ABSTRACT
Activation of the STAT family of transcription factors is regulated by cytokines and growth factors. STAT tyrosine and serine phosphorylation are linked to the transcriptional activation and function of STAT. We have previously described a unique pathway inducing keratinocyte proliferation, which is mediated by insulin stimulation and depends on protein kinase C delta (PKCdelta). In this study, we assessed STAT3 activation downstream of this pathway and characterized the role of PKCdelta activation in STAT3 tyrosine and serine phosphorylation and keratinocyte proliferation. Following insulin stimulation, STAT3 interacted with PKCdelta but not with any other PKC isoform expressed in skin. Activated forms of PKCdelta and STAT3 were essential for insulin-induced PKCdelta-STAT3 activation in keratinocyte proliferation. Abrogation of PKCdelta activity inhibited insulin-induced STAT3 phosphorylation, PKCdelta-STAT3 association and nuclear translocation. In addition, overexpression of STAT3 tyrosine mutant eliminated insulin-induced PKCdelta activation and keratinocyte proliferation. Finally, overexpression of a STAT3 serine mutant abrogated insulin-induced STAT3 serine phosphorylation and STAT3-induced keratinocyte proliferation, whereas STAT3 tyrosine phosphorylation was induced and nuclear localization remained intact. This study indicates that PKCdelta activation is a primary regulator of STAT3 serine phosphorylation and that PKCdelta is essential in directing insulin-induced signaling in keratinocyte proliferation.
Subject(s)
Hypoglycemic Agents/pharmacology , Insulin/pharmacology , Keratinocytes/enzymology , Protein Kinase C-delta/metabolism , STAT3 Transcription Factor/metabolism , Signal Transduction/physiology , Animals , Cell Proliferation/drug effects , Cells, Cultured , Enzyme Activation/drug effects , Hypoglycemic Agents/metabolism , Insulin/metabolism , Keratinocytes/cytology , Mice , Mice, Inbred BALB C , Phosphorylation/drug effects , Protein Processing, Post-Translational/drug effects , Protein Processing, Post-Translational/physiology , Signal Transduction/drug effectsABSTRACT
In mammalian epidermis, alpha6beta4 integrin is expressed exclusively on the basal layer localized to the hemidesmosomes, where it interacts extracellularly with the laminin-5 ligand. During differentiation, loss of alpha6beta4 is associated with keratinocyte detachment from the basement membrane and upward migration. The protein kinase C (PKC) family of isoforms participates in regulation of integrin function and is linked to skin differentiation. Exposure of primary murine keratinocytes to PKC activators specifically downregulates alpha6beta4 expression. Utilizing recombinant adenoviruses, we selectively overexpressed skin PKC isoforms in primary keratinocytes. PKCdelta and PKCzeta induced downregulation of alpha6beta4 protein expression, leading to reduced keratinocyte attachment to laminin-5 and enhanced gradual detachment from the underlying matrix. In contrast, PKCalpha upregulated alpha6beta4 protein expression, leading to increased keratinocyte attachment to laminin-5 and to the underlying matrix. Altogether, these results suggest distinct roles for specific PKC isoforms in alpha6beta4 functional regulation during the early stages of skin differentiation.
Subject(s)
Gene Expression Regulation, Enzymologic/physiology , Integrin alpha6beta1/metabolism , Keratinocytes/metabolism , Protein Kinase C/classification , Protein Kinase C/metabolism , Skin/metabolism , Animals , Animals, Newborn , Cell Adhesion/physiology , Cell Differentiation/physiology , Cells, Cultured , Isoenzymes/classification , Isoenzymes/metabolism , Keratinocytes/cytology , Mice , Mice, Inbred BALB C , Skin/cytologyABSTRACT
Tumor necrosis factor-alpha (TNF-alpha) is a multifunctional cytokine that interferes with insulin signaling, but the molecular mechanisms of this effect are unclear. Because certain protein kinase C (PKC) isoforms are activated by insulin, we examined the role of PKC in TNF-alpha inhibition of insulin signaling in primary cultures of mouse skeletal muscle. TNF-alpha, given 5 min before insulin, inhibited insulin-induced tyrosine phosphorylation of insulin receptor (IR), IR substrate (IRS)-1, insulin-induced association of IRS-1 with the p85 subunit of phosphatidylinositol 3-kinase (PI3-K), and insulin-induced glucose uptake. Insulin and TNF-alpha each caused tyrosine phosphorylation and activation of PKCs delta and alpha, but when TNF-alpha preceded insulin, the effects were less than that produced by each substance alone. Insulin induced PKCdelta specifically to coprecipitate with IR, an effect blocked by TNF-alpha. Both PKCalpha and -delta are constitutively associated with IRS-1. Whereas insulin decreased coprecipitation of IRS-1 with PKCalpha, it increased coprecipitation of IRS-1 with PKCdelta. TNF-alpha blocked the effects of insulin on association of both PKCs with IRS-1. To further investigate the involvement of PKCs in inhibitory actions of TNF-alpha on insulin signaling, we overexpressed specific PKC isoforms in mature myotubes. PKCalpha overexpression inhibited basal and insulin-induced IR autophosphorylation, whereas PKCdelta overexpression increased IR autophosphorylation and abrogated the inhibitory effect of TNF-alpha on IR autophosphorylation and signaling to PI3-K. Blockade of PKCalpha antagonized the inhibitory effects of TNF-alpha on both insulin-induced IR tyrosine phosphorylation and IR signaling to PI3-K. We suggest that the effects of TNF-alpha on IR tyrosine phosphorylation are mediated via alteration of insulin-induced activation and association of PKCdelta and -alpha with upstream signaling molecules.
