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PDF file, 48K, PCR primers used in study.

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PDF file, 3359K, Effect of Epim on ductal length, terminal endbuds, and lobuloalveolar structure formation.

PDF file, 845K, Epim overexpression leads to enlargement of organoid cyst and upregulation of Pgr during bud formation.

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The expression and processing of filaggrin, a filament-associated protein in the skin epidermis, is closely associated with keratinocyte cornification. The large precursor profilaggrin (Pro-FLG) is initially detected at the granular layer in keratohyalin granules, subsequently processed into ten to twelve filaggrin monomers (mFLGs) for keratin assembly, and ultimately degraded into smaller peptides that behave as natural moisturizing factor (NMF) at the outermost epidermis. We previously reported that a t-SNARE protein epimorphin (EPM) often translocates across the membrane upon external stimuli and severely perturbs epidermal terminal differentiation. Using HaCaT keratinocytes with inducible expression and recombinant EPM and FLG, we investigated the effect of extracellular EPM on the expression profile of filaggrin. In response to ectopic stimulation with extracellular EPM, Pro-FLG expression decreased with elimination of keratohyalin granules in the cells, with filaggrin mRNA remained constant and profilaggrin processing was not accelerated. Additionally, using a recombinant form of mFLG engineered for intracellular localization, we found that extracellular EPM hindered proteolytic cleavage of mFLG for production of NMF. Taken together, extracellularly extruded EPM, an epidermal cornification blocker, not only decreases Pro-FLG expression but also reduces the production of NMF in HaCaT keratinocytes.

Mammary epithelia undergo dramatic morphogenesis after puberty. During pregnancy, luminal epithelial cells in ductal trees are arranged to form well-polarized cystic structures surrounded by a myoepithelial cell layer, an active supplier of the basement membrane (BM). Here, we identified a novel regulatory mechanism involved in this process by using a reconstituted BM-based three-dimensional culture and aggregates of a model mouse cell line, EpH4, that had either been manipulated for inducible expression of the t-SNARE protein syntaxin4 in intact or signal peptide-connected forms, or that were genetically deficient in syntaxin4. We found that cells extruded syntaxin4 upon stimulation with the lactogenic hormone prolactin, which in turn accelerated the turnover of E-cadherin. In response to extracellular expression of syntaxin4, cell populations that were less affected by the BM actively migrated and integrated into the cell layer facing the BM. Concurrently, the BM-facing cells, which were simultaneously stimulated with syntaxin4 and BM, acquired unique epithelial characteristics to undergo dramatic cellular arrangement for cyst formation. These results highlight the importance of the concerted action of extracellular syntaxin4 extruded in response to the lactogenic hormone and BM components in epithelial morphogenesis.

Background: Long-term peritoneal dialysis results in functional and histopathological alterations of the peritoneal membrane, leading to peritoneal fibrosis (PF). The mechanism of PF has not been fully elucidated, and at present there is no effective therapy for PF. Epimorphin is a mesenchymal protein that not only regulates morphogenesis in organ development but is implicated in tissue repair. However, the role of epimorphin in PF has not yet been clarified. Methods: PF was induced in C57/Bl6 mice by intraperitoneal injection of chlorhexidine gluconate (CG-injected mice) three times a week for 3 weeks. The parietal peritoneum was subsequently dissected and assessed by Masson’s trichrome staining, and epimorphin expression was analysed by immunohistochemistry and real-time reverse transcription polymerase chain reaction (RT-PCR). Furthermore, epimorphin-positive regions were analysed by multiple immunofluorescence staining using fibrosis-associated markers. In addition, normal rat fibroblast cells (NRK-49F) were treated with transforming growth factor-β (TGF-β) in the presence or absence of epimorphin. The expression of fibrosis-associated markers was assessed by real-time RT-PCR. Results: In CG-injected mice, Masson’s trichrome staining showed marked thickening of the submesothelial compact zone. Weak epimorphin expression was observed in the narrow submesothelial compact zone beneath the mesothelial cells in control mice; however, epimorphin expression was stronger in the submesothelial compact zone in CG-injected mice. Epimorphin expression was observed mainly in α-smooth muscle actin (α-SMA)-positive myofibroblasts. Epimorphin suppressed the TGF-β-induced upregulation of α-SMA and platelet-derived growth factor receptor-β in cultured cells. Conclusions: Our results suggest that epimorphin may be a therapeutic target for fibrotic diseases of the peritoneum.

Prolonged and intense stress chronically increases blood concentration of glucocorticoids, which in turn causes downregulation of glucocorticoid receptor (GR) in the central nervous system (CNS). This process has been suggested to be involved in the pathogenesis of major depressive disorder (MDD). Here, we found that basic fibroblast growth factor (bFGF) increased the expression of GR in the rat cerebral cortex and cultured cortical neurons and restored the reduced GR expression caused by glucocorticoid exposure. Among intracellular signaling pathways stimulated by bFGF, extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway was responsible for the upregulation of GR. The bFGF-induced GR was functional as a transcription factor to enhance transcription of a target gene. Because high stress augments bFGF levels in the brain, it is likely that bFGF plays a compensating role for reduced GR expression after stress and thus should be studied as a therapeutic target for the treatment of MDD.

