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Keratinocyte differentiation requires integrating signaling among intracellular ionic changes, kinase cascades, sequential gene expression, cell cycle arrest, and programmed cell death. We now show that Cl(-) intracellular channel 4 (CLIC4) expression is increased in both mouse and human keratinocytes undergoing differentiation induced by Ca(2+), serum and the protein kinase C (PKC)-activator, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Elevation of CLIC4 is associated with signaling by PKCdelta, and knockdown of CLIC4 protein by antisense or shRNA prevents Ca(2+)-induced keratin 1, keratin 10 and filaggrin expression and cell cycle arrest in differentiating keratinocytes. CLIC4 is cytoplasmic in actively proliferating keratinocytes in vitro, but the cytoplasmic CLIC4 translocates to the nucleus in keratinocytes undergoing growth arrest by differentiation, senescence or transforming growth factor beta (TGFbeta) treatment. Targeting CLIC4 to the nucleus of keratinocytes via adenoviral transduction increases nuclear Cl(-) content and enhances expression of differentiation markers in the absence of elevated Ca(2+). In vivo, CLIC4 is localized to the epidermis in mouse and human skin, where it is predominantly nuclear in quiescent cells. These results suggest that CLIC4 participates in epidermal homeostasis through both alterations in the level of expression and subcellular localization. Nuclear CLIC4, possibly by altering the Cl(-) and pH of the nucleus, contributes to cell cycle arrest and the specific gene expression program associated with keratinocyte terminal differentiation.

Keratinocyte differentiation requires integrating signaling among intracellular ionic changes, kinase cascades, sequential gene expression, cell cycle arrest, and programmed cell death. We now show that Cl¯ intracellular channel 4 (CLIC4) expression is increased in both mouse and human keratinocytes undergoing differentiation induced by Ca2+, serum and the protein kinase C (PKC)-activator, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Elevation of CLIC4 is associated with signaling by PKCδ, and knockdown of CLIC4 protein by antisense or shRNA prevents Ca2+-induced keratin 1, keratin 10 and filaggrin expression and cell cycle arrest in differentiating keratinocytes. CLIC4 is cytoplasmic in actively proliferating keratinocytes in vitro, but the cytoplasmic CLIC4 translocates to the nucleus in keratinocytes undergoing growth arrest by differentiation, senescence or transforming growth factor β (TGFβ) treatment. Targeting CLIC4 to the nucleus of keratinocytes via adenoviral transduction increases nuclear Cl¯ content and enhances expression of differentiation markers in the absence of elevated Ca2+. In vivo, CLIC4 is localized to the epidermis in mouse and human skin, where it is predominantly nuclear in quiescent cells. These results suggest that CLIC4 participates in epidermal homeostasis through both alterations in the level of expression and subcellular localization. Nuclear CLIC4, possibly by altering the Cl¯and pH of the nucleus, contributes to cell cycle arrest and the specific gene expression program associated with keratinocyte terminal differentiation. [ABSTRACT FROM AUTHOR]

The introduction of the techniques of molecular biology as tools to study skin carcinogenesis has provided more precise localization of biochemical pathways that regulate the tumor phenotype. This approach has identified genetic changes that are characteristic of each of the specific stages of squamous cancer pathogenesis: initiation, exogenous promotion, premalignant progression, and malignant conversion. Initiation can result from mutations in a single gene, and the Harvey allele of the ras gene family has been identified as a frequent site for initiating mutations. Heterozygous activating mutations in c-rasHa are dominant, and affected keratinocytes hyperproliferate and are resistant to signals for terminal differentiation. An important pathway impacted by c-rasHa activation is the protein kinase C (PKC) pathway, a major regulator of keratinocyte differentiation. Increased activity of PKCα and suppression of PKCδ by tyrosine phosphorylation contribute to the phenotypic consequences of rasHa gene activation in keratinocytes. Tumor promoters disturb epidermal homeostasis and cause selective clonal expansion of initiated cells to produce multiple benign squamous papillomas. Resistance to differentiation and enhanced growth rate of initiated cells impart a growth advantage when the epidermis is exposed to promoters. The frequency of premalignant progression varies among papillomas, and subpopulations at high risk for progression have been identified. These high-risk papillomas overexpress the α6β4 integrin and are deficient in transforming growth factor β1 and β2 peptides, two changes associated with a very high proliferation rate in this subset of tumors. The introduction of an oncogenic rasHa gene into epidermal cells derived from transgenic mice with a null mutation in the TGFβ1 gene have an accelerated rate of malignant progression when examined in vivo. Thus members of the TGFβ gene family contribute a tumor-suppressor function in carcinogenesis. Accelerated malignant progression is also found with v-rasHa transduced keratinocytes from skin of mice with a null mutation in the p53 gene. The similarities in risk for malignant conversion by initiated keratinocytes from TGFβ and p53 null geneotypes suggest that a common, growth-related pathway may underly the tumor-suppressive functions of these proteins in the skin carcinogenesis model. [ABSTRACT FROM AUTHOR]

