared a lactating MEC culture model that produced milk components and formed less-permeable tight junctions (TJs) to investigate the molecular mechanism of curcumin on milk production, TJs, and inflammation in vitro. RESULTS: Curcumin downregulated milk production in lactation MECs concurrently with inactivation of lactogenesis-relating signaling (STAT5 and glucocorticoid receptor). The maintenance of a less-permeable TJ barrier was also confirmed, although the TJ protein claudin-4 increased. Curcumin inactivated NFκB and STAT3 signaling, which are closely involved in inflammatory responses in weaning and mastitis mammary glands. The expression levels of IL-1β and TNF-α were also decreased by curcumin treatment. Furthermore, curcumin blocked activation of inflammatory signaling by lipopolysaccharide treatment in MECs, similar to those in MECs that were treated with diclofenac sodium. The drastic phosphorylation of ERK was induced by curcumin treatment in the absence of EGF. U0126, an inhibitor of ERK phosphorylation, attenuated the adverse effects of curcumin on lactating MECs. CONCLUSION: The results of the present study suggests that curcumin downregulates milk production via inactivation of STAT5 and GR signaling with concurrent suppression of inflammatory responses via STAT3 and NFκB signaling in MECs. These findings provide new insights into the role of curcumin as a mild suppressor of milk production without inflammatory damages in breastfeeding mothers.
  • Shota Yamamura, Nanami Goda, Hiroki Akizawa, Nanami Kohri, Ahmed Z Balboula, Ken Kobayashi, Hanako Bai, Masashi Takahashi, Manabu Kawahara
    Developmental biology 468 (1-2) 14 - 25 2020/12/01 [Refereed][Not invited]
     
    A mammalian embryo experiences the first cell segregation at the blastocyst stage, in which cells giving form to the embryo are sorted into two lineages; trophectoderm (TE) and inner cell mass (ICM). This first cell segregation process is governed by cell position-dependent Hippo signaling, which is a phosphorylation cascade determining whether Yes-associated protein 1 (YAP1), one of the key components of the Hippo signaling pathway, localizes within the nucleus or cytoplasm. YAP1 localization determines the transcriptional on/off switch of a key gene, Cdx2, required for TE differentiation. However, the control mechanisms involved in YAP1 nucleocytoplasmic shuttling post blastocyst formation remain unknown. This study focused on the mechanisms involved in YAP1 release from TE nuclei after blastocoel contraction in bovine blastocysts. The blastocysts contracted by blastocoel fluid aspiration showed that the YAP1 translocation from nucleus to cytoplasm in the TE cells was concomitant with the protruded actin cytoskeleton. This YAP1 release from TE nuclei in the contracted blastocysts was prevented by actin disruption and stabilization. In contrast, Y27632, which is a potent inhibitor of Rho-associated coiled-coil containing protein kinase 1/2 (ROCK) activity, was found to promote YAP1 nuclear localization in the TE cells of contracted blastocysts. Meanwhile, lambda protein phosphatase (LPP) treatment inducing protein dephosphorylation could not prevent YAP1 release from TE nuclei in the contracted blastocysts, indicating that YAP1 release from TE nuclei does not depend on the Hippo signaling pathway. These results suggested that blastocyst contraction causes YAP1 release from TE nuclei through actin cytoskeleton remodeling in a Hippo signaling-independent manner. Thus, the present study raised the possibility that YAP1 subcellular localization is controlled by actin cytoskeletal organization after the blastocyst formation. Our results demonstrate diverse regulatory mechanisms for YAP1 nucleocytoplasmic shuttling in TE cells.
  • Norihiro Suzuki, Yusaku Tsugami, Haruka Wakasa, Takahiro Suzuki, Takanori Nishimura, Ken Kobayashi
    Molecular nutrition & food research e2000853  2020/11/14 [Refereed]
     
    SCOPE: Peppermint is traditionally used as an antigalactagogue in breastfeeding women. However, the suppressive mechanism remains unclear. The authors investigate whether and how peppermint influences milk production at the cellular and molecular levels. METHODS AND RESULTS: A lactating mammary epithelial cell (MEC) culture model that produces major milk components is prepared. Peppermint oil (PMO) suppresses β-casein production in conjunction with the induced enlargement of cytoplasmic lipid droplets (CLDs). PMO also significantly inactivates STAT5 and mTOR in the lactogenic signaling pathway. Menthol treatment, which is a primary PMO component, leads to decreases in β-casein production, enlarged CLDs, the inactivated STAT5 and mTOR. Eucalyptol and menthyl acetate, other components of peppermint, does not show suppressive effects on lactating MECs. The inactivation of STAT5 and mTOR upon menthol administration is also evident in alveolar MECs of lactating mice. Furthermore, lactating MECs expresses TRPM8 and TRPA1, which are menthol receptors known as cold receptors. Agonists of TRPM8 and TRPA1 suppresses β-casein production and inactivation of STAT5 and mTOR in the lactating MECs. CONCLUSION: These findings indicate that peppermint has potential as an antigalactagogue. Menthol is suggested to be an active compound in peppermint. TRPM8 and TRPA1 may function as receptors for menthol.
  • Shiori Tsutsui, Haruka Wakasa, Yusaku Tsugami, Takahiro Suzuki, Takanori Nishimura, Ken Kobayashi
    Journal of mammary gland biology and neoplasia 25 (3) 219 - 232 2020/09 [Refereed][Not invited]
     
    The mammary gland structurally and functionally remodels during pregnancy, during lactation and after weaning. There are three types of fibrillar collagens, types I, III, and V, in mammary stromal tissue. While the importance of the fibrillar structure of collagens for mammary morphogenesis has been suggested, the expression patterns of each type of fibrillar collagen in conjunction with mammary remodeling remain unclear. In this study, we investigated their expression patterns during pregnancy, parturition, lactation and involution. Type I collagen showed a well-developed fibril structure during pregnancy, but the fibrillar structure of type I collagen then became sparse at parturition and during lactation, which was concurrent with the downregulation of its mRNA and protein levels. The well-developed fibrillar structure of type I collagen reappeared after weaning. On the other hand, type V collagen showed a well-developed fibrillar structure and upregulation in the lactation period but not in the periods of pregnancy and involution. Type III collagen transiently developed a dense fibrillar network at the time of parturition and exhibited drastic increases in mRNA expression. These results indicate that each type of fibrillar collagen is distinctly involved in structural and functional remodeling in mammary glands during pregnancy, parturition, lactation, and involution after weaning. Furthermore, in vitro studies of mammary epithelial cells showed regulatory effects of type I collagen on cell adhesion, cell proliferation, ductal branching, and β-casein secretion. Each type of fibrillar collagen may have different roles in defining the cellular microenvironment in conjunction with structural and functional mammary gland remodeling.
  • Ryoyo Ikebuchi, Maika Fujimoto, Taiki Moriya, Yutaka Kusumoto, Ken Kobayashi, Michio Tomura
    Journal of reproductive immunology 140 103137 - 103137 0165-0378 2020/08 [Refereed][Not invited]
     
    Immune cells are present in the breast milk of several mammalian species; however, their immunological function and transmigration mechanisms to milk remain unknown. Some researchers hypothesize that milk leukocytes have a mammary gland (MG) origin and transmigrate thorough the paracellular pathway, but mammary alveolar epithelial cells strictly regulate the paracellular movement of milk components during lactation via barrier structures, such as tight junctions (TJs). To investigate this discrepancy, we compared leukocyte populations in mouse MG and milk and explored TJ protein expression profiles in MG leukocytes. The main subsets of milk leukocytes were CD8+ and CD4+ T cells displaying the memory phenotype. The proportions of myeloid, B, and dendritic cells were significantly lower in milk than in the MG. CD8+ T cells expressed genes encoding the TJ proteins claudin-3, -7, -12, and ZO-1 at higher levels when compared with myeloid and B cells in the MG among lactating mice. Alveolar epithelial cells in the MG expressed claudin-3, -4, and -7. Administration of FTY720, an inhibitory agonist of sphingosine 1-phosphate receptor 1 that stabilizes TJ permeability, increased the myeloid cell proportion in milk. Different leukocyte populations in the MG and milk suggest active and selective mechanisms of cell transmigration to milk. Both TJ-forming components in alveolar epithelial cells from the MG and TJ protein expression profiles in leukocytes from the MG appear to regulate milk leukocyte populations. T cells are the main population in mouse breast milk and express similar profiles of TJ proteins as those in mammary alveolar epithelial cells.
  • Yoshiki Kaihoko, Yusaku Tsugami, Norihiro Suzuki, Takahiro Suzuki, Takanori Nishimura, Ken Kobayashi
    Cell and tissue research 380 (3) 513 - 526 0302-766X 2020/06 [Refereed][Not invited]
     
    Milk osmolarity maintains an isotonic status for suckling infants during lactation. However, it remains unclear how the water content in milk is regulated in lactating mammary glands. In lactating mammary alveoli and ducts, mammary epithelial cells (MECs) are in direct contact with milk. In this study, we focus on two types of water channels, aquaporin 3 (AQP3) and AQP5, in alveolar and ductal MECs before and after parturition. AQP3 showed diffuse localization in the cytoplasm of ductal MECs but concentrated localization in the basolateral membrane of alveolar MECs during the late pregnancy and lactation periods. Translocation of AQP5 from the cytoplasm toward the apical membrane occurred in ductal MECs immediately before parturition. Subsequently, we examined the hormonal influences on the expression of AQP3 and AQP5 in cultured MECs in vitro. Progesterone and estrogen distinctly increased AQP3 and AQP5 in cultured MECs, respectively. Cotreatment with prolactin and dexamethasone significantly decreased both AQP3 and AQP5. Prolactin also facilitated the translocation of AQP5 into the apical membrane of MECs. In cultured MECs, AQP3 was homogeneously expressed in MECs, whereas AQP5 showed different expression levels between MECs regardless of the hormonal treatment. Different activation states of the prolactin/STAT5 pathway were also observed between ductal and alveolar MECs. These findings suggest that the expression pattern of AQP3 and AQP5 is distinctly regulated by lactogenic hormones in alveolar and ductal MECs before and after parturition. AQP5 expressed in ductal MECs may function as a water channel to regulate milk osmolarity in mice.
  • Yusaku Tsugami, Norihiro Suzuki, Takahiro Suzuki, Takanori Nishimura, Ken Kobayashi
    Journal of agricultural and food chemistry 68 (21) 5847 - 5853 0021-8561 2020/05/27 [Refereed][Not invited]
     