A recent study revealed that corticotropin-releasing hormone (CRH) in the cerebral cortex (CTX) plays a regulatory role in emotional behaviors in rodents. Given the functional interaction between brain-derived neurotrophic factor (BDNF) and the CRH-signaling pathway in the hypothalamic-pituitary-adrenal axis, we hypothesized that BDNF may regulate gene expression of CRH and its related molecules in the CTX. Findings of real-time quantitative PCR (RT-qPCR) indicated that stimulation of cultured rat cortical neurons with BDNF led to marked elevations in the mRNA levels of CRH and CRH-binding protein (CRH-BP). The BDNF-induced up-regulation of CRH-BP mRNA was attenuated by inhibitors of tropomyosin related kinase (Trk) and MEK, but not by an inhibitor for PI3K and Phospholipase C gamma (PLCγ). The up-regulation was partially blocked by an inhibitor of lysine-specific demethylase (KDM) 6B. Fluorescent imaging identified the vesicular pattern of pH-sensitive green fluorescent protein-fused CRH-BP (CRH-BP-pHluorin), which co-localized with mCherry-tagged BDNF in cortical neurons. In addition, live-cell imaging detected drastic increases of pHluorin fluorescence in neurites upon membrane depolarization. Finally, we confirmed that tetrodotoxin partially attenuated the BDNF-induced up-regulation of CRH-BP mRNA, but not that of the protein. These observations indicate the following: In cortical neurons, BDNF led to gene expression of CRH-BP and CRH. TrkB, MEK, presumably ERK, and KDM6B are involved in the BDNF-induced gene expression of CRH-BP, and BDNF is able to induce the up-regulation in a neuronal activity-independent manner. It is suggested that CRH-BP is stored into BDNF-containing secretory granules in cortical neurons, and is secreted in response to membrane depolarization.

HaCaT cells have been widely used as undifferentiated epidermal keratinocytes, since these non-tumorigenic cells can be readily maintained in conventional medium and partly retain epidermal differentiation potential upon stimulation with high concentration of calcium. In contrast to primary epidermal keratinocytes, however, these cells never form tight junction (TJ), a specific structure in highly differentiated keratinocytes, solely by the differentiation stimulation. Here, we show that HaCaT cells secrete a considerable amount of high mobility group box-1 protein (HMGB1), one of major inflammatory mediator, which appeared to be responsible, at least in part, for such aberrant differentiation response. So far, inhibition of c-Jun N-terminal kinase (JNK) in high calcium medium has been supposed to be the only way to induce TJ formations in HaCaT cells; however, SP600125, a potent inhibitor of JNK showed cytostatic effects and clearly attenuated epidermal differentiation and stratification. In contrast, dipotassium glycyrrhizate (GK2), a soluble analogue of HMGB1-blocker Glycyrrhizin, down-regulated interferon-β, a typical inflammatory cytokine induced by secreted HMGB1, and accelerated differentiation responses to the calcium treatment in these cells. In addition, GK2-treatmenrt resulted in the formation of double cell layers in cultured HaCaT cells, where the stratified upper cells transiently accumulated TJ proteins at the cell–cell contact sites. These results highlight the importance of attenuation of secreted HMGB1-signals in cultured HaCaT cells for studies of functional keratinocytes. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10616-019-00367-6) contains supplementary material, which is available to authorized users.

Epithelial morphogenesis in the mammary gland proceeds as a consequence of complex cell behaviors including apoptotic cell death and epithelial-mesenchymal transition (EMT); the extracellular matrix (ECM) protein laminin is crucially involved. Syntaxins mediate intracellular vesicular fusion, yet certain plasmalemmal members have been shown to possess latent extracellular functions. In this study, the extracellular subpopulation of syntaxin-4, extruded in response to the induction of differentiation or apoptosis in mammary epithelial cells, was detected. Using a tetracycline-repressive transcriptional system and clonal mammary epithelial cells, SCp2, we found that the expression of cell surface syntaxin-4 elicits EMT-like cell behaviors. Intriguingly, these cells did not up-regulate key transcription factors associated with the canonical EMT such as snail, slug, or twist, and repressed translation of E-cadherin. Concurrently, the cells completely evaded the cellular aggregation/rounding triggered by a potent EMT blocker laminin-111. We found that the recombinant form of syntaxin-4 not only bound to laminin but also latched onto the glycosaminoglycan (GAG) side chains of syndecan-1, a laminin receptor that mediates epithelial morphogenesis. Thus, temporal extracellular extrusion of syntaxin-4 emerged as a novel regulatory element for laminin-induced mammary epithelial cell behaviors. J. Cell. Biochem. 118: 686-698, 2017. © 2016 Wiley Periodicals, Inc.

Spatial and temporal epithelial-mesenchymal transition (EMT) is a critical event for the generation of asymmetric epithelial architectures. We found that only restricted cell populations in the morphogenic mammary epithelia extrude syntaxin-4, a plasmalemmal t-SNARE protein, and that epithelial cell clusters with artificial heterogenic presentation of extracellular syntaxin-4 undergo asymmetric morphogenesis. A previous study revealed that inducible expression of cell surface syntaxin-4 causes EMT-like cell behaviors in the clonal mammary epithelial cells, where laminin-mediated signals were abolished so that cells readily succumb to initiate EMT. The present study added new mechanistic insight into syntaxin-4-driven EMT-like cell behaviors. Extracellular syntaxin-4 directly perturbs E-cadherin-mediated epithelial cell-cell adhesion and activates Smad signals. We found that the epithelial cells activated Smad2/3 upon induction of expression of extracellular syntaxin-4, leading to the upregulation of certain transcriptional targets of these TGF-β signaling mediators. Intriguingly, however, mRNA expression of canonical EMT initiators, such as Snail and Slug, was unchanged. In addition, E-cadherin protein was steeply decreased, yet its transcriptional expression remained constant for a couple of days. We found that extracellular syntaxin-4 directly bound to E-cadherin and sequestered β-catenin from cell-cell contact sites, perturbing intercellular adhesive property. The functional ablation of E-cadherin by syntaxin-4 was further validated by L cells with stably expressing E-cadherin, in which cells shows intercellular adhesive property solely by E-cadherin. These results underline the role of local exportation of syntaxin-4 for onset of complex epithelial morphogenesis.

