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ABSTRACT: The induction of epidermal differentiation by extracellular Ca2+ involves activation of both tyrosine kinase and protein kinase C (PKC) signaling cascades. To determine if the differentiation-dependent activation of tyrosine kinase signaling can influence the PKC pathway, we examined the tyrosine phosphorylation status of PKC isoforms in primary mouse keratinocytes stimulated to terminally differentiate with Ca2+. Elevation of extracellular Ca2+ induced tyrosine phosphorylation of PKC-delta, but not the other keratinocyte PKC isoforms (alpha, epsilon, eta, zeta). We have previously demonstrated that activation of the epidermal growth factor receptor (EGFR) pathway induces PKC-delta tyrosine phosphorylation in basal keratinocytes (Denning M F, Dlugosz A A, Threadgill D W, Magnuson T, Yuspa S H (1996) J Biol Chem 271: 5325-5331). When basal keratinocytes were stimulated to differentiate by Ca2+, the level of cell-associated transforming growth factor-alpha (TGF-alpha) increased 30-fold, while no increase in secreted TGF-alpha was detected. Furthermore, Ca2+-induced tyrosine phosphorylation of PKC-delta and phosphotyrosine-association of the receptor adapter protein Shc was diminished in EGFR -/- keratinocytes, suggesting that EGFR activation may occur during keratinocyte differentiation. Tyrosine phosphorylated PKC-delta was also detected in mouse epidermis, suggesting that this differentiation-associated signaling pathway is physiological. These results establish a requirement for the EGFR in Ca2+-induced tyrosine phosphorylation of PKC-delta, and document the production of cell-associated TGF-alpha in differentiated keratinocytes which may function independent of its usual mitogenic effects.
Experimental Dermatology 07/2000; 9(3):192-9. · 3.54 Impact Factor
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ABSTRACT: We have assessed the role of epidermal growth factor receptor (EGFR) signaling in biological responses to the v-ras(Ha) oncogene using primary keratinocytes from Egfr -/- mice and wild-type littermates. On the basis of several criteria, Egfr -/- keratinocytes were unresponsive to either acute or chronic exposure to several EGFR ligands but were stimulated to proliferate in response to several other mitogens. Although conditioned medium from primary keratinocytes transduced with v-ras(Ha) retrovirus (v-ras(Ha) keratinocytes) was a potent mitogen for wild-type but not Egfr -/- keratinocytes, v-ras(Ha) transduction of primary keratinocytes of either genotype resulted in a strong mitogenic response, arguing against an obligatory role for EGFR activation in v-ras(Ha)-mediated stimulation of keratinocyte proliferation. Infection with high-titer v-ras(Ha) retrovirus altered the keratin expression pattern in keratinocytes of both genotypes, suppressing differentiation-specific keratins K1 and K10 while activating aberrant expression of K8 and K18. In wild-type but not Egfr -/- cultures, K1 and K10 were also suppressed following infection at lower retroviral titers, presumably as a result of paracrine EGFR activation on uninfected cells present in these cultures. Squamous papillomas produced by grafting Egfr -/- v-ras(Ha) keratinocytes onto nude mice were only 21% of the size of wild-type v-ras(Ha) tumors, and a striking redistribution of S-phase cells was detected by immunostaining for bromodeoxyuridine. In Egfr -/- v-ras(Ha) papillomas, the fraction of total labeled nuclei detected in suprabasal layers was increased from 19 to 39%. In contrast, the basal layer labeling index of Egfr -/- papillomas was reduced to 34%, compared to 43% in wild-type tumors. Our results indicate that, although autocrine EGFR signaling is not required for keratinocyte responses to oncogenic ras in culture or benign tumor formation in nude mouse grafts, disruption of this pathway impairs growth of v-ras(Ha) papillomas by a mechanism that may involve alterations in keratinocyte cell cycle progression and/or migration in vivo.
Cancer Research 09/1997; 57(15):3180-8. · 7.86 Impact Factor
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ABSTRACT: 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 Ca2+, 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 alpha, epsilon, and/or delta 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.