    Soy products contain abundant genistein and daidzein isoflavones. Orally ingested soy isoflavones are partially metabolized to isoflavan by enteric bacteria. Their levels in the blood increase after soy products are eaten. In this study, we investigated the influence of genistein, daidzein, and equol by intraperitoneal administration in lactating mice. Genistein decreased the secretion of α- and β-casein and downregulated the gene expression of Csn1, Csn2, Csn3, and Wap while inactivating the signal transducer and activator of transcription 5 (STAT5) and ERK1/2. In contrast, equol increased Csn1-3 expression while inactivating STAT3. Daidzein did not show inhibitory effects on milk production. The effects of genistein and equol were also confirmed in lactating mammary epithelial cells (MECs), which were cultured in the presence of soy isoflavones and equol at physiological concentrations for 7 days. These findings indicate that genistein, daidzein, and equol influence milk production in MECs in vivo and in vitro in distinctly different ways.
  • Aogu Kumai, Yusaku Tsugami, Haruka Wakasa, Norihiro Suzuki, Takahiro Suzuki, Takanori Nishimura, Ken Kobayashi
    Advanced biosystems 4 (4) e1900187 - 1900187 2366-7478 2020/04 [Refereed][Not invited]
     
    Isoflavones are a class of flavonoids present in legumes and are called phytoestrogens because of their estrogen-like activity. Endogenous estrogen is well known to regulate mammary gland morphogenesis during pregnancy. Each isoflavone also has different physiological activities. However, it is difficult to investigate the direct effect of each isoflavone in mammary morphogenesis in vivo because isoflavones are metabolized into different isoflavones by enteric bacteria. In this study, investigated are the direct influences of coumestrol, daidzein, and genistein on mammary structure development and future milk production ability of mammary epithelial cells (MECs) using in vitro culture models. Mouse MECs are cultured in Matrigel with basic fibroblast growth factor and epidermal growth factor to induce ductal branching and alveolar formation, respectively. Coumestrol and genistein inhibit ductal branching and alveolar formation by affecting the proliferation and migration of MECs with the induction of apoptosis. Daidzein hardly influences mammary structure development. Furthermore, pretreatment with coumestrol adversely affects the induction of milk production ability of MECs. These results suggest that each isoflavone differentially influences mammary morphogenesis and future milk production by affecting MEC behaviors. These results also suggest that the culture models are effective to study mammary epithelial morphogenesis in vitro.
  • Kobayashi, K., Tsugami, Y., Suzuki, N., Suzuki, T., and Nishimura, T.
    Toxicology in Vitro 63 104741 - 104741 2020/03 [Refereed][Not invited]
     
    Nicotine from tobacco smoke is absorbed into the bloodstream and transferred into breast milk in breastfeeding mothers. Smoking causes a decrease in breast milk volume, adverse changes to the milk composition, and a shortened lactation period. Breast milk is produced by mammary epithelial cells (MECs) in mammary glands during lactation. However, it remains unclear whether nicotine directly affects milk production in lactating MECs. To address this issue, we prepared a culture model with high milk production ability and less-permeable tight junctions (TJs) by seeding mouse MECs on a cell culture insert. Lactating MECs showed expression of α2, α3, β2, and β4 of nicotinic acetylcholine receptors. The high concentration of nicotine at 10-100 μM inhibited β-casein secretion and caused abnormal localization of TJ proteins. We subsequently investigated whether nicotine at a physiological concentration could affect lactating MECs. Nicotine at 1.0 μM directly inhibited α- and β-casein secretion in lactating MECs concurrently with inactivation of STAT5 and glucocorticoid receptor without affecting the TJ barrier. Nicotine treatment also induced MEC apoptosis concurrently with inactivation of Akt. These results support the adverse effects of nicotine on breastfeeding in smoking mothers.
  • Yusaku Tsugami, Norihiro Suzuki, Manabu Kawahara, Takahiro Suzuki, Takanori Nishimura, Ken Kobayashi
    Animal Science Journal 91 (1) 1344-3941 2020/01 [Refereed][Not invited]
  • Kangmin Seo, Takahiro Suzuki, Ken Kobayashi, Takanori Nishimura
    Animal Science Journal 90 (3) 423 - 434 1344-3941 2019/03 [Refereed][Not invited]
  • Potential of Gouda cheese whey to improve epidermal conditions by regulating proliferation and differentiation of keratinocytes
    Shinagawa F, Takata S, Toba Y, Ikuta M, Hioki S, Suzuki T, Nishimura T, Nakamura R, Kobayashi K
    International Dairy Journal 87 100 - 106 2018/12 [Refereed][Not invited]
  • Kumura Haruto, Ohtsuyama Takeru, Matsusaki Yoh-hey, Taitoh Miho, Koyanagi Haruka, Kobayashi Ken, Hayakawa Toru, Wakamatsu Jun-ichi, Ishizuka Satoshi
    JOURNAL OF FOOD PROCESSING AND PRESERVATION 42 (10) 0145-8892 2018/10 [Refereed][Not invited]
  • IL-1β directly inhibits milk lipid production in lactating mammary epithelial cells concurrently with enlargement of cytoplasmic lipid droplets
    Matsunaga K, Tsugami Y, Kumai A, Suzuki T, Nishimura T, Kobayashi K
    Experimental Cell Research 370 (2) 365 - 372 2018/09 [Refereed][Not invited]
  • Ken Kobayashi, Yusaku Tsugami, Kota Matsunaga, Takahiro Suzuki, Takahiro Nishimura
    Journal of Mammary Gland Biology and Neoplasia 23 (1-2) 75 - 88 1083-3021 2018/06 [Refereed][Not invited]
  • Hiroki Akizawa, Ken Kobayashi, Hanako Bai, Masashi Takahashi, Shinjiro Kagawa, Hiroaki Nagatomo, Manabu Kawahara
    Reproduction 155 (6) 563 - 571 1470-1626 2018/06 [Refereed][Not invited]
     
    The first segregation at the blastocyst stage is the symmetry-breaking event to characterize two cell components; namely, inner cell mass (ICM) and trophectoderm (TE). TEA domain transcription factor 4 (TEAD4) is a well-known regulator to determine TE properties of blastomeres in rodent models. However, the roles of bovine TEAD4 in blastocyst development have been unclear. We here aimed to clarify the mechanisms underlining TE characterization by TEAD4 in bovine blastocysts. We first found that theTEAD4mRNA expression level was greater in TE than in ICM, which was further supported by TEAD4 immunofluorescent staining. Subsequently, we examined the expression patterns of TE-expressed genes;CDX2,GATA2andCCN2, in theTEAD4-knockdown (KD) blastocysts. These expression levels significantly decreased in theTEAD4KD blastocysts compared with controls. Of these downregulated genes, theCCN2expression level decreased the most. We further analyzed the expression levels of TE-expressed genes;CDX2,GATA2andTEAD4in theCCN2KD blastocysts. Strikingly, theCCN2KD blastocysts showed the downregulation ofCDX2,GATA2andTEAD4. Furthermore, the ratio of TE-to-ICM cell numbers in theCCN2KD blastocysts significantly decreased compared to controls. To our knowledge, this is the first study showing the regulation ofCCN2expression thoroughTEAD4in mammalian embryos. Not only that, this study also provides evidence that reciprocal regulation ofTEAD4andCCN2is required for TE development with appropriate gene expression in bovine blastocysts.
  • Yusaku Tsugami, Kota Matsunaga, Takahiro Suzuki, Takanori Nishimura, Ken Kobayashi
    JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 65 (50) 11118 - 11124 0021-8561 2017/12 [Refereed][Not invited]
     
    During lactation, mammary epithelial cells (MECs) form the blood-milk barrier by less-permeable tight junctions (TJs) to prevent the leakage of milk components. Phytoestrogens affect the proliferation, differentiation, and apoptosis of MECs. However, it remains unclear whether phytoestrogens are involved in the blood-milk barrier. Therefore, we investigated the influence of phytoestrogens (coumestrol, genistein, and daidzein) by using an in vitro mouse-MEC-culture model. The results showed that coumestrol and genistein changed the expression of TJ proteins (claudins-3 and -4 and occludin), weakened barrier function, and reduced beta-casein production. Daidzein also weakened barrier function without inhibiting beta-casein production. Additionally, coumestrol and genistein induced apoptosis in MECs. These results indicate that phytoestrogens weaken the blood-milk barrier by directly affecting TJs and the cellular viability of lactating MECs in different ways.
  • Yusaku Tsugami, Kota Matsunaga, Takahiro Suzuki, Takanori Nishimura, Ken Kobayashi
    MOLECULAR NUTRITION & FOOD RESEARCH 61 (10) 201700156  1613-4125 2017/10 [Refereed][Not invited]
     
