Cross-talk between epidermal growth factor receptor and protein kinase C during calcium-induced differentiation of keratinocytes.

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.

Essential role for Sonic hedgehog during hair follicle morphogenesis.

The hair follicle is a source of epithelial stem cells and site of origin for several types of skin tumors. Although it is clear that follicles arise by way of a series of inductive tissue interactions, identification of the signaling molecules driving this process remains a major challenge in skin biology. In this study we report an obligatory role for the secreted morphogen Sonic hedgehog (Shh) during hair follicle development. Hair germs comprising epidermal placodes and associated dermal condensates were detected in both control and Shh -/- embryos, but progression through subsequent stages of follicle development was blocked in mutant skin. The expression of Gli1 and Ptc1 was reduced in Shh -/- dermal condensates and they failed to evolve into hair follicle papillae, suggesting that the adjacent mesenchyme is a critical target for placode-derived Shh. Despite the profound inhibition of hair follicle morphogenesis, late-stage follicle differentiation markers were detected in Shh -/- skin grafts, as well as cultured vibrissa explants treated with cyclopamine to block Shh signaling. Our findings reveal an essential role for Shh during hair follicle morphogenesis, where it is required for normal advancement beyond the hair germ stage of development.

Differentiation of cultured human epidermal keratinocytes at high cell densities is mediated by endogenous activation of the protein kinase C signaling pathway.

Normal human epidermal keratinocytes (NHEK) grown in serum-free medium on a plastic substrate spontaneously differentiate at high cell densities in vitro. Because protein kinase C (PKC) regulates murine keratinocyte differentiation triggered by a variety of stimuli, we examined the role of this signaling pathway in density-dependent activation of NHEK differentiation. Relative to subconfluent cultures, confluent NHEK expressed markedly higher levels of multiple differentiation markers assayed by immunoblotting, including keratin 1, loricrin, filaggrin, involucrin, TGK, and SPR-1. Expression of several of these markers continued to increase for several days after cells reached confluency. The total level of several PKC isoforms was not substantially altered in NHEK harvested at different cell densities, based on immunoblotting; however, subcellular fractionation revealed that PKCalpha underwent a redistribution to the particulate fraction in confluent and postconfluent NHEK cultures, suggesting that this isozyme was activated under these conditions and may be involved in triggering the terminal differentiation program. Supporting this concept, inhibition of PKC function using bryostatin 1 or GF 109203X blocked the induction of keratinocyte differentiation markers at high cell densities. These data suggest that endogenous activation of PKC is responsible for cell density-mediated stimulation of NHEK differentiation, establishing a critical role for this pathway in regulating human as well as murine keratinocyte differentiation.

Targeted disruption of the epidermal growth factor receptor impairs growth of squamous papillomas expressing the v-ras(Ha) oncogene but does not block in vitro keratinocyte responses to oncogenic ras.

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.

Definition by specific antisense oligonucleotides of a role for protein kinase C alpha in expression of differentiation markers in normal and neoplastic mouse epidermal keratinocytes.

Epidermal keratinocyte differentiation is a tightly regulated, stepwise process that requires protein kinase C (PKC) activation. Studies using cultured mouse keratinocytes induced to differentiate with Ca2+ have indirectly implicated the alpha isoform of PKC in upregulation of "late" (granular cell) epidermal differentiation markers. Activation of this isoform is also implicated in the suppression of "early" differentiation markers keratin (K) 1 and 10 that characterizes the neoplastic phenotype produced by the v-Ha-ras oncogene. We used antisense oligonucleotides (AS) to directly address the role of PKC alpha in regulating expression of these markers in normal and v-Ha-ras-transduced primary keratinocytes and a keratinocyte cell line (SP-1) containing an activating mutation of the c-Ha-ras gene. Transfection of PKC alpha AS reduced the PKC alpha protein level in a dose-dependent manner, with a maximum effect at doses of 100 nM or higher. Immunoblot analysis with antibodies against PKC alpha, PKC delta, PKC epsilon, and PKC eta confirmed that PKC alpha AS selectively reduced the level of PKC alpha but not the other isoforms. In vitro kinase assays also revealed suppression of Ca(2+)-dependent PKC activity, which is the PKC alpha activity in this cell type, after transfection of PKC alpha AS. When PKC alpha AS-treated normal keratinocytes were stimulated to terminally differentiate with Ca2+, induction of the late differentiation markers loricrin, filaggrin, and SPR-1, as well as transglutaminase K mRNA, was suppressed when compared with their induction in scrambled AS-treated controls. In neoplastic v-Ha-ras-transduced keratinocytes and SP-1 cells, transfection of PKC alpha AS, but not the scrambled AS control, selectively downregulated PKC alpha and restored differentiation-specific expression of K1. These findings directly confirm that PKC alpha is an important component of the signaling pathway regulating terminal differentiation of normal keratinocytes and that activation of PKC alpha contributes to the altered differentiation program of neoplastic murine keratinocytes.