In the skin epidermis, keratinocytes undergo anchorage-dependent cornification, which gives rise to stratified multilayers, each with a distinct differentiation feature. The active formation of the cornified cell envelope (CCE), an important element in the skin barrier, occurs in keratinocytes of the upper epidermal layers and impacts their terminal differentiation. In the present study, we identified the extracellularly extruded syntaxin-4 as a potent differentiation regulator of epidermal keratinocytes. We found that differentiation stimuli led to the acceleration of syntaxin-4 exposure at the keratinocyte cell surface and that the artificial control of extracellular syntaxin-4, either by the forced expression of several syntaxin-4 mutants with structural alterations at the putative functional core site (AIEPQK), or by using antagonistic circular peptides containing this core sequence, dramatically influenced the CCE formation, with spatial misexpression of TGase1 and involucrin. We also found that the topical application of a peptide that exerted the most prominent antagonistic activity for syntaxin-4, named ST4n1, evidently prevented the formation of the hyperplastic and hyperkeratotic epidermis generated by physical irritation in HR-1 mice skin. Collectively, these results demonstrate that extracellularly extruded syntaxin-4 is a potent regulator of CCE differentiation, and that ST4n1 has potential as a clinically applicable reagent for keratotic skin lesions.

Biological evaluation of hair growth/differentiation activity in vitro has been a formidable challenge, primarily due to the lack of relevant model cell systems. To solve this problem, we generated a stable model cell line in which successive differentiation via epidermal progenitors to hair components is easily inducible and traceable. Mouse induced pluripotent stem (iPS) cell–derived cells were selected to stably express a tetracycline (Tet)-inducible bone morphogenic protein-4 (BMP4) expression cassette and a luciferase reporter driven by a hair-specific keratin 31 gene (krt31) promoter (Tet-BMP4-KRT31-Luc iPS). While Tet- BMP4-KRT31-Luc iPS cells could be maintained as stable iPS cells, the cells differentiated to produce luciferase luminescence in the presence of all-trans retinoic acid (RA) and doxycycline (Dox), and addition of a hair differentiation factor significantly increased luciferase fluorescence. Thus, this cell line may provide a reliable cell-based screening system to evaluate drug candidates for hair differentiation activity.

Activation of the epithelial-mesenchymal transition (EMT) program promotes cell invasion and metastasis, and is reversed through mesenchymal-epithelial transition (MET) after formation of distant metastases. Here, we show that an imbalance of gene products encoded by the transcriptional factor C/EBPβ, LAP (liver-enriched activating protein) and LIP (liver-enriched inhibitory protein), can regulate both EMT- and MET-like phenotypic changes in mouse mammary epithelial cells. By using tetracycline repressive LIP expression constructs, we found that SCp2 cells, a clonal epithelial line of COMMA1-D cells, expressed EMT markers, lost the ability to undergo alveolar-like morphogenesis in 3D Matrigel, and acquired properties of benign adenoma cells. Conversely, we found that inducible expression of LAP in SCg6 cells, a clonal fibroblastic line of COMMA1-D cells, began to express epithelial keratins with suppression of proliferation. The overexpression of the C/EBPβ gene products in these COMMA1-D derivatives was suppressed by long-term cultivation on tissue culture plastic, but gene expression was maintained in cells grown on Matrigel or exposed to proteasome inhibitors. Thus, imbalances of C/EBPβ gene products in mouse mammary epithelial cells, which are affected by contact with basement membrane, are defined as a potential regulator of metastatic potential.

Epimorphin/syntaxin-2 is a membrane-tethered protein localized extracellularly (Epim) and intracellularly (Stx-2). The extracellular form Epim stimulates morphogenic processes in a range of tissues, including in murine mammary glands where its overexpression in luminal epithelial cells is sufficient to drive hyperplasia and neoplasia. We analyzed WAP-Epim transgenic mice to gain insight into how Epim promotes malignancy. Ectopic overexpression of Epim during postnatal mammary gland development led to early side-branching onset, precocious bud formation, and increased proliferation of mammary epithelial cells. Conversely, peptide-based inhibition of Epim function reduced side branching. Because increased side branching and hyperplasia occurs similarly in mice upon overexpression of the progesterone receptor isoform-a (Pgr-a), we investigated whether Epim exhibits these phenotypes through Pgr modulation. Epim overexpression indeed led to a steep upregulation of both total Pgr mRNA and Pgr-a protein levels. Notably, the Pgr antagonist RU486 abrogated Epim-induced ductal side branching, mammary epithelial cell proliferation, and bud formation. Evaluation of Epim signaling in a three-dimensional ex vivo culture system showed that its action was dependent on binding to its extracellular receptor, integrin-αV, and on matrix metalloproteinase 3 activity downstream of Pgr-a. These findings elucidate a hitherto unknown transcriptional regulator of Pgr-a, and shed light on how overexpression of Epim leads to malignancy. Cancer Res; 73(18); 5719–29. ©2013 AACR.

