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Adiponectin modulates focal adhesion disassembly in activated hepatic stellate cells: implication for reversing hepatic fibrosis.

ABSTRACT: Previous evidence indicates that adiponectin possesses antifibrogenic activity in inhibiting liver fibrosis. Therapeutic strategies, however, are limited by adiponectin quaternary structure and effective concentrations in circulation. Here we postulate a novel molecular mechanism, whereby adiponectin targets focal adhesion kinase (FAK) activity and disrupts key features of the fibrogenic response. Adiponectin-null (Ad(-/-)) mice and wild-type littermates were exposed to either saline or carbon tetrachloride (CCl4) for 6 wk. CCl4-gavaged mice were also injected with attenuated adenoviral adiponectin (Ad-Adn) or Ad-LacZ for 2 wk. Hepatic stellate cells (HSCs) were treated with or without adiponectin to elucidate signal transduction mechanisms. In vivo delivery of Ad-Adn markedly attenuates CCl4-induced expression of key integrin proteins and markers of HSC activation: ?v, ?3, ?1, ?2(I) collagen, and ?-smooth muscle actin. Confocal experiments of liver tissues demonstrated that adiponectin delivery also suppressed vinculin and p-FAK activity in activated HSCs. In vitro, adiponectin induced dephosphorylation of FAK, mediated by a physical association with activated tyrosine phosphatase, Shp2. Conversely, Shp2 knockdown by siRNA significantly attenuated adiponectin-induced FAK deactivation, and expression of TIMP1 and ?2(I) collagen was abolished in the presence of adiponectin and si-FAK. Finally, we documented that either adiponectin or the synthetic peptide with adiponectin properties, ADP355, suppressed p-FAK in synthetic matrices with stiffness measurements of 9 and 15 kPa, assessed by immunofluorescent imaging and quantitation. The in vivo and in vitro data presented indicate that disassembly of focal adhesion complexes in HSCs is pivotal for hepatic fibrosis therapy, now that small adiponectin-like peptides are available.

SUBMITTER: Kumar P

PROVIDER: S-EPMC6190967 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Adiponectin modulates focal adhesion disassembly in activated hepatic stellate cells: implication for reversing hepatic fibrosis.

Kumar Pradeep P Smith Tekla T Rahman Khalidur K Mells Jamie E JE Thorn Natalie E NE Saxena Neeraj K NK Anania Frank A FA

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20140825 12

Previous evidence indicates that adiponectin possesses antifibrogenic activity in inhibiting liver fibrosis. Therapeutic strategies, however, are limited by adiponectin quaternary structure and effective concentrations in circulation. Here we postulate a novel molecular mechanism, whereby adiponectin targets focal adhesion kinase (FAK) activity and disrupts key features of the fibrogenic response. Adiponectin-null (Ad(-/-)) mice and wild-type littermates were exposed to either saline or carbon t ...[more]

PMID: 25154876

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Project description:The survival of endothelial cells is dependent on interactions between the matrix and integrins mediated through focal adhesions. Focal adhesion kinase (FAK) is thought to play a key role in maintaining focal adhesion function and cell survival, whereas caspase-mediated FAK proteolysis is implicated in focal adhesion disassembly during apoptosis. We examined the relationship between changes in FAK phosphorylation and proteolysis during apoptosis of primary porcine aortic endothelial cells (PAEC) induced by staurosporine, a widely used apoptogenic agent in diverse cell types. Staurosporine-induced PAEC apoptosis was detected after 1 h and was preceded by disruption and loss of FAK localization to focal adhesions within a few minutes, whereas staurosporine-induced cleavage of FAK occurred only after 8-24 h. Staurosporine induced a very rapid dephosphorylation of FAK at Tyr(861) and Tyr(397) and caused dissociation of phosphorylated FAK from focal adhesions as early as 30 s. The effect of staurosporine was very potent with striking inhibition of Tyr(861) and Tyr(397) phosphorylation and focal adhesion disruption occurring in the range 10-100 nM. Selective inhibition of a known target of staurosporine, protein kinase C, using GF109203X, and of phosphoinositide 3'-kinase using wortmannin, did not reduce FAK tyrosine phosphorylation at Tyr(861) and Tyr(397), or cause disruption of focal adhesions. Cycloheximide, the protein synthesis inhibitor, induced PAEC apoptosis more slowly than staurosporine, but did not induce FAK dephosphorylation or rapid focal adhesion disruption, and instead caused a slower loss of focal adhesions and a marked increase in FAK proteolysis. These studies show that FAK dephosphorylation and focal adhesion disassembly are very early events mediating the onset of staurosporine-induced endothelial cell apoptosis and are dissociated from FAK proteolysis. Cycloheximide induces apoptosis through a pathway involving FAK proteolysis without dephosphorylation.

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.

Dataset Information

Adiponectin modulates focal adhesion disassembly in activated hepatic stellate cells: implication for reversing hepatic fibrosis.

Publications

Adiponectin modulates focal adhesion disassembly in activated hepatic stellate cells: implication for reversing hepatic fibrosis.

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