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Liver Epithelial Focal Adhesion Kinase Modulates Fibrogenesis and Hedgehog Signaling

ABSTRACT: Focal adhesion kinase (FAK) is an important mediator of extracellular matrix-integrin mechano-signal transduction that regulates cell motility, survival, and proliferation. As such, FAK is being investigated as a potential therapeutic target for malignant and fibrotic diseases, and numerous clinical trials of FAK inhibitors are underway. The function of FAK in non-malignant non-motile epithelial cells is not well understood. We previously showed that hepatocytes demonstrated activated FAK near stiff collagen tracts in fibrotic liver. In this study, we examined the role of liver epithelial FAK by inducing fibrotic liver disease in mice with liver epithelial FAK deficiency. We found that mice that lack FAK in liver epithelial cells develop more severe liver injury and worse fibrosis as compared to controls. Increased fibrosis in liver epithelial FAK-deficient mice is linked to the activation of several pro-fibrotic pathways, including the hedgehog-smoothened pathway. FAK-deficient hepatocytes produce increased Indian hedgehog in a matrix stiffness-dependent manner. Furthermore, there is increased expression of the hedgehog receptor, smoothened, in macrophages and biliary cells of hepatocyte-specific FAK-deficient fibrotic liver. These results indicate that liver epithelial FAK has important regulatory roles in the response to liver injury and progression of fibrosis.

ORGANISM(S): Mus musculus

PROVIDER: GSE157156 | GEO | 2020/09/15

REPOSITORIES: GEO

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Project description:Older age is a major risk factor for damage to many tissues, including liver. Aging undermines resiliency (i.e., the ability to recover from injury) and impairs liver regeneration. The mechanisms whereby aging reduces resiliency are poorly understood. Hedgehog is a signaling pathway with critical mitogenic and morphogenic functions during development. Recent studies indicate that Hedgehog regulates metabolic homeostasis in adult liver. The present study evaluates the hypothesis that Hedgehog signaling becomes dysregulated in hepatocytes during aging, resulting in decreased resiliency and therefore, impaired regeneration and enhanced vulnerability to damage. Methods: Partial hepatectomy (PH) was performed on young and old wild type mice and Smoothened (Smo)-floxed mice treated with AAV8-TBG luciferase (control) or AAV8-TBG-Cre vectors to conditionally delete Smo and disrupt Hedgehog signaling specifically in hepatocytes. Changes in signaling were correlated with changes in regenerative responses and compared among groups. Results: Old livers had fewer hepatocytes proliferating after PH. RNA sequencing identified Hedgehog as a top down-regulated pathway in old hepatocytes before and after the regenerative challenge. Deleting Smo in healthy young hepatocytes before PH prevented Hedgehog pathway activation after PH and inhibited regeneration. GO analysis demonstrated that both old and Smo-deleted young hepatocytes had activation of pathways involved in innate immune responses and suppression of several signaling pathways that control liver growth and metabolism including insulin-like growth factor, Wnt and NOTCH. Hedgehog inhibition promoted telomere shortening and mitochondrial dysfunction in hepatocytes, consequences of aging that promote inflammation and impair tissue growth and metabolic homeostasis. Conclusion: Hedgehog signaling is dysregulated in old hepatocytes. This accelerates aging, resulting in decreased resiliency and therefore, impaired liver regeneration and enhanced vulnerability to damage.