The proteins in the syntaxin family are known to mediate fusion of cytoplasmic vesicles to the target membrane, yet subpopulations of certain syntaxins, including syntaxin4, translocate across the cell membrane in response to external stimuli. Here, we show that extracellularly presented syntaxin4 impacts cell behavior and differentiation in teratocarcinoma F9 cells. While undifferentiated F9 cells extruded a small subpopulation of extracellular syntaxin4 at the lateral cell membrane, the induction of differentiation with all-trans retinoic acid (RA) abolished this localized expression pattern. We found that the cells that were stimulated in a non-directional fashion by extracellular syntaxin4 displayed a flattened shape and retained a substrate-bound morphology even under a long-term, serum-starved cultivation. Such a cellular response was also elicited by a circular peptide composed of the potential functional core of syntaxin4 (AIEPQK; amino acid residues 103~108) (ST4n1). While the proliferation and metabolism were not affected in these cells, cell-cell interaction became weakened and the expression of vinculin, a regulator of both intercellular and cell-substrate adhesion molecules, was altered. We also found that the expressions of several differentiation markers were up-regulated in cells stimulated with extracellular syntaxin4 and that syntaxin3, another family member, was most prominent. Intriguingly, forced expression of syntaxin3 induced the spread morphology in F9 cells, indicating that syntaxin3 partly mediates the function of extracellular syntaxin4. These results demonstrate the involvement of a non-directional stimulation of extracellular syntaxin4 in the functional and morphological differentiation of F9 cells.

The melanin granules are synthesized in melanocytes in the epidermal basal layer and the hair matrix. For the effective passage of melanin granules to the adjacent keratinocytes, melanocytes utilize unique cytoplasmic delivery system in which cytoskeletal network is prominently involved. Here, we show that the t-SNARE protein syntaxin3, a member of a family of key mediators of the cytoplasmic vesicle fusion and potent modulators of cytoskeletal dynamics, dramatically affects melanocyte cell behavior. Although plasmalemmal syntaxin3 has been detected also on the melanosomes of normal human melanocytes, we noticed that mouse melanoma B16 cells had completely lost endogenous syntaxin3. In response to the forcible expression of syntaxin3, B16 cells formed well-developed dendritic filopodia and accumulated melanin granules in the cytoplasm. We found that exogenous syntaxin3 was not expressed at the plasma membrane, but rather, localized with non-fibrous F-actin and melanin-packed melanosomes in the cytoplasm, by which the assembly/polymerization of actin was dramatically impacted and the melanosome secretion was severely suppressed. The syntaxin3-triggered phenotypic changes were also induced by a syntaxin3 mutant lacking SNARE and transmembrane domains, and they were completely reverted by the subsequent knockdown of exogenous syntaxin3. This t-SNARE protein may act as a regulator of the actin dynamics, rather than a direct vesicle fusion mediator, to determine the fundamental properties of melanocytes.

Background Upon skin injuries, dermal fibroblasts actively produce transforming growth factor-β (TGF-β), which leads to the formation of α-smooth muscle actin (αSMA)-positive granulation tissues. The hyperplasia or incomplete regression of these tissues subsequently causes scar formation in the skin, where sulfated glycosaminoglycans (GAGs), side chains of unique proteoglycans, are supposed to play important roles. Objective The aim of this study is to clarify the effects of sulfated GAGs on dermal cell behaviors triggered by the TGF-β signaling, along with its possible regulators basic fibroblast growth factor (bFGF) and cell surface epimorphin. bFGF and epimorphin might regulate the TGF-β-induced αSMA expression, they could exert such effects only in specific cellular contexts, given that they lack conventional signal sequences for extracellular localization. Methods Human scar-derived dermal fibroblasts (HSFs) were treated with TGF-β alone, TGF-β plus bFGF, and TGF-β plus cell surface expression of epimorphin. The effects of GAGs on the expression of αSMA and the cellular morphology were then investigated. Results A highly sulfated chondroitin sulfate (CS-E) or its substitute (heparinoid) had marked inhibitory effects on TGF-β-mediated changes in HSF behaviors. We found that heparinoid can directly associate with TGF-β, bFGF and epimorphin. We also found that bFGF downregulated αSMA, which was attenuated by heparinoid, whereas epimorphin augmented αSMA expression, which was further amplified by heparinoid. Conclusions TGF-β, bFGF and epimorphin in the extracellular microenvironment cooperatively affect HSF behaviors under the control of a highly sulfated chondroitin sulfate. These results provide important evidence towards understanding the regulation of TGF-β-induced HSF behaviors.

Epithelial morphogenesis in the mammary gland proceeds as a consequence of complex cell behaviors including apoptotic cell death and epithelial-mesenchymal transition (EMT); the extracellular matrix (ECM) protein laminin is crucially involved. Syntaxins mediate intracellular vesicular fusion, yet certain plasmalemmal members have been shown to possess latent extracellular functions. In this study, the extracellular subpopulation of syntaxin-4, extruded in response to the induction of differentiation or apoptosis in mammary epithelial cells, was detected. Using a tetracycline-repressive transcriptional system and clonal mammary epithelial cells, SCp2, we found that the expression of cell surface syntaxin-4 elicits EMT-like cell behaviors. Intriguingly, these cells did not up-regulate key transcription factors associated with the canonical EMT such as snail, slug, or twist, and repressed translation of E-cadherin. Concurrently, the cells completely evaded the cellular aggregation/rounding triggered by a potent EMT blocker laminin-111. We found that the recombinant form of syntaxin-4 not only bound to laminin but also latched onto the glycosaminoglycan (GAG) side chains of syndecan-1, a laminin receptor that mediates epithelial morphogenesis. Thus, temporal extracellular extrusion of syntaxin-4 emerged as a novel regulatory element for laminin-induced mammary epithelial cell behaviors. J. Cell. Biochem. 118: 686-698, 2017. © 2016 Wiley Periodicals, Inc.