Journal of Investigative Dermatology 04/1996; 106(3):482-9. · 6.31 Impact Factor
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ABSTRACT: Autocrine epidermal growth factor receptor activation by transforming growth factor alpha (TGF alpha) has been implicated in growth stimulation during epithelial neoplasia. Using keratinocytes isolated from mice with genetic defects in TGF alpha expression, we tested whether TGF alpha is required for transformation by the v-rasHa oncogene. Introduction of v-rasHa into primary epidermal cultures using a retroviral vector stimulated growth of both control (TGF alpha +/+, BALB/c) and TGF alpha-deficient (TGF alpha -/-, wa-1) keratinocytes. Moreover, v-rasHa elicited characteristic changes in marker expression (keratin 1 was suppressed; keratin 8 was induced), previously shown to be associated with epidermal growth factor (EGF) receptor activation, in both TGF alpha +/+ and TGF alpha -/- keratinocytes. v-rasHa markedly increased secreted (> 10-fold) and cell-associated (2-3-fold) TGF alpha levels in keratinocytes from TGF alpha +/+ and BALB/c mice, but not TGF alpha -/- or wa-1 mice. Based on Northern blot analysis, v-rasHa induced striking up-regulation of transcripts encoding the additional EGF family members amphiregulin, heparin-binding EGF-like growth factor, and betacellulin in cultured keratinocytes from all four mouse strains. Interestingly, in addition to the normal 4.5-kilobase TGF alpha transcript, wa-1 keratinocytes expressed two additional TGF alpha transcripts, 4.7 and 5.2 kilobases long. All three transcripts were up-regulated in response to v-rasHa, as well as exogenous TGF alpha or keratinocyte growth factor treatment, and were also detected in RNA isolated from wa-1 brain and skin. In vivo, v-rasHa keratinocytes from control as well as TGF alpha-deficient mice produced squamous tumors when grafted onto nude mice, and these lesions expressed high levels of amphiregulin, heparin-binding EGF-like growth factor, and betacellulin mRNA, regardless of their TGF alpha status. These findings indicate that TGF alpha is not essential for epidermal neoplasia induced by the v-rasHa oncogene and suggest that another EGF family member(s) may contribute to autocrine growth stimulation of ras-transformed keratinocytes.
Cancer Research 06/1995; 55(9):1883-93. · 7.86 Impact Factor
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ABSTRACT: Primary mouse keratinocytes expressing the v-rasHa oncogene (v-rasHa keratinocytes) produce squamous papillomas when grafted onto nude mice and respond abnormally to signals for terminal differentiation both in vivo and in vitro. Since protein kinase C (PKC) activators and v-rasHa induce similar phenotypic changes in cultured keratinocytes, and cellular diacylglycerol levels are constitutively elevated in ras-transformed keratinocytes, we tested whether PKC is a downstream target for oncogenic ras in this cell type. Ca(2+)-dependent PKC activity was increased in lysates from cultured v-rasHa keratinocytes when compared to control cells; in contrast, Ca(2+)-independent activity decreased. Similar to PKC activators, v-rasHa blocked Ca(2+)-mediated expression of the early epidermal differentiation markers keratins K1 and K10 while inducing aberrant expression of K8. Pretreatment of v-rasHa keratinocytes with bryostatin to block PKC function restored Ca(2+)-mediated expression of K1 and K10 and blocked abnormal expression of K8, suggesting that these responses are mediated by the PKC pathway. Furthermore, expression of K1 is restored at bryostatin doses which specifically down-modulate PKC-alpha, the only Ca(2+)-dependent PKC isozyme detected in cultured keratinocytes. In contrast to the inhibition of K1 and K10, Ca(2+)-induced expression of the late epidermal differentiation markers loricrin, filaggrin, and keratinocyte transglutaminase was accelerated by v-rasHa, as previously reported in normal keratinocytes treated with PKC activators. Pretreatment of v-rasHa keratinocytes with bryostatin blocked expression of late markers in these cells, and this response was correlated with down-regulation of PKC-alpha. The results of this study suggest that oncogenic ras alters keratinocyte differentiation by altering the function of the PKC signaling pathway, and that PKC-alpha is the specific isozyme involved in down-modulating expression of keratins K1 and K10 and up-regulating expression of loricrin, filaggrin, and keratinocyte transglutaminase.
