Tuesday, April 16, 2024
4/16/2024
PROJECT SUMMARY Hepatic stellate cell (HSC) activation is a hallmark of liver fibrosis associated with increased collagen production and extracellular matrix deregulation. Limited antifibrotic therapies are available. Enhanced HSC activation is observed in hepatotoxin-related liver fibrosis [carbon-tetrachloride (CCL4) or streptozotocin/high fat diet (STAM™)-induced] as well as in human liver fibrosis. A-kinase anchoring protein 12 (AKAP12) scaffolds protein kinases A/(PKA/PKC) and cyclin-D1 (CCND1) and controls cell proliferation/migration/invasiveness. AKAP12 phosphorylation by PKCa inhibits its binding to CCND1 in the cytoplasm, allowing CCND1 nuclear translocation and cell cycle progression. We recently published that HSC activation caused by liver injury (due to alcohol treatment or bile duct ligation) induces AKAP12 phosphorylation and inhibits its scaffolding activity towards CCND1 and PKCa. This effect is observed in HSCs but not in other liver cells. We discovered a novel function of AKAP12 in scaffolding the collagen chaperone, heat shock protein 47 (HSP47) that was inhibited by AKAP12 phosphorylation during HSC activation. Based on the above, we have investigated AKAP12’s phosphorylation and scaffolding activity during liver fibrogenesis. Our data demonstrate that CCL4 or STAM™ models of liver fibrosis exhibit extensive AKAP12 phosphorylation mainly in HSCs. This was associated with a drastic drop in AKAP12’s scaffolding activity towards CCND1 and HSP47. Increased AKAP12 phosphorylation and loss of its CCND1/HSP47 scaffolding were also evident in fibrotic human liver. Alterations in AKAP12’s interactions with kinases, PKCa, G-protein coupled receptor kinase 2 (GRK2) and death-associated protein kinase 1 (DAPK1) were found in activated HSCs and fibrotic liver. We recently published that AKAP12 was anti-fibrogenic in normal HSCs but acquired pro-fibrogenic properties during HSC activation. We suspected that this was caused by site- specific phosphorylation of AKAP12. Using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-directed mutagenesis in HSCs, we now provide evidence that phospho-site editing restores the loss of scaffolding activity of AKAP12 towards CCND1 and HSP47 in activated HSCs. Moreover, the phospho- mutations strongly inhibit growth and HSC activation. AKAP12 phospho-mutations did not affect its interaction with CCND1 in hepatocytes. We therefore hypothesize that site-specific phosphorylation in fibrotic HSCs may prevent AKAP12 from scaffolding CCND1 or HSP47, thereby facilitating their growth-promoting and collagen chaperoning/maturation functions, respectively. To test this hypothesis, we propose to identify specific kinases that phosphorylate AKAP12 in HSCs and examine how this influences its scaffolding-dependent co-localization with CCND1, HSP47 and the kinases themselves. We will explore the possibility that blocking AKAP12 phospho- sites may restore its CCND1/HSP47-scaffolding activity and thereby control growth and collagen maturation. If deciphered, AKAP12 phosphorylation may serve as a novel therapeutic target for liver fibrosis. .
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