Cultured Mouse Keratinocytes Research Articles

Regeneration of Multilineage Skin Epithelia by Differentiated Keratinocytes

Although homeostasis of rapidly renewing tissues like skin epithelia is maintained by stem cells, the committed progeny of stem cells in the basal layer of epidermis retain regenerative potential and are capable of forming epidermis in response to environmental cues. It is not clear, however, at what point within the epidermal lineage keratinocytes lose this regenerative potential. In this study, we examined the extent of tissue formation by post-mitotic differentiated keratinocytes. We show that cultures of mouse keratinocytes that were, by all measures, differentiated were able to reform a self-renewing, hair-bearing skin when transplanted onto suitable sites in vivo. Genetic labeling and lineage-tracing studies in combination with an involucrin-driven Cre/lox reporter system confirmed that transplanted differentiated keratinocytes were indeed the source of the regenerated skin. More importantly, analysis of early stages of skin regeneration showed hallmarks of dedifferentiation of transplanted differentiated keratinocytes. These data indicate that commitment to differentiation does not prohibit cells from re-entering the cell cycle, de-differentiating, and acquiring "stemness". These findings suggest that epidermis can use different strategies for homeostasis and tissue regeneration.

Identification of a Novel Family of Laminin N-terminal Alternate Splice Isoforms

The laminins are a family of heterotrimeric basement membrane proteins that play roles in cellular adhesion, migration, and tissue morphogenesis. Through in silico analysis of the laminin-encoding genes, we identified a novel family of alternate splice isoforms derived from the 5′-end of the LAMA3 and LAMA5 genes. These isoforms resemble the netrins in that they contain a laminin N-terminal domain followed by a short stretch of laminin-type epidermal growth factor-like repeats. We suggest the terms LaNt (laminin N terminus) α3 and LaNt α5, for the predicted protein products of these mRNAs. RT-PCR confirmed the presence of these transcripts at the mRNA level. Moreover, they exhibit differential, tissue-specific, expression profiles. To confirm the existence of LaNt α3 protein, we generated an antibody to a unique domain within the putative polypeptide. This antibody recognizes a protein at the predicted molecular mass of 64 kDa by immunoblotting. Furthermore, immunofluorescence analyses revealed a basement membrane staining in epithelial tissue for LaNt α3 and LaNt α3 localized along the substratum-associated surface of cultured keratinocytes. We have also tested the functionality LaNt α3 through RNAi-mediated knockdown. Keratinocytes exhibiting specific knockdown of LaNt α3 displayed impaired adhesion, stress resistance, and reduced ability to close scratch wounds in vitro.

Activation of P2X7-mediated apoptosis Inhibits DMBA/TPA-induced formation of skin papillomas and cancer in mice

BackgroundThe study tested the hypothesis that apoptosis can prevent and control growth of neoplastic cells. Previous studies in-vitro have shown that the pro-apoptotic P2X7 receptor regulates growth of epithelial cells. The specific objective of the present study was to understand to what degree the P2X7 system controls development and growth of skin cancer in vivo, and what cellular and molecular mechanisms are involved in the P2X7 action.MethodsSkin neoplasias in mice (papillomas, followed by squamous spindle-cell carcinomas) were induced by local application of DMBA/TPA. Experiments in-vitro utilized cultured epidermal keratinocytes generated from wild-type or from P2X7-null mice. Assays involved protein immunostaining and Western blots; mRNA real-time qPCR; and apoptosis (evaluated in situ by TUNEL and quantified in cultured keratinocytes as solubilized DNA or by ELISA). Changes in cytosolic calcium or in ethidium bromide influx (P2X7 pore formation) were determined by confocal laser microscopy.Results(a) Co-application on the skin of the P2X7 specific agonist BzATP inhibited formation of DMBA/TPA-induced skin papillomas and carcinomas. At the completion of study (week 28) the proportion of living animals with cancers in the DMBA/TPA group was 100% compared to 43% in the DMBA/TPA+BzATP group. (b) In the normal skin BzATP affected mainly P2X7-receptor – expressing proliferating keratinocytes, where it augmented apoptosis without evoking inflammatory changes. (c) In BzATP-treated mice the degree of apoptosis was lesser in cancer than in normal or papilloma keratinocytes. (d) Levels of P2X7 receptor, protein and mRNA were 4–5 fold lower in cancer tissues than in normal mouse tissues. (e) In cultured mouse keratinocytes BzATP induced apoptosis, formation of pores in the plasma membrane, and facilitated prolonged calcium influx. (f) The BzATP-induced apoptosis, pore-formation and augmented calcium influx had similar dose-dependence for BzATP. (g) Pore formation and the augmented calcium influx were depended on the expression of the P2X7 receptor, while the BzATP-induced apoptosis depended on calcium influx. (h) The BzATP-induced apoptosis could be blocked by co-treatment with inhibitors of caspase-9 and caspase-3, but not of caspase-8.Conclusion(a) P2X7-dependent apoptosis is an important mechanism that controls the development and progression of epidermal neoplasia in the mouse. (b) The P2X7-dependent apoptosis is mediated by calcium influx via P2X7 pores, and involves the caspase-9 (mitochondrial) pathway. (c) The diminished pro-apoptotic effect of BzATP in mouse cancer keratinocytes is possibly the result of low expression of the P2X7 receptor. (d) Activation of P2X7-dependent apoptosis, e.g. with BzATP could be a novel chemotherapeutic growth-preventive modality for papillomas and epithelial cancers in vivo.