    Scope: Isoflavones are a class of polyphonic compounds present in legumes and are called phytoestrogens because of their estrogen-like activity. Estrogen influences the behavior of mammary epithelial cells (MECs) during pregnancy and lactation. In this study, we investigated the direct influences of isoflavones and their metabolites in milk production ability of MECs. Methods and results: Mouse MECs were cultured with prolactin and dexamethasone (glucocorticoid analog) to induce milk production ability. Subsequently, lactating MECs were treated with each isoflavone. Coumestrol, biochanin A, genistein, and formononetin decreased the intracellular and secreted beta-casein. On the other hand, p-ethylphenol, daidzein, and equol did not significantly influence beta-casein production at any concentration. Coumestrol, biochanin A and genistein down-regulated the mRNA expression of whey acidic protein (WAP), lactoferrin and alpha-lactalbumin. In contrast, p-ethylphenol, daidzein and equol up-regulated beta-casein and/or WAP with alpha-lactalbumin. Furthermore, coumestrol and genistein down-regulated the expression of prolactin receptor and signal transducer and activator of transcription 5 (STAT5) accompanied by a decrease in STAT5 phosphorylation. Conclusion: Isoflavones and their metabolites influence the milk production ability of MECs through different interactions with prolactin/STAT5 signaling. Simultaneous intake of multiple isoflavones by consumption of legumes may induce promotive or adverse effects on lactating MECs.
  • Heidi K. Baumgartner, Michael C. Rudolph, Palaniappian Ramanathan, Valerie Burns, Patricia Webb, Benjamin G. Bitler, Torsten Stein, Ken Kobayashi, Margaret C. Neville
    JOURNAL OF MAMMARY GLAND BIOLOGY AND NEOPLASIA 22 (2) 141 - 157 1083-3021 2017/06 [Refereed][Not invited]
     
    Claudins are a large family of membrane proteins whose classic function is to regulate the permeability of tight junctions in epithelia. They are tetraspanins, with four alpha-helices crossing the membrane, two extracellular loops, a short cytoplasmic N-terminus and a longer and more variable C-terminus. The extracellular ends of the helices are known to undergo side-to-side (cis) interactions that allow the formation of claudin polymers in the plane of the membrane. The extracellular loops also engage in head-to-head (trans) interactions thought to mediate the formation of tight junctions. However, claudins are also present in intracellular structures, thought to be vesicles, with less well-characterized functions. Here, we briefly review our current understanding of claudin structure and function followed by an examination of changes in claudin mRNA and protein expression and localization through mammary gland development. Claudins-1, 3, 4, 7, and 8 are the five most prominent members of the claudin family in the mouse mammary gland, with varied abundance and intracellular localization during the different stages of post-pubertal development. Claudin-1 is clearly localized to tight junctions in mammary ducts in non-pregnant non-lactating animals. Cytoplasmic puncta that stain for claudin-7 are present throughout development. During pregnancy claudin-3 is localized both to the tight junction and basolaterally while claudin-4 is found only in sparse puncta. In the lactating mouse both claudin-3 and claudin-8 are localized at the tight junction where they may be important in forming the paracellular barrier. At involution and under challenge by lipopolysaccharide claudins -1, -3, and -4 are significantly upregulated. Claudin-3 is still colocalized with tight junction molecules but is also distributed through the cytoplasm as is claudin-4. These largely descriptive data provide the essential framework for future mechanistic studies of the function and regulation of mammary epithelial cell claudins.
  • Haruto Kumura, Chiharu Saito, Yuko Taniguchi, Taiki Machiya, Yutaroh Takahashi, Ken Kobayashi, Akira Kimura
    Advances in Dairy Research 05 (03) 2017 [Refereed]
  • Ken Kobayashi, Shoko Oyama, Chinatsu Kuki, Yusaku Tsugami, Kota Matsunaga, Takahiro Suzuki, Takanori Nishimura
    MOLECULAR AND CELLULAR ENDOCRINOLOGY 440 (C) 16 - 24 0303-7207 2017/01 [Refereed][Not invited]
     
    Beta-casein is a secretory protein contained in milk. Mammary epithelial cells (MECs) synthesize and secrete beta-casein during lactation. However, it remains unclear how the beta-casein secretion pathway is developed after parturition. In this study, we focused on prolactin (PRL), epidermal growth factor (EGF), and glucocorticoids, which increase in blood plasma and milk around parturition. MECs cultured with PRL, EGF and dexamethasone (DEX: glucocorticoid analog) developed the beta-casein secretion pathway. In the absence of PRL, MECs hardly expressed beta-casein. EGF enhanced the expression and secretion of beta-casein in the presence of PRL and DEX. DEX treatment rapidly increased secreted beta-casein concurrent with enhancing beta-casein expression. DEX also up-regulated the expression of SNARE proteins, such as SNAP-23, VAMP-8 and Syntaxin-12. Furthermore, PRL and DEX regulated the expression ratio of alpha(st-), beta- and kappa-casein. These results indicate that PRL, EGF and glucocorticoids have distinct roles in the establishment of beta-casein secretion pathway. (C) 2016 Elsevier Ireland Ltd. All rights reserved.
  • Ken Kobayashi, Yusaku Tsugami, Kota Matsunaga, Shoko Oyama, Chinatsu Kuki, Haruto Kumura
    BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 1863 (8) 2006 - 2016 0167-4889 2016/08 [Refereed][Not invited]
     
    Alveolar mammary epithelial cells (MECs) in mammary glands are highly specialized cells that produce milk for suckling infants. Alveolar MECs also form less permeable tight junctions (TJs) to prevent the leakage of milk components after parturition. In the formation process of less permeable TJs, MECs show a selective downregulation of Cldn4 and a localization change of Cldn3. To investigate what induces less permeable TJs through these compositional changes in Cldns, we focused on two lactogenesis-related hormones: prolactin (Prl) and glucocorticoids. Prl caused a downregulation of Cldn3 and Cldn4 with the formation of leaky TJs in MECs in vitro. Prl-treated MECs also showed low beta-casein expression with the activation of STAT5 signaling. By contrast, dexamethasone (Dex), a glucocorticoid analogue, upregulated Cldn3 and Cldn4, concurrent with the formation of less permeable TJs and the activation of glucocorticoid signaling without the expression of beta-casein. Cotreatment with Prl and Dex induced the selective downregulation of Cldn4 and the concentration of Cldn3 in the region of Tjs concurrent with less permeable TJ formation and high beta-casein expression. The inhibition of Prl secretion by bromocriptine in lactating mice induced the upregulation of Cldn3 and Cldn4 concurrent with the downregulation of milk production. These results indicate that the coactivation of Prl and glucocorticoid signaling induces lactation-specific less permeable TJs concurrent with lactogenesis. (C) 2016 Elsevier B.V. All rights reserved.
  • Keisuke Sasaki, Aiko Tanaka, Hiroaki Nagatomo, Hidehiko Ogawa, Ken Kobayashi, Masashi Takahashi, Manabu Kawahara
    Journal of Genital System & Disorders 05 (01) 2325-9728 2016 [Refereed][Not invited]
  • Ken Kobayashi, Chinatsu Kuki, Shoko Oyama, Haruto Kumura
    Experimental Cell Research 340 (2) 295 - 304 0014-4827 2016/01 [Refereed][Not invited]
  • Jennifer E. Kloepper, Olivier R. Baris, Karen Reuter, Ken Kobayashi, Daniela Weiland, Silvia Vidali, Desmond J. Tobin, Catherin Niemann, Rudolf J. Wiesner, Ralf Paus
    JOURNAL OF INVESTIGATIVE DERMATOLOGY 135 (3) 679 - 689 0022-202X 2015/03 [Refereed][Not invited]
     
    Here, we studied how epithelial energy metabolism impacts overall skin development by selectively deleting intraepithelial mtDNA in mice by ablating a key maintenance factor (Tfam(EKO)), which induces loss of function of the electron transport chain (ETC). Quantitative (immuno)histomorphometry demonstrated that Tfam(EKO) mice showed significantly reduced hair follicle (HF) density and morphogenesis, fewer intrafollicular keratin15+ epithelial progenitor cells, increased apoptosis, and reduced proliferation. Tfam(EKO) mice also displayed premature entry into (aborted) HF cycling by apoptosis-driven HF regression (catagen). Ultrastructurally, Tfam(EKO) mice exhibited severe HF dystrophy, pigmentary abnormalities, and telogen-like condensed dermal papillae. Epithelial HF progenitor cell differentiation (Plet1, Lrig1 Lef1and beta-catenin), sebaceous gland development (adipophilin, Scd1, and oil red), and key mediators/markers of epithelial mesenchymal interactions during skin morphogenesis (NCAM, versican, and alkaline phosphatase) were all severely altered in Tfam(EKO) mice. Moreover, the number of mast cells, major histocompatibility complex class II+, or CD11b+ immunocytes in the skin mesenchyme was increased, and essentially no subcutis developed. Therefore, in contrast to their epidermal counterparts, pilosebaceous unit stem cells depend on a functional ETC. Most importantly, our findings point toward a frontier in skin biology: the coupling of HF keratinocyte mitochondrial function with the epithelial-mesenchymal interactions that drive overall development of the skin and its appendages.
  • Takaaki Uejyo, Chinatsu Kuki, Shoko Oyama, Haruto Kumura, Ken Kobayashi
    CELL AND TISSUE RESEARCH 359 (2) 643 - 653 0302-766X 2015/02 [Refereed][Not invited]
     
    The mammary gland is a highly specialized organ that is able to repeat development and regression (involution) of alveolar structures for milk production. Mammary involution consists in two phases. The first phase is reversible and lasts until approximately 48 h after weaning in mice. Interestingly, an extended milking interval can change the milk-secretory activity of alveolar epithelial cells (AECs) before the first phase of involution begins. In this study, we investigate the changes in the ability of AECs to secrete milk during the involution progression. Careful observation of the number and locations of cleaved caspase-3 positive AECs revealed that the first phase of involution occurred approximately 24 h after weaning and the second phase began between 48 and 72 h after weaning. However, initial changes in the milk production ability of AECs began just 1 h after weaning and milk production gradually ceased within 24 h. In addition, activation of STAT3 and inactivation of STAT5 had occurred in some AECs by 6 h after weaning and more broadly by 24 h. In addition, milk production processes such as nutrient uptake, synthesis, and secretion ceased by 24 h post-weaning. Interestingly, enlarged cytoplasmic lipid droplets were observed in AECs 12 h after weaning even though the expression levels of genes relevant to triglyceride production (Srebp1 and AQP3) were down-regulated. These results indicate that several changes in the milk production ability of AECs occur during expanded suckling intervals and prior to involution.
  • Shota Miyata, Yozo Oda, Chika Matsuo, Haruto Kumura, Ken Kobayashi
    JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 62 (49) 11854 - 11861 0021-8561 2014/12 [Refereed][Not invited]
     
