MCPIP1 Inhibits Hepatic Stellate Cell Activation in Autocrine and Paracrine Manners, Preventing Liver Fibrosis.

BACKGROUND & AIMS: Hepatic fibrosis is characterized by enhanced deposition of extracellular matrix (ECM), which results from the wound healing response to chronic, repeated injury of any etiology. Upon injury, hepatic stellate cells (HSCs) activate and secrete ECM proteins, forming scar tissue, which leads to liver dysfunction. Monocyte-chemoattractant protein-induced protein 1 (MCPIP1) possesses anti-inflammatory activity, and its overexpression reduces liver injury in septic mice. In addition, mice with liver-specific deletion of Zc3h12a develop features of primary biliary cholangitis. In this study, we investigated the role of MCPIP1 in liver fibrosis and HSC activation. METHODS: We analyzed MCPIP1 levels in patients' fibrotic livers and hepatic cells isolated from fibrotic murine livers. In vitro experiments were conducted on primary HSCs, cholangiocytes, hepatocytes, and LX-2 cells with MCPIP1 overexpression or silencing. RESULTS: MCPIP1 levels are induced in patients' fibrotic livers compared with their nonfibrotic counterparts. Murine models of fibrosis revealed that its level is increased in HSCs and hepatocytes. Moreover, hepatocytes with Mcpip1 deletion trigger HSC activation via the release of connective tissue growth factor. Overexpression of MCPIP1 in LX-2 cells inhibits their activation through the regulation of TGFB1 expression, and this phenotype is reversed upon MCPIP1 silencing. CONCLUSIONS: We demonstrated that MCPIP1 is induced in human fibrotic livers and regulates the activation of HSCs in both autocrine and paracrine manners. Our results indicate that MCPIP1 could have a potential role in the development of liver fibrosis.

Hepatic Mcpip1 regulates adaptation to food restriction in mice.

Monocyte-chemoattractant protein-induced protein 1 (MCPIP1, or Regnase-1) is an endoribonuclease that degrades translationally active mRNA molecules. MCPIP1 is mostly known for its anti-inflammatory actions, but it is also an important regulator of adipogenesis and lipid metabolism. Its overexpression impairs adipogenesis by reducing mRNA levels of C/EBPß and PPARγ, key transcription factors regulating this process. Although adipocytes overexpressing MCPIP1 are characterised by impaired glucose uptake, the function of MCPIP1 in hepatocyte metabolism remains unknown. In this study, conditional deletion of Zc3h12a in murine liver epithelial cells was used to characterise the role of Mcpip1 in adaptation to 24-hour food restriction. We found that Mcpip1 deficiency in liver epithelial cells (Mcpip1fl/flAlbCre mice) resulted in higher blood glucose levels in response to fasting in comparison to Mcpip1fl/fl counterparts. Hepatic proteome analysis showed 26 down-regulated and 117 up-regulated proteins in Mcpip1fl/flAlbCre animals that were involved in cellular adhesion, extracellular matrix and metabolic processes. In conclusion, our studies provide new insight into the hepatic function of Mcpip1 and its involvement in metabolic control.

Hepatic MCPIP1 protein levels are reduced in NAFLD patients and are predominantly expressed in cholangiocytes and liver endothelium.

