Role of A-Kinase Anchoring Proteins in Hepatotoxin- Induced Liver Fibrosis

项目总结 肝星状细胞(HSC)活化是与胶原生成增加相关的肝纤维化的标志 以及放松对细胞外基质的调控。有限的抗纤维化治疗是可用的。增强的HSC激活是 在肝毒素相关性肝纤维化[四氯化碳(CCl4)或链脲佐菌素/高脂饮食]中观察到 (STAM™)诱导的)以及在人的肝纤维化中。A-激酶锚定蛋白12支架蛋白 蛋白激酶A/(PKA/PKC)和细胞周期蛋白-D_1(CCND_1)参与调控细胞的增殖、迁移和侵袭。AKAP12 蛋白激酶Cα的磷酸化抑制其与胞浆中ccnd1的结合，使ccnd1发生核转位 和细胞周期进程。我们最近发表了由肝损伤(由于酒精引起)引起的HSC激活 治疗或胆管结扎)诱导AKAP12磷酸化并抑制其支架活性 Ccnd1和PKCα。这种效应在HSCs中观察到，但在其他肝细胞中观察不到。我们发现了一种新的功能 AKAP12抑制的胶原伴侣热休克蛋白47(HSP47)在支架中的表达 肝星状细胞激活过程中的磷酸化。在此基础上，我们对AKAP12的S磷酸化进行了研究 和肝纤维化形成过程中的支架活性。我们的数据表明，肝脏的CCl_4或STAM™模型 纤维化表现为广泛的AKAP12磷酸化，主要见于HSC。这与一次急剧下降有关。 AKAP12‘S对CCND 1和HSP47的支架活性。AKAP12磷酸化增强和ITS丢失 肝纤维化组织中也可见CCND1/HSP47支架。AKAP12与S相互作用的变化 蛋白激酶Cα、G蛋白偶联受体激酶2和死亡相关蛋白激酶1 在活化的HSCs和肝纤维化中均有表达。我们最近发表了AKAP12在正常人群中的抗纤维化作用 但在HSC激活过程中，HSC获得了促纤维化特性。我们怀疑这是由场地- AKAP12的特异性磷酸化。使用成簇的规则间隔的短回文重复 (CRISPR)在造血干细胞中的定向突变，我们现在提供了磷酸位点编辑修复丢失的证据 激活的HSCs中AKAP12对CCND1和HSP47的支架活性。此外，磷酸盐- 突变会强烈地抑制生长和HSC的激活。AKAP12磷酸化突变不影响其相互作用 在肝细胞中与CCND_1结合。因此，我们假设纤维化的HSCs中的位点特异性磷酸化可能 防止AKAP12支架CCND1或HSP47，从而促进其生长和胶原蛋白 分别为陪伴/成熟功能。为了验证这一假设，我们建议识别特定的激酶。 使HSCs中的AKAP12磷酸化，并研究这如何影响其支架依赖的共定位 与CCND1、HSP47和激酶本身有关。我们将探索阻断AKAP12磷酸化的可能性。 部位可以恢复其CCND1/HSP47-支架活性，从而控制生长和胶原成熟。如果 破译，AKAP12的磷酸化可能成为治疗肝纤维化的新靶点。 。

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 PKCα 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 PKCα. 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, PKCα, 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. .