Homocysteine Induces Monocyte Chemoattractant Protein-1 Expression in Hepatocytes Mediated via Activator Protein-1 Activation

Departments of Animal Science and Physiology, University of Manitoba, Canadian Centre for Agri-Food Research in Health and Medicine, St. Boniface Hospital Research Centre, Winnipeg, Canada.
Journal of Biological Chemistry (Impact Factor: 4.57). 02/2008; 283(3):1282-92. DOI: 10.1074/jbc.M707886200
Source: PubMed


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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    • "Electrophoretic mobility shift assay (EMSA) To determine whether NADPH oxidase subunit expression was regulated by NF-kB, the binding activity of NF-kB with DNA was measured by EMSA. In brief, nuclear proteins were prepared from mouse liver as previously described (Woo et al. 2008; Wu et al. 2009). Nuclear proteins (10 µg) were incubated with excess 32 P-end-labeled oligonucleotides containing a consensus sequence specific for the NF-kB DNA binding site (5′-AGTTGAGGGGACTTTCCC AGGC- 3′) (Promega, Madison, Wisconsin, USA). "
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    ABSTRACT: Diets high in saturated fat and cholesterol facilitate weight gain, a predisposing factor that contributes to the onset of obesity and metabolic disorders. Hepatic oxidative stress is commonly reported in various animal models of obesity and has been associated with enhanced expression of NADPH oxidase. We have previously reported several antioxidant mechanisms through which folic acid confers protection during hyperhomocysteinemia-induced oxidative stress. The objective of the present study was to investigate whether folic acid supplementation ameliorates high-fat diet induced oxidative stress in the liver, and to identify the underlying mechanisms. Male C57BL/6J mice were fed a control diet, a high-fat diet, or a high-fat diet supplemented with folic acid for 12 weeks. A high-fat diet led to increased body mass, hepatic lipid peroxidation, and liver injury. There was a significant increase in hepatic NADPH oxidase activity, which was associated with enhanced expression of several NADPH-oxidase subunits. Folic acid supplementation had a protective effect against high-fat diet induced hepatic oxidative stress and liver injury. Further analysis revealed that the antioxidant effect of folic acid was attributed, in part, to transcriptional regulation of NADPH oxidase. These results suggested that folic acid supplementation may be hepatoprotective from liver injury associated with a high-fat diet.
    Preview · Article · Feb 2012 · Canadian Journal of Physiology and Pharmacology
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    • "For another cytokine, MCP-1 there was a 20 percent increase in protein levels, but this was not statistically significant. Other studies have demonstrated a 20 to 40 percent increase in MCP-1 by MC [8] and hepatocytes [29] exposed to comparable concentrations of Hcy. Hence, our observations are similar to the aforementioned reports, but in the current study, Hcy-induced MCP-1 changes were not significant. "
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    ABSTRACT: Homocysteine (Hcy) and inflammatory cytokines have been linked to adverse outcomes in persons with cardiovascular and kidney diseases and recent reports suggest that cytokine-mediated inflammatory infiltrates may be an important contributor to the pathogenesis the aforementioned diseases. Although some reports suggest that Hcy directly influences inflammatory cytokine production, this proposition has not been supported by data from other studies. The objective of the current study was to a) utilize an in vitro cellular model to identify cytokines that may be affected by Hcy and b) examine the role of mitogen activated protein kinase (MAPK) and phosphatidyl inositol 3- (PI3) Kinase in Hcy modulated cytokine production. Primary rat glomerular mesangial cells (MC, passage 8 to 15), isolated by standard sieving methodology, were exposed to Hcy (15, 50 or 100 muM) with L-cysteine (L-Cys; 100 muM) serving as a control. An antibody array was used to identify cytokines that were modulated when MCs were exposed to Hcy. Gene expression was assessed by quantitative RT-PCR, while western blotting analysis was used to assess cellular protein levels in the presence and absence of inhibitors of MAPK and PI3 Kinase. Finally, leukocyte adhesion assay was used to examine the effect of Hcy on leukocyte adhesion to glomerular MCs that were maintained in media without, and with, kinase inhibitors. We identified macrophage inflammatory protein 2 (MIP-2) as a key cytokine that manifested increases in both protein and mRNA following exposure of glomerular MC to pathophysiologic Hcy levels (50 muM). Further analyses revealed that Hcy-induced MIP-2 was dependent on activation of p38 MAPK and PI3 kinase. MIP-2 enhanced leukocyte adhesion to MC and this MIP-2-enhanced leukocyte adhesion was also dependent on activation of p38 MAPK and PI3K. Finally, we demonstrate that leukocyte adhesion to MC is specifically inhibited by anit-MIP2 antibody. The data suggest that Hcy participates in inflammatory cytokines production by glomerular MC and that Hcy-induced MIP-2 mediates leukocyte adhesion to MC.
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    • "Moreover, Hwang et al. [34] also found a reduction of IkB of about 25% in kidney of hyperhomocysteinemic rats. However, Woo et al. [37] reported that Hcy was not able to induce NF-jB activation in primary hepatocytes . Here we found that treatment of primary hepatocytes with Hcy did not resulted in IjBa degradation. "
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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.
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