Endocytosis regulates TGF-β-induced Akt signal transduction in hepatocytes

TGF-β is a mayor cytokine in liver physiology. It regulates both cell proliferation and apoptosis of hepatocytes, and is the main drive of fibrosis, a typical complication of chronic liver diseases. An important feature of the signal transduction events is endocytosis of the receptor complexes. For TGF-β this remains a controversial issue, since independent approaches have shown either no effect or an important participation of endocytosis in its signal transduction.

We addressed the importance of endocytosis in the regulation of TGF-β signaling in the cell line Hepatoma 1–6 and in primary mouse hepatocytes. In both, TGF-β signaling is strongly induced, as shown by P-Smad2 Western blot, Smad2 nuclear accumulation and Smad binding element-luciferase reporter assays. Endocytosis via clathrin coated vesicles of Alexa633-labeled transferrin was followed by confocal microscopy. TGF-β induced Akt phosphorylation was detected in primary hepatocytes. Inhibition of endocytosis by over-expression of dominant negative Dynamin had no influence on the Smad2 pathway in both cell types. However, only in primary hepatocytes, TGF-β-induced activation of the Akt pathway was strongly reduced by endocytosis inhibition. Other signaling pathways known to be induced by TGF-β (eg p38, JNK) were also investigated. Finally, the relevance of endocytosis in TGF-β-induced apoptosis was investigated in primary mouse hepatocytes, indicating that abrogation of Akt pathway by both endocytosis inhibition and Wortmannin enhanced the pro-apoptotic effect of TGF-β.

In summary, for a more detailed understanding of TGF-β signaling in hepatocytes, regulatory mechanisms involving endocytosis have to be considered, specially regarding the development of anti-TGF-β therapeutic approaches.