The expression of differentiation markers in aquaporin-3 deficient epidermis

Aquaporin-3 (AQP3) is a water/glycerol transporting protein expressed strongly at the plasma membrane of keratinocytes. There is evidence for involvement of AQP3-facilitated water and glycerol transport in keratinocyte migration and proliferation, respectively. Here, we investigated the involvement of AQP3 in keratinocyte differentiation. Studies were done using AQP3 knockout mice, primary cultures of mouse keratinocytes (AQP3 knockout), neonatal human keratinocytes (AQP3 knockdown), and human skin. Cells were cultured with high Ca(2+) or 1alpha,25-dihydroxyvitamin D(3) (VD(3)) to induce differentiation. The expression of differentiation marker proteins and differentiating responses were comparable in control and AQP3-knockout or knockdown keratinocytes. Topical application of all-trans retinoic acid (RA), a known regulator of keratinocyte differentiation and proliferation, induced comparable expression of differentiation marker proteins in wildtype and AQP3 null epidermis, though with impaired RA-induced proliferation in AQP3 null mice. Immunostaining of human and mouse epidermis showed greater AQP3 expression in cells undergoing proliferation than differentiation. Our results showed little influence of AQP3 on keratinocyte differentiation, and provide further support for the proposed involvement of AQP3-facilitated cell proliferation.

Expression of HPV 58 Long and Short L1 Capsid Proteins in Primary Mouse Keratinocyte Cultures

We studied expression of HPV 58 long and short L1 proteins in primary mouse keratinocyte (KC) cultures by transient transfection of the L1 expression constructs. Following transient transfection, long and short L1 open reading frames (ORFs) were transcribed continuously for 9 days; however, no significant difference was detected between the long and short L1 mRNA levels measured by quantitative RT-PCR. Western blot analysis showed that both long and short L1 proteins were continuously detected in L1-transfected KCs for 9 days post-transfection and the significantly increased signals of the L1 proteins over time were associated with KC differentiation. Moreover, L1 protein was more abundant in KCs transfected with the short L1 ORF than the long L1 ORF. In vitro translation of the L1 mRNAs indicated further that the short L1 mRNA had significantly higher translation efficiency than the long L1 mRNA in cell-free lysate system. The L1 proteins expressed from the two L1 mRNAs in KCs were similarly stable. Thus, approximate 40% lower level of expression of the L1 protein in KCs transfected with the long L1 ORF was probably due to a stem-loop structure with high DeltaG value downstream the first AUG codon in its mRNA secondary structure. This stem-loop structure might prevent efficient binding of the ribosome to mRNA and therefore reduced translation.