Macrophage-stimulating protein induces proliferation and migration of murine keratinocytes.

Macrophage stimulating protein (MSP) is a chemotactic factor for murine peritoneal macrophages. The receptor for human MSP was recently identified as the ron gene product, a transmembrane protein tyrosine kinase cloned from a human keratinocyte cDNA library. Here we report that MSP induced proliferation of murine primary keratinocytes and established keratinocyte cell lines in a concentration-dependent manner. The growth efficacy of MSP was comparable to that of epidermal growth factor and keratinocyte growth factor. In three of four cell lines tested in a chemotaxis chamber, MSP also stimulated migration of keratinocytes on a collagen type IV substratum. The action of MSP was mediated by specific binding of MSP to the STK gene product, a murine homologue of the RON MSP receptor. Binding of MSP to keratinocyte STK induced phosphorylation of the 150 kDa STK beta chain. Herbimycin A, a protein tyrosine kinase inhibitor, blocked MSP-mediated phosphorylation of the STK receptor as well as proliferation of keratinocytes, suggesting the importance of tyrosine kinase activity for transduction of the message delivered by MSP. Previously, the only known target cell for MSP was the resident peritoneal macrophage. These studies establish the keratinocyte as a new target cell for MSP. The action of MSP on keratinocytes may have implications for tissue repair, wound healing, and tumor growth.

Differentiation of mouse keratinocytes is accompanied by PKC-dependent changes in AP-1 proteins.

The conversion of cultured basal keratinocytes to the spinous and granular cell phenotypes seen in the skin can be stimulated by raising the levels of extracellular calcium. Here we show that AP-1 DNA binding activity is very low in primary cultures of basal keratinocytes, but that this activity is induced 24-48 h after increasing the concentration of extracellular calcium from 0.05 to 0.12 mM. As such, the induction of AP-1 DNA binding activity correlates with events occurring during the terminal stages of keratinocyte differentiation. Calcium-induced AP-1 DNA binding complexes consist of Fra-1, Fra-2, c-Jun, JunB and JunD and are independent of c-Fos, since the induction of DNA binding activity and the composition of the AP-1 binding complexes are identical in differentiating keratinocytes derived from c-fos null and wild type mice. The formation of calcium-induced AP-1 binding complexes is regulated by protein kinase C (PKC) and requires a functional PKCalpha isozyme, as determined through pharmacological down-modulation of specific PKC isozymes in differentiating keratinocytes. Moreover, PKC activation is required for the increased expression of Fra-2, JunB and JunD in the nucleus of differentiating cells in vitro. This observation provides a link between the obligate activation of PKC during keratinocyte differentiation and the nuclear response required to alter gene expression. In vivo expression patterns suggest that the predominant AP-1 heterodimer in the granular layer consists of Fra-2 and JunB while a JunD and Fra-1 complex predominates the spinous layer of mouse epidermis. These findings suggest distinct functions for different AP-1 proteins in the regulation of events related to keratinocyte maturation.

Staurosporine induces a complete program of terminal differentiation in neoplastic mouse keratinocytes via activation of protein kinase C.

Staurosporine (stsp) is a kinase inhibitor which induces cornified envelope assembly and terminal differentiation in normal and neoplastic mouse keratinocytes. In the tumorigenic cell lines 308 and SP-1 experiments were performed to determine if this effect was due only to activation of transglutaminases (TGases) already residing within the cell, or whether stsp was capable of inducing a full program of differentiation. Assessment of keratinocyte differentiation-specific protein expression in neoplastic cells revealed that expression of the suprabasal marker SPR-1 and the granular markers loricrin and filaggrin were induced by stsp. Protein expression was controlled by changes in mRNA expression, determined by Northern blotting. Transcripts for the TGase isoforms TGK and TGE were also induced by stsp in SP-1 cells, whereas only TGK expression was increased in 308 cells, which appear not to express TGE. Protein kinase C (PKC) activation is required for differentiation in normal mouse keratinocytes. To determine if stsp induces differentiation in neoplastic cells by regulation of this signaling pathway cells were treated with the specific PKC inhibitor GF 109203X or with different concentrations of bryostatin 1 to down-regulate specific isoforms of PKC prior to and during treatment with stsp. Stsp-induced protein cross-linking and marker expression were inhibited by GF 109203X, suggesting paradoxical activation of PKC by stsp. PKC alpha, epsilon and delta, but not PKC eta and zeta, were down-regulated by treating both cell types with bryostatin; pre-treatment of cells with bryostatin inhibited stsp-induced protein cross-linking and marker expression, suggesting a necessity for the alpha, delta and/or epsilon isoforms in stsp-induced differentiation.