Background AHF/trichohyalin is a large structural protein abundant in the inner root sheath (IRS) of anagenic hair follicles, which has been thought to mediate the keratin filamentous assembly. However, its functional mechanism is largely unknown. Objective This study aimed at the identification of the key domain in AHF for keratin association and the establishment of a plausible mechanism for the modulation of the keratin meshwork. Methods Several keratinocyte cell lines were introduced with the full length or several mutants of AHF, together with IRS-specific keratin krt31, and the profile of the AHF granules and the cellular behaviors were carefully analyzed. Results Full length of AHF formed small round granules that clearly bound to and aligned on the exogenous keratin filaments in the keratinocytes, severely affected cellular growth, mobility and shape. Intriguingly, the removal of only 6 amino acids around the C-terminal tail of AHF resulted not only in the complete loss of its keratin adherent ability but also in a dramatic enlargement of the granules. Conclusion We propose a model for cytoskeletal modulation in the IRS of anagenic hair follicles: AHF latches onto the keratin bundles by its C-terminus and rearranges the keratin meshwork by intrinsic cohesive activity for the granule formation.

The mesoderm-derived segmental somite differentiates into dermomyotome and sclerotome, the latter of which undergoes vertebrogenesis to spinal cartilage and ultimately to vertebral bones. However, analysis and manipulation of the developing mammalian vertebrae in the same embryo has been infeasible because of their placental-dependent embryogenesis. Here, we report a novel culture system of the mouse embryonic tailbud, by which the developmental processes of mammalian vertebral cartilage are traceable and manipulatable in the same sample. The anaplastic segmental somites/sclerotomes in the tailbud of 13 gestational day (g.d.) embryo that are structurally continuous to the vertebral column underwent progressive vertebrogenesis when (1) the ectoderm-derived nascent epidermis was microsurgically removed prior to cultivation, and (2) the sample was incubated at the air-medium interface. After cultivation for 5 days, the size and shape of the instructed vertebral cartilage showed features comparable to well-differentiated body vertebra along with the expression of the cartilage marker collagen type II, suggesting that aggressive differentiation of the sclerotomal cell lineage was achieved. In the presence of recombinant bone morphogenic protein (BMP) and Noggin, or adenoviral particles for extracellular epimorphin, dramatic alteration of the vertebral morphology ensued in the explants. Thus, this model system provides an approach to study the detailed molecular mechanisms of mammalian vertebrogenesis and enables pretreatment strategies of precartilagious fragments for improving the efficacy of subsequent transplantation.

Background and Aims: Many investigations have demonstrated that cell injuries caused by generation of reactive oxygen species (ROS) is a common mechanism of various hepatic disorders. Recently, we have demonstrated that epimorphin, originally cloned as a mesenchymal protein, protects cultured intestinal epithelial cells from ROS. We therefore examine whether epimorphin protects primary cultured hepatocytes from ROS-induced cell injury. Methods: We explored the cell viability and the intracellular ROS levels of purified murine hepatocytes after exposure to 0.5 mM H2O2 with or without pretreatment of epimorphin. Then, we observed mitochondrial permeability transition (MPT) and depolarization using confocal microscopy to make clear the mechanism that epimorphin inhibited cell injuries after exposure to H2O2. In addition, to clarify the signaling pathways related to cell survival, we carried out Western blotting analysis with phosphorylated stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) polyclonal antibody to evaluate the inhibition of JNK by epimorphin. Finally, we evaluated the cell viability in hepatocytes administered JNK inhibitor. Results: Epimorphin protected primary cultured hepatocytes from H2O2-induced cell injuries independent of intracellular ROS levels. Epimorphin also inhibited onset of MPT, depolarization of the mitochondrial membrane potential, and eventually cell killing. The cell protective function of epimorphin after exposure to H2O2 was not dependent on Akt signaling but on JNK signaling. Conclusion: Epimorphin can protect hepatocytes from MPT-dependent cell injury induced by ROS. Since hepatic disorders could be caused by MPT-dependent cell injuries with excessive ROS, epimorphin might open a new therapeutic avenue for hepatic disorders.

The maintenance of organ homeostasis and the control of an appropriate response to environmental alterations require the intimate coordination of cellular functions and tissue organization. An important component of this coordination could be provided by proteins that can have distinct but linked functions on both sides of the plasma membrane. We present a model that proposes that unconventional secretion provides a mechanism through which single proteins can integrate complex tissue functions.