Cancer Research 01/1995; 54(24):6413-20. · 7.86 Impact Factor
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ABSTRACT: Epidermal growth factor receptor (EGFR) ligands are fundamental regulators of epithelial growth, differentiation, and neoplastic transformation. In addition to being potent mitogens for murine epidermal keratinocytes in vitro, transforming growth factor alpha (TGF alpha) and EGF elicit distinctive changes in keratin expression: Ca(2+)-mediated induction of the differentiation-specific keratins K1 and K10 is blocked, while simple epithelial keratins K8 and K18 are expressed aberrantly (C. Cheng et al., Cell Growth, & Differ., 4: 317-327, 1993). We have evaluated several additional growth factors to determine the specificity of this response for EGFR ligands. TGF alpha, keratinocyte growth factor (KGF), and acidic fibroblast growth factor (aFGF), but not basic fibroblast growth factor (bFGF) or insulin-like growth factor type I, block Ca(2+)-mediated expression of K1 while inducing K8. Since KGF and aFGF (but not bFGF) are ligands for the KGF receptor (KGFR), we explored the possibility that the TGF alpha/EGFR pathway is an intermediary in signaling through the KGFR. TGF alpha mRNA was increased in cells treated with KGF, aFGF, or TGF alpha but not bFGF or insulin-like growth factor type I. Similar changes were detected at the protein level; TGF alpha in conditioned medium (CM) from control, KGF-, TGF alpha-, and aFGF-treated cultures was 54 (+/- 8, SEM), 365 (+/- 50), 146 (+/- 20), and 120 (+/- 50) pg/ml, respectively. KGF and TGF alpha also increased expression of cell-associated TGF alpha measured in keratinocyte lysates. KGF increased TGF alpha secretion and mRNA levels in human as well as mouse keratinocytes. CM from KGF-treated cultures stimulated cell growth when added to cultures of normal keratinocytes. Preincubation with neutralizing antibodies to both TGF alpha and KGF, but not KGF antibody alone, blocked cell growth in cultures treated with KGF CM, suggesting that the predominant keratinocyte mitogen in KGF CM is TGF alpha. In support of this hypothesis, treatment of keratinocytes for 5 min with either KGF CM or purified TGF alpha resulted in EGFR autophosphorylation. Furthermore, after approximately 24 h, KGF as well as TGF alpha induced EGFR down-regulation based on Western blot analysis and 125I-EGF binding. Induction of TGF alpha in KGF-treated keratinocytes, coupled to activation and down-modulation of the EGFR, suggests that TGF alpha may be a proximal effector of KGF action for at least certain aspects of epidermal growth and differentiation.
Cell growth & differentiation: the molecular biology journal of the American Association for Cancer Research 12/1994; 5(12):1283-92.
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ABSTRACT: Cytokeratins 8 and 18 (Endo A and B) are among the earliest expressed embryonic genes and the major components of the cytoskeleton in simple epithelia of the adult. Recent data indicate that these cytokeratins are aberrantly expressed in several epithelial tumor types and that expression in cultured mouse keratinocytes is linked to activation of the rasHa oncogene. Furthermore, up-regulation of K8/K18 in keratinocytes is associated with reciprocal suppression of K1. We now show that the aberrant expression of K8 and K18 and suppression of K1 in cultured keratinocytes transduced with the v-rasHa gene are mediated by a factor secreted into the culture medium. Furthermore, transforming growth factor alpha (TGF-alpha) and epidermal growth factor elicit an identical pattern of K8/K18 expression and K1 suppression in normal keratinocytes. The factor in medium from v-rasHa keratinocytes is TGF-alpha, as a specific blocking antibody for rat and mouse TGF-alpha prevents the expression of K8 and restores expression of K1. The tyrosine kinase inhibitor genistein also prevents K8 induction in v-rasHa keratinocytes and in normal keratinocytes treated with TGF-alpha- or v-rasHa-conditioned medium. However, simply stimulating proliferation of keratinocytes by cholera toxin does not result in expression of K8 or suppression of K1. Finally, tumor grafts from neoplastic epidermal cells overexpressing TGF-alpha via retroviral transduction of human TGF-alpha complementary DNA in vitro show coordinate expression of K8 and human TGF-alpha. These studies indicate that K8 expression in keratinocytes, and derivative neoplastic cells, in vivo and in vitro is regulated by epidermal growth factor receptor ligands. Since the expression of cytokines and K8/K18 in early embryogenesis is often coincident, cytokines may be the physiological mediators of K8/K18 expression in embryonic cells.
Cell growth & differentiation: the molecular biology journal of the American Association for Cancer Research 05/1993; 4(4):317-27.
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Recent results in cancer research. Fortschritte der Krebsforschung. Progrès dans les recherches sur le cancer 02/1993; 128:299-308.
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ABSTRACT: Introduction of a v-rasHa oncogene into cultured mouse keratinocytes by transduction with a defective retrovirus is sufficient to transform keratinocytes to the benign phenotype. Transduced keratinocytes overexpress TGF alpha and hyperproliferate in culture medium with 0.05 mM Ca2+. Whereas normal keratinocytes respond to elevated medium Ca2+ by cessation of proliferation and induction of terminal differentiation, v-rasHa keratinocytes are not induced to differentiate by Ca2+. We now demonstrate that v-rasHa keratinocytes have elevated basal levels of phosphatidylinositol, inositol phosphates and diacylglycerols in 0.05 mM Ca2+ medium. Basal turnover of phosphatidylcholine is not altered by the rasHa oncogene. The generation of inositol phosphates is even further stimulated in v-rasHa cells by an increase in extracellular Ca2+ or by exposure to aluminum fluoride. Thus, the v-rasHa gene product does not stimulate the inositol phospholipid pathway maximally and additional phosphatidylinositol is available for turnover in response to inducers of phospholipase C activity. TGF alpha and medium conditioned by v-rasHa keratinocytes, both of which stimulate proliferation of normal cells in 0.05 mM Ca2+, transiently increased phosphatidylinositol turnover in normal keratinocytes but did not inhibit Ca(2+)-induced terminal differentiation. In contrast, sustained elevation in basal phosphatidylinositol metabolism was produced by aluminum fluoride. Combined exposure to aluminum fluoride and exogenous TGF alpha caused hyperproliferation, resistance to Ca(2+)-induced differentiation and morphological changes identical to those of v-rasHa keratinocytes. These results provide a link between the biological consequences of v-rasHa gene expression and biochemical changes which are known to alter the keratinocyte phenotype.
