Effect of Myh9 knockdown and PEG950 treatment on in vitro differentiated hepatocyte gene expression
Ontology highlight
ABSTRACT: Myh9 knockdown reduces the strength of the apical actomyosin cortex from hepatocytes, which causes isotropic growth of the bile canaliculus (BC) and ultimately results in the formation of hepatocyte cysts with a large central apical lumen. Treatment of hepatocytes with the osmotically active bioinert polymer PEG950 causes and increase in intraluminal pressure, which causes expansion of the bile canaliculus (BC) and ultimately results in the formation of hepatocyte cysts with a large central apical lumen. In these cysts, hepatocytes have common apico-basal polarity, such as observed for simple epithelia like cholangiocytes. To investigate whether this change in apical lumen morphology and polarization is associated with alterations in gene expression we compared hepatocytes with and without Myh9 knockdown and with and without PEG950 treatment.
Project description:Rab35 knockdown results in a loss of apical bulkheads from hepatocytes, which causes isotropic growth of the bile canaliculus (BC) and ultimately results in the formation of hepatocyte cysts. In these cysts, hepatocytes have common apico-basal polarity, such as observed for simple epithelia like cholangiocytes. To investigate whether this change in polarization is associated with alterations in gene expression we compared hepatocytes with and without Rab35 knockdown in a previous RNA-sequencing experiment and found changes in gene expression after 5 days of culture. In this experiment, we want to investigate when Rab35 knockdown starts to affect gene expression. For this, we performed a time-course RNA-sequencing experiment in hepatocytes with and without Rab35 knockdown.
Project description:Formation of epithelial tissues requires the generation of apical-basal polarity and the co-ordination of this polarity between neighboring cells to form a central lumen. MDCK cell line has proven to be a powerful model to study mammalian polarized epithelia in vitro. MDCK cells plated in extracellular matrix (ECM) form cysts, a spherical structure of polarized cells enclosing a central lumen which resembles epithelial tubular structures. The morphogenetic process requires drastic changes in cell architecture, which are regulated by change in gene expression. We used microarrays to identify genes up-regulated in lumen formation. The identification of up-regulated genes could lead us to characterize novel pathways needed for this process. MDCKII cells were plated in two different conditions: Cells cultured in confluence in plastic dishes, forming polarized monolayers (2D); or cells cultured in plastic dishes covered with Matrigel (ECM) forming three dimensional cysts (3D). Comparison of both transcriptomic profiles would lead us to identify up-regulated genes in the 3D condition, which would be good candidates to be key regulators of novel processes involved in lumen morphogenesis.
Project description:Non-muscle myosin IIA plays an important role in cell adhesion, cell migration and tissue architecture. We previously showed that low activity of the heavy chain of non-muscle myosin II Myh9 is beneficial to Lgr5+ intestinal stem cell maintenance. However, the function of Myh9 in adult mouse intestinal epithelium is largely unclear. In this study, we used the inducible Villin-creERT2 knockout approach to delete Myh9 in adult mouse intestinal epithelium and observed that homozygous deletion of Myh9 causes colitis-like morphologic changes in intestine, leads to a high sensitivity to dextran sulfate sodium and promotes the colitis related adenomas formation in colon. Myh9 deletion disturbs cell junctions and impairs intestinal lumen barrier integrity, promoting the necroptosis of epithelial cells. Consistently, these changes can be partially rescued by Ripk3 knockout. Our results indicate that Myh9 is required for the maintenance of intestinal epithelium integrity and the prevention of cell necroptosis.
Project description:We observed de novo formation of peripheral bile ducts by transdifferentiation of hepatocytes in mice born without peripheral bile ducts. To assess the authenticity and maturity of the hepatocyte-derived peripheral cholangiocytes forming the new bile ducts, we compared their global gene expression profile to that of peripheral cholangiocytes isolated from normal mice. We also included hepatocytes isolated from mice born without peripheral bile ducts as a control.
Project description:Formation of epithelial tissues requires the generation of apical-basal polarity and the co-ordination of this polarity between neighboring cells to form a central lumen. MDCK cell line has proven to be a powerful model to study mammalian polarized epithelia in vitro. MDCK cells plated in extracellular matrix (ECM) form cysts, a spherical structure of polarized cells enclosing a central lumen which resembles epithelial tubular structures. The morphogenetic process requires drastic changes in cell architecture, which are regulated by change in gene expression. We used microarrays to identify genes up-regulated in lumen formation. The identification of up-regulated genes could lead us to characterize novel pathways needed for this process.
Project description:The liver is the central organ critically regulating the balance of the metabolically potent yet toxic bile acids in the body. While genomic association studies have pointed to hepatic Sel1L – a critical component of mammalian Hrd1 ER-associated degradation (ERAD) machinery – as an influencer of serum bile acid levels, physiological relevance and mechanistic insights of ERAD in bile homeostasis remain unexplored. Using hepatocyte-specific Sel1L-deficient mouse models, we report that hepatic Sel1L-Hrd1 ERAD critically manages bile homeostasis in the body. Mice with hepatocyte-specific Sel1L developed intrahepatic cholestasis, with significant overload of bile acids in the liver and circulation under basal condition, and were hypersensitive to dietary bile acid challenge. By contrast, biliary bile acid and phosphatidylcholine levels were reduced, pointing to an export defect from hepatocytes. Unbiased proteomics analysis followed by biochemical assays revealed significant accumulation of the bile-stabilizing phosphatidylcholine exporter ATP-binding cassette 4 (Abcb4) in the ER of Sel1L-deficient livers, a gene associated with Progressive Familial Intrahepatic Cholestasis type III. Indeed, Abcb4 was a substrate of Sel1L-Hrd1 ERAD. Hence, hepatic Sel1L-Hrd1 ERAD maintains bile equilibrium via quality control of Abcb4 maturation in the ER.