Defect of Hepatocyte Growth Factor Activator Inhibitor Type 1/Serine Protease Inhibitor, Kunitz Type 1 (Hai-1/Spint1) Leads to Ichthyosis-Like Condition and Abnormal Hair Development in Mice

Hepatocyte growth factor activator inhibitor type 1 (HAI-1)/serine protease inhibitor, Kunitz type 1 (SPINT1) is a membrane-bound, serine proteinase inhibitor initially identified as an inhibitor of hepatocyte growth factor activator. It also inhibits matriptase and prostasin, both of which are membrane-bound serine proteinases that have critical roles in epidermal differentiation and function. In this study, skin and hair phenotypes of mice lacking the Hai-1/Spint1 gene were characterized. Previously, we reported that the homozygous deletion of Hai-1/Spint1 in mice resulted in embryonic lethality attributable to impaired placental development. To test the role of Hai-1/Spint1 in mice, the placental function of Hai-1/Spint1-mutant mice was rescued. Injection of Hai-1/Spint1(+/+) blastocysts with Hai-1/Spint1(-/-) embryonic stem cells successfully generated high-chimeric Hai-1/Spint1(-/-) embryos (B6Hai-1(-/-High)) with normal placentas. These embryos were delivered without apparent developmental abnormalities, confirming that embryonic lethality of Hai-1/Spint1(-/-) mice was caused by placental dysfunction. However, newborn B6Hai-1(-/-High) mice showed growth retardation and died by 16 days. These mice developed scaly skin because of hyperkeratinization, reminiscent of ichthyosis, and abnormal hair shafts that showed loss of regular cuticular septation. The interfollicular epidermis showed acanthosis with enhanced Akt phosphorylation. Immunoblot analysis revealed altered proteolytic processing of profilaggrin in Hai-1/Spint1-deleted skin with impaired generation of filaggrin monomers. These findings indicate that Hai-1/Spint1 has critical roles in the regulated keratinization of the epidermis and hair development.

Multiple Signaling Pathways Are Responsible for Prostaglandin E2–Induced Murine Keratinocyte Proliferation

Although prostaglandin E2 (PGE2) has been shown by pharmacologic and genetic studies to be important in skin cancer, the molecular mechanism(s) by which it contributes to tumor growth is not well understood. In this study, we investigated the mechanisms by which PGE2 stimulates murine keratinocyte proliferation using in vitro and in vivo models. In primary mouse keratinocyte cultures, PGE2 activated the epidermal growth factor receptor (EGFR) and its downstream signaling pathways as well as increased cyclic AMP (cAMP) production and activated the cAMP response element binding protein (CREB). EGFR activation was not significantly inhibited by pretreatment with a c-src inhibitor (PP2), nor by a protein kinase A inhibitor (H-89). However, PGE2-stimulated extracellularly regulated kinase 1/2 (ERK1/2) activation was completely blocked by EGFR, ERK1/2, and phosphatidylinositol 3-kinase (PI3K) pathway inhibitors. In addition, these inhibitors attenuated the PGE2-induced proliferation, nuclear factor-kappa B, activator protein-1 (AP-1), and CREB binding to the promoter regions of the cyclin D1 and vascular endothelial growth factor (VEGF) genes and expression of cyclin D1 and VEGF in primary mouse keratinocytes. Similarly, in vivo, we found that WT mice treated with PGE2 and untreated cyclooxygenase-2-overexpressing transgenic mice had higher levels of cell proliferation and expression of cyclin D1 and VEGF, as well as higher levels of activated EGFR, nuclear factor-kappa B, AP-1, and CREB, than vehicle-treated WT mice. Our findings provide evidence for a link between cyclooxygenase-2 overexpression and EGFR-, ERK-, PI3K-, cAMP-mediated cell proliferation, and the tumor-promoting activity of PGE2 in mouse skin.

UVB light upregulates prostaglandin synthases and prostaglandin receptors in mouse keratinocytes

