A PKC-Sp1 signaling pathway induces early differentiation of human keratinocytes through upregulation of TSG101

Abstract

The TSG101 protein has been implicated in multiple biological functions including regulation of gene transcription, vesicular trafficking, cellular growth and differentiation. However, the cellular signals that control TSG101 functions are unclear. Here, we demonstrate that TSG101 is upregulated during keratinocyte differentiation in both human foreskin tissue and reconstructed organotypic skin cultures. In addition, we found that TSG101 siRNA inhibits calcium-induced early differentiation of human foreskin keratinocytes, indicating an essential and downstream role for TSG101 in this process. Furthermore, the PKC agonist TPA promotes expression of TSG101 and keratin 10 in keratinocytes under low calcium conditions, while co-treatment with the PKC inhibitor GF 109203X blocks TPA-induced TSG101 and keratin 10 upregulation. Previous work has established that the TSG101 gene is controlled by a TATA-less promoter that harbors a Sp1-binding site. Here we show that both calcium and TPA activate PKC, stimulate phosphorylation of Sp1, and augment the activity of the TSG101 promoter in a manner dependent on its Sp1-binding site. Release of calcium from intracellular stores with thapsigargin, an endoplasmic reticulum Ca2+-ATPase inhibitor that elevates intracellular free Ca2+ without activating PKC, does not affect Sp1 phosphorylation and TSG101 promoter activity. Taken together, these data suggest that an intracellular calcium store independent PKC-Sp1 signaling pathway induces early keratinocyte differentiation through upregulation of TSG101.