    Propolis is a natural honeybee hive product with the potential for use in the treatment of dermatological conditions, such as cutaneous abrasions, burns, and acne. In this study, we investigated whether propolis stimulates hair growth in mice. Ethanol-extracted propolis, which contains various physiologically active substances such as caffeic acid and kaempferol, stimulated anagen induction in shaved back skin. Anagen induction occurred without any detectable abnormalities in the shape of the hair follicles (HFs), hair stem cells in the bulge, proliferating hair matrix keratinocytes in the hair bulb, or localization of versican in the dermal papilla. Propolis treatment also stimulated migration of hair matrix keratinocytes into the hair shaft in HFs during late anagen in the depilated back skin. Organotypic culture of skin containing anagen stage HFs revealed significant stimulation of hair matrix keratinocyte proliferation by propolis. Furthermore, propolis facilitated the proliferation of epidermal keratinocytes. These results indicate that propolis stimulates hair growth by inducing hair keratinocyte proliferation.
  • Ken Kobayashi, Shoko Oyama, Takaaki Uejyo, Chinatsu Kuki, Md Morshedur Rahman, Haruto Kumura
    VETERINARY RESEARCH 44 119  0928-4249 2013/12 [Refereed][Not invited]
     
    Mastitis, the inflammation of mammary glands resulting from bacterial infection, disrupts milk production in lactating mammary glands. In this study, we injected lipopolysaccharide (LPS), one of the endotoxins from Escherichia coli into mouse mammary glands to disrupt milk production, and we investigated the influence of LPS on nutrient uptake, synthesis, and secretion processes for milk component production in alveolar epithelial cells (AEC). The expression of genes relevant to the three-staged milk component production process (nutrient uptake, synthesis, and secretion of milk components) were down-regulated within 12 h after LPS injection in AEC. The internalization of glucose transporter 1 (GLUT-1) from the basolateral membrane to the cytoplasm occurred in accordance with the down-regulation of gene expression 3 h after LPS injection. The abnormal localization of adipophilin and beta-casein was also observed in the LPS-injected mammary glands. SLC7A1, an amino acid transporter, was up-regulated 3 and 6 h after LPS injection. Furthermore, the inactivation of signal transducer and activator of transcription 5 (STAT5) and the activation of STAT3 and nuclear factor-kappa B (NFkappaB) occurred 3 h after LPS injection. These results indicate that the nutrient uptake, synthesis, and secretion of milk components in AEC are rapidly shut down in the lactating mammary glands after LPS injection.
  • Ken Kobayashi, Takaaki Uejyo, Shoko Oyama, Md. Morshedur Rahman, Haruto Kumura
    Cell and Tissue Research 354 (2) 495 - 506 0302-766X 2013/11 [Refereed][Not invited]
     
    The mammary alveolus is a highly specialized structure that secretes milk for suckling infants during lactation. The secreting alveolus consists in alveolar epithelial cells (AECs) and myoepithelial cells and is surrounded by microvascular endothelial cells, adipocytes and several immune cell types such as macrophages and neutrophils. During normal lactation, these cells play distinct roles needed to maintain the secretory ability of the mammary alveolus. However, inflammation resulting from pathogenic bacterial infections causes structural and functional regression of the secreting alveolus in the lactating mammary gland. We initiated artificial inflammation in the mammary glands of lactating mice by injecting lipopolysaccharide (LPS), as a mammary inflammation model and investigated, by immunohistochemical analysis, the early response of the cells constituting and surrounding the alveolus. Some AECs sloughed away from the alveolar epithelial layer and showed progression of apoptosis detected by immunostaining of cleaved caspase-3 after LPS injection. Adipocytes exhibited transient shrinkage and re-accumulation of lipid droplets, although the numbers of adipocytes did not demonstrate a significant difference. Activation of F4/80-positive cells around the mammary alveolus was observed 3 h after LPS injection. However, the recruitment of CD11b-positive cells into the alveolar lumen was not observed until 12 h after LPS injection. Myoepithelial cells were contracted after LPS injection. LPS injection around the alveolus did not induce any detectable structural changes in capillaries surrounding the alveolus. Thus, cell-specific behavior and tissue remodeling of the alveolus occur after LPS injection in a time-dependent manner. © 2013 Springer-Verlag Berlin Heidelberg.
  • Marta Bertolini, Katja C. Meyer, Radomir Slominski, Ken Kobayashi, Ralf J. Ludwig, Ralf Paus
    EXPERIMENTAL DERMATOLOGY 22 (9) 593 - 598 0906-6705 2013/09 [Refereed][Not invited]
     
    Although vibrissae hair follicles (VHFs) have long been a key research model in the life sciences, their immune system (IS) is essentially unknown. Therefore, we have characterized basic parameters of the VHF-IS of C57BL/6J mice by quantitative (immuno-)histomorphometry. Murine anagen VHF harbour few CD4+ and CD8+ T cells in the distal mesenchyme and sinuses but hardly any gamma-delta T cells in their distal epithelium. MHC class II+ Langerhans cells are seeded in the VHF infundibulum, which is also surrounded by MHC class II+ and CD11b+ cells (macrophages). The number of Langerhans cells then declines sharply in the VHF bulge, and the VHF bulb lacks MHC class II+ cells. Mast cells densely populate the VHF connective tissue sheath, where they strikingly cluster around the bulge. Both the bulge and the bulb of VHF display signs of immune privilege, that is, low MHC class I and MHC class II expression and local immunoinhibitor expression (CD200, TGF beta 1). This immunophenotyping study fills an important gap in the immunobiology of murine skin and identifies differences between the IS of VHF, mouse pelage and human terminal HFs. This facilitates utilizing murine VHF as a versatile organ culture model for general immunology and immune privilege research in situ.
  • Ken Kobayashi, Shoko Oyama, Atsushi Numata, Md. Morshedur Rahman, Haruto Kumura
    PLoS ONE 8 (4) e62187  1932-6203 2013/04/23 [Refereed][Not invited]
     
    Mastitis, inflammation of the mammary gland, is the most costly common disease in the dairy industry, and is caused by mammary pathogenic bacteria, including Escherichia coli. The bacteria invade the mammary alveolar lumen and disrupt the blood-milk barrier. In normal mammary gland, alveolar epithelial tight junctions (TJs) contribute the blood-milk barrier of alveolar epithelium by blocking the leakage of milk components from the luminal side into the blood serum. In this study, we focused on claudin subtypes that participate in the alveolar epithelial TJs, because the composition of claudins is an important factor that affects TJ permeability. In normal mouse lactating mammary glands, alveolar TJs consist of claudin-3 without claudin-1, -4, and -7. In lipopolysaccharide (LPS)-induced mastitis, alveolar TJs showed 2-staged compositional changes in claudins. First, a qualitative change in claudin-3, presumably caused by phosphorylation and participation of claudin-7 in alveolar TJs, was recognized in parallel with the leakage of fluorescein isothiocyanate-conjugated albumin (FITC-albumin) via the alveolar epithelium. Second, claudin-4 participated in alveolar TJs with claudin-3 and claudin-7 12 h after LPS injection. The partial localization of claudin-1 was also observed by immunostaining. Coinciding with the second change of alveolar TJs, the severe disruption of the blood-milk barrier was recognized by ectopic localization of β-casein and much leakage of FITC-albumin. Furthermore, the localization of toll-like receptor 4 (TLR4) on the luminal side and NFκB activation by LPS was observed in the alveolar epithelial cells. We suggest that the weakening and disruption of the blood-milk barrier are caused by compositional changes of claudins in alveolar epithelial TJs through LPS/TLR4 signaling. © 2013 Kobayashi et al.
  • H. Kumura, T. Sawada, Y. Oda, M. Konno, K. Kobayashi
    JOURNAL OF DAIRY SCIENCE 95 (7) 3629 - 3633 0022-0302 2012/07 [Refereed][Not invited]
     
    Among the lipids in bovine milk, minor components such as conjugated linoleic acids and phospholipids are more attractive than triacylglycerols from the standpoint of biological activity. To explore novel functions of bovine milk polar lipids (MPL), topical application to murine dorsal skin was introduced as an assay system. The acetone-insoluble lipid fraction derived from bovine milk was dispersed in ethanol and applied to 9-wk-old C57BL/6N female mice for 3 wk. In combination with visual assessment of the dorsal pigmentation, the progression of the hair cycle was estimated by calculating the ratio of subcutis to dermis thickness. The administration of MPL led to earlier progression of the hair cycle compared with administration of the vehicle. In some cases, the extent of MPL-induced hair cycle progression was comparable to that in animals treated with minoxidil, the most well-known reagent that initiates anagen. These results indicate that the MPL preparation contains a dermal penetrative component that can regulate the hair cycle and, thus, this preparation possesses potential for cosmetic use.
  • Ken Kobayashi, Haruto Kumura
    HISTOCHEMISTRY AND CELL BIOLOGY 136 (5) 587 - 594 0948-6143 2011/11 [Refereed][Not invited]
     