BACKGROUND AND AIMS: NAFLD is characterized by the excessive accumulation of fat in hepatocytes. NAFLD can range from simple steatosis to the aggressive form called NASH, which is characterized by both fatty liver and liver inflammation. Without proper treatment, NAFLD may further progress to life-threatening complications, such as fibrosis, cirrhosis, or liver failure. Monocyte chemoattractant protein-induced protein 1 (MCPIP1, alias Regnase 1) is a negative regulator of inflammation, acting through the cleavage of transcripts coding for proinflammatory cytokines and the inhibition of NF-κB activity. METHODS: In this study, we investigated MCPIP1 expression in the liver and peripheral blood mononuclear cells (PBMCs) collected from a cohort of 36 control and NAFLD patients hospitalized due to bariatric surgery or primary inguinal hernia laparoscopic repair. Based on liver histology data (hematoxylin and eosin and Oil Red-O staining), 12 patients were classified into the NAFL group, 19 into the NASH group, and 5 into the control (non-NAFLD) group. Biochemical characterization of patient plasma was followed by expression analysis of genes regulating inflammation and lipid metabolism. The MCPIP1 protein level was reduced in the livers of NAFL and NASH patients in comparison to non-NAFLD control individuals. In addition, in all groups of patients, immunohistochemical staining showed that the expression of MCPIP1 was higher in the portal fields and bile ducts in comparison to the liver parenchyma and central vein. The liver MCPIP1 protein level negatively correlated with hepatic steatosis but not with patient body mass index or any other analyte. The MCPIP1 level in PBMCs did not differ between NAFLD patients and control patients. Similarly, in patients' PBMCs there were no differences in the expression of genes regulating ß-oxidation (ACOX1, CPT1A, and ACC1) and inflammation (TNF, IL1B, IL6, IL8, IL10, and CCL2), or transcription factors controlling metabolism (FAS, LCN2, CEBPB, SREBP1, PPARA, and PPARG). CONCLUSION: We have demonstrated that MCPIP1 protein levels are reduced in NAFLD patients, but further research is needed to investigate the specific role of MCPIP1 in NAFL initiation and the transition to NASH.

Role of MCPIP1 protein in lipid metabolism, liver homeostasis and non-alcoholic fatty liver disease / Rola bialka MCPIP1 w metabolizmie lipidów, utrzymaniu homeostazy watroby oraz rozwoju niealkoholowej stluszczeniowej choroby watroby.

Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive accumulation of lipids in hepatocytes. Among NAFLD patients, in 25% of them this disease progress to nonalcoholic steatohepatitis, which is characterized additionally by the development of inflammation and fibrosis of liver. Currently, it is estimated that 24% of the world's population suffers from NAFLD. MCPIP1 protein is an RNase described as a negative regulator of inflammation. Also, MCPIP1 plays a role in lipid metabolism because it inhibits the process of adipogenesis and mice with a deletion of Zc3h12a gene are characterized by dyslipidemia and reduced body fat content. In the case of ischemia-reperfusion injury in liver, MCPIP1 is protective against the inflammation and damage of this organ. Lipid accumulation by hepatocytes is associated with a decrease of Mcpip1 level. In addition, MCPIP1 may influence the PPARγ-mediated lipogenesis process. Presence of Mcpip1 in both myeloid leukocytes and liver epithelial cells is crucial for the maintenance of liver homeostasis.

Selected transgenic murine models of human autoimmune liver diseases.

Murine models of human diseases are of outmost importance for both studying molecular mechanisms driving their development and testing new treatment strategies. In this review, we first discuss the etiology and risk factors for autoimmune liver disease, including primary biliary cholangitis, autoimmune hepatitis and primary sclerosing cholangitis. Second, we highlight important features of murine transgenic models that make them useful for basic scientists, drug developers and clinical researchers. Next, a brief description of each disease is followed by the characterization of selected animal models.

Role of Mcpip1 in obesity-induced hepatic steatosis as determined by myeloid and liver-specific conditional knockouts.