Prostaglandins belong to a class of cyclic lipid-derived mediators synthesized from arachidonic acid via COX-1, COX-2 and various prostaglandin synthases. Members of this family include prostaglandins such as PGE 2, PGF 2α, PGD 2 and PGI 2 (prostacyclin) as well as thromboxane. In the present studies we analyzed the effects of UVB on prostaglandin production and prostaglandin synthase expression in primary cultures of undifferentiated and calcium-differentiated mouse keratinocytes. Both cell types were found to constitutively synthesize PGE 2, PGD 2 and the PGD 2 metabolite PGJ 2. Twenty-four hours after treatment with UVB (25 mJ/cm 2), production of PGE 2 and PGJ 2 increased, while PGD 2 production decreased. This was associated with increased expression of COX-2 mRNA and protein. UVB (2.5–25 mJ/cm 2) also caused marked increases in mRNA expression for the prostanoid synthases PGDS, mPGES-1, mPGES-2, PGFS and PGIS, as well as expression of receptors for PGE 2 (EP1 and EP2), PGD 2 (DP and CRTH2) and prostacyclin (IP). UVB was more effective in inducing COX-2 and DP in differentiated cells and EP1 and IP in undifferentiated cells. UVB readily activated keratinocyte PI-3-kinase (PI3K)/Akt, JNK and p38 MAP signaling pathways which are known to regulate COX-2 expression. While inhibition of PI3K suppressed UVB-induced mPGES-1 and CRTH2 expression, JNK inhibition suppressed mPGES-1, PGIS, EP2 and CRTH2, and p38 kinase inhibition only suppressed EP1 and EP2. These data indicate that UVB modulates expression of prostaglandin synthases and receptors by distinct mechanisms. Moreover, both the capacity of keratinocytes to generate prostaglandins and their ability to respond to these lipid mediators are stimulated by exposure to UVB.

Sequential Down-regulation of E-Cadherin with Squamous Cell Carcinoma Progression: Loss of E-Cadherin via a Prostaglandin E2-EP2–Dependent Posttranslational Mechanism

The incidence of skin cancer is on the rise, with over 1 million new cases yearly. Although it is known that squamous cell cancers (SCC) are caused by UV light, the mechanism(s) involved remains poorly understood. In vitro studies with epithelial cells or reports examining malignant skin lesions suggest that loss of E-cadherin-mediated cell-cell contacts may contribute to SCCs. Other studies show a pivotal role for cyclooxygenase-dependent prostaglandin E2 (PGE2) synthesis in this process. Using chronically UV-irradiated SKH-1 mice, we show a sequential loss of E-cadherin-mediated cell-cell contacts as lesions progress from dysplasia to SCCs. This E-cadherin down-regulation was also evident after acute UV exposure in vivo. In both chronic and acute UV injury, E-cadherin levels declined at a time when epidermal PGE2 synthesis was enhanced. Inhibition of PGE2 synthesis by indomethacin in vitro, targeted deletion of EP2 in primary mouse keratinocyte (PMK) cultures or deletion of the EP2 receptor in vivo abrogated this UV-induced E-cadherin down-regulation. In contrast, addition of PGE2 or the EP2 receptor agonist butaprost to PMK produced a dose- and time-dependent decrease in E-cadherin. We also show that UV irradiation, via the PGE2-EP2 signaling pathway, may initiate tumorigenesis in keratinocytes by down-regulating E-cadherin-mediated cell-cell contacts through its mobilization away from the cell membrane, internalization into the cytoplasm, and shuttling through the lysosome and proteasome degradation pathways. Further understanding of how UV-PGE2-EP2 down-regulates E-cadherin may lead to novel chemopreventative strategies for the treatment of skin and other epithelial cancers.

Endothelin-2/vasoactive Intestinal Contractor via ROCK regulates transglutaminase 1 on differentiation of mouse keratinocytes

We previously found that endothelin-2/vasoactive intestinal contractor (ET-2/VIC) greatly increased in mouse epidermis after birth. In the present study, we evaluated whether ET-2/VIC expression was associated with the calcium-induced differentiation of cultured mouse keratinocytes. The differentiation induction was revealed by morphological change, cornified envelope (CE) formation, and involucrin and transglutaminase 1 (TG 1) expressions. ET-2/VIC gene expression and peptide production subsequently increased in the induction of the differentiation. We also found that Y-27632, a Rho-associated coiled–coil forming protein serine/threonine kinase (ROCK) inhibitor, suppressed up-regulation of ET-2/VIC gene expression, the induction of morphological change, the CE formation, and TG 1 expression, but not involucrin expression. These results indicate new three findings, (1) ET-2/VIC expression increases and has potential as a differentiation marker, (2) ET-2/VIC expression is mediated by ROCK, and (3) the ROCK regulated TG 1 expression, on the calcium-induced differentiation of mouse keratinocytes.