    Tight junctions (TJs) are the most apical junctional complexes and restrict the fluid flux through the paracellular pathway. In the mammary glands, the tightness of TJs occurs shortly after parturition to prevent the leakage of milk components from the lumen and the loosening of TJs is induced immediately after weaning. Claudins are transmembrane proteins, and their composition at the apical-most regions determines the permeability of TJs. In this study, we investigated the localization and expression patterns of claudin-3 and -4 in the mammary glands around the lactation period because it is unclear how claudins construct mammary TJs in the apical-most regions. Our results showed that claudin-3 and -4 change not only their level of expression but also their localization in the processes of parturition, lactation, and weaning. Claudin-3 was concentrated in the apical-most regions during lactation, whereas claudin-4 gradually decreased at the beginning of lactation and increased drastically immediately after weaning. The qualitative change of claudin-3 was also identified by western blotting analysis as an additional band around the lactation period. In addition, parts of the mammary epithelial cells showed intensive positive reactions to claudin-4 in the lateral membrane and cytoplasm after weaning, concurrently with the involution mammary glands. These results indicate that claudin-3 in the apical-most regions maintains the impermeable TJs during lactation, and claudin-4 contributes to the permeability changes of TJs immediately after parturition and weaning.
  • Ken Kobayashi, Hideki Miwa, Masato Yasui
    MOLECULAR AND CELLULAR ENDOCRINOLOGY 337 (1-2) 36 - 42 0303-7207 2011/04 [Refereed][Not invited]
     
    The amniotic epithelium is in direct contact with the amniotic fluid and restricts fluid flux via the paracellular pathway by means of tight junctions (TJs). Several factors affect TJs to modulate the paracellular flux. Progesterone contributes to the antenatal formation and disappearance of TJs in uterine and mammary epithelial tissues. In this study, we investigated whether progesterone positively or negatively influences amniotic TJs. The administration of RU-486, a progesterone receptor (PR) antagonist, into pregnant mice adversely affects the localization and expression of claudin-3 and claudin-4 in the amniotic epithelium. RU-486 administration also increased the permeability of the amniotic membrane. In organ-cultured amniotic membranes, progesterone induced increases in claudin-3 and claudin-4 expression in a dose-dependent manner but did not influence their localization. PRs were also present in the amniotic epithelium during midpregnancy but they disappeared during late pregnancy. These results indicate that the progesterone/PR pathway maintains TJs in the amniotic epithelium during midpregnancy. (C) 2011 Elsevier Ireland Ltd. All rights reserved.
  • Ken Kobayashi, Kazuo Umezawa, Masato Yasui
    BIOLOGY OF REPRODUCTION 84 (2) 248 - 254 0006-3363 2011/02 [Refereed][Not invited]
     
    The amniotic epithelium is in direct contact with the amniotic fluid and has tight junctions. The amniotic tight junctions function as a barrier to restrict fluid flux via the amniotic membrane during midpregnancy in the mouse. However, during late pregnancy, amniotic fluid volume significantly decreases in association with the disruption of amniotic tight junctions. The disruption of amniotic tight junctions is caused by apoptosis in the amniotic epithelium on Embryonic Day 17 (E17). In this study, we examine the molecular mechanisms underlying apoptosis of the amniotic epithelium of the mouse. We found that from E16, the number of activated macrophages that express high levels of NOS2 and tumor necrosis factor (TNF) increase in amniotic fluid. TNF receptor type 1 (TNFR1) was detectable from E16 onward. On E17, amniotic epithelial cells expressing TNFR1 became TUNEL positive, suggesting that TNF/TNFR1 signaling may initiate apoptosis. To further confirm the role of TNF/TNFR1 signaling, WP9QY, a TNFR1 antagonist, was injected into the amniotic cavity and was found to significantly reduce the numbers of apoptotic cells in the E17 amniotic epithelium. Furthermore, dehydroxymethylepoxyquinomicin, a specific nuclear factor-kappa B inhibitor, was found to inhibit TNF production in macrophages and amniotic apoptosis in vivo. Finally, we showed that injection of TNF into the amniotic cavity induces early onset of apoptosis. These results indicate that amniotic apoptosis is induced by the TNF pathway via TNFR1 expressed in the amniotic epithelial cells and that activation of macrophages may trigger amniotic apoptosis.
  • Ken Kobayashi, Hideki Miwa, Masato Yasui
    JOURNAL OF PHYSIOLOGY-LONDON 588 (24) 4859 - 4869 0022-3751 2010/12 [Refereed][Not invited]
     
    In chorioamnionitis, intra-amniotic infections render the amniotic fluid an adverse environment for the fetus and increase the risk of fetal mortality and morbidity. It remains unclear how infection crosses the amniotic barrier, which is made up of tight junctions (TJs). In this study, we investigated whether amniotic TJs are disrupted in inflammatory conditions such as chorioamnionitis. Amniotic TJs were disrupted by single applications of interleukin (IL)-1 beta, IL-6, tumour necrosis factor-alpha (TNF-alpha), and prostaglandin E2. In organ-cultured amniotic membranes, these inflammatory mediators decreased the claudin-3 and claudin-4 levels at the apical junction at different times. Injecting IL-6 into the amniotic cavity concurrently induced the disruption of amniotic TJs by decreasing the claudin-3 and claudin-4 levels at the apical junction, and the dysfunction of the amniotic barrier; in contrast, injecting TNF-alpha weakened the amniotic barrier by inducing apoptosis of the amniotic epithelial cells, with no decrease in claudin-3 and claudin-4 at the apical junction. Furthermore, inflammation in the amniotic membrane, which was induced by the administration of lipopolysaccharide to pregnant mice, concurrently caused dysfunction of the amniotic barrier and disruption of TJs, involving the decrease of claudin-3 and claudin-4 levels at the apical junction and apoptosis in the amniotic epithelium. These results indicate that the adverse effects of the inflammatory mediators on amniotic TJs cause severe dysfunction of the amniotic barrier.
  • Ken Kobayashi, Masato Yasui
    CELL AND TISSUE RESEARCH 342 (2) 307 - 316 0302-766X 2010/11 [Refereed][Not invited]
     
    The amniotic membrane encloses the amniotic fluid and plays roles in the regulation of amniotic fluid flux through the intramembranous pathway during pregnancy. Aquaporins (AQPs) 1, 3, 8, and 9 are expressed in amniotic membranes. AQPs are water channel proteins that facilitate the rapid flux of water or small molecules across the plasma membrane. Recently, additional roles of AQPs in facilitating cell migration, proliferation, and apoptosis have been suggested, with AQPs being distributed in the appropriate subcellular regions for their functions. The cellular and subcellular distributions of AQPs in the amniotic membrane however remain unclear. We have examined the cellular and subcellular localization of AQPs in amniotic membranes during pregnancy in mice. After embryonic day 12 (El 2), AQP1 was distributed in the plasma membrane of finely branched cell processes in the amniotic fibroblasts. AQP3 was present in both epithelial cells and fibroblasts between El and E12. The distribution of AQP3 in the epithelial cells dynamically changed as follows: at El4 in the lateral membrane and apical junction; at El 6 in the lateral membrane alone; at El 7 in the lateral membrane and cytoplasm. AQP8 was expressed in the epithelial cells and complementarily localized in the apical junction and the lateral membrane. AQP9 was detected only in the apoptotic cells of the epithelium. These cellular and subcellular localizations of amniotic AQPs indicate that each AQP plays distinct functional roles, such as in water and urea transport, cell migration, cell proliferation, and apoptosis, for amniotic fluid homeostasis or tissue remodeling of amniotic membranes.
  • Ken Kobayashi, Teruhisa Suzuki, Yukio Nomoto, Yasuhiro Tada, Masao Miyake, Akihiro Hazama, Ikuo Wada, Tatsuo Nakamura, Koichi Omori
    BIOMATERIALS 31 (18) 4855 - 4863 0142-9612 2010/06 [Refereed][Not invited]
     
    In some types of tracheal disease, tracheal resection is required. For patients with tracheal resection, artificial grafts, made from collagen sponge with a spiral polypropylene stent and mesh, have been clinically used by our group. However, epithelial regeneration was confirmed to be slow. In the present study, we investigated the potential of gingival fibroblasts (GFBs) and adipose-derived stem cells (ASCs) as autologous transplanted cells in combination with artificial graft for tracheal epithelial regeneration. In in vitro co-culturing with tracheal epithelial cells, GFBs stimulated epithelial cell differentiation and reconstruction of a pseudostratified epithelium. ASCs stimulated epithelial cell proliferation and reconstruction of a multi-layered epithelium. Subsequently, we prepared three kinds of bioengineered scaffolds from GFBs and/or ASCs and implanted them into rat tracheal defects. The bioengineered scaffolds containing GFBs were covered with tracheal epithelial cells after 1 week, and highly ciliated epithelium was formed after 2 weeks of transplantation. The bioengineered scaffold containing ASCs induced thick epithelium, and then pseudostratified epithelium containing goblet cells was formed. Furthermore, the application of both GFBs and ASCs had synergistic effects on tracheal epithelial regeneration, suggesting that bioengineered scaffolds containing GFBs and ASCs are useful for hastening tracheal epithelial regeneration. (C) 2010 Elsevier Ltd. All rights reserved.
  • K. Kobayashi, I. Kadohira, M. Tanaka, Y. Yoshimura, K. Ikeda, M. Yasui
    PLACENTA 31 (2) 158 - 162 0143-4004 2010/02 [Refereed][Not invited]
     
    Amnion is the innermost layer of the fetal membrane and has been suggested to regulate the volume of amniotic fluid via the amniotic epithelium The transepithelial pathway is generally restricted by tight junctions (TJs) Thus far, human amniotic TJs have not been identified. In this study, we determined whether the human amniotic epithelium contains TJs Reverse transcription polymerase chain reaction (RT-PCR) and western blotting analyses showed that the human amniotic epithelium has TJ components, Such as occludin, ZO-1, and at least 2 types of claudins, i e, claudin-4 and claudin-7 The TJ components were found to localize in the lateral membranes and cytoplasm at 35 weeks of gestation; these components disappeared from the lateral membrane at 37 weeks of gestation Organ culturing of the amnion at 37 weeks gestation induced the relocalization of the TJ proteins from the cytoplasm to the lateral membranes Furthermore, in cultured amniotic epithelial cells, dexamethasone induced the downregulation of the protein expression of TJs. These findings suggest that the human amniotic epithelium has TJs that disrupt during late pregnancy. The disruption may be induced by several factors such as glucocorticoids present in the amniotic fluid during late pregnancy (C) 2009 Elsevier Ltd All rights reserved.
  • Wataru Okano, Yukio Nomoto, Ikuo Wada, Ken Kobayashi, Masao Miyake, Tatsuo Nakamura, Koichi Omori
    ANNALS OF OTOLOGY RHINOLOGY AND LARYNGOLOGY 118 (11) 796 - 804 0003-4894 2009/11 [Refereed][Not invited]
     