Monocyte chemoattractant protein-induced protein 1 (MCPIP1, alias Regnase 1) is a negative regulator of inflammation, acting through cleavage of transcripts coding for proinflammatory cytokines and by inhibition of NFκB activity. Moreover, it was demonstrated that MCPIP1 regulates lipid metabolism both in adipose tissue and in hepatocytes. In this study, we investigated the effects of tissue-specific Mcpip1 deletion on the regulation of hepatic metabolism and development of nonalcoholic fatty liver disease (NAFLD). We used control Mcpip1fl/fl mice and animals with deletion of Mcpip1 in myeloid leukocytes (Mcpip1fl/fl LysMCre ) and in hepatocytes (Mcpip1fl/fl AlbCre ), which were fed chow or a high-fat diet (HFD) for 12 weeks. Mcpip1fl/fl LysMCre mice fed a chow diet were characterized by a significantly reduced hepatic expression of genes regulating lipid and glucose metabolism, which subsequently resulted in low plasma glucose level and dyslipidemia. These animals also displayed systemic inflammation, demonstrated by increased concentrations of cytokines in the plasma and high Tnfa, Il6, IL1b mRNA levels in the liver and brown adipose tissue (BAT). Proinflammatory leukocyte infiltration into BAT, together with low expression of Ucp1 and Ppargc1a, resulted in hypothermia of 22-week-old Mcpip1fl/fl LysMCre mice. On the other hand, there were no significant changes in phenotype in Mcpip1fl/fl AlbCre mice. Although we detected a reduced hepatic expression of genes regulating glucose metabolism and ß-oxidation in these mice, they remained asymptomatic. Upon feeding with a HFD, Mcpip1fl/fl LysMCre mice did not develop obesity, glucose intolerance, nor hepatic steatosis, but were characterized by low plasma glucose level and dyslipidemia, along with proinflammatory phenotype. Mcpip1fl/fl AlbCre animals, following a HFD, became hypercholesterolemic, but accumulated lipids in the liver at the same level as Mcpip1fl/fl mice, and no changes in the level of soluble factors tested in the plasma were detected. We have demonstrated that Mcpip1 protein plays an important role in the liver homeostasis. Depletion of Mcpip1 in myeloid leukocytes, followed by systemic inflammation, has a more pronounced effect on controlling liver metabolism and homeostasis than the depletion of Mcpip1 in hepatocytes.

Fatty Acids and a High-Fat Diet Induce Epithelial-Mesenchymal Transition by Activating TGFß and ß-Catenin in Liver Cells.

Nonalcoholic fatty liver disease is defined as the accumulation of excessive fat in the liver in the absence of excessive alcohol consumption or any secondary cause. Although the disease generally remains asymptomatic, chronic liver inflammation leads to fibrosis, liver cirrhosis, and even to the development of hepatocellular carcinoma (HCC). Fibrosis results from epithelial-mesenchymal transition (EMT), which leads to dedifferentiation of epithelial cells into cells with a mesenchymal-like phenotype. During EMT, epithelial cells with high expression of E-cadherin, influenced by growth factors, cytokines, and inflammatory processes, undergo morphological changes via enhanced expression of, e.g., vimentin, fibronectin, and N-cadherin. An inducer of EMT and, consequently, of fibrosis development is transforming growth factor beta (TGFß), a pleiotropic cytokine associated with the progression of hepatocarcinogenesis. However, the understanding of the molecular events that direct the development of steatosis into steatohepatitis and liver fibrosis remains incomplete. Our study revealed that both prolonged exposure of hepatocarcinoma cells to fatty acids in vitro and high-fat diet in mice (20 weeks) result in inflammation. Prolonged treatment with fatty acids increased the levels of TGFß, MMP9, and ß-catenin, important EMT inducers. Moreover, the livers of mice fed a high-fat diet exhibited features of liver fibrosis with increased TGFß and IL-1 levels. Increased expression of IL-1 correlated with a decrease in monocyte chemoattractant protein-induced protein 1 (MCPIP1), a negative regulator of the inflammatory response that regulates the stability of proinflammatory transcripts encoding IL-1. Our study showed that a high-fat diet induced EMT by increasing the levels of EMT-activating transcription factors, including Zeb1, Zeb2, and Snail and changed the protein profile to a profile characteristic of the mesenchymal phenotype.

Deletion of Mcpip1 in Mcpip1fl/flAlbCre mice recapitulates the phenotype of human primary biliary cholangitis.

