ABSTRACT: Transforming growth factor ? (TGF?) induces hepatic stellate cell (HSC) differentiation into tumor-promoting myofibroblast, although underlying mechanism remains incompletely understood. Focal adhesion kinase (FAK) is activated in response to TGF? stimulation, so it transmits TGF? stimulus to extracellular signal-regulated kinase and P38 mitogen-activated protein kinase signaling. However, it is unknown whether FAK can, in return, modulate TGF? receptors. In this study, we tested whether FAK phosphorylated TGF? receptor 2 (TGF?R2) and regulated TGF?R2 intracellular trafficking in HSCs. The FAKY397F mutant and PF-573,228 were used to inhibit the kinase activity of FAK, the TGF?R2 protein level was quantitated by immunoblotting, and HSC differentiation into myofibroblast was assessed by expression of HSC activation markers, alpha-smooth muscle actin, fibronectin, or connective tissue growth factor. We found that targeting FAK kinase activity suppressed the TGF?R2 protein level, TGF?1-induced mothers against decapentaplegic homolog phosphorylation, and myofibroblastic activation of HSCs. At the molecular and cellular level, active FAK (phosphorylated FAK at tyrosine 397) bound to TGF?R2 and kept TGF?R2 at the peripheral plasma membrane of HSCs, and it induced TGF?R2 phosphorylation at tyrosine 336. In contrast, targeting FAK or mutating Y336 to F on TGF?R2 led to lysosomal sorting and degradation of TGF?R2. Using RNA sequencing, we identified that the transcripts of 764 TGF? target genes were influenced by FAK inhibition, and that through FAK, TGF?1 stimulated HSCs to produce a panel of tumor-promoting factors, including extracellular matrix remodeling proteins, growth factors and cytokines, and immune checkpoint molecule PD-L1. Functionally, targeting FAK inhibited tumor-promoting effects of HSCs in vitro and in a tumor implantation mouse model. Conclusion: FAK targets TGF?R2 to the plasma membrane and protects TGF?R2 from lysosome-mediated degradation, thereby promoting TGF?-mediated HSC activation. FAK is a target for suppressing HSC activation and the hepatic tumor microenvironment.
