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

ABSTRACT: TGF? induces the differentiation of hepatic stellate cells (HSCs) into tumor-promoting myofibroblasts but underlying mechanisms remain incompletely understood. Because endocytosis of TGF? receptor II (T?RII), in response to TGF? 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?RII and reduced SMAD3 phosphorylation induced by TGF?1. Mechanistic studies revealed that the N-terminal portion of Diaph1 interacted with both T?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?RII in HSCs compared to the expression of wild-type Rab5a. Functionally, TGF? stimulation promoted HSCs to express tumor-promoting factors, and ?-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.

SUBMITTER: Liu D

PROVIDER: S-EPMC7686927 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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

Liu Donglian D Fu Xinhui X Wang Yuanguo Y Wang Xianghu X Wang Hua H Wen Jialing J Kang Ningling N

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20200418 6

TGFβ induces the differentiation of hepatic stellate cells (HSCs) into tumor-promoting myofibroblasts but underlying mechanisms remain incompletely understood. Because endocytosis of TGFβ receptor II (TβRII), in response to TGFβ 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 th ...[more]

PMID: 32304339

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Project description: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.

Project description:The pathogenesis of cirrhosis, a disabling outcome of defective liver repair, involves deregulated accumulation of myofibroblasts derived from quiescent hepatic stellate cells (HSCs), but the mechanisms that control transdifferentiation of HSCs are poorly understood. We investigated whether the Hedgehog (Hh) pathway controls the fate of HSCs by regulating metabolism.Microarray, quantitative polymerase chain reaction, and immunoblot analyses were used to identify metabolic genes that were differentially expressed in quiescent vs myofibroblast HSCs. Glycolysis and lactate production were disrupted in HSCs to determine if metabolism influenced transdifferentiation. Hh signaling and hypoxia-inducible factor 1? (HIF1?) activity were altered to identify factors that alter glycolytic activity. Changes in expression of genes that regulate glycolysis were quantified and localized in biopsy samples from patients with cirrhosis and liver samples from mice following administration of CCl(4) or bile duct ligation. Mice were given systemic inhibitors of Hh to determine if they affect glycolytic activity of the hepatic stroma; Hh signaling was also conditionally disrupted in myofibroblasts to determine the effects of glycolytic activity.Transdifferentiation of cultured, quiescent HSCs into myofibroblasts induced glycolysis and caused lactate accumulation. Increased expression of genes that regulate glycolysis required Hh signaling and involved induction of HIF1?. Inhibitors of Hh signaling, HIF1?, glycolysis, or lactate accumulation converted myofibroblasts to quiescent HSCs. In diseased livers of animals and patients, numbers of glycolytic stromal cells were associated with the severity of fibrosis. Conditional disruption of Hh signaling in myofibroblasts reduced numbers of glycolytic myofibroblasts and liver fibrosis in mice; similar effects were observed following administration of pharmacologic inhibitors of Hh.Hedgehog signaling controls the fate of HSCs by regulating metabolism. These findings might be applied to diagnosis and treatment of patients with cirrhosis.

Dataset Information

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

Publications

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

Similar Datasets

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