Primary biliary cholangitis (PBC) is an autoimmune disease characterized by progressive destruction of the intrahepatic bile ducts. The immunopathology of PBC involves excessive inflammation; therefore, negative regulators of inflammatory response, such as Monocyte Chemoattractant Protein-1-Induced Protein-1 (MCPIP1) may play important roles in the development of PBC. The aim of this work was to verify whether Mcpip1 expression protects against development of PBC. Genetic deletion of Zc3h12a was used to characterize the role of Mcpip1 in the pathogenesis of PBC in 6-52-week-old mice. We found that Mcpip1 deficiency in the liver (Mcpip1fl/flAlbCre) recapitulates most of the features of human PBC, in contrast to mice with Mcpip1 deficiency in myeloid cells (Mcpip1fl/flLysMCre mice), which present with robust myeloid cell-driven systemic inflammation. In Mcpip1fl/flAlbCre livers, intrahepatic bile ducts displayed proliferative changes with inflammatory infiltration, bile duct destruction, and fibrosis leading to cholestasis. In plasma, increased concentrations of IgG, IgM, and AMA autoantibodies (anti-PDC-E2) were detected. Interestingly, the phenotype of Mcpip1fl/flAlbCre mice was robust in 6-week-old, but milder in 12-24-week-old mice. Hepatic transcriptome analysis of 6-week-old and 24-week-old Mcpip1fl/flAlbCre mice showed 812 and 8 differentially expressed genes, respectively, compared with age-matched control mice, and revealed a distinct set of genes compared to those previously associated with development of PBC. In conclusion, Mcpip1fl/flAlbCre mice display early postnatal phenotype that recapitulates most of the features of human PBC.

New therapeutic strategies in nonalcoholic fatty liver disease: a focus on promising drugs for nonalcoholic steatohepatitis.

The prevalence of nonalcoholic fatty liver disease (NAFLD) is increasing worldwide. Globally, it is currently the most common liver disease and is estimated to affect up to 25% of the population. In the first stage, NAFLD is characterized by simple hepatic steatosis (NAFL, nonalcoholic fatty liver) that might progress to nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis or hepatocellular carcinoma. In this review, we discuss the global burden of NAFLD, together with future perspectives on how this epidemic could be restrained. There is also an urgent need for the development of new medical strategies for NAFLD patients. We aim to present the beneficial effects of life-style modifications that should be advised to both non-obese and obese NAFLD patients. Since there are currently no medications directly used for the treatment of more advanced NAFLD stages, the central part of this review summarizes ongoing and recently completed clinical trials testing promising drugs for NASH resolution. The marketing of new therapeutic agents would greatly increase the odds of reducing the global burden of NAFLD.

Integrative genomics reveal a role for MCPIP1 in adipogenesis and adipocyte metabolism.

Obesity is considered a serious chronic disease, associated with an increased risk of developing cardiovascular diseases, non-alcoholic fatty liver disease and type 2 diabetes. Monocyte chemoattractant protein-1-induced protein-1 (MCPIP1) is an RNase decreasing stability of transcripts coding for inflammation-related proteins. In addition, MCPIP1 plays an important role in the regulation of adipogenesis in vitro by reducing the expression of key transcription factors, including C/EBPß. To elucidate the role of MCPIP1 in adipocyte biology, we performed RNA-Seq and proteome analysis in 3T3-L1 adipocytes overexpressing wild-type (WTMCPIP1) and the mutant form of MCPIP1 protein (D141NMCPIP1). Our RNA-Seq analysis followed by confirmatory Q-RT-PCR revealed that elevated MCPIP1 levels in 3T3-L1 adipocytes upregulated transcripts encoding proteins involved in signal transmission and cellular remodeling and downregulated transcripts of factors involved in metabolism. These data are consistent with our proteomic analysis, which showed that MCPIP1 expressing adipocytes exhibit upregulation of proteins involved in cellular organization and movement and decreased levels of proteins involved in lipid and carbohydrate metabolism. Moreover, MCPIP1 adipocytes are characterized by decreased level of insulin receptor, reduced insulin-induced Akt phosphorylation, as well as depleted Glut4 level and impaired glucose uptake. Overexpression of Glut4 in 3T3-L1 cells expressed WTMCPIP1 rescued adipogenesis. Interestingly, we found decreased level of MCPIP1 along with an increase in body mass index in subcutaneous adipose tissue. The presented data show a novel role of MCPIP1 in modulating insulin sensitivity in adipocytes. Overall, our findings demonstrate that MCPIP1 is an important regulator of adipogenesis and adipocyte metabolism.