Synergistic Pathogenic Effects of Combined Mouse Monoclonal Anti-Desmoglein 3 IgG Antibodies on Pemphigus Vulgaris Blister Formation

Pemphigus vulgaris (PV) is an autoimmune blistering disease caused by anti-desmoglein 3 (Dsg3) IgG antibodies. Previously, we generated an active mouse model for PV by adoptive transfer of splenocytes from immunized or naive Dsg3(-/-) mice. In this study, we isolated 10 anti-Dsg3 IgG mAbs (NAK-series) from PV model mice generated by transfer of naive Dsg3(-/-) splenocytes. We characterized their epitopes using domain-swapped and point-mutated Dsg1/Dsg3 molecules and examined their pathogenic activities in blister formation in three different assays. In a passive transfer model using neonatal mice, eight of 10 NAK mAbs showed pathogenic activity when injected together with half the minimum pathogenic dose of anti-Dsg1 IgG autoantibodies from pemphigus foliaceus (PF) patients. None of the mAbs could induce the PV phenotype when individual hybridoma clones were inoculated by peritoneal injection into adult Rag2(-/-) mice. NAK mAbs displayed a range of potency in an in vitro dissociation assay using primary cultured mouse keratinocytes. Interestingly, when multiple hybridoma clones recognizing different epitopes were inoculated in combination, recipient mice developed the PV phenotype. In vitro dissociation assays confirmed that combined NAK mAbs had synergistic pathogenic effects. These findings indicate that although an individual anti-Dsg3 IgG is not sufficient to cause blistering in adult mice, several together can induce the PV phenotype. These mAbs will provide a valuable tool to investigate the molecular mechanisms of blister formation, mimicking the effects of the polyclonal IgG antibodies found in patients.

Protein Kinase C Negatively Regulates Akt Activity and Modifies UVC-induced Apoptosis in Mouse Keratinocytes

Skin keratinocytes are subject to frequent chemical and physical injury and have developed elaborate cell survival mechanisms to compensate. Among these, the Akt/protein kinase B (PKB) pathway protects keratinocytes from the toxic effects of ultraviolet light (UV). In contrast, the protein kinase C (PKC) family is involved in several keratinocyte death pathways. During an examination of potential interactions among these two pathways, we found that the insulin-like growth factor (IGF-1) activates both the PKC and the Akt signaling pathways in cultured primary mouse keratinocytes as indicated by increased phospho-PKC and phospho-Ser-473-Akt. IGF-1 also selectively induced translocation of PKCdelta and PKCepsilon from soluble to particulate fractions in mouse keratinocytes. Furthermore, the PKC-specific inhibitor, GF109203X, increased IGF-1-induced phospho-Ser-473-Akt and Akt kinase activity and enhanced IGF-1 protection from UVC-induced apoptosis. Selective activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) reduced phospho-Ser-473-Akt, suggesting that activation of PKC inhibits Akt activity. TPA also attenuated IGF-1 and epidermal growth factor-induced phospho-Ser-473-Akt, reduced Akt kinase activity, and blocked IGF-1 protection from UVC-induced apoptosis. The inhibition of Akt activity by TPA was reduced by inhibitors of protein phosphatase 2A, and TPA stimulated the association of phosphatase 2A with Akt. Individual PKC isoforms were overexpressed in cultured keratinocytes by transduction with adenoviral vectors or inhibited with PKC-selective inhibitors. These studies indicated that PKCdelta and PKCepsilon were selectively potent at causing dephosphorylation of Akt and modifying cell survival, whereas PKCalpha enhanced phosphorylation of Akt on Ser-473. Our results suggested that activation of PKCdelta and PKCepsilon provide a negative regulation for Akt phosphorylation and kinase activity in mouse keratinocytes and serve as modulators of cell survival pathways in response to external stimuli.

Chemoprevention of Skin Carcinogenesis by Phenylretinamides: Retinoid Receptor–Independent Tumor Suppression