Hairs in mammals undergo well-programmed cyclic development, which is strictly controlled by the surrounding hair follicle cells. Among hair follicle epithelia the inner root sheath (IRS) directly envelops the hair shaft to mechanically support its cyclic growth, but the molecular mechanism underlying its sharp regulation remains obscure. Here, we identify regulatory elements for the expression of the IRS-specific protein AHF (Anagenic Hair Follicle antigen), the putative mouse orthologue of trichohyalin (THH), which plays a key role in the assembly of keratin intermediate filaments (IFs) during the hair cycle. AHF becomes abundantly present in the growing anagenic hair follicle and is suddenly diminished as the tissue enters into the subsequent resting stages under the control of ubiquitin-dependent proteolysis. Using primary human keratinocytes, we found that bone morphogenic protein-4 facilitates THH transcription, and intriguingly, a nuclear lamina component plays a key role in the posttranslational stabilization of THH protein. Silencing of the lamin A/C gene leads to rapi THH degradation, whereas exogenously introduced lamin C, but not lamin A, protects THH from proteolytic elimination. These results shed light on the strict molecular mechanisms which control stage- and compartment-specific IF assemblies in support of the cyclic development of the hair shaft.

Extensive studies have identified a large number of the molecular components of cellular tight junctions (TJ), including the claudins, occludin and ZO-1/2, and also many of the physical interactions between these molecules. However, the regulatory mechanisms of TJ formation are as yet poorly understood. In HaCaT, a human epidermal keratinocyte cell line, TJ were newly formed when cells were cultured in the presence of SP600125, a JNK inhibitor. Moreover, claudin-4 was newly phosphorylated during this process. We found that claudin-4 contains a sequence which is phosphorylated by atypical PKC (aPKC). Kinase assay demonstrated that the 195th serine (serine195) of mouse claudin-4 was phosphorylated by aPKC in vitro. The 194th serine (serine194) of human claudin-4 corresponding to serine195 of mouse claudin-4 was phosphorylated in HaCaT cells when TJ were formed, and the phosphorylated claudin-4 co-localized with ZO-1 at TJ. aPKC activity was required for both the claudin-4 phosphorylation and TJ formation in HaCaT. Furthermore, overexpression of mutant claudin-4 protein S195A, which was not phosphorylated by aPKC, perturbed the TJ formation mediated by SP600125. These findings suggest that aPKC regulates TJ formation through the phosphorylation of claudin-4.

Epimorphin (also known as syntaxin2) is a stromal signaling factor that is temporally secreted via a non-classical route to regulate the morphogenesis of various epithelia, including skin epidermis. In this study, we show that epimorphin signaling also regulates the differentiation program in the keratinocyte. The extracellular presentation of this molecule is detectable predominantly in the dermal compartments of the skin and its secretion is increased by cell stress such as UVB irradiation, which generates an epimorphin signaling gradient in the epidermis. Artificial stimulation of functionally normal keratinocyte HaCaT cells with extracellular epimorphin triggered initiation of their differentiation program with a dramatic depression of metabolic turnover. Intriguingly, however, sustained epimorphin signaling appeared to severely attenuate the terminal cornification in the cells induced by calcium influx and anchorage-dependent anoikis. In the organotypic culture of HaCaT cells, overexpression of epimorphin impaired the successive differentiation program in the stratified epidermis-like structures; the cells underwent aberrant multicellular arrangement with a presentation of mid-differentiation markers throughout all the cell layers. These results demonstrate that inadequate epimorphin elicits an abnormal differentiation response in keratinocytes, and indicate a causal function of the epimorphin signaling gradient for the establishment of differentiated epidermal structure in the skin.

Epimorphin, a mesenchymal morphogenic protein expressed by hepatic stellate cells, is considered important to liver morphogenesis in both healthy and pathologic conditions. However, the stellate cell phenotype, quiescent versus activated, that expresses epimorphin is unknown. We studied the relationship between epimorphin expression and stellate cell status in carbon tetrachloride-induced acute and chronic injury to mouse liver and in mouse liver regeneration following 70% partial hepatectomy. Epimorphin-positive cells in sinusoids expressed desmin, indicating that they are stellate cells. Epimorphin-positive cells were more numerous and larger in pericentral than periportal sinusoids in normal liver. In early-phase acute liver injury and liver regeneration, epimorphin expression transiently decreased while alphaSMA-positive stellate cells increased. In the recovery phase of acute and chronic injury as well as the late phase of liver regeneration, epimorphin expression was strikingly enhanced while alphaSMA-positive stellate cells decreased. This expression pattern was seen in both Balb/c and C57BL6 mouse strains irrespective of their differences in response to the hepatotoxin. In conclusion, stellate cells express epimorphin in their quiescent state and in the recovery phase, respectively associated with maintenance and reconstruction of microscopic liver structure.

Epimorphin/syntaxin-2 (EPM) is a plasma membrane–anchored protein that has at least two distinct functions depending on its membrane topology: vesicle fusion when localized to the cytoplasmic surface and morphogenic signaling when localized to the extracellular surface. Transgenic mice that express full-length extracellular EPM fused to the NH2-terminal signal sequence of interleukin-2, under the control of the whey acidic protein (WAP) gene promoter, exhibit aberrant mammary gland morphogenesis associated with increased expression of CCAAT enhancer binding protein β (C/EBPβ). Here we report that aged nulliparous and uniparous female WAP-EPM transgenic mice develop alveolar hyperplasias and well-differentiated adenocarcinomas that express high levels of C/EBPβ, keratin-14, matrix metalloproteinase-3, and β-catenin. This study reveals another pathway in which overexpression and alteration of a normal morphogenic process promote the development of cancer in the mammary gland.

