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Role of A-Kinase Anchoring Protein Phosphorylation in Alcohol-Induced Liver Injury and Hepatic Stellate Cell Activation.

ABSTRACT: Alcoholic liver injury is associated with hepatic stellate cell (HSC) activation. A-kinase anchoring protein 12 (AKAP12) scaffolds protein kinase C and cyclin-D1, which is regulated by its phosphorylation, and spatiotemporally controls cell proliferation, invasiveness, and chemotaxis. HSC activation induces AKAP12 expression, but the role of AKAP12's scaffolding activity in liver function is unknown. Because AKAP12 phosphorylation is enhanced in ethanol-treated HSCs, we examined AKAP12's scaffolding functions in alcohol-mediated HSC activation and liver injury. AKAP12 expression, interaction, and phosphorylation were assayed in in vitro and in vivo ethanol models and human subjects by real-time PCR, coimmunoprecipitation, immunoblotting, and phosphorylated proteomics/Phos-tag. Ethanol induced AKAP12 phosphorylation in the liver and in primary HSCs, but not in hepatocytes. AKAP12's scaffolding activity for protein kinase C/cyclin-D1 decreased in ethanol-treated HSCs but not hepatocytes. AKAP12 negatively regulated HSC activation, which was reversed by ethanol-mediated AKAP12 phosphorylation. AKAP12 interacted with heat shock protein 47 (HSP47), which chaperones collagen and induces its secretion. Ethanol inhibited AKAP12-HSP47 and induced HSP47-collagen interaction. Ethanol-induced phosphorylated AKAP12 was unable to bind to HSP47 compared with its unphosphorylated counterpart, thereby proving that ethanol-mediated phosphorylation of AKAP12 inhibited the HSP47-AKAP12 scaffold. Silencing AKAP12 facilitated the chaperoning of collagen by HSP47. Hence, AKAP12 scaffolds HSP47 and regulates collagen-HSP47 interaction. Ethanol quenches AKAP12's scaffolding activity through phosphorylation and facilitates HSC activation.

SUBMITTER: Ramani K

PROVIDER: S-EPMC5842034 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Role of A-Kinase Anchoring Protein Phosphorylation in Alcohol-Induced Liver Injury and Hepatic Stellate Cell Activation.

Ramani Komal K Tomasi Maria Lauda ML Berlind Joshua J Mavila Nirmala N Sun Zhaoli Z

The American journal of pathology 20180102 3

Alcoholic liver injury is associated with hepatic stellate cell (HSC) activation. A-kinase anchoring protein 12 (AKAP12) scaffolds protein kinase C and cyclin-D1, which is regulated by its phosphorylation, and spatiotemporally controls cell proliferation, invasiveness, and chemotaxis. HSC activation induces AKAP12 expression, but the role of AKAP12's scaffolding activity in liver function is unknown. Because AKAP12 phosphorylation is enhanced in ethanol-treated HSCs, we examined AKAP12's scaffol ...[more]

PMID: 29305319

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Project description:Background & aimsSteatohepatitis drives fibrogenesis in alcohol-related liver disease. Recent studies have suggested that hepatic stellate cells (HSCs) may regulate the parenchymal cell injury and inflammation that precedes liver fibrosis, although the mechanism remains incompletely defined. Neuropilin-1 (NRP-1) and synectin are membrane proteins implicated in HSC activation. In this study, we disrupted NRP-1 and synectin as models to evaluate the role of HSC activation on the development of steatohepatitis in response to alcohol feeding in mice.MethodsMice with HSC-selective deletion of NRP (ColCre/Nrp1loxP) or synectin (ColCre/synectinloxP) vs. paired Nrp1loxP or synectinloxP mice were fed a control diet or the chronic/binge alcohol feeding model. Several markers of steatosis and inflammation were evaluated.ResultsColCre/Nrp1loxP mice showed less fibrosis, as expected, but also less inflammation and steatosis, with lower hepatic triglyceride content. Similar results were observed in the synectin model. Hepatocytes treated with supernatant of HSCs from ColCre/Nrp1loxP mice compared to supernatant from Nrp1loxP mice were protected against ethanol-induced lipid droplet formation. An adipokine and inflammatory protein array from the supernatant of HSCs with NRP-1 knockdown showed a significant reduction in Igfbp3 (a major insulin-like growth factor-binding protein with multiple metabolic functions) and an increase in SerpinA12 (a serine-protease inhibitor) secretion compared to wild-type HSCs. Recombinant Igfbp3 induced lipid droplets, triglyceride accumulation, and lipogenic genes in hepatocytes in vitro, while SerpinA12 was protective against ethanol-induced steatosis. Finally, Igfbp3 was increased, and SerpinA12 was decreased in serum and liver tissue from patients with alcoholic hepatitis.ConclusionSelective deletion of NRP-1 from HSCs attenuates alcohol-induced steatohepatitis through regulation of Igfbp3 and SerpinA12 signaling.Lay summaryHepatic stellate cells are known for their role in fibrosis (scarring of the liver). In this study, we describe their role in the modulation of fat deposition and inflammation in the liver, which occurs secondary to alcohol damage.

Project description:Exposure to alcohol and its metabolites can initiate hepatic injury and fibrogenesis. Fibrosis is mediated through hepatic stellate cell (HSC) activation, leading to global changes in mRNA and microRNA (miR) expression. miRs are expressed in cells or shuttled to exosomes which can be detected in tissue culture media (TCM) and biological fluids. The mechanisms and function underlying the differential expression and processing of miRs and their downstream effects during hepatic injury remain poorly understood.Expression of primary (pri)-miR17-92. and individual members of this cluster, miR17a, 18a, 19a, 20a, 19b, and 92, were examined in primary HSCs and human LX2 cells exposed to alcohol-conditioned media (CM), liver tissue from a rodent model of alcoholic injury, and in exosomes from TCM and plasma of rodent models and patients with alcoholic liver disease (ALD). miR expression was examined in HSCs transduced with an AAV2 vector carrying GFP-miR19b or GFP-control transgene under the collagen promoter.Profibrotic markers were enhanced in primary HSCs and LX2 cells exposed to alcohol-CM, concomitant with decreased miR19b expression and a significant increase in pri-miR17-92. Increased pri-miR17-92 was confirmed in a rodent model of alcohol-induced liver injury. Individual members of the cluster were inversely proportionate in cells and exosomes. AAV2-mediated miR19b overexpression inhibited miR17-92 and altered expression of individual cluster members in cells and exosomes. Expression of individual miR17-92 cluster members in plasma exosomes isolated from patients with ALD was similar to that seen in a rodent model of alcoholic injury and in vitro.Reintroduction of miR19b inhibits HSC activation and modulates expression of pri-miR17-92 and the inverse expression of individual cluster members in cells and exosomes. Better understanding of miR17-92 processing may provide mechanistic insights into the role of individual miRs and exosomes during hepatic injury, revealing new therapeutic targets.

Dataset Information

Role of A-Kinase Anchoring Protein Phosphorylation in Alcohol-Induced Liver Injury and Hepatic Stellate Cell Activation.

Publications

Role of A-Kinase Anchoring Protein Phosphorylation in Alcohol-Induced Liver Injury and Hepatic Stellate Cell Activation.

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