Multistage carcinogenesis in the skin.

The multistage evolution of squamous cell cancer on mouse skin has provided a model to dissect the biological and genetic changes that contribute to specific stages of carcinogenesis. Keratinocyte cell culture models have been developed that reproduce the genetic and epigenetic events in multistage skin carcinogenesis, and these provide insights into the biochemistry of the process. When the v-rasHa oncogene is transduced into normal mouse keratinocytes, the resultant papilloma phenotype is characterized by a high rate of cell proliferation, an aberrant program of differentiation marker expression, and resistance to terminal differentiation. These changes are attributed to differential effects on isoforms of protein kinase C coupled to activation of the epidermal growth factor receptor. Premalignant progression requires additional genetic changes in the v-rasHa-transduced cells. The acquisition of these changes is suppressed by transforming growth factor beta (TGF beta), and the absence of TGF beta in premalignant tumors indicates a high risk for malignant progression. Keratinocytes that are genetically null for the TGF beta 1 gene rapidly progress to squamous cell carcinomas when transduced with the v-rasHa oncogene. In culture, TGF beta 1 null keratinocytes exhibit a high rate of gene amplification that can be suppressed by concentrations of exogenous TGF beta well below those required to inhibit proliferation. This model is well-suited to identifying critical genetic changes that contribute to premalignant progression.

Activation of the epidermal growth factor receptor signal transduction pathway stimulates tyrosine phosphorylation of protein kinase C delta.

The expression of an oncogenic rasHa gene in epidermal keratinocytes stimulates the tyrosine phosphorylation of protein kinase C delta and inhibits its enzymatic activity (Denning, M. F., Dlugosz, A. A., Howett, M. K., and Yuspa, S. H. (1993) J. Biol. Chem. 268, 26079-26081). Keratinocytes expressing an activated rasHa gene secrete transforming growth factor alpha (TGFalpha) and have an altered response to differentiation signals involving protein kinase C (PKC). Because the neoplastic phenotype of v-rasHa expressing keratinocytes can be partially mimicked in vitro by chronic treatment with TGF alpha and the G protein activator aluminum fluoride (AlF4-), we determined if TGF alpha or AlF4- could induce tyrosine phosphorylation of PKCdelta. Treatment of primary keratinocyte cultures for 4 days with TGFalpha induced tyrosine phosphorylation of PKCdelta, whereas AlF4- only slightly stimulated PKCdelta tyrosine phosphorylation. The PKCdelta that was tyrosine-phosphorylated in response to TGFalpha had reduced activity compared with the nontyrosine-phosphorylated PKCdelta. Treatment of keratinocytes expressing a normal epidermal growth factor receptor (EGFR) with TGFalpha or epidermal growth factor for 5 min induced PKCdelta tyrosine phosphorylation. This acute epidermal growth factor treatment did not induce tyrosine phosphorylation of PKCdelta in keratinocytes isolated from waved-2 mice that have a defective epidermal growth factor receptor. In addition, the level of PKCdelta tyrosine phosphorylation in v-rasHa-transduced keratinocytes from EGFR null mice was substantially lower than in v-rasHa transduced wild type cells, suggesting that activation of the EGFR is important for PKC delta tyrosine phosphorylation in ras transformation. However, purified EGFR did not phosphorylate recombinant PKC delta in vitro, whereas members of the Src family (c-Src, c-Fyn) and membrane preparations from keratinocytes did. Furthermore, clearing c-Src or c-Fyn from keratinocyte membrane lysates decreased PKCdelta tyrosine phosphorylation, and c-Src and c-Fyn isolated from keratinocytes treated with TGFalpha had increased kinase activity. Acute or chronic treatment with TGFalpha did not induce significant PKCdelta translocation in contrast to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, which induced both translocation and tyrosine phosphorylation of PKCdelta. This suggests that TGFalpha-induced tyrosine phosphorylation of PKC delta results from the activation of a tyrosine kinase rather than physical association of PKCdelta with a membrane-anchored tyrosine kinase. Taken together, these results indicate that PKCdelta activity is inhibited by tyrosine phosphorylation in response to EGFR-mediated signaling and activation of a member of the Src kinase family may be the proximal tyrosine kinase acting on PKCdelta in keratinocytes.

