Regulation of TGF‐α expression in human keratinocytes: PKC‐dependent and ‐independent pathways

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Abstract
Transforming growth factor–α (TGF‐α) is an autocrine growth factor for epidermal keratinocytes that can induce its own expression (autoinduction). Because the regulation of this process may be important for the control of epidermal growth, we examined the roles of EGF receptor tyrosine kinase and protein kinase C (PKC) in TGF‐α autoinduction in cultured human keratinocytes. Antiphosphotyrosine immunoblot analysis demonstrated that EGF and TGF‐α rapidly and markedly stimulated tyrosine phosphorylation of a 170 kDa protein in growth factor–deprived keratinocytes. This protein was identified as the EGF receptor by immuno‐precipitation using anti‐EGF receptor mAbs. Tyrosine phosphorylation and TGF‐α mRNA accumulation in response to EGF and TGF‐α were both inhibited by a monoclonal antibody against the EGF receptor and by the EGF receptor tyrosine kinase inhibitor RG50864, demonstrating the involvement of the tyrosine kinase activity of the receptor in TGF‐α autoinduction. The monoclonal antibody inhibited keratinocyte growth and TGF‐α autoinduction with similar potency (IC50 ∼ 0.1 μg/ml). TGF‐α and the PKC activator tetradecanoyl phorbol 12‐myristyl, 13‐acetate (TPA) had similar effects on TGF‐α steady‐state mRNA levels, suggesting that PKC activation might be a downstream mediator of TGF‐α autoinduction. However, down‐regulation of more than 90% of keratinocyte PKC activity by bryostatin pretreatment abrogated the induction of TGF‐α mRNA in response to TPA without affecting the autoinductive response or EGF‐stimulated tyrosine phosphorylation. These results indicate that EGF receptor and PKC stimulate TGF‐α gene expression by different pathways, and suggest that PKC is not required for TGF‐α autoinduction in this system. Moreover, the fact that EGF‐stimulated tyrosine phosphorylation and TGF‐α autoinduction were not potentiated after PKC down‐regulation suggests that PKC does not exert a tonic inhibitory influence on EGF receptor tyrosine kinase activity in normal human keratinocytes.

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