    Objectives: Although our group has had mostly successful results with clinical application of a tracheal prosthesis, delayed epithelial regeneration remains a problem. In our previous studies using rats, it was demonstrated that tracheal fibroblasts accelerated proliferation and differentiation of the tracheal epithelium in vitro and in vivo. The purpose of this study was to evaluate the effects of fibroblasts on epithelial regeneration in larger tracheal defects in rabbits. Methods: We developed a bioengineered scaffold, the luminal surface of which was coated with fibroblasts. This scaffold was implanted into tracheal defects in 12 rabbits (bioengineered group), and scaffolds without fibroblasts were implanted in 12 rabbits (control group). The regenerated epithelium was histologically examined by light microscopy, scanning electron microscopy, and immunohistochemical studies. Results: In the bioengineered group, a stratified squamous epithelium was observed on the surface 7 days after transplantation. However, in the control group, the scaffolds were exposed. Fourteen days after implantation, a columnar ciliated epithelium was observed in the bioengineered group. The average thickness of the regenerated epithelium in the bioengineered group was significantly greater than that in the control group. Conclusions: This study indicated that fibroblasts had a stimulatory effect that hastened regeneration of the epithelium in large tracheal defects.
  • K. Kobayashi, T. Inai, Y. Shibata, M. Yasui
    PLACENTA 30 (10) 840 - 847 0143-4004 2009/10 [Refereed][Not invited]
     
    The amniotic membrane encloses and retains amniotic fluid during pregnancy. In general, fluid flux is regulated by epithelial tissues, which have tight junctions (TJs). However, TJs have not yet been identified in the amniotic epithelium. In this study, we have determined whether the mouse amniotic epithelium contains TJs. Freeze-fracture electron microscopy revealed the presence of strand-like TJs in the amniotic epithelium. Amniotic TJs were composed of occludin; zona occludens (ZO)-1; and claudins 1, 3, 4, and 7. These claudins underwent developmental changes during pregnancy. The localization patterns of the claudins and their detergent solubility drastically changed between embryonic day (E) 16 and E17; the volume of the amniotic fluid also decreased sharply. Furthermore, in vitro assessment of amniotic membrane permeability showed that the amniotic membrane was more permeable on E17 than on E16. On E17, TJ components were sparsely distributed in parts of the amniotic epithelium. The results of Annexin V-fluorescein staining and Terminal dUTP nick-end labeling (TUNEL) assay revealed ongoing apoptosis in all the cells in such regions. The above findings suggest that TJs in the amniotic epithelium maintain amniotic fluid volume during pregnancy, while apoptosis of amniotic epithelial cells between E16 and E17 causes disruption of the TJs. (C) 2009 Elsevier Ltd. All rights reserved.
  • Koichi Omori, Yasuhiro Tada, Teruhisa Suzuki, Yukio Nomoto, Takashi Matsuzuka, Ken Kobayashi, Tatsuo Nakamura, Shinichi Kanemaru, Masaru Yamashita, Ryo Asato
    ANNALS OF OTOLOGY RHINOLOGY AND LARYNGOLOGY 117 (9) 673 - 678 0003-4894 2008/09 [Refereed][Not invited]
     
    Objectives: The objective of the present study was to demonstrate the efficacy of the clinical application of in situ tissue engineering using a scaffolding technique for laryngeal and tracheal tissue. Methods: We have developed a tissue scaffold made from a Marlex mesh tube covered by collagen sponge. Based on successful animal experimental studies, in situ tissue engineering with a scaffold implant was applied to repair the larynx and trachea in 4 patients. Results: In 1 patient with subglottic stenosis, the thyroid cartilage, cricoid cartilage, and cervical trachea with scarring and a granulation were resected and reconstructed by use of the scaffold. In 3 patients with thyroid cancer, the trachea and cri coid cartilage with tumor invasion were resected and the scaffold was implanted into the defect. Postoperative endoscopy during the observation period of 8 to 34 months showed a well-epithelialized airway lumen without any obstruction. Conclusions: Our current technique of in situ tissue engineering using a scaffold shows great potential for use in the regeneration of airway defects.
  • Teruhisa Suzuki, Ken Kobayashi, Yasuhiro Tada, Yukie Suzuki, Ikuo Wada, Tatuo Nakamura, Koichi Omori
    ANNALS OF OTOLOGY RHINOLOGY AND LARYNGOLOGY 117 (6) 453 - 463 0003-4894 2008/06 [Refereed][Not invited]
     
    Objectives: Our group has developed and clinically applied an artificial graft made from a collagen sponge scaffold for the regeneration of tracheal tissue. However, the artificial graft requires about 2 months for epithelial regeneration. The purpose of the present study was to accelerate the regeneration process of the trachea through the effective use of a bioengineered scaffold. Adipose-derived stem cells (ASCs) with multilineage differentiation capability were used. In our study, we implanted a bioengineered scaffold that included autologous ASCs into tracheal defects in rats. Methods: Collagen gel, including ASCs labeled with monomeric yellow fluorescent protein, was layered onto the surface of the collagen sponge to form a bioengineered scaffold. This scaffold was implanted into the tracheal defects in rats. A control scaffold without ASCs was also implanted. Results: On day 14 after implantation, a pseudostratified columnar epithelium with well-differentiated ciliated and goblet cells and neovascularization was observed in rats that received the implant with the bioengineered scaffold that included ASCs. Conclusions: These results suggested that implanted ASCs accelerated neovascularization and epithelialization on the regenerated trachea. Thus, our newly developed bioengineered scaffold contributes,to tracheal regeneration.
  • Yasuhiro Tada, Teruhisa Suzuki, Toshiaki Takezawa, Yukio Nomoto, Ken Kobayashi, Tatuo Nakamura, Koichi Omori
    ANNALS OF OTOLOGY RHINOLOGY AND LARYNGOLOGY 117 (5) 359 - 365 0003-4894 2008/05 [Refereed][Not invited]
     
    Objectives: The purpose of the present study was to evaluate the effectiveness of a novel bipotential collagen scaffold as a bioengineered trachea for the regeneration of the tracheal epithelium.Methods: The bipotential collagen scaffold was developed by conjugating a collagen vitrigel membrane to a collagen sponge in order to promote both epithelial cell growth and mesenchymal cell infiltration. The bipotential collagen scaffold was transplanted into tracheal defects in rats, and a conventional collagen sponge was implanted as a control model. Histologic examinations were undertaken to evaluate the results.Results: The bioengineered trachea was covered with epithelium in the vitrigel model, but not in the control model, at 7 days after implantation. At 14 days after implantation, the bioengineered trachea was covered with epithelium involving the basal cell layer in the vitrigel model. At 28 days after implantation, a columnar ciliated epithelium was observed only in the vitrigel model.Conclusions: Our technique for trachea reconstruction using a novel bipotential collagen scaffold affords a feasible approach for accelerating epithelial regeneration on the intraluminal surface of the host tracheal defect.
  • Yukio Nomoto, Ken Kobayashi, Yasuhiro Tada, Ikuo Wada, Tatsuo Nakamura, Koichi Omori
    ANNALS OF OTOLOGY RHINOLOGY AND LARYNGOLOGY 117 (1) 59 - 64 0003-4894 2008/01 [Refereed][Not invited]
     
    Objectives: Our group applied a tracheal prosthesis, which was composed of polypropylene as the frame and collagenous sponge as the scaffold, to the first human case and had successful results. The objective of this study was to find a way to acquire more rapid re-epithelialization with fibroblasts on this tracheal prosthesis. Methods: Tracheal epithelial cells, which were isolated from the trachea of rats, were suspended in a collagenous gel. The collagenous gel with fibroblasts was layered on a collagenous sponge. The grafts of this "bioengineered trachea" were implanted into tracheal defects of rats, and the regenerated epithelium on the grafts was histologically examined. Results: Seven days after implantation, stratified squamous epithelium covered almost all of the surface of the gel, and some of the implanted fibroblasts in the gel were lined up just below the epithelium. Fourteen days after implantation, columnar and cuboidal ciliated epithelium covered almost all of the surface of the defects, and the implanted fibroblasts had disappeared. The numbers of regenerated epithelial cells at 14 days after implantation were larger than those of control models without fibroblasts, with statistical significance. Conclusions: The results suggested that the grafts of bioengineered trachea composed of collagenous sponge and collagenous gel with tracheal fibroblasts accelerated epithelial differentiation and proliferation in vivo.
  • Ken Kobayashi, Teruhisa Suzuki, Yukio Nomoto, Yasuhiro Tada, Masao Miyake, Akihiro Hazama, Tatsuo Nakamura, Koichi Omori
    TISSUE ENGINEERING 13 (9) 2175 - 2184 1076-3279 2007/09 [Refereed][Not invited]
     