Epimorphin is representative of a unique class of stromal membrane-anchored proteins that plays distinct functions depending on its membrane topology. When exposed extracellularly, this molecule acts as a morphoregulator for various tissues including hair follicle epithelia. Previous study identified its functional domain (the pep7 domain: SIEQSCDQDE) for hair follicular morphogenesis followed by the successful generation of a chemically modified active peptide. Here, we report optimization of this peptide by the introduction of sequential mutations and subsequent structural determination. We found that three residues from the C-terminus are dispensable, and alternation of the seventh amino acid to an Alanine residue enhanced activity. To favour the biologically active conformation, epsilon-Acp (NH(CH(2))(5)CO) linked to a Cysteine residue was connected at the N-terminus followed by the introduction of an intramolecular disulphide bridge, the modification process of which could be included in the peptide synthesis. The obtained modified peptide, termed 'EPM (epimorphin-derived) peptide', has a Mw of 950 Da and exerts an inductive effect on hair follicle regeneration at a concentration of approximately 0.00001% or even lower. The action of this EPM peptide was more apparent in mice treated with 1% minoxidil, suggesting its potential clinical benefit as a new type of hair-regenerating agent.

Conceptus implantation to the uterine endometrium represents a complex series of events, including synchronized development of conceptus and uterus through up- and/or down-regulation of numerous gene products. In a previous study using the DNA microarray technique, we had discovered evidence that increase in a transcript for mesenchymal morphogen, epimorphin, was noted as the conceptus attached to the matrix in vitro (Qin et al., 2003). In the present study, the expression and potential function of epimorphin in developing conceptuses was investigated through the use of reverse transcription-polymerase chain reaction (RT-PCR), whole mount in situ hybridization/immunohistochemistry, and in vitro blastocyst culture. RT-PCR and in situ hybridization analysis revealed that epimorphin mRNA was expressed weakly in murine conceptuses during early developmental stages (1 cell to post-adhesion blastocyst stages) and higher levels of epimorphin transcripts were observed in both inner cell mass (ICM) and trophectoderm of outgrowing blastocysts. Immunohistochemical analysis confirmed that epimorphin was localized in outgrowing trophoblast cells and ICM. Treating blastocysts in culture with a 115 kDa form of recombinant epimorphin promoted trophoblast outgrowth (P

Background and Aim: Epimorphin is suggested to play a key role in the morphogenesis of epithelial cells. We focused on epimorphin and attempted to clarify the relationship between expression of this molecule and inflammatory bowel disease (IBD). Methods: Colonic specimens were taken from 23 patients with ulcerative colitis (UC), 15 with Crohn's disease (CD), six with non-IBD colitis, and 10 normal controls. Distribution and expression of epimorphin protein were examined by immunohistochemistry and western blot analysis, and expression of epimorphin messenger (m)RNA by reverse transcription–polymerase chain reaction. Distribution of basic fibroblast growth factor (bFGF) was also examined by immunohistochemistry. Results: In the normal colonic mucosa, epimorphin was observed around the epithelial cells, in some fibroblasts, vascular endothelial cells, and macrophages. In UC patients, epimorphin around epithelial cells disappeared or decreased, and the number of epimorphin-containing cells (mean + standard error/mm2) was significantly increased in active UC patients (1550.8 ± 144.2) compared with patients in remission (692.8 ± 45.8), CD patients (790.0 ± 31.2), non-IBD colitis patients (710.8 ± 29.2), and controls (664.8 ± 39.6) (P

The mammary gland develops its adult form by a process referred to as branching morphogenesis. Many factors have been reported to affect this process. We have used cultured primary mammary epithelial organoids and mammary epithelial cell lines in three-dimensional collagen gels to elucidate which growth factors, matrix metalloproteinases (MMPs) and mammary morphogens interact in branching morphogenesis. Branching stimulated by stromal fibroblasts, epidermal growth factor, fibroblast growth factor 7, fibroblast growth factor 2 and hepatocyte growth factor was strongly reduced by inhibitors of MMPs, indicating the requirement of MMPs for three-dimensional growth involved in morphogenesis. Recombinant stromelysin 1/MMP3 alone was sufficient to drive branching in the absence of growth factors in the organoids. Plasmin also stimulated branching; however, plasmin-dependent branching was abolished by both inhibitors of plasmin and MMPs, suggesting that plasmin activates MMPs. To differentiate between signals for proliferation and morphogenesis, we used a cloned mammary epithelial cell line that lacks epimorphin, an essential mammary morphogen. Both epimorphin and MMPs were required for morphogenesis, but neither was required for epithelial cell proliferation. These results provide direct evidence for a crucial role of MMPs in branching in mammary epithelium and suggest that, in addition to epimorphin, MMP activity is a minimum requirement for branching morphogenesis in the mammary gland.

Epimorphin was recently described as a mesenchymal factor modulating morphogenesis of murine mammary ducts, skin, liver, and lung in vitro. In this study epimorphin was analyzed in a human, pancreatic adenocarcinoma cell line (A818-6) which develops single layer epithelial hollow spheres resembling normal pancreatic ductal structures in vitro. Soluble 34- and 31-kD isoforms of epimorphin were found in the culture supernatant of A818-6 cells. In lysates of A818-6 cells we detected the 34-and 31-kD isoforms and the dimers, and in lysates of fibroblasts the 150-kD tetramers of epimorphin additionally. A neutralizing monoclonal antibody against epimorphin (MC-1) efficiently blocked the development of hollow sphere structures from A818-6 cells. Coculture of A818-6 cells with fibroblasts stimulated the development of hollow sphere structures in general and increased differentiation in 5–6-d-old hollow spheres. A818-6 hollow sphere development in the presence of fibroblasts was also blocked by MC-1. In this novel system for human duct-like differentiation of pancreatic epithelial cells, we provide evidence for an autocrine and paracrine function of epimorphin as a major mediator for morphogenesis.

