A decisive function of transforming growth factor-β/Smad signaling in tissue morphogenesis and differentiation of human HaCaT keratinocytes

The mechanism by which transforming growth factor-beta (TGF beta) regulates differentiation in human epidermal keratinocytes is still poorly understood. To assess the role of Smad signaling, we engineered human HaCaT keratinocytes either expressing small interfering RNA against Smads2, 3, and 4 or overexpressing Smad7 and verified impaired Smad signaling as decreased Smad phosphorylation, aberrant nuclear translocation, and altered target gene expression. Besides abrogation of TGF beta-dependent growth inhibition in conventional cultures, epidermal morphogenesis and differentiation in organotypic cultures were disturbed, resulting in altered tissue homeostasis with suprabasal proliferation and hyperplasia upon TGF beta treatment. Neutralizing antibodies against TGF beta, similar to blocking the actions of EGF-receptor or keratinocyte growth factor, caused significant growth reduction of Smad7-overexpressing cells, thereby demonstrating that epithelial hyperplasia was attributed to TGF beta-induced "dermis"-derived growth promoting factors. Furthermore impaired Smad signaling not only blocked the epidermal differentiation process or caused epidermal-to-mesenchymal transition but induced a switch to a complex alternative differentiation program, best characterized as mucous/intestinal-type epithelial differentiation. As the same alternative phenotype evolved from both modes of Smad-pathway interference, and reduction of Smad7-overexpression caused reversion to epidermal differentiation, our data suggest that functional TGF beta/Smad signaling, besides regulating epidermal tissue homeostasis, is not only essential for terminal epidermal differentiation but crucial in programming different epithelial differentiation routes.