Protein diaphanous homolog 1 (Diaph1) promotes myofibroblastic activation of hepatic stellate cells by regulating Rab5a activity and TGFβ receptor endocytosis

TGF beta induces the differentiation of hepatic stellate cells (HSCs) into tumor-promoting myofibroblasts but underlying mechanisms remain incompletely understood. Because endocytosis of TGF beta receptor II (T beta RII), in response to TGF beta stimulation, is a prerequisite for TGF signaling, we investigated the role of protein diaphanous homolog 1 (known as Diaph1 or mDia1) for the myofibroblastic activation of HSCs. Using shRNA to knockdown Diaph1 or SMIFH2 to target Diaph1 activity of HSCs, we found that the inactivation of Diaph1 blocked internalization and intracellular trafficking of T beta RII and reduced SMAD3 phosphorylation induced by TGF beta 1. Mechanistic studies revealed that the N-terminal portion of Diaph1 interacted with both T beta RII and Rab5a directly and that Rab5a activity of HSCs was increased by Diaph1 overexpression and decreased by Diaph1 knockdown. Additionally, expression of Rab5aQ79L (active Rab5a mutant) increased whereas the expression of Rab5aS34N (inactive mutant) reduced the endosomal localization of T beta RII in HSCs compared to the expression of wild-type Rab5a. Functionally, TGF beta stimulation promoted HSCs to express tumor-promoting factors, and alpha-smooth muscle actin, fibronection, and CTGF, markers of myofibroblastic activation of HSCs. Targeting Diaph1 or Rab5a suppressed HSC activation and limited tumor growth in a tumor implantation mouse model. Thus, Dipah1 and Rab5a represent targets for inhibiting HSC activation and the hepatic tumor microenvironment.