We have shown previously that epimorphin (EPM), a protein expressed on the surface of myoepithelial and fibroblast cells of the mammary gland, acts as a multifunctional morphogen of mammary epithelial cells. Here, we present the molecular mechanism by which EPM mediates luminal morphogenesis. Treatment of cells with EPM to induce lumen formation greatly increases the overall expression of transcription factor CCAAT/enhancer binding protein (C/EBP)β and alters the relative expression of its two principal isoforms, LIP and LAP. These alterations were shown to be essential for the morphogenetic activities, since constitutive expression of LIP was sufficient to produce lumen formation, whereas constitutive expression of LAP blocked EPM-mediated luminal morphogenesis. Furthermore, in a transgenic mouse model in which EPM expression was expressed in an apolar fashion on the surface of mammary epithelial cells, we found increased expression of C/EBPβ, increased relative expression of LIP to LAP, and enlarged ductal lumina. Together, our studies demonstrate a role for EPM in luminal morphogenesis through control of C/EBPβ expression.

Studies of the epidermis have been carried out in various models, such as the monolayer culture in vitro model and three-dimensional (3D) skin models, that are spatially organized to display the architectural features seen in human skin. These models have furthered our understanding of epidermal cell biology and provided quite a few lines of evidence on proliferation, cellular metabolism, morphological status, and state of differentiation. In this chapter, we describe a novel method using epithelial cell aggregates embedded in a collagen gel, instead of individual cells, for building cell-cell and cell-matrix interactions. Analyzing cell behaviors during epidermal differentiation would be helpful. Our method would help to analyze a possible regulatory mechanism underlying epidermal differentiation.

A mesenchymal protein, epimorphin, is known to bind directly to the cell surface through its central portion and to act as a signaling molecule for epithelial morphogenesis. Utilizing several recombinant polypeptides and synthetic peptides, we identified the cellular recognition sequence of epimorphin in the central portion of this molecule (amino acids 105-123, NGNRTSVDLRIRRTQHSVL; termed NL-peptide sequence). Interestingly, although a model cell type bound to the NL-peptide as strong as to the full-length epimorphin, this peptide itself didn't induce the cellular functional responses so far tested. We found that the NL-peptide behaved as an antagonist for the endogenous epimorphin and severely perturbed lung branching morphogenesis in organ culture. These results not only revealed a part of the functional mechanism of epimorphin but also demonstrated that cell/ epimorphin interaction through the NL-peptide sequence is a critical step for lung epithelial morphogenesis.

Studies of the epidermis have been carried out in various models, such as the monolayer culture in vitro model and three-dimensional (3D) skin models, that are spatially organized to display the architectural features seen in human skin. These models have furthered our understanding of epidermal cell biology and provided quite a few lines of evidence on proliferation, cellular metabolism, morphological status, and state of differentiation. In this chapter, we describe a novel method using epithelial cell aggregates embedded in a collagen gel, instead of individual cells, for building cell-cell and cell-matrix interactions. Analyzing cell behaviors during epidermal differentiation would be helpful. Our method would help to analyze a possible regulatory mechanism underlying epidermal differentiation.

A B S T R A C T Background: The physical properties of the hair are predominantly determined by the assembly of keratin bundles. The keratin-associated proteins (Krtaps) are thought to be involved in keratin bundle assembly, however, the functional role of the individual member still remains largely unknown. Objective: The aim of this study is to clarify the role of a unique class of Krtaps, Krtap11-1, in the development and physical properties of the hair. Methods: The expression regulation of Krtap11-1 was analyzed and its binding partners in the hair cortex were determined. Also, the effects of the forcible expression of this protein on the hair follicle development were analyzed in culture. Results: The expression pattern of Krtap11-1 was concentrically asymmetric in the faulty hair that develops in Foxn1nu mice. In cultured keratinocytes, the expression of Krtap11-1 transgene product was strictly regulated by the keratinization process and proteasome-dependent protein elimination. While the association with keratin as well as the cohesive self-assembly of Krtap11-1 appeared to be stabilized by disulfide cross-links, the biotinylated Krtap11-1 probe enabled the adherence to certain type I keratins in the hair cortex, including K31, 33 and 34, in the absence of disulfide formation. When embryonic upper lip rudiments were forcibly introduced with Krtap11-1, the hair follicles formed irregularly arranged globular hair keratin-clumps surrounded by multilayered epithelial cells in culture. Conclusion: Krtap11-1 may play an important role on keratin-bundle assembly in the hair cortex and this study provides insight into the physical properties of the hair shaft.

Ultra-violet B (UVB)-induced oxidative stress crucially perturbs the epidermal homeostasis, and the skin is endowed with protective mechanisms to take action against such damage. Here, we show the possible involvement of t-SNARE protein syntaxin3, a membrane fusion mediator of cytoplasmic vesicles, and which is released from dying keratinocytes, to play a role in this response. UVB irradiation, which generates reactive oxidative stress in cells, was shown to lead to the keratinocyte cell death accompanied by a release of cytoplasmic syntaxin3. We found that such extracellularly sourced syntaxin3 completely blocked the processing of a crucial effector for apoptotic cell death, caspase-3, and thus facilitated the survival of keratinocytes damaged by oxidative stress. These results demonstrate the latent prosurvival function of syntaxin3 and underline the importance of intracellular molecular elements for the maintenance of homeostasis in epidermal keratinocytes.