The decline in cellular iron is crucial for differentiation in keratinocytes.

Iron is a vital metal for most biological functions in tissues, and its concentration is exquisitely regulated at the cellular level. During the process of differentiation, keratinocytes in the epidermis undergo a noticeable reduction in iron content. Conversely, psoriatic lesions, characterized by disruptions in epidermal differentiation, frequently reveal an excessive accumulation of iron within keratinocytes that have undergone differentiation. In this study, we clarified the significance of attenuated cellular iron content in the intricate course of epidermal differentiation. We illustrated this phenomenon through the utilization of hinokitiol, an iron chelator derived from the heartwood of Taiwanese hinoki, which forcibly delivers iron into cells independent of the intrinsic iron-regulation systems. While primary cultured keratinocytes readily succumbed to necrotic cell death by this iron chelator, mild administration of the hinokitiol-iron complex modestly disrupts the process of differentiation in these cells. Notably, keratinocyte model cells HaCaT and anaplastic skin rudiments exhibit remarkable resilience against the cytotoxic impact of hinokitiol, and the potent artificial influx of iron explains a suppressive effect selectively on epidermal differentiation. Moreover, the augmentation of iron content induced by the overexpression of divalent metal transporter 1 culminates in the inhibition of differentiation in HaCaT cells. Consequently, the diminution in cellular iron content emerges as an important determinant influencing the trajectory of keratinocyte differentiation.

CAMSAP3, a microtubule orientation regulator, plays a vital role in manifesting differentiation-dependent characteristics in keratinocytes.

Microtubules constitute pivotal structural elements integral to cellular architecture and physiological functionality. Within the epidermis of the skin, microtubules undergo a noteworthy transition in orientation, shifting from centrosomal to non-centrosomal configurations during the processes of differentiation and stratification. This transition aligns with a discernible increase in the expression of CAMSAP3, a protein that binds to the minus end of microtubules, thereby regulating their orientation. In this study, we identified microtubule-bound CAMSAP3 within HaCaT keratinocytes, revealing an upregulation during the mitotic phase and accumulation at the intercellular bridge during cytokinesis. Building upon this observation, we scrutinized cellular responses upon a tetracycline/doxycycline-inducible CAMSAP3 expression in CAMSAP3-deficient HaCaT cells. Remarkably, CAMSAP3 deficiency induced shifts in microtubule orientation, resulting in cell cycle exit and delayed cytokinesis in a subset of the cells. Furthermore, our inquiry unveiled that CAMSAP3 deficiency adversely impacted the formation and stability of Adherens Junctions and Tight Junctions. In contrast, these perturbations were rectified upon the re-expression of CAMSAP3, underscoring the pivotal role of CAMSAP3 in manifesting differentiation-dependent characteristics in stratified keratinocytes. These observations emphasize the significance of CAMSAP3 in maintaining epidermal homeostasis.

A crucial stem cell plasticity regulation pathway: identification of key elements using the NCCIT human embryonic carcinoma cell line.

Upon removal of stemness factors, a small subpopulation of embryonic stem cells (ESCs) spontaneously extrudes the t-SNARE protein syntaxin-4, which upregulates the cell adhesion molecule P-cadherin and induces the onset of epithelial-mesenchymal transition (EMT)-like behaviors with loss of stemness in each cell. In this study, we identified a series of molecular elements responsible for this phenomenon using several small-molecule inhibitors and the human embryonic carcinoma cell line, NCCIT. We found that the syntaxin-4-triggered morphological changes and a decrease in stemness signatures were independently induced by the activation of Rho-associated kinase (ROCK) and the abrogation of PI3K/Akt signaling. We also found that the extracellular expression of syntaxin-4 inactivated focal adhesion kinase (FAK) in association with the augmented expression of P-cadherin, and comparable controls of either of these downstream elements of syntaxin-4 accelerated both ROCK-induced F-actin stress fiber formation and P13K/Akt-suppressed loss of stemness signatures. Cells expressing P-cadherin inactivated FAK but FAK inhibition did not affect P-cadherin expression, demonstrating a causal relationship between P-cadherin and FAK in the event of syntaxin-4 induction. These results reveal a novel signaling axis in stem cells and shed new light on the crucial elements for stem cell plasticity and the maintenance of stemness.

Syntaxin4, P-cadherin, and CCAAT enhancer binding protein ß as signaling elements in the novel differentiation pathway for cultured embryonic stem cells.