Proteins from mouse epidermis cytosol extracts react on immunoblots with a polyclonal rabbit antiserum raised against rat skin calcium-binding protein (SCaBP), a parvalbumin of the panniculus carnosus. Three mouse epidermal proteins with molecular weights between 10-12K, which are distinct from SCaBP, are recognized by the antiserum. The synthesis of these proteins in keratinocyte culture is modulated by Ca++, as is the differentiation of the keratinocytes. Proliferating mouse keratinocytes in medium containing 0.07 mM Ca++ (low CaCa++) undergo terminal differentiation when the Ca++ concentration is elevated to 1.8 mM (high Ca++). Synthesis of the 3 antigens can be demonstrated when soluble extracts of keratinocytes labeled with [35S]methionine in low Ca++ medium are immunoprecipitated with anti-SCaBP serum. These antigens are not synthesized in cultures of dermal fibroblasts. When keratinocytes are switched to high Ca++ medium, synthesis of these antigens is greatly diminished over the course of 48-72 h. However, the antigens persist in differentiating cells. When proliferating keratinocytes in low Ca++ medium are exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), differentiation is induced in a subpopulation of cells, and specific antigen synthesis is transiently inhibited. The inhibition correlates with the time when many cells are differentiating in response to TPA. When proliferating keratinocytes are pulse-labeled with 32PO4, the 11 K antigen is phosphorylated and the phosphorylation is not enhanced by TPA exposure. All 3 antigens are synthesized in a reticulocyte lysate preparation with added newborn mouse epidermis messenger RNA or mRNA from keratinocytes cultured in low Ca++ medium. Thus, these antigens are likely to represent unique proteins rather than processed or degraded ones. The coordinately regulated expression of these antigens associated with the differentiation state of the keratinocyte proliferation and differentiation. [ABSTRACT FROM AUTHOR]

CLIC4/mtCLIC, a chloride intracellular channel protein, localizes to the mitochondria and cytoplasm of keratinocytes and participates in the apoptotic response to stress. We now show that multiple stress inducers cause the translocation of cytoplasmic CLIC4 to the nucleus. Immunogold electron microscopy and confocal analyses indicate that nuclear CLIC4 is detected prior to the apoptotic phenotype. CLIC4 associates with the Ran, NTF2, and Importin-α nuclear import complexes in immunoprecipitates of lysates from cells treated with apoptotic/stress-inducing agents. Deletion or mutation of the nuclear localization signal in the C terminus of CLIC4 eliminates nuclear translocation, whereas N terminus deletion enhances nuclear localization. Targeting CLIC4 to the nucleus via adenoviral transduction accelerates apoptosis when compared with cytoplasmic CLIC4, and only nuclear-targeted CLIC4 causes apoptosis in Apaf null mouse fibroblasts or in Bcl-2-overexpressing keratinocytes. These results indicate that CLIC4 nuclear translocation is an integral part of the cellular response to stress and may contribute to the initiation of nuclear alterations that are associated with apoptosis. [ABSTRACT FROM AUTHOR]

Staurosporine (stsp) induces assembly of cornified envelopes in mouse keratinocyte cultures. To clarify whether this effect is the consequence of a coordinated differentiation program similar to that observed in epidermis, we assessed the expression of multiple differentiation-specific markers in stsp-treated keratinocytes. In medium containing 0.05 mM Ca&sup2+; in which the basal cell phenotype is normally maintained, stsp induced dose-dependent increases in keratin 1, epidermal and keratinocyte transglutaminases, SPR-1, loricrin, and profilaggrin mRNA. Based on nuclear run-on analysis, stsp-mediated marker expression was found to be due at least in part to increased transcription. Since protein kinase C (PKC) activation is required for keratinocyte differentiation, we tested whether stsp influenced this signaling pathway. Stsp induced the translocation of multiple PKC isoforms from the cytosol to membrane and/or cytoskeletal fractions, inducing isozyme downregulation within 24 h. Moreover, AP-1 DNA binding activity was elevated in stsp-treated keratinocytes, consistent with the notion that this agent influences keratinocyte-specific gene expression via the PKC pathway. Stsp-mediated marker expression was inhibited by the PKC inhibitor GF 109203X. In cells pre-treated with bryostatin 1 to selectively down-modulate specific PKC isoforms, stsp-induced loricrin, filaggrin, and SPR-1 expression was suppressed when PKC α, ϵ, and/or δ were downregulated, suggesting that these isozymes may be necessary for marker expression in response to this agent. Thus, in addition to its effects on cornified envelope assembly, stsp induces a coordinate program of differentiation-specific keratinocyte gene expression that is mediated at least in part by the PKC signaling pathway. [ABSTRACT FROM AUTHOR]