Fenretinide [N-(4-hydroxyphenyl)retinamide or 4-HPR] is a synthetic retinoid analogue with antitumor and chemopreventive activities. N-(4-Methoxyphenyl)retinamide (4-MPR) is the most abundant metabolite of 4-HPR detected in human serum following 4-HPR therapy. We have shown in in vitro studies that 4-HPR and 4-MPR can act independent of the classic nuclear retinoid receptor pathway and that 4-HPR, but not 4-MPR, can also activate nuclear retinoid receptors. In this study, we have compared the chemopreventive effects of topically applied 4-HPR and 4-MPR with the primary biologically active retinoid, all-trans retinoic acid (ATRA), in vivo in the mouse skin two-stage chemical carcinogenesis model. All three retinoids suppressed tumor formation but the effect of 4-HPR and 4-MPR, and not of ATRA, was sustained after their discontinuation. The tumor-suppressive effects of 4-HPR and 4-MPR were quantitatively and qualitatively similar, suggesting that the two may be acting through the same retinoid receptor-independent mechanism(s). We further explored this effect in vitro by analyzing primary cultures of mouse keratinocytes treated with the same retinoids. All three could induce apoptosis with a 48-hour treatment and only ATRA and 4-HPR induced an accumulation of cells in the G1 phase of the cell cycle. This finding is consistent with our previous results showing that the effects of phenylretinamides on the cell cycle are retinoid receptor dependent whereas apoptosis induction is not. A microarray-based comparison of gene expression profiles for mouse skin treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) alone and TPA + 4-HPR or TPA + 4-MPR reveals a high degree of coincidence between the genes regulated by the two phenylretinamides. We propose that 4-HPR may exert therapeutic and chemopreventive effects by acting primarily through a retinoid receptor-independent mechanism(s) and that 4-MPR may contribute to the therapeutic effect of 4-HPR by acting through the same retinoid receptor-independent mechanism(s).

986. Targeting Transgene Expression to Suprabasal Layers of Epidermis as a Strategy To Evade Immune Responses

Top of pageAbstract Transfer of new genetic material to epidermis offers an opportunity for corrective gene therapy for a variety of inherited skin and systemic deficiency disorders for which the genetic basis has been identified. Host immune responses to therapeutic gene products however remains a potentially serious complication for cutaneous gene therapy especially in patients carrying a null mutation in the affected allele. Using an ex vivo method of cutaneous gene transfer we have shown that expression of an antigenic transgene product in keratinocyte progenitor population resulted in rejection of transgenic skin in 4-5 weeks post grafting. The loss of transgenic cells was associated with Th2 type immune responses characterized by greater levels of interleukin 4 secreted by lymphocytes, an intense eosinophil infiltration in transgenic tissue, and a predominance of anti-GFP IgG1 in serum. To eliminate or control transgene-specific immune responses, we explored the effect of confining neoantigen expression specifically to differentiating epidermal keratinocytes in the suprabasal layers of epidermis. Cultures of mouse keratinocytes were transduced with a transcriptionally-targeted retrovector in which GFP expression was controlled by involucrin promoter (INV-GFP). Reconstitution of mouse skin with INV-GFP-transduced keratinocytes resulted in prolonged GFP expression in the suprabasal layers of epidermis. Interestingly, characterization of transgene-specific immune responses indicated elicitation of humoral responses and release of Th2 cytokines by T cells, however, tissue inflammation was significantly suppressed. We are currently investigating the mechanism of suppression of inflammation and persistence of transgene in this model. Our data suggest that targeting transgene expression to suprabasal layers of epidermis may be used as a strategy to restricting immune-mediated rejection of transgenic keratinocytes.

Expression of RNA-Binding Protein Musashi in Hair Follicle Development and Hair Cycle Progression

Epithelial stem cells reside in the hair follicle (HF) bulge region and possess the ability to differentiate into a variety of cutaneous epithelial cells. The evolutionarily conserved Musashi family of RNA-binding proteins is associated with maintenance and/or asymmetric cell division of neural progenitor cells, and a mammalian Musashi protein is expressed in various epithelial stem/progenitor cells, including gut, stomach, and mammary gland. Thus, we hypothesized that Musashi might be expressed in stem cells and early progenitor cells of HF epithelium. Reverse transcriptase-polymerase chain reaction and immunoblotting identified Musashi-1 (Msi-1) and Musashi-2 (Msi-2) mRNA and protein in cultured mouse keratinocytes, but only Msi-1 was identified in human keratinocytes. In mice, immunohistochemical studies showed that Msi-1 and Msi-2 were expressed in the epidermis and HFs from E14.5 until adulthood. In the early anagen phase, Msi-1 and Msi-2 were expressed in the bulge and secondary germ cells and subsequently in inner root sheath (IRS) cells, especially the middle IRS cells, during the late anagen phase. In human skin, Msi-1 was detected in fetal HF cells but not in adult HFs. These observations suggest that Musashi functions not only in the asymmetric division of early progenitor cells but also in the differentiation of IRS cells during HF development and hair cycle progression.