Pluripotent stem cells possess the potential to differentiate into all three germ layers. However, upon removal of the stemness factors, pluripotent stem cells, such as embryonic stem cells (ESCs), exhibit EMT-like cell behavior and lose stemness signatures. This process involves the membrane translocation of the t-SNARE protein syntaxin4 (Stx4) and the expression of the intercellular adhesion molecule P-cadherin. The forced expression of either of these elements induces the emergence of such phenotypes even in the presence of stemness factors. Interestingly, extracellular Stx4, but not P-cadherin, appears to induce a significant upregulation of the gastrulation-related gene brachyury, along with a slight upregulation of the smooth muscle cell-related gene ACTA2 in ESCs. Furthermore, our findings reveal that extracellular Stx4 plays a role in preventing the elimination of CCAAT enhancer binding protein ß (C/EBPß). Notably, the forced overexpression of C/EBPß led to the downregulation of brachyury and a significant upregulation of ACTA2 in ESCs. These observations suggest that extracellular Stx4 contributes to early mesoderm induction while simultaneously activating an element that alters the differentiation state. The fact that a single differentiation cue can elicit multiple differentiation responses may reflect the challenges associated with achieving sensitive and directed differentiation in cultured stem cells.

Role of syntaxin3 an apical polarity protein in poorly polarized keratinocytes: regulation of asymmetric barrier formations in the skin epidermis.

The skin epidermis exhibits an asymmetric structure composed of multilayered keratinocytes and those in the outer layers form two-way physical barriers, cornified cell envelope (CCE), and tight junctions (TJs). While undifferentiated keratinocytes in the basal layer continuously deliver daughter cells outward, which undergo successive differentiation with losing their polarized characteristics, they retain the expression of several polarity proteins. In the present study, we revealed that the t-SNARE protein syntaxin3, a critical element for the formation of the apical compartment in simple epithelial cells, is required to confer the ability to organize the physical barriers on "poorly polarized" keratinocytes in epidermal outer layers. HaCaT keratinocytes with genetic ablation of syntaxin3 readily succumbed to hydrogen peroxide-induced cell death. Additionally, they lost the ability to organize TJ and CCE structures, accompanied by notable downregulation of transglutaminase1 and caspase14 (a cornification regulator) expression. These syntaxin3-knockout cells appeared to restore oxidative stress tolerance and functional TJ formation ability, in response to the inducible re-expression of exogenous syntaxin3. While plausible mechanisms underlying these phenomena remain unclear, syntaxin3, an apical polarity protein in the simple epithelia, has emerged as a potentially crucial element for barrier formation in poorly polarized keratinocytes in polarized epidermal tissue.

Extracellular epimorphin impairs expression and processing of profilaggrin in HaCaT keratinocytes.

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 10 to 12 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 epimorphin (EPM) extruded upon external stimuli 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. As expression and processing of Pro-FLG in primary keratinocytes are accompanied with apoptotic cell death, we employed HaCaT keratinocytes that grow and express filaggrin mRNA in standard culture medium. 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. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-022-00566-8.

Suppression of P-cadherin expression as a key regulatory element for embryonic stem cell stemness.

In embryonic stem (ES) cell colonies, a small subpopulation that changes cell shape and loses pluripotency often appears in two-dimensional (2D) cultures, even in the presence of a stemness factor. We have previously shown that membrane translocation of the syntaxin4, t-SNARE protein contributes to this phenomenon. Here, we show that ES cells in three-dimensional (3D) aggregates do not succumb to extruded syntaxin4 owing to suppressed expression of P-cadherin protein. While extracellular expression of syntaxin4 led to the striking upregulation of P-cadherin mRNA in both 2D and 3D-ES cells, morphological changes and appreciable expression of P-cadherin protein were detected only in 2D-ES cells. Importantly, the introduction of an expression cassette for P-cadherin practically reproduced the effects induced by extracellular syntaxin4, where the transgene product was clearly detected in 2D-, but not 3D-ES cells. An expression construct for P-cadherin-Venus harboring an in-frame insertion of the P2A sequence at the joint region gave fluorescent signals only in the cytoplasm of 2D-ES cells, demonstrating translational regulation of P-cadherin. These results provide the mechanistic insight into the uncontrollable differentiation in 2D-ES cells and shed light on the validity of the "embryoid body protocol commonly used for ES cell handling" for directional differentiation.Key words: differentiation, embryoid body, ES cells, P-cadherin, syntaxin4.

Increased expression of epimorphin in a peritoneal fibrosis mouse model.

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-ß (TGF-ß) 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-ß-induced upregulation of α-SMA and platelet-derived growth factor receptor-ß in cultured cells. CONCLUSIONS: Our results suggest that epimorphin may be a therapeutic target for fibrotic diseases of the peritoneum.

Cooperation of membrane-translocated syntaxin4 and basement membrane for dynamic mammary epithelial morphogenesis.

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.

Restoration of calcium-induced differentiation potential and tight junction formation in HaCaT keratinocytes by functional attenuation of overexpressed high mobility group box-1 protein.

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-ß, 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.

