Epigenetic regulation of IPF fibroblast phenotype by glutaminolysis.

OBJECTIVE: Excessive extra-cellular-matrix production and uncontrolled proliferation of the fibroblasts are characteristics of many fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). The fibroblasts have enhanced glutaminolysis with up-regulated glutaminase, GLS1, which converts glutamine to glutamate. Here, we investigated the role of glutaminolysis and glutaminolysis-derived metabolite α-ketoglutarate (α-KG) on IPF fibroblast phenotype and gene expression. METHODS: Reduced glutamine conditions were carried out either using glutamine-free culture medium or silencing the expression of GLS1 with siRNA, with or without α-KG compensation. Cell phenotype has been characterized under these different conditions, and gene expression profile was examined by RNA-Seq. Specific profibrotic genes (Col3A1 and PLK1) expression were examined by real-time PCR and western blots. The levels of repressive histone H3K27me3, which demethylase activity is affected by glutaminolysis, were examined and H3K27me3 association with promoter region of Col3A1 and PLK1 were checked by ChIP assays. Effects of reduced glutaminolysis on fibrosis markers were checked in an animal model of lung fibrosis. RESULTS: The lack of glutamine in the culture medium alters the profibrotic phenotype of activated fibroblasts. The addition of exogenous and glutaminolysis-derived metabolite α-KG to glutamine-free media barely restores the pro-fibrotic phenotype of activated fibroblasts. Many genes are down-regulated in glutamine-free medium, α-KG supplementation only rescues a limited number of genes. As α-KG is a cofactor for histone demethylases of H3K27me3, the reduced glutaminolysis alters H3K27me3 levels, and enriches H3K27me3 association with Col3A1 and PLK1 promoter region. Adding α-KG in glutamine-free medium depleted H3K27me3 association with Col3A1 promoter region but not that of PLK1. In a murine model of lung fibrosis, mice with reduced glutaminolysis showed markedly reduced fibrotic markers. CONCLUSIONS: This study indicates that glutamine is critical for supporting pro-fibrotic fibroblast phenotype in lung fibrosis, partially through α-KG-dependent and -independent mechanisms, and supports targeting fibroblast metabolism as a therapeutic method for fibrotic diseases.

MeCP2 epigenetically regulates alpha-smooth muscle actin in human lung fibroblasts.

BACKGROUND: A critical feature for fibroblasts differentiation into myofibroblasts is the expression of alpha-smooth muscle actin (α-SMA) during the tissue injury and repair process. The epigenetic mechanism, DNA methylation, is involved in regulating α-SMA expression. It is not clear how methyl-CpG-binding protein 2 (MeCP2) interacts with CpG-rich region in α-SMA, and if the CpG methylation status would affect MeCP2 binding and regulation of α-SMA expression. METHODS: The association of MeCP2 with α-SMA CpG rich region were examined by chromatin immunoprecipitation (ChIP) assays in primary fibroblasts from idiopathic pulmonary fibrosis (IPF) and non-IPF control individuals, and in the lung fibroblasts treated with profibrotic cytokine transforming growth factor ß1 (TGF-ß1). The regulation of α-SMA by MeCP2 was examined by knocking down MeCP2 with small interfering RNA (siRNA). To explore the effects of the DNA methylation status of the CpG rich region on α-SMA expression, the cells were treated with DNA methyltransferase inhibitor, 5'-azacytidine (5'-aza). The expression of α-SMA was examined by Western blot and quantitative polymerase chain reaction, the association with MeCP2 was assessed by ChIP assays, and the methylation status was checked by bisulfate sequencing. RESULTS: The human lung fibroblasts with increased α-SMA showed an enriched association of MeCP2, while knockdown MeCP2 by siRNA reduced α-SMA upregulation by TGF-ß1. The 5'-Aza-treated cells have decreased α-SMA expression with reduced MeCP2 association. However, bisulfite sequencing revealed that most CpG sites are unmethylated despite the different expression levels of α-SMA after being treated by TGF-ß1 or 5'-aza. CONCLUSION: Our data indicate that the methyl-binding protein MeCP2 is critical for α-SMA expression in human lung myofibroblast, and the DNA methylation status at the CpG rich region of α-SMA is not a determinative factor for its inducible expression.

Diagnostic value of serum PIVKA-II levels for BCLC early hepatocellular carcinoma and correlation with HBV DNA.

BACKGROUND: It is reported that prothrombin induced by vitamin K absence-II (PIVKA-II) has a better performance of diagnosis for HCC, and has also been known to be an independent risk factor for vascular invasion. Few studies study the relationship between PIVKA-II and HBV DNA. OBJECTIVE: To determine the clinical value of serum Prothrombin induced by vitamin K absence-II (PIVKA-II) in early hepatocellular carcinoma (HCC), and to explore its relationship with vascular invasion and HBV DNA. METHODS: In a Chinese cohort, we conducted a case-control study to compare the performances of a-fetoprotein (AFP) and PIVKA-II serum levels for diagnosis of HCC and early HCC. Fifty one healthy controls, 37 chronic hepatitis patients, 43 cirrhotic patients and 143 HCC cases of which 48 (33.57%) had early stage HCC (n= 19 very early, n= 29 early) were enrolled. We explored the correlation between PIVKA-II serum level and several pathological features such as vascular invasion. The serum levels of and AFP were measured by chemiluminescence assay (CLIA) and electrochemiluminescence assay (ECLA). RESULTS: The serum levels of both PIVKA-II and AFP in HCC group were higher than that in chronic hepatitis, cirrhosis and healthy control groups. The sensitivity, specificity, positive predictive value, negative predictive value and kappa of PIVKA-II were higher than AFP in the diagnosis of HCC. Serum PIVKA-II level was correlated with tumor size, tumor cell differentiation and BCLC staging (P< 0.05). For the diagnosis of early HCC, the combination of PIVKA-II (AUC 0.812; 95% CI, 0.702-0.894) and AFP (0.797; 95% CI, 0.686-0.883) slightly improve the diagnostic performance for early HCC(AUC 0.849; 95% CI, 0.745-0.923). PIVKA-II > 166 mAU/ml is an independent risk factor for vascular invasion. The serum HBV DNA level in cirrhosis and HCC patients was significantly higher than in chronic hepatitis patients. We detected a negative association between serum PIVKA-II and serum HBV DNA levels. CONCLUSIONS: PIVKA-II was more efficient than AFP for the diagnosis of early HCC and has no correlation with serum HBV DNA levels.