    The tracheal epithelium maintains the health of the respiratory tract through mucociliary clearance and regulation of ion and water balance. When the trachea is surgically removed, artificial grafts have been clinically used by our group to regenerate the trachea. In such cases, the tracheal epithelium needs 2 months for functional regeneration. Previous study has shown that fibroblasts facilitate tracheal epithelial regeneration. In this study, heterotopic fibroblasts originating from the dermis, nasal, and gingival mucosa were cocultured with tracheal epithelial cells to evaluate their potential as autologous transplanted cells for tracheal epithelial regeneration. The epithelia induced by the heterotopic fibroblasts showed differences in structure, cilia development, mucin secretion, and expression of ion and water channels. These results indicated that nasal fibroblasts could not induce mature tracheal epithelium and that dermal fibroblasts induced epidermis-like epithelium. Only the gingival fibroblasts (GFBs) could induce morphologically and functionally normalized tracheal epithelium comparable to the epithelium induced by tracheal fibroblasts. Epithelial cell proliferation and migration were also upregulated by GFBs. These results indicate that GFBs are useful as autologous transplant cells for tracheal epithelial regeneration.
  • エゾシカ外皮の鞣しに伴う変化
    福永重治, 杉浦智樹, 小林 謙, 山川育生, 竹ノ内一昭, 中村富美男
    北海道畜産学会報 49 43 - 50 2007 [Refereed][Not invited]
  • Ken Kobayashi, Yukio Nomoto, Teruhisa Suzuki, Yasuhiro Tada, Masao Miyake, Akihiro Hazama, Shinichi Kanemaru, Tatsuo Nakamura, Koichi Omori
    TISSUE ENGINEERING 12 (9) 2619 - 2628 1076-3279 2006/09 [Refereed][Not invited]
     
    Several artificial grafts for covering deficient trachea have been produced through tissue engineering. Recently, our group clinically used an artificial trachea made from collagen sponge for patients with noncircumferential tracheal resection. However, the slowness of epithelial regeneration on the surface of the artificial trachea was confirmed as one particular problem. In this study, we co-cultured tracheal epithelial cells with fibroblasts and examined effects of fibroblasts on epithelial regeneration in vitro. Fibroblasts activated epithelial cell proliferation and migration. In co-culture with fibroblasts, epithelial cells reconstructed pseudostratified epithelium, which was composed of ciliated, goblet, and basal cells. Furthermore, a basement membrane was reconstructed between epithelial cells and fibroblasts, and integrin beta 4 was also observed there. Fibroblasts rapidly increased mucin secretion by epithelial cells. These results indicate that stimulatory effects of fibroblasts on epithelial cell migration, proliferation, and differentiation would reduce the time required for covering of epithelial cells on the defect of luminal surface and hasten regeneration of morphologically and functionally normalized epithelium involving the reconstruction of basement membrane.
  • Yukio Nomoto, Teruhisa Suzuki, Yasuhiro Tada, Ken Kobayashi, Masao Miyake, Akihiro Hazama, Ikuo Wada, Shinichi Kanemaru, Tatsuo Nakamura, Koichi Omori
    ANNALS OF OTOLOGY RHINOLOGY AND LARYNGOLOGY 115 (7) 501 - 506 0003-4894 2006/07 [Refereed][Not invited]
     
    Objectives: The slowness of epithelialization on the artificial trachea that has been successfully used in humans is a problem. The purpose of this study was to develop a way to regenerate the epithelium on the surface of this artificial trachea. Methods: In an in vitro study, isolated rat tracheal epithelial cells were seeded on a collagenous get that was stratified on a collagenous sponge. Histologic and immunohistochemical examinations were made. III an in vivo study, we transplanted grafts with green fluorescent protein-positive tracheal epithelial cells onto the tracheal defects of normal rats. At 3, 7. 14, and 30 days after the operation, histologic and immunohistochemical examinations were made. Results: In the in vitro study, the 3 layers - the epithelium, gel, and sponge - could be observed. The epithelium expressed cytokeratin 14, cytokeratin 18, and occludin. In the in vivo study, the artificial trachea was covered with epithelium at 3 days after operation, and then the epithelium differentiated from single- or double-stratified squamous epithelium into columnar ciliated epithelium. Green fluorescent protein-positive cells were found 3 days after operation. Conclusions: We believe that the method used in our experiment is an effective way to regenerate the epithelium on the surface of an artificial trachea. With further experimentation, this method should be suitable for clinical application.
  • コラーゲンの産生異常がニワトリ羽毛の初期発生に及ぼす影響
    小林 謙, 福永重治, 竹之内一昭, 加藤(森)ゆうこ, 中村富美男
    北海道畜産学会報 48 53 - 58 2006 [Refereed][Not invited]
  • ニワトリ初生羽の発生に伴う真皮コラーゲンの形態変化
    小林 謙, 福永重治, 竹之内一昭, 加藤(森)ゆうこ, 中村富美男
    北海道畜産学会報 47 65 - 71 2005 [Refereed][Not invited]
  • Ken Kobayashi, Shigeharu Fukunaga, Kazuaki Takenouchi, Yuko Kato-Mori, Fumio Nakamura
    Animal Science Journal 76 (3) 273 - 282 1344-3941 2005 [Refereed][Not invited]
     
    The regulatory function of type VI collagen during early feather development in embryonic chickens was investigated at the cellular and organ levels. Immunohistochemical studies of embryonic chicken skin showed that type VI collagen was distributed in spatial-specific and temporal-specific manners related to early feather development. To clarify the role of type VI collagen, we studied the feather development in intact, reconstituted and reconstituted gel skin cultures. When ethyl-3,4-dihydroxybenzoate (EDHB) was added to the medium of intact skin as an inhibitor of type VI collagen synthesis, the feather buds did not elongate and the number of neural cell adhesion molecule (NCAM)-positive cells was reduced. However, the magnitudes of both suppressive effects of EDHB were reduced by the addition of liquid type VI collagen. Similar improvement was also observed in the reconstituted skin with liquid type VI collagen and in the reconstituted gel skin with solid type VI collagen at a low concentration. Moreover, type VI collagen promoted feather bud development in the absence of EDHB. However, a high concentration of solid type VI collagen in the reconstituted gel skin arrested the feather bud elongation, and antitype VI collagen antibodies caused feather buds to become longer and smaller in the reconstituted skin. At the cellular level, type VI collagen affected the proliferation, migration and NCAM expression of mesenchymal cells. These results suggest that type VI collagen regulates early feather development by controlling mesenchymal cell behavior.
  • Ken Kobayashi, Shigeharu Fukunaga, Kazuaki Takenouchi, Fumio Nakamura
    Animal Science Journal 75 (5) 441 - 449 1344-3941 2004/10 [Refereed][Not invited]
     
    To clarify the role of fibronectin (FN) during the early period of feather development, reconstituted skin consisting of intact epithelium and isolated mesenchymal cells from embryonal chick skin was used. In early feather development, FN was localized around mesenchymal cells of the dermal condensation. Isolated mesenchymal cells had associated with FN on their surfaces. FN on the cell surface dissociated following EDTA treatment, and EDTA-treated cells re-associated with exogenous FN. The intact epithelium also bound to exogenous FN at the placode. When FN-associated or FN-reassociated mesenchymal cells were used, the reconstituted skin formed feather rudiments only at the positions where the epithelial placode existed originally, and the locality of tenascin transferred from the placode to the mesenchyme during the period of feather bud formation. However, in reconstituted skin using FN-dissociated mesenchymal cells, feather rudiments did not form. Additionally, the epithelial placodes disappeared, and tenascin was distributed uniformly on the surface of the epithelium and not localized in the mesenchyme. These findings suggest that FN associated on the surfaces of mesenchymal cells maintains the functions of mesenchymal cells as dermal condensation and mediates epithelial-mesenchymal interactions during the early period of feather development. The results also suggest that reconstituted skin is a useful tool for functional studies on the extracellular matrix.
  • ウシ脊髄における正常型細胞性プリオンタンパク質（PrPc）の局在
    中村富美男, 田中誠一, 小林 謙, 關 巌, 李 昇遠, 福永重治
    北海道畜産学会報 45 25 - 31 2003 [Refereed][Not invited]
  • Nakamura F, Seki I, Kobayashi K, Tanaka M, Fukunaga S
    Animal Science Journal 73 (6) 553 - 556 1344-3941 2002/12 [Refereed][Not invited]

MISC

Association Memberships

  • 日本乳房炎研究会   THE JAPANESE SOCIETY FOR REGENERATIVE MEDICINE   The American Society for Cell Biology   THE MOLECULAR BIOLOGY SOCIETY OF JAPAN   JAPANESE SOCIETY OF ANIMAL SCIENCE   

Research Projects

  • 物理的刺激を起点として乳腺上皮細胞の乳分泌を調節する機構の解明
    日本学術振興会：基盤研究（B）
    Date (from‐to) : 2024/04 -2028/03
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2023/06 -2025/03 
    Author : 小林 謙
  • 乳腺上皮細胞に発現する機械刺激受容体Piezo2が乳産生を制御する機構の解明
    秋山記念生命科学振興財団：研究助成(一般)
    Date (from‐to) : 2024/07 -2025/03
  • 物理化学的刺激を感知するTRPチャネルが乳腺組織の形態形成と乳分泌を制御する機構
    日本学術振興会：基盤研究（B）
    Date (from‐to) : 2021/04 -2024/03
  • 血流と搾乳を模した2系統の灌流培養システムを用いて母乳分泌調節性食品の同定と作用機序解明を培養下で可能にする方法の確立
    一般財団法人 糧食研究会：
    Date (from‐to) : 2024
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2021/07 -2023/03 
    Author : 小林 謙
     