RNase MCPIP1 regulates hepatic peroxisome proliferator-activated receptor gamma via TXNIP/PGC-1alpha pathway.

Monocyte chemoattractant protein-1-induced protein-1 (MCPIP1) acts as an endonuclease that degrades selected mRNAs, viral RNAs and pre-miRNAs. MCPIP1 inhibits adipogenesis by degradation of C/EBPß mRNA and adipogenesis-related miRNA, however its role in the regulation of hepatic lipid homeostasis is unknown. In this study, we investigated the role of MCPIP1 in the regulation of lipid metabolism in hepatocytes. C57BL/6 mice were fed a high-fat diet (HFD) for 2-20 weeks and next primary hepatocytes and adipose tissue were isolated. For in vitro experiments we used murine primary hepatocytes, control HepG2 cells and HepG2 with overexpressed or silenced MCPIP1. We found that Mcpip1 levels were lower in primary hepatocytes isolated from HFD-fed mice than in control cells starting at 4 weeks of a HFD. Level of Mcpip1 was also depleted in visceral fat isolated from obese and glucose-intolerant mice characterized by fatty liver disease. We showed that MCPIP1 overexpression in HepG2 cells treated with oleate induces the level and activity of peroxisome proliferator-activated receptor γ (PPARγ). This phenotype was reverted upon silencing of MCPIP1 in HepG2 cells and in primary hepatocytes lacking Mcpip1 protein. MCPIP1 activated the PPARγ transcription factor via the thioredoxin-interacting protein (TXNIP)/peroxisome proliferator-activated receptor γ coactivator 1- α (PGC-1α) pathway. MCPIP1 contributes to lipid metabolism in hepatocytes by regulating the TXNIP/PGC-1α/PPARγ pathway.

A Novel Monoallelic Nonsense Mutation in the NFKB2 Gene Does Not Cause a Clinical Manifestation.

NF-κB signaling, acting through NFKB1 dependent canonical and NFKB2 dependent non-canonical pathways plays a critical role in inflammatory and immune responses. Recent studies have associated mutations in these two genes with a common variable immunodeficiency (CVID). While evaluating a female patient seeking a diagnosis explaining her recurrent infections, we found a novel heterozygous c.1831C > T (p.Arg611∗) nonsense mutation in the NFKB2 gene which introduces a Stop codon in the ankyrin repeat domain of p100. Whole exome sequencing (WES) analysis, followed by Sanger sequencing, identified this previously unknown mutation in two other family members. Penetrance of the c.1831C > T variant was assessed by flow-cytometry and protein expression in peripheral blood mononuclear cells (PBMC); whereas, activation of the NF-κB2 signaling pathway was examined through immunoblotting and real-time PCR. Heterozygous c.1831C > T variant led to the expansion of lymphocyte B subpopulations with concomitant reduction of plasmablasts, low IgG levels, and accumulation of p52 in PBMC. On the other hand, tested subjects had normal levels of IgM, IgA, IgE and no impairment in lymphocytes proliferation. Although evaluated patients did not fulfill all clinical features of CVID, their health should be monitored in the future for possible late manifestation of the disease. In conclusion, we showed that NFKB2 haplodeficiency caused by c.1831C > T nonsense mutation is asymptomatic, possibly due to the compensatory mechanisms and allele redundancy.