Homocysteine induces monocyte chemoattractant protein-1 expression in hepatocytes mediated via activator protein-1 activation
ABSTRACT Hyperhomocysteinemia is characterized by abnormally high concentrations of homocysteine (Hcy) in the plasma. It is a metabolic disorder associated with dysfunction of several organs such as atherosclerosis, altered lipid metabolism, and liver injury. In this study we investigated the effect of Hcy on transcriptional regulation of monocyte chemoattractant protein-1 (MCP-1), a potent chemokine, expression in hepatocytes. Hyperhomocysteinemia was induced in rats by a high-methionine diet for 4 weeks. MCP-1 mRNA and protein levels were significantly elevated in the liver tissue homogenate and in hepatocytes of hyperhomocysteinemic rats. The role of transcription factors in MCP-1 expression was examined by electrophoretic mobility shift assay. Activation of activator protein (AP)-1 but not nuclear factor kappaB was detected in the liver tissue of those rats. Incubation of rat hepatocytes with Hcy (50-200 microm) caused a significant increase in AP-1 activation followed by an increase in intracellular MCP-1 mRNA expression and an elevation of MCP-1 protein secreted into the culture medium. Hcy markedly increased the DNA binding activity of human recombinant AP-1 (c-Fos and c-Jun proteins). The presence of a sulfhydryl group in Hcy was essential for Hcy-induced AP-1 activation. When hepatocytes were transfected with decoy AP-1 oligodeoxynucleotide to inhibit AP-1 activation, Hcy-induced MCP-1 mRNA expression was abolished. Further analysis revealed that increased hepatic MCP-1 expression was positively correlated with the serum MCP-1 level. These results suggest that Hcy-induced MCP-1 expression in the liver is mediated via AP-1 activation, which may contribute to chronic inflammation associated with hyperhomocysteinemia.
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ABSTRACT: Hepatic steatosis is a clinical feature observed in severe hyperhomocysteinemic patients. In mice, cystathionine beta synthase (CBS) deficiency, the most common cause of severe hyperhomocysteinemia, is also associated with steatosis, fibrosis and inflammation. Proinflammatory cytokines usually induce apoptosis. However, hyperhomocysteinemia does not increase apoptosis in liver of CBS-deficient mice compared to wild type mice. The aim of the study was to analyze the activation state of the NF-kappaB pathway in liver of CBS-deficient mice and to investigate its possible involvement in anti-apoptotic signals. We analyzed the level of I kappaB alpha in liver of CBS-deficient mice. A co-culture of primary hepatocytes and Kupffer cells was also used in order to investigate how I kappaB alpha degradation occurs in response to homocysteine. We found lower I kappaB alpha level not only in liver of CBS-deficient mice but also in hepatocyte/Kupffer cell co-culture. The homocysteine-mediated I kappaB alpha enhanced proteolysis occurred via calcium-dependent calpains, which was supported by an increased level of calpain activity and a reduced expression of calpastatin in liver of CBS-deficient mice. Intraperitoneal administration of the inhibitor PDTC normalized the expression of two genes induced by NF-kappaB activation, heme oxygenase-1 and cellular inhibitor of apoptosis 2. Moreover, PDTC administration induced an increase of caspase-3 activity in liver of CBS-deficient mice. Our results suggest that hyperhomocysteinemia induces calpain-mediated I kappaB alpha degradation which is responsible for anti-apoptotic signals in liver.Molecular Genetics and Metabolism 03/2009; 97(2):114-20. DOI:10.1016/j.ymgme.2009.02.005 · 2.83 Impact Factor
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ABSTRACT: Hyperhomocysteinemia (HHcy) is a risk factor for vascular disease but its underlying molecular pathology is not understood. Homocysteine is metabolically linked to the epigenetic process of DNA methylation. Tissue-specific changes in DNA methylation have been observed in HHcy but little is known about vascular tissue. The objective of this study was to determine if changes in the epigenetic regulation of glucocorticoid receptor (GR) expression (encoded by Nr3c1) in aorta are associated with HHcy. C57BL/6 mice heterozygous for disruption of the cystathionine-β-synthase gene (Cbs+/-) and controls (Cbs+/+) were fed a control or high methionine/low folate (HH) diet to induce HHcy. Cbs+/- and Cbs+/+ fed the HH diet had higher plasma total homocysteine levels (19.9 ± 3.2 and 7.0 ± 0.9 μM, respectively) than Cbs+/+ mice fed the control diet (2.7 ± 0.2 μM), and this was accompanied by lower Nr3c1 mRNA and lower GR protein in aorta. The Nr3c1 gene contains multiple first exons producing heterogeneous RNA transcripts expressed in a tissue-specific manner. We identified expression of two transcripts in aorta. Bisulfite pyrosequencing found increased methylation of the promoter regions for these transcripts at sites corresponding to Sp1 and Nrf1 binding sites. Chromatin immunoprecipitation found lower binding of Nrf1 to the Nr3c1 promoter but higher expression of Nrf1 protein in aorta from mice with HHcy. These findings show methylation and silencing of vascular Nr3c1 expression and suggest a role for epigenetic regulation of gene expression in HHcy.Epigenetics: official journal of the DNA Methylation Society 05/2012; 7(5):514-21. DOI:10.4161/epi.19836 · 5.11 Impact Factor
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ABSTRACT: An elevated level of homocysteine called hyperhomocysteinemia (HHcy) is associated with pathological cardiac remodeling. Hydrogen sulfide (H2S) acts as a cardioprotective gas; however, the mechanism by which H2S mitigates homocysteine-mediated pathological remodeling in cardiomyocytes is unclear. We hypothesized that H2S ameliorates HHcy-mediated hypertrophy by inducing cardioprotective miR-133a in cardiomyocytes. To test the hypothesis, HL1 cardiomyocytes were treated with (1) plain medium (control, CT), (2) 100 µM of homocysteine (Hcy), (3) Hcy with 30 µM of H2S (Hcy + H2S), and (4) H2S for 24 h. The levels of hypertrophy markers: c-fos, atrial natriuretic peptide (ANP), and beta-myosin heavy chain (β-MHC), miR-133a, and its transcriptional inducer myosin enhancer factor-2C (MEF2C) were determined by Western blotting, RT-qPCR, and immunofluorescence. The activity of MEF2C was assessed by co-immunoprecipitation of MEF2C with histone deacetylase-1(HDAC1). Our results show that H2S ameliorates homocysteine-mediated up-regulation of c-fos, ANP, and β-MHC, and down-regulation of MEF2C and miR-133a. HHcy induces the binding of MEF2C with HDAC1, whereas H2S releases MEF2C from MEF2C-HDAC1 complex causing activation of MEF2C. These findings elicit that HHcy induces cardiac hypertrophy by promoting MEF2C-HDAC1 complex formation that inactivates MEF2C causing suppression of anti-hypertrophy miR-133a in cardiomyocytes. H2S mitigates hypertrophy by inducing miR-133a through activation of MEF2C in HHcy cardiomyocytes. To our knowledge, this is a novel mechanism of H2S-mediated activation of MEF2C and induction of miR-133a and inhibition of hypertrophy in HHcy cardiomyocytes.Molecular and Cellular Biochemistry 03/2015; 404(1-2). DOI:10.1007/s11010-015-2383-5 · 2.39 Impact Factor