Carcinogenesis 01/1993; 13(12):2367-73. · 5.70 Impact Factor
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Progress in clinical and biological research 02/1992; 376:103-15.
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ABSTRACT: The induction of cancer on mouse skin by initiation-promotion protocols occurs through stages in which a benign squamous papilloma is an obligate precursor of squamous cell carcinoma. Activation of the Ha-ras gene is sufficient to produce the papilloma phenotype, while additional genetic changes are required for malignant conversion. The introduction of Ha-ras into normal keratinocytes suppresses the expression of differentiation markers, keratin K1 and K10, and loricrin (a cornified envelope precursor) and, to a lesser extent, filaggrin, at the level of transcription. However, cells initiated by Ha-ras express a nonepidermal keratin, K8. The transcription of K8 in these cells is sensitive to the level of medium Ca2+, being abundant in 0.5 mM Ca2+ and not detected in 0.05 mM Ca2+. Epidermal differentiation is regulated by signalling, which involves changes in phosphatidylinositol turnover and intracellular Ca2+. Cells initiated by Ha-ras do not differ from normal keratinocytes in their intracellular Ca2+ response patterns, at least in response to changes in extracellular Ca2+ and serum factors. However, c-Ha-ra keratinocytes have a high basal level of phosphatidylinositol (PI) turnover, which is additive with several other inducers of this pathway, including Ca2+ and aluminum fluoride. Additional studies suggest that high turnover of the PI pathway is incompatible with differentiation-specific gene expression in keratinocytes. We suggest this negative relationship is mediated through elevated diacylglycerol production and chronic down-modulation of protein kinase C. Protein kinase C is known to be essential for expression of differentiation-related genes in keratinocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
Environmental Health Perspectives 07/1991; 93:3-10. · 7.04 Impact Factor
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ABSTRACT: Cultured mouse keratinocytes can be initiated in vitro by the introduction of a v-rasHa gene by viral transduction. Previous studies indicated that v-rasHa-transduced keratinocytes have a high proliferation rate in medium with 0.05 mM Ca2+ and resist terminal differentiation in medium with greater than 0.1 mM Ca2+, a culture condition in which normal cells mature into squames. The current studies demonstrate that v-rasHa keratinocytes do not express transcripts or protein for epidermal early differentiation markers keratins 1 and 10 when cells are challenged with 0.12 mM Ca2+, which is a signal for expression of these genes in normal cells. Both transcript and protein for the late differentiation marker loricrin are also diminished in v-ras keratinocytes, but filaggrin, also a late differentiation-related gene product, is expressed in nearly normal amounts but at a different Ca2+ optimum. Modification of intracellular Ca2+ with ionomycin failed to restore the expression of any suprabasal keratinocyte markers. In contrast to the effects on normal products of keratinocyte differentiation, the introduction of the v-rasHa gene facilitated the expression of keratins 8 (K8) and 18 (K18). These keratins are characteristic of embryonic cells and cells of simple adult epithelia but not stratified squamous epithelia such as skin. Like normal differentiation markers, the expression of K8 and K18 was dependent both on the v-ras oncogene and the Ca2+ concentration of the culture medium, with greater than 0.1 mM Ca2+ being optimal. At the optimal Ca2+ level, the majority of v-ras keratinocytes expressed K8 and K18 after 96 h, and many cells had reduced amounts of the normal keratinocyte cytokeratin K14. These studies indicate that the v-ras gene causes substantial reprogramming of epidermal physiology, producing an unusual phenotype devoid of early suprabasal markers but at least partially permissive for late marker expression. Furthermore, the Ca2(+)-dependent expression of K8 and K18 suggests that a normal signalling pathway used in keratinocyte differentiation is diverted to an abnormal endpoint.
Molecular Carcinogenesis 02/1990; 3(6):363-73. · 3.16 Impact Factor