    黄色ブドウ球菌は伝染性の乳房炎を引き起こす。黄色ブドウ球菌による乳房炎は難治性であり、その治療のための抗生物質の投与は黄色ブドウ球菌の薬剤耐性化を誘発する。そのため、黄色ブドウ球菌の乳房炎を効果的に治療する方策が求められている。しかし、黄色ブドウ球菌の乳房炎がなぜ難治性なのか、その感染戦略の全容はわかっていない。そこで本研究では、“黄色ブドウ球菌は乳腺上皮細胞に寄生し、その感染防御バリアを細胞内部から崩壊させることで難治性乳房炎を引き起こす”と仮説を立て、仮説を実証するため、以下の実験を行った。 まず、マウスから乳腺上皮細胞を単離し、cell culture insert上にコンフルエントになるまで培養した後、生体同様に乳産生とタイトジャンクション形成を行うよう、プロラクチンやデキサメタゾンを含む培地を用いて分化誘導した。この乳腺上皮細胞の培地に蛍光色素で標識した黄色ブドウ球菌を添加すると、培地中や乳腺上皮細胞のみならず、乳腺上皮細胞内にも侵入する様子が観察された。また、感染した細胞では細胞の形態が変化しており、アクチン線維のネットワークの変化が起きていた。続いて、細胞外の黄色ブドウ球菌を抗生物質で除去し、その3日後に乳腺上皮細胞の乳産生能力とタイトジャンクションバリア機能を評価した。その結果、代表的な乳タンパク質であるカゼインの産生量減少とタイトジャンクション構成タンパク質の質的変化が確認された。また、黄色ブドウ球菌由来毒素であるリポタイコ酸が培地中に存在した場合においても乳腺上皮細胞の乳産生能力とタイトジャンクションの変化が起きていた。 以上の結果より、黄色ブドウ球菌は細胞内外から乳腺上皮細胞の性質を変化させることが示唆された。
  • 乳腺上皮細胞のうま味受容体が母乳中のグルタミン酸濃度を感知する機構の解明
    日本うま味調味料協会技術部会：研究助成金
    Date (from‐to) : 2022 -2023
  • 泌乳期乳腺におけるアセチルコリンが乳腺上皮細胞の乳産生能力を調節する機構の解明
    公益財団法人 喫煙科学研究財団：一般研究助成
    Date (from‐to) : 2021 -2023
  • 辛味成分が乳腺上皮細胞のTRPA1を介して母乳分泌を調節する機構の解明
    公益財団法人 浦上食品・食文化振興財団：研究助成
    Date (from‐to) : 2023
  • 乳腺上皮細胞の灌流培養システムを用いた培養乳の生産に関する研究
    公益財団法人 寿原記念財団：研究助成
    Date (from‐to) : 2022
  • 泌乳期における非糖質甘味料の摂取が血液乳関門に及ぼす影響
    公益財団法人 日本食品化学研究振興財団：研究助成
    Date (from‐to) : 2022
  • 培養乳の持続的生産を可能にする乳腺上皮細胞の培養モデルに関する基礎研究
    公益財団法人 アサヒグループ学術振興財団：学術研究助成
    Date (from‐to) : 2022
  • 食品由来成分の母乳移行性を評価する血液乳関門モデルの作製
    一般財団法人 東洋水産財団：学術奨励金
    Date (from‐to) : 2022
  • 乳腺上皮細胞の温度応答性分子群が乳腺組織の形態形成と機能発現を制御する機構の解明
    日本学術振興会：基盤研究（B）
    Date (from‐to) : 2018/04 -2021/03 
    Author : 小林謙
  • マメ科植物に含有する多様な植物エストロゲンが泌乳期乳腺の血液乳関門に及ぼす影響
    一般財団法人 旗影会：研究助成
    Date (from‐to) : 2021
  • 甘味と旨味が乳腺上皮細胞の母乳分泌を調節する機構の解明
    公益財団法人 サッポロ生物科学振興財団：研究助成
    Date (from‐to) : 2021
  • 暑熱ストレスが乳分泌細胞の乳産生能力を低下させる機序の解明
    公益財団法人 栗林育英学術財団：研究助成
    Date (from‐to) : 2020
  • 乳腺上皮細胞に発現する甘味受容体TAS1R3が乳産生を制御する機構に関する研究
    公益財団法人 秋山記念生命科学振興財団：
    Date (from‐to) : 2020
  • 大豆イソフラボンとその代謝産物が母乳分泌に与える効果の検証
    公益財団法人 タカノ農芸化学研究助成財団：研究助成
    Date (from‐to) : 2020
  • 温度受容体であるTRPチャネルの活性化が線維芽細胞の皮膚恒常性維持機能へ及ぼす影響
    一般財団法人 ホーユー科学財団：研究助成
    Date (from‐to) : 2017 -2019
  • 食品由来成分が母乳分泌へ与える影響を数値化する培養モデルの開発
    公益財団法人 飯島藤十郎記念食品科学振興財団：学術研究助成
    Date (from‐to) : 2019
  • グルコースセンサーの不適切な活性化が授乳期の母乳トラブルを引き起こす機序の解明
    公益財団法人 三島海雲記念財団
    Date (from‐to) : 2019
  • 妊娠期における非糖質系甘味料の摂取が乳腺発達と分娩後の乳産生に及ぼす影響
    公益財団法人 日本食品化学研究振興財団：研究助成
    Date (from‐to) : 2019
  • ニコチンが乳腺上皮細胞の乳汁分泌能に及ぼす影響
    公益財団法人 喫煙科学研究財団：若手研究助成
    Date (from‐to) : 2016 -2018
  • 現代の食生活の中で母乳育児を改善するために母乳分泌調節性食品（Galactogogue）をスクリーニングする方法の確立とその実用性の検証
    一般財団法人 糧食研究会：研究助成
    Date (from‐to) : 2018
  • クルクミンが母乳産生を改善する効果の検証
    公益財団法人 山崎香辛料振興財団：研究助成
    Date (from‐to) : 2018
  • 母乳分泌を調節する食品成分の同定とその作用機構解明
    公益財団法人 食生活研究会：研究助成金
    Date (from‐to) : 2018
  • 温度感受性TRPチャネルを介した乳汁分泌調節機序に関する研究
    日本学術振興会：基盤研究 (C)
    Date (from‐to) : 2014/04 -2017/03 
    Author : 小林謙
  • マメ科植物に含有する多様な植物エストロゲンが乳腺発達と乳産生に及ぼす影響
    一般社団法人 旗影会：研究助成
    Date (from‐to) : 2017
  • 母乳分泌に影響を及ぼす生理活性物質の探索と作用メカニズムに関する研究
    公益社団法人 アサヒグループ学術振興財団：学術研究助成
    Date (from‐to) : 2017
  • レモングラスとペパーミントに含まれる有効成分が母乳分泌を調節する効果
    公益社団法人 日本アロマ環境協会：研究助成
    Date (from‐to) : 2017
  • 授乳期における非糖質系甘味料の摂取が乳腺上皮細胞の乳汁分泌能に及ぼす影響
    公益財団法人 日本食品化学研究振興財団：研究助成
    Date (from‐to) : 2016
  • ローヤルゼリーとプロポリスが発毛・育毛に及ぼす相乗効果の検証
    山田養蜂場：みつばち研究助成基金
    Date (from‐to) : 2013 -2015
  • 泌乳期乳腺の乳成分産生を調節するメントールに関する研究
    公益財団法人 戸部眞紀財団：研究助成
    Date (from‐to) : 2015
  • マメ科牧草に含まれるイソフラボンが泌乳期乳腺の乳汁分泌に与える影響
    公益財団法人 栗林育英学術財団：研究助成
    Date (from‐to) : 2015
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2012/04 -2014/03 
    Author : KOBAYASHI KEN
     
    Alveolar epithelial cells (AECs) secrete milk components for suckling infants in lactating mammary glands. AECs developed tight junctions (TJs) to prevent back-flow of milk from the alveolar lumen to the body. In this study, influences of tight junctions on milk secretion ability in lactating mammary alveolar epithelial cells were investigated. The results showed that TJs in lactation mammary glands consist of claudin-3 where claudin-4 causes weakness of TJ barrier. Furthermore, the expression level of claudin-4 was regulated by several physiological active substances, which have been known to regulate milk secretion ability in AECs. Therefore, close relationship of TJs with milk secretion ability in AEC is suggested.
  • 乳腺胞上皮細胞と脂肪細胞に着目した泌乳期乳腺におけるグルコース吸収経路の解明
    公益財団法人 秋山記念生命科学振興財団：研究助成（奨励）
    Date (from‐to) : 2014
  • マメ科牧草含有の植物エストロゲンが泌乳期乳腺の乳分泌能に及ぼす影響
    一般財団法人旗影会：研究助成
    Date (from‐to) : 2014
  • 飼料添加物に用いられる成分が乳腺上皮細胞に及ぼす影響
    日本ニュートリション株式会社：受託研究
    Date (from‐to) : 2013
  • ホエーの入浴剤としての効能に関する基礎研究
    公益財団法人ノーステック財団：研究シーズ発掘補助金
    Date (from‐to) : 2013
  • 乳期乳腺におけるカゼインミセルサイズの調節因子に関する研究
    一般財団法人 糧食研究会：一般公募研究助成
    Date (from‐to) : 2013
  • ハチミツが毛包発達に及ぼす作用に関する基礎的検証
    山田養蜂場：みつばち研究助成基金
    Date (from‐to) : 2011 -2012
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2008/04 -2011/03 
    Author : KOBAYASHI Ken
     
    The amniotic epithelium is in direct contact with the amniotic fluid and regulates fluid flux via paracellular pathway by tight junctions. In this study, we investigated the regulatory mechanism of tight junctions and claudins as the basic research for premature birth and chorioamnionitis. The results have been shown that several physiological active substances such as hormone and inflammatory cytokines regulate the permeability of amniotic tight junctions through claudins.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2009 -2010 
    Author : SUZUKI Teruhisa, KOBAYASHI Ken, OKANO Wataru, SUZUKI Masahiro
     
    Freshly isolated adipose-derived stromal vascular fraction cells (SVF) have further advantages over ASC in that they are more readily collected and do not require any culturing prior to use. The study was to identify the enhancement of the regeneration process in the trachea obtained through the use of SVF.SVF show high levels of expression of the stem cell-associated markers CD90, CD105, and CD29, and possess pluripotency like ASC. Our results show that SVF have potential clinical applications as implantable stem cells for use in the regenerative medicine.
  • 三次元培養モデルを用いた腹膜透析調整機序に関する研究
    バクスター株式会社：バクスターPD研究基金
    Date (from‐to) : 2007 -2008

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