Mismatch repair protein Msh2 contributes to UVB-induced cell cycle arrest in epidermal and cultured mouse keratinocytes

Nucleotide excision repair (NER), cell cycle regulation and apoptosis are major defence mechanisms against the carcinogenic effects of UVB radiation. NER eliminates UVB-induced DNA photolesions via two subpathways: global genome repair (GGR) and transcription-coupled repair (TCR). In a previous study, we found UVB-induced accumulation of tetraploid (4N) keratinocytes in the epidermis of Xpc −/− mice (no GGR), but not in Xpa −/− (no TCR and no GGR) or in wild-type (WT) mice. We inferred that this arrest in Xpc −/− mice is caused by erroneous replication past photolesions, leading to ‘compound lesions’ known to be recognised by mismatch repair (MMR). MMR-induced futile cycles of breakage and resynthesis at sites of compound lesions may then sustain a cell cycle arrest. The present experiments with Xpc −/− Msh2 −/− mice and derived keratinocytes show that the MMR protein Msh2 indeed plays a role in the generation of the UVB-induced arrested cells: a Msh2-deficiency lowered significantly the percentage of arrested cells in vivo (40–50%) and in vitro (30–40%). Analysis of calyculin A (CA)-induced premature chromosome condensation (PCC) of cultured Xpc −/− keratinocytes showed that the delayed arrest occurred in late S phase rather than in G 2-phase. Taken together, the results indicate that in mouse epidermis and cultured keratinocytes, the MMR protein Msh2 plays a role in the UVB-induced S-phase arrest. This indicates that MMR plays a role in the UVB-induced S-phase arrest. Alternatively, Msh2 may have a more direct signalling function.

Epidermal growth factor receptor-mediated activation of Stat3 during multistage skin carcinogenesis.

In the present study, we have investigated the possible role of signal transducers and activators of transcription (STATs), particularly Stat3, in mouse skin tumor promotion and multistage carcinogenesis. Stat1, Stat3, and Stat5 were activated in mouse epidermis after treatment with different classes of tumor promoters, including 12-O-tetradecanoylphorbol-13-acetate (TPA), okadaic acid, and chrysarobin. In addition, Stat1, Stat3, and Stat5 were constitutively activated in skin tumors generated by the two-stage carcinogenesis regimen using 7,12-dimethylbenz(a)anthracene as initiator and TPA as promoter. Several approaches were used to examine the possible role of epidermal growth factor receptor (EGFR) in modulating Stat3 activity during tumor promotion. In primary cultures of mouse keratinocytes, addition of exogenous EGF led to activation of Stat3 as shown by an elevation in tyrosine phosphorylation and nuclear translocation. In epidermis of transgenic mice expressing transforming growth factor alpha under control of the keratin 14 promoter, Stat3 was constitutively activated. Abrogation of EGFR function in mouse epidermis using an EGFR kinase inhibitor or by overexpressing a dominant negative form of EGFR led to a reduction in Stat3 activation in response to TPA treatment. Immunoprecipitation analyses using lysates from TPA-treated epidermis and skin papillomas showed enhanced interaction between the EGFR and Stat3. Finally, Stat3 deficiency in mouse epidermis significantly reduced the proliferative response after TPA treatment. Collectively, the current results suggest that Stat3 activation may be a critical event during mouse skin tumor promotion, possibly through regulation of keratinocyte proliferation. In addition, Stat3 activation in tumor promoter-treated epidermis and in skin papillomas may occur, at least in part, via interaction with and phosphorylation by the EGFR. Finally, constitutive activation of Stat3 in both papillomas and squamous cell carcinomas suggest a role in both the development of autonomous growth and the progression of epithelial tumors in mouse skin.

