Distinct role of endocytosis for Smad and non-Smad TGF-β signaling regulation in hepatocytes

Background & Aims: In injured liver, TGF-beta affects all hepatic cell types and participates in wound healing and fibrogenesis. TGF-beta downstream signaling is highly complex and cell type dependent, involving Smad and non-Smad signaling cascades thus requiring tight regulation. Endocytosis has gained relevance as important mechanism to control signaling initiation and termination. In this study, we investigated endocytic mechanisms for TGF-beta mediated Smad and non-Smad signaling in hepatocytes. Methods: Endocytosis in hepatocytes was elucidated using chemical inhibitors, RNAi, viral gene transfer and caveolin-1-/- mice. TGF-beta signaling was monitored by Western blot, reporter assays and gene expression analysis. Results: In hepatocytes, Smad activation is to a large degree accomplished AP-2 complex dependent on the hepatocyte surface without the necessity of clathrin coated pit formation or an endocytic step. In contrast, non-Smad/AKT pathway activation required functional dynamin mediated endocytosis and the presence of caveolin-1, an essential protein for caveolae formation. Furthermore, these two TGF-beta signaling initiation platforms discriminate distinct signaling routes that integrate at the transcriptional level as shown for TGF-beta target genes, Id1, Smad7, and CTGF. Endocytosis inhibition increased canonical Smad signaling and culminated in a superinduction of Id1 and Smad7 expression, whereas caveolin-1 mediated AKT pathway activation was required for maximal CTGF induction. Conclusions: Endocytosis is critical for TGF-beta signaling regulation in hepatocytes and determines gene expression signature and (patho)physiological outcome. (C) 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.