Project description:Hepatocytes are highly polarized epithelia. Loss of hepatocyte polarity is associated with various liver diseases, including cholestasis. However, the molecular underpinnings of hepatocyte polarization remain poorly understood. Previously, we have shown that loss of β-catenin at adherens junctions (AJs) is compensated by β-catenin and dual loss of both catenins in dual knockouts (DKO) in mice liver leads to progressive intrahepatic cholestasis. However, the clinical relevance of this observation, and further phenotypic characterization of the phenotype, is important. Here, we identify simultaneous loss of β- and γ-catenin in a subset of liver samples from patients of progressive familial intrahepatic cholestasis and primary sclerosing cholangitis. Hepatocytes in DKO mice exhibited defect in apical-basolateral localization of polarity proteins, impaired bile canaliculi formation, and loss of microvilli. Loss of polarity in DKO livers manifested as epithelial-to-mesenchymal transition, increased hepatocyte proliferation, and suppression of hepatocyte differentiation, which was associated with up-regulation of TGFβ signaling and repression of Hnf4α expression and activity. In conclusion, concomitant loss of the two catenins in the liver may be playing a pathogenic role in subsets of cholangiopathies. Our findings also support a previously unknown role β- and γ-catenin in the maintenance of hepatocyte polarity. Improved understanding of the regulation of hepatocyte polarization processes by β and γ-catenin could potentially benefit development of new therapies for cholestasis.
Project description:The HGF/c-Met system is an essential inducer of hepatocyte growth and proliferation. Although a fundamental role for the HGF receptor c-Met has been demonstrated in acute liver regeneration its cell specific role in hepatocytes during chronic liver injury and fibrosis progression has not been determined yet. In order to better characterize the role of c-Met in hepatocytes we generated a hepatocyte-specific c-Met knockout mouse (c-MetM-bM-^HM-^Fhepa) using the Cre-loxP system and studied its relevance after bile-duct ligation. Two strategies for c-Met deletion in hepatocytes were tested. Early deletion during embryonic development was lethal, while post-natal Cre-expression was successful leading to the generation of viable c-MetM-bM-^HM-^Fhepa mice. Bile-duct ligation in these mice resulted in extensive necrosis and lower proliferation rates of hepatocytes. Gene array analysis of c-MetM-bM-^HM-^Fhepa mice revealed a significant reduction of anti-apoptotic genes in c-Met deleted hepatocytes. These findings could be functionally tested as c-MetM-bM-^HM-^Fhepa mice showed a stronger apoptotic response after bile-duct ligation and Jo-2 stimulation. This phenotype was associated with increased expression of pro-inflammatory cytokines (TNF-a and IL-6) and an enhanced recruitment of neutrophils. Activation of these mechanisms triggered a stronger pro-fibrogenic response as evidenced by increased TGF-b1, a-SMA, collagen-1a mRNA expression and enhanced collagen-fiber staining in c-MetM-bM-^HM-^Fhepa mice. For gene array analysis c-MetDhepa and c-MetloxP/loxP controls were stimulated for 2 hours with 2M-BM-5g recombinant mouse HGF.Three animals per group were treated in parallel, before and after i.p. injection of recombinant HGF or NaCl.
Project description:We report that the decreased expression of mitochondrial genes we observe in hepatocyte-specific Nampt knockout mice is normalized in primary hepatocytes, and that hepatocyte isolations causes major changes to the hepatocyte transcriptome for both knockouts and wildtype mice.
Project description:Primary hepatocytes have been widely explored as cell sources for the study of in vitro drug metabolism and pharmacokinetics (DMPK). Aiming toward establishing an in vitro drug screening method, the current study illustrated a comprehensive increase in the DMPK-related gene expression of nanopillar (NP)-cultured 3D-spheroid. To examine the expressional changes in DMPK-related genes under four different conditions, namely, NP-, sandwich (SW)-, monolayer (ML)-cultured rat hepatocytes, and freshly isolated hepatocytes, genome-wide gene-expression analysis using a DNA microarray was performed. Among the DMPK-related genes, cytochrome P450, UDP-glucuronosyltransferase, and transporter genes were focused on. Principal component analysis showed that the global gene expression profile in sample from NP culture is closer to that from freshly isolated hepatocytes than that from SW culture. The expressions of almost all Cyp 1 to 3 and Ugt genes of NP-cultured 3-D spheroid were higher than those of ML and SW. The expression of Abcc2 gene whose translation product has a critical role in excretion of metabolized bile acids in hepatocyte to bile canaliculi was three times higher in NP than in ML. From these results, 3-D spheroid formed by the NP culture was suggested to possess higher ability of metabolism and excretion than conventional 2-D monolayer culture. The NP culture has a potential as an alternative culturing technique for evaluating metabolism and toxicity toward the development of new drugs. Gene expression in rat hepatocyte was measured under four different conditions, namely, Nanopillar (NP)-, sandwich (SW)-, monolayer (ML)-cultured rat hepatocytes, and freshly isolated hepatocytes. Three independent experiments were performed at 95 hours of post-seeding.