Abstract
Elevated levels of circulating asymmetric and symmetric dimethylarginines (ADMA and SDMA) predict and potentially contribute to end organ damage in cardiovascular diseases. Alanine-glyoxylate aminotransferase 2 (AGXT2) regulates systemic levels of ADMA and SDMA, and also of beta-aminoisobutyric acid (BAIB)-a modulator of lipid metabolism. We identified a putative binding site for hepatic nuclear factor 4 α (HNF4α) in AGXT2 promoter sequence. In a luciferase reporter assay we found a 75% decrