Focal Adhesion Kinase Promotes Hepatic Stellate Cell Activation by Regulating Plasma Membrane Localization of TGFβ Receptor 2

Abstract

Transforming growth factor β (TGFβ) 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 TGFβ stimulation, so it transmits TGFβ stimulus to extracellular signal-regulated kinase and P38 mitogenactivated protein kinase signaling. However, it is unknown whether FAK can, in return, modulate TGFβ receptors. In this study, we tested whether FAK phosphorylated TGFβ receptor 2 (TGFβR2) and regulated TGFβR2 intracellular trafficking in HSCs. The FAKY397F mutant and PF-573,228 were used to inhibit the kinase activity of FAK, the TGFβR2 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 TGFβR2 protein level, TGFβ1-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 TGFβR2 and kept TGFβR2 at the peripheral plasma membrane of HSCs, and it induced TGFβR2 phosphorylation at tyrosine 336. In contrast, targeting FAK or mutating Y336 to F on TGFβR2 led to lysosomal sorting and degradation of TGFβR2. Using RNA sequencing, we identified that the transcripts of 764 TGFβ target genes were influenced by FAK inhibition, and that through FAK, TGFβ1 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 tumorpromoting effects of HSCs in vitro and in a tumor implantation mouse model. Conclusion: FAK targets TGFβR2 to the plasma membrane and protects TGFβR2 from lysosome-mediated degradation, thereby promoting TGFβ-mediated HSC activation. FAK is a target for suppressing HSC activation and the hepatic tumor microenvironment. (Hepatology Communications 2020;4:268-283).269 express α-smooth muscle actin (α-SMA), fibronectin and connective tissue growth factor (CTGF), markers of HSC activation, (4,5) and paracrine factors that promote liver metastatic growth. (6) Understanding how TGFβ signaling events are regulated, such as how TGFβ receptors distribute and traffic in HSCs, will help identify targets to inhibit HSC activation and the metastasis-promoting liver microenvironment.Focal adhesion kinase (FAK) is a 125-kDa nonreceptor tyrosine (Y) kinase. It consists of an N-terminal FERM domain, a middle kinase domain, and a C-terminal FAT domain. (7,8) Inactive FAK exists as an auto-inhibited monomer, and its autophosphorylation at Y397 creates a binding site for SH2 domain of Src, so that Src is recruited to induce phosphorylation of FAK at additional sites, leading to full activation of FAK kinase. (7,8) In addition, FAK functions as a protein scaffold for sign...

EZH2 Engages TGFβ Signaling to Promote Breast Cancer Bone Metastasis via Integrin β1-FAK Activation

“…In the present study, we found that integrin β1-FAK is involved in the classical TGFβ/Smad-dependent pathway rather than the p38 MAPK pathway. Also, our data on the binding between integrin β1 and TGFβRI suggested It was reported that FAK and integrin β3 bind to TGFβRII in stellate hepatic cells 39 and breast cancer cells 40 . Here, we detected that in EZH2 high expressing breast cancer cells, FAK and integrin β1 bound to TGFβRI rather than TGFβRII and that FAK phosphorylated TGFβRI.…”

“…It was reported that FAK and integrin β3 bind to TGFβRII in stellate hepatic cells 39 and breast cancer cells 40 . Here, we detected that in EZH2 high expressing breast cancer cells, FAK and integrin β1 bound to TGFβRI rather than TGFβRII and that FAK phosphorylated TGFβRI.…”

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The involvement of TGF-β1 /FAK/α-SMA pathway in the antifibrotic impact of rice bran oil on thioacetamide-induced liver fibrosis in rats

“…Integrins are the principal adhesion receptors that transfer signals between ECM pathways and cytoplasmic domains across the cell plasma membrane. FAK is also activated in response to TGF-β1 activation and also other cytokines and growth factors [ 9 , 59 ]. Consistent with the current study’s findings, TGF-β1 activates HSCs to produce fibronectin, α-SMA, and CTGF, markers of HSC activation.…”