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.

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.

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.

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.

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.

Krtap11-1, a hair keratin-associated protein, as a possible crucial element for the physical properties of hair shafts.

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.

Effluent syntaxin3 from dying cells affords protection against apoptosis in epidermal keratinocytes.

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.

Extracellular syntaxin4 triggers the differentiation program in teratocarcinoma F9 cells that impacts cell adhesion properties.

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.

Epimorphin is a novel regulator of the progesterone receptor isoform-a.

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.

Ectopic expression of syntaxin3 affects behaviors of B16 melanoma by controlling actin dynamics.

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.

Extracellular epimorphin modulates epidermal differentiation signals mediated by epidermal growth factor receptor.

BACKGROUND: The epidermal stratification/differentiation program is initiated in keratinocytes by a basement membrane-detachment cue and subsequently controlled by spatially and temporally regulated signaling molecules. The vital signals for the developmental behavior of the epidermis include those mediated by epidermal growth factor receptor (EGFR); however, regulatory elements responsible for activation have not yet been fully elucidated. OBJECTIVE: The objectives of this study were (1) to assess the effects of EGFR activation on epidermal differentiation, (2) to study the effects of epimorphin on the level of EGFR signaling degree dependent on matrix engagement, and (3) to address the impact of epimorphin modulation on EGFR-driven epidermal differentiation in a three-dimensional (3D) organotypic skin model. METHODS: We constructed skin-equivalent models with a well-stratified differentiated epidermis and utilized them to evaluate the epidermal behaviors. RESULTS: Extracellularly secreted epimorphin was identified as a strong candidate for signaling pathway involvement. In a 3D epidermis model, EGF stimulation was sufficient for epidermal stratification. However, overactivation of EGFR led to irregular multicellular arrangements with an abnormal differentiation profile, which appeared to be reorganized back to the normal epidermal phenotype by extracellular epimorphin. Extracellular epimorphin interestingly attenuated EGF-stimulated EGFR phosphorylation, cell growth, and migration in adherent cells. In contrast to the results of adhesion culture, extracellular epimorphin reinforced EGFR activation in suspended cells. CONCLUSIONS: These results demonstrate that epimorphin modulates the signaling pathways mediated by EGFR for epidermal tissue organization.

Identification of the C-terminal tail domain of AHF/trichohyalin as the critical site for modulation of the keratin filamentous meshwork in the keratinocyte.

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.

A culture system for the live analysis of successive developmental processes and the morphological control of mammalian vertebral cartilage.

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.

Molecular elements of the regulatory control of keratin filament modulator AHF/trichohyalin in the hair follicle.

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.

Phosphorylation of claudin-4 is required for tight junction formation in a human keratinocyte cell line.

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.