Staurosporine induces a sequential program of mouse keratinocyte terminal differentiation through activation of PKC isozymes.

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.

Targeted disruption of mouse EGF receptor: effect of genetic background on mutant phenotype.

Gene targeting was used to create a null allele at the epidermal growth factor receptor locus (Egfr). The phenotype was dependent on genetic background. EGFR deficiency on a CF-1 background resulted in peri-implantation death due to degeneration of the inner cell mass. On a 129/Sv background, homozygous mutants died at mid-gestation due to placental defects; on a CD-1 background, the mutants lived for up to 3 weeks and showed abnormalities in skin, kidney, brain, liver, and gastrointestinal tract. The multiple abnormalities associated with EGFR deficiency indicate that the receptor is involved in a wide range of cellular activities.

Autocrine transforming growth factor alpha is dispensible for v-rasHa-induced epidermal neoplasia: potential involvement of alternate epidermal growth factor receptor ligands.

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.

Differential regulation by anti-tumor-promoting 12-deoxyphorbol-13-phenylacetate reveals distinct roles of the classical and novel protein kinase C isozymes in biological responses of primary mouse keratinocytes.

12-Deoxyphorbol-13-phenylacetate (dPP) is the prototype for a new class of phorbol derivatives that function as protein kinase C (PKC) activators with potent anti-tumor-promoting activity. To explore the mechanism of action of dPP, we have conducted detailed analyses of the translocation and down-regulation patterns of individual PKC isozymes in mouse primary keratinocytes upon dPP treatment. PKC-alpha, -delta, and -epsilon were very quickly (within 2-5 min) translocated from the soluble fraction to the Triton X-100-soluble particulate fraction. PKC-delta and -epsilon were translocated with 2 orders of magnitude higher potency than was PKC-alpha. After translocation, PKC-alpha, -delta, -eta, and -epsilon were down-regulated; the down-regulation of PKC-epsilon contrasts with its retention after phorbol-12-myristate-13-acetate or bryostatin treatment. As was the case with translocation, dPP down-regulated the novel PKC isozymes (delta, epsilon, and eta) with 2 orders of magnitude higher potency (ED50, about 1-2 nM), compared with PKC-alpha (ED50, about 100 nM). dPP induced transglutaminase activity, ornithine decarboxylase activity, and cornification with potencies similar to that for PKC-alpha translocation. On the other hand, dPP caused inhibition of EGF binding with a potency similar to that for the translocation of the novel PKC isozymes. Although the generality of its selectivity in different cell types remains to be determined, at least in keratinocytes dPP is a powerful tool for dissecting the involvement of the classical and novel PKC isozymes in biological responses. The unique regulatory pattern of PKC-epsilon could contribute to the anti-tumor-promoting activity of dPP.

Specific protein kinase C isozymes mediate the induction of keratinocyte differentiation markers by calcium.

The maturation of epidermal keratinocytes is a tightly regulated, stepwise process which requires protein kinase C (PKC) activation. We investigated the effect of elevated extracellular Ca2+, a potent differentiation signal which increases cellular sn-1,2-diacylglycerol levels, on the PKC isozyme profile of cultured murine keratinocytes. Five PKC isozymes (alpha, delta, epsilon, zeta, and eta) were detected by immunoblotting. During Ca(2+)-induced differentiation, total cellular PKC alpha decreased, PKC epsilon and eta increased 3-5-fold, and the level of other PKC isozymes was relatively unchanged. PKC alpha underwent a progressive translocation from the soluble to the particulate fraction following elevation of extracellular Ca2+. The kinetics of PKC alpha translocation corresponded with the induction of keratinocyte differentiation markers. Both PKC delta and epsilon were selectively lost from the soluble fraction of keratinocytes exposed to elevated extracellular Ca2+, resulting in an increase in the proportion of these isoforms in the particulate fraction. PKC eta increased in both the soluble and particulate fractions, while PKC zeta did not change in amount or distribution during keratinocyte differentiation. Selective down-regulation of PKC isoforms by either 12-deoxyphorbol-13-phenylacetate or bryostatin 1 inhibited Ca(2+)-induced expression of differentiation markers at doses most specific for the down-regulation of PKC alpha. Taken together, these observations suggest that the induction of keratinocyte differentiation by Ca2+ results in the activation of specific PKC isozymes.

Alterations in murine keratinocyte differentiation induced by activated rasHa genes are mediated by protein kinase C-alpha.

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.

Keratinocyte growth factor receptor ligands induce transforming growth factor alpha expression and activate the epidermal growth factor receptor signaling pathway in cultured epidermal keratinocytes.

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.