Project description:Background: HGF/c-Met signaling plays a pivotal role in hepatocyte survival and tissue remodeling during liver regeneration. Treatment with HGF has been shown to accelerate resolution of fibrotic liver lesions in experimental animal models. To formally address the importance of c-Met signaling in hepatocytes in the context of chronic liver injury, we have used hepatocyte-specific Metfl/fl;Alb-Cre+/- conditional knockout mice (KO) and a model of liver fibrosis. Methods: CCl4 was administrated biweekly over a period of 4 weeks (injury phase), and the animals were followed over the next 4 weeks (healing phase). Macroscopic and microscopic changes during the injury and healing phases were monitored by IHC. Deposition of ECM was assessed by Sirius red staining and hydroxiproline content. Activation of hepatic stellate cells (HSC) was estimated by a-SMA using WB and IHC. Expression levels of the selected key fibrotic molecules were evaluated by RT-qPCR and WB. Time-dependent global transcriptomic changes from whole livers and isolated hepatocytes were examined using gene expression microarrays. Results: Loss of HGF/c-Met signaling in hepatocytes altered the hepatic microenvironment and dramatically aggravated hepatic fibrogenesis. Increased liver damage was associated with decreased hepatocyte proliferation, progressive accumulation of HSCs, and delayed fibrinolysis causing increased collagen deposit. Dystrophic calcification of necrotic areas impaired phagocytosis, resulting in sustained inflammatory and fibrogenic signaling further augmenting severity of fibrogenesis. Global gene-expression analysis demonstrated upregulation of key fibrogenic molecules, such as Tgf-Ã¢ and Pdgf-Ã¢, paralleled by a decreased expression of genes important for cell cycle, stress response and regeneration, which could be attributed to the c-Met deficiency in hepatocytes. Additionally, key chemotactic and inflammatory cytokines, including Ccl2, SDF1/Cxcr4 and Spp1, were upregulated in Metfl/fl;Alb-Cre+/- hepatocytes. However, the major pro-fibrotic signaling originated from the non-parenchymal cell compartments, as revealed by cell type-specific gene expression signatures. Conclusion: These results indicate that lack of c-Met signaling in hepatocytes disrupts the balance between extracellular matrix production and degradation and establish a protective role for c-Met against adverse microenvironment leading to the development of fibrotic liver disease. In the present study, we reported a detailed and comprehensive dynamic characterization of the cellular and molecular alterations involved in fibrosis in the liver of c-Met transgenic mice. Liver samples from female animals were collected at various time-points after fibrosis induction using CCL4 ranging from 0 weeks to 3 weeks. Tissue samples were divided into two parts; one was fixed in 10% formalin for histological evaluation and the other was used for RNA analysis.

Project description:Immunotherapeutics represent highly promising agents with the potential to improve patient outcomes in a variety of cancer types. Unfortunately, single-agent immunotherapy has achieved limited clinical benefit to date in patients suffering from pancreatic ductal adenocarcinoma (PDAC). This may be due to the presence of a uniquely immunosuppressive tumor microenvironment (TME) present in PDACs, which creates a barrier to effective immune surveillance. Critical obstacles to immunotherapy in PDAC tumors include the dense desmoplastic stroma that acts as a barrier to T-cell infiltration and the high numbers of tumor-associated immunosuppressive cells. We have identified hyperactivated focal adhesion kinase (FAK) activity in neoplastic PDAC cells as a significant regulator of the fibrotic and immunosuppressive TME. We found that FAK activity was elevated in human PDAC tissues and correlates with high levels of fibrosis and poor CD8+ cytotoxic T-cell infiltration. Single-agent FAK inhibition (VS-4718) dramatically limited tumor progression, resulting in a doubling of survival in the p48-Cre/LSL-KrasG12D/p53Flox/+ (KPC) mouse model of human PDAC. This alteration in tumor progression was associated with dramatically reduced tumor fibrosis, decreased numbers of tumor-infiltrating immature myeloid cells and immunosuppressive macrophages. We postulated that these desirable effects of FAK inhibition on the TME might render PDAC tumors more sensitive to immunotherapy. Accordingly, we found that VS-4718 rendered the previously unresponsive KPC mouse model responsive to anti-PD1 and anti-CTLA4 antagonists leading to a nearly tripling of survival times. These data suggest that FAK inhibition increases immune surveillance by overcoming the fibrotic and immunosuppressive PDAC TME thus rendering tumors more responsive to immunotherapy. We treated KP orthotopic tumor-bearing mice with vehicle and FAK inhibitor (FAKi) for 14 days, then extracted total RNA from tumor tissues.

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Liver Epithelial Focal Adhesion Kinase Modulates Fibrogenesis and Hedgehog Signaling

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