Basic fibroblast growth factor increased glucocorticoid receptors in cortical neurons through MAP kinase pathway.

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.

Membrane-tethered syntaxin-4 locally abrogates E-cadherin function and activates Smad signals, contributing to asymmetric mammary epithelial morphogenesis.

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-ß 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 ß-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.

Corticotropin-releasing hormone-binding protein is up-regulated by brain-derived neurotrophic factor and is secreted in an activity-dependent manner in rat cerebral cortical neurons.

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.

Generation of iPS-derived model cells for analyses of hair shaft differentiation.

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.

Sulfated glycosaminoglycans and non-classically secreted proteins, basic FGF and epimorphin, coordinately regulate TGF-ß-induced cell behaviors of human scar dermal fibroblasts.

BACKGROUND: Upon skin injuries, dermal fibroblasts actively produce transforming growth factor-ß (TGF-ß), 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-ß signaling, along with its possible regulators basic fibroblast growth factor (bFGF) and cell surface epimorphin. bFGF and epimorphin might regulate the TGF-ß-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-ß alone, TGF-ß plus bFGF, and TGF-ß 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-ß-mediated changes in HSF behaviors. We found that heparinoid can directly associate with TGF-ß, 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-ß, 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-ß-induced HSF behaviors.

Membrane translocation of t-SNARE protein syntaxin-4 abrogates ground-state pluripotency in mouse embryonic stem cells.

Embryonic stem (ES) and induced pluripotent stem (iPS) cells are attractive tools for regenerative medicine therapies. However, aberrant cell populations that display flattened morphology and lose ground-state pluripotency often appear spontaneously, unless glycogen synthase kinase 3ß (GSK3ß) and mitogen-activated protein kinase kinase (MEK1/2) are inactivated. Here, we show that membrane translocation of the t-SNARE protein syntaxin-4 possibly is involved in this phenomenon. We found that mouse ES cells cultured without GSK3ß/MEK1/2 inhibitors (2i) spontaneously extrude syntaxin-4 at the cell surface and that artificial expression of cell surface syntaxin-4 induces appreciable morphological changes and mesodermal differentiation through dephosphorylation of Akt. Transcriptome analyses revealed several candidate elements responsible for this, specifically, an E-to P-cadherin switch and a marked downregulation of Zscan4 proteins, which are DNA-binding proteins essential for ES cell pluripotency. Embryonic carcinoma cell lines F9 and P19CL6, which maintain undifferentiated states independently of Zscan4 proteins, exhibited similar cellular behaviors upon stimulation with cell surface syntaxin-4. The functional ablation of E-cadherin and overexpression of P-cadherin reproduced syntaxin-4-induced cell morphology, demonstrating that the E- to P-cadherin switch executes morphological signals from cell surface syntaxin-4. Thus, spontaneous membrane translocation of syntaxin-4 emerged as a critical element for maintenance of the stem-cell niche.

Extracellularly Extruded Syntaxin-4 Binds to Laminin and Syndecan-1 to Regulate Mammary Epithelial Morphogenesis.

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.

A newly established culture method highlights regulatory roles of retinoic acid on morphogenesis and calcification of mammalian limb cartilage.

During mammalian embryogenesis, sclerotome-derived chondrocytes in the limb bud are arranged into a complicated bone shape with specific areas undergoing hypertrophy and calcification, creating a region-specific mineralized pattern in the cartilage. To follow chondrogenesis progression in vitro, we isolated limb cartilage from mice on embryonic day 13 (E13) and cultured it at the air-liquid interface after microsurgical removal of the ectoderm/epidermis. Explants underwent proper morphogenesis, giving rise to complete templates for limb bones in vitro. We found that region-specific calcification patterns resembling limbs of prepartum mature embryos could be induced in explants using culture medium containing high concentrations of CaCl2 (Ca), ascorbic acid (AA), and ß-glycerophosphoric acid (BGP). In this culture system, excess amounts of all-trans retinoic acid (RA) severely disrupted morphogenesis and calcification patterns in limb cartilage. These effects were more pronounced in forearms than in phalanges. Although dissociated, the nascent chondrocytes in culture did not give rise to cartilage units even though augmented calcification was induced in these cell aggregates in the presence of RA. Taken together, our newly established culture system revealed that RA independently regulates three-dimensional morphogenesis and calcification.

Extracellularly Extruded Syntaxin-4 Is a Potent Cornification Regulator of Epidermal Keratinocytes.

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.

CCAAT/enhancer binding protein beta (C/EBPß) isoform balance as a regulator of epithelial-mesenchymal transition in mouse mammary epithelial cells.

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/EBPß, 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/EBPß 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/EBPß gene products in mouse mammary epithelial cells, which are affected by contact with basement membrane, are defined as a potential regulator of metastatic potential.