Expression of a novel secretory form ( Crb1s) of mouse Crumbs homologue Crb1 in skin development

Drosophila Crumbs and the mammalian homologues encoded by the Crb genes are transmembrane proteins required for determination of retinal cell polarity. We cloned a novel variant of mouse Crb1 and termed it Crb1s. Since the 3 ′-end of exon 6 remained unspliced, Crb1s coded for a short secretory protein lacking the transmembrane and cytoplasmic domains required for the function of Crb1. The Crb1 expression was confined to brain and eye, whereas Crb1s was detectable in various tissues including skin, lung, and kidney in adult mice. Active expression of Crb1s, but not Crb1, was observed during the skin development, in which localization of the Crb1s protein was altered from the basal layer to the upper layers. Cultured mouse keratinocytes synthesized the Crb1s protein and secreted a 80 kDa processed form to the supernatant. After Ca 2+-induced differentiation, Crb1s became associated with focal adhesions and cell–cell contacts. Crb1s may play a role distinct from that of Crb1 in epidermal tissue morphogenesis.

Nitric oxide induces expression of cyclooxygenase-2 in mouse skin through activation of NF-kappaB.

Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are frequently overexpressed in tumor tissues or transformed cells. In the present work, we assessed the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on expression of iNOS and COX-2 in mouse skin. Topical application to the dorsal skin of female ICR mice of 10 nmol TPA led to maximal induction of iNOS and COX-2 protein expression at approximately 2 and 4 h, respectively. When applied topically onto shaven backs of mice 30 min prior to TPA, the NOS inhibitor aminoguanidine (AG) inhibited the expression of COX-2 protein at the pharmacologically effective dose. Pretreatment with a more specific iNOS inhibitor, N(G)-nitro-l-arginine-methyl ester, also suppressed TPA-induced COX-2 expression. Immunohistochemical analysis of TPA-treated mouse skin using an anti-nitrotyrosine antibody reveals enhanced levels of nitrotyrosine protein localized in epidermal and dermal layers. Topical application of NO donors, such as sodium nitroprusside (SNP) and S-nitroso-N-acetyl-d,l-penicillamine, induced expression of COX-2 in mouse skin, which was attenuated by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide. SNP treatment stimulated NF-kappaB activation in mouse skin, which was associated with the degradation of IkappaBalpha. Topical application of inhibitors of NF-kappaB, such as pyrrolidine dithiocarbamate or N-alpha-p-tosyl-l-lysine chloromethylketone, inhibited the SNP-induced COX-2 expression. SNP induced a weak but concentration-related increase in COX-2 expression in cultured mouse keratinocytes, which was abolished by treatment with SN50, a specific inhibitor of nuclear translocation of NF-kappaB. Mouse keratinocytes treated with SNP exhibited an elevated NF-kappaB-driven COX-2 promoter activity. Topical application of AG (10 micro mol) prior to each TPA treatment after initiation reduced the multiplicity of papillomas by 44% at 22 weeks. In conclusion, up-regulation of COX-2 by NO may be mediated by activation of NF-kappaB in mouse skin, which provides a molecular mechanism by which COX-2 is induced during tumor promotion.

Genetic deficiency or pharmacological inhibition of cyclooxygenase‐1 or ‐2 induces mouse keratinocyte differentiation in vitro and in vivo

Previously we demonstrated that genetic deficiency of the cyclooxygenases (COX-1 or COX-2) altered keratinocyte differentiation in mouse skin [Tiano et. al. (2002) Cancer Res. 62, 3395-3401]. In this study, we show that topical application of SC-560 (a COX-1 selective inhibitor) or celecoxib (COX-2 selective) to TPA-treated wild-type skin caused fivefold increases in the number of basal keratinocytes expressing the early differentiation marker keratin 1 (K1). In contrast to skin, COX-2 not COX-1 was the major isoform expressed in cultured primary keratinocytes. COX-1 was predominantly expressed in detached, differentiated cells, whereas COX-2 was found in the attached, proliferating cells. High Ca++ medium induced K1 and COX-1 in wild-type keratinocytes but did not change COX-2 expression. As observed in skin, COX-1-/- and COX-2-/- primary keratinocytes expressed fivefold more K1 than wild-type cells. K1 levels in cultured wild-type keratinocytes were also increased by treatment with celecoxib and indomethacin. However, unlike its in vivo effect, SC-560, possibly due to low COX-1 expression in cultured mouse keratinocytes, did not increase K1 levels. Furthermore, no increases in apoptotic cell numbers were observed in COX-deficient keratinocytes or COX-inhibitor treated wild-type cells. Thus, a major effect of COX inhibitors and COX-deficiency is the induction of keratinocyte differentiation.