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

A-激酶锚定蛋白在肝毒素诱导的肝纤维化中的作用

• 批准号: 9893423
• 负责人: Komal Ramani
• 金额: $ 20.88万
• 依托单位: CEDARS-SINAI MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-23 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9893423
• 关键词: A kinase anchoring protein; Affect; Alcohols; Binding; CCL4 gene; CCND1 gene; Carbon Tetrachloride; Cell Cycle Progression; Cell Proliferation; Chemotaxis; Clustered Regularly Interspaced Short Palindromic Repeats; Collagen; Coupled; Cyclic AMP-Dependent Protein Kinases; Cyclin D1; Cytoplasm; DAP kinase; Data; Direct Repeats; Drops; Event; Exhibits; Extracellular Matrix; Fibrosis; G protein coupled receptor kinase; Gravin; Growth; Hepatic Fibrogenesis; Hepatic Stellate Cell; Hepatocyte; High Fat Diet; Hsp47 protein; Human; Ligation; Link; Liver; Liver Fibrosis; Mediating; Modeling; Modification; Molecular Chaperones; Mus; Mutagenesis; Mutation; Nuclear Translocation; Outcome; PRKCA gene; Phosphopeptides; Phosphorylation; Phosphotransferases; Post-Translational Protein Processing; Production; Property; Protein Kinase; Proteins; Proteomics; Publishing; Role; Scaffolding Protein; Site; Streptozocin; Testing; Work; alcohol abuse therapy; base; bile duct; cell growth; cell motility; crosslink; hepatotoxin; liver injury; loss of function; migration; mouse model; mutant; new therapeutic target; novel; nucleocytoplasmic transport; phosphoproteomics; prevent; receptor; scaffold; senescence; spatiotemporal; therapeutic target

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. .

项目摘要 肝星状细胞（HSC）活化是肝纤维化的标志，与胶原蛋白产生增加有关 和细胞外基质失调。有限的抗纤维化治疗是可用的。增强的HSC活化是 在肝毒素相关肝纤维化[四氯化碳（CCL 4）或链脲佐菌素/高脂饮食]中观察到 (STAM™）诱导的]以及在人肝纤维化中。A-激酶锚定蛋白12（AKAP 12）支架蛋白 激酶A/（PKA/PKC）和细胞周期蛋白-D1（CCND 1），并控制细胞增殖/迁移/侵袭。AKAP 12 PKCα的磷酸化抑制其与细胞质中CCND 1的结合，使CCND 1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）引起的HSC活化 治疗或胆管结扎）诱导AKAP 12磷酸化并抑制其支架活性， CCND 1和PKCα。这种效应在HSC中观察到，但在其他肝细胞中没有观察到。我们发现了一个新的功能 AKAP 12在支架胶原蛋白伴侣，热休克蛋白47（HSP 47），被AKAP 12抑制 HSC活化过程中的磷酸化。在此基础上，我们研究了AKAP 12的磷酸化 以及肝纤维化过程中的支架活性。我们的数据表明，CCL 4或STAM™肝脏模型 纤维化表现出主要在HSC中广泛的AKAP 12磷酸化。这是与急剧下降， AKAP 12对CCND 1和HSP 47的支架活性。AKAP 12磷酸化增加和其磷酸化的丧失 CCND 1/HSP 47支架在纤维化的人肝脏中也是明显的。AKAP 12与 激酶，PKCα，G蛋白偶联受体激酶2（GRK 2）和死亡相关蛋白激酶1（DAPK 1） 在活化的HSC和纤维化的肝脏中发现。我们最近发表了AKAP 12在正常人中具有抗纤维化作用。 但在HSC活化过程中获得促纤维化特性。我们怀疑这是由现场造成的- AKAP 12的特异性磷酸化。使用重复的规则间隔短回文重复 在HSC中的CRISPR定向诱变，我们现在提供了磷酸化位点编辑恢复丢失的证据。 AKAP 12对活化HSC中CCND 1和HSP 47的支架活性。此外，磷酸- 突变强烈抑制生长和HSC活化。AKAP 12磷酸化突变不影响其相互作用 CCND 1在肝细胞中。因此，我们假设纤维化HSC中的位点特异性磷酸化可能 防止AKAP 12从支架CCND 1或HSP 47，从而促进其生长促进和胶原 伴侣/成熟功能。为了验证这一假设，我们建议确定特定的激酶 磷酸化HSC中AKAP 12，并研究这如何影响其支架依赖性共定位 CCND 1、HSP 47和激酶本身。我们将探索阻断AKAP 12磷酸化的可能性， 位点可以恢复其CCND 1/HSP 47-支架活性，从而控制生长和胶原成熟。如果 AKAP 12磷酸化可能作为肝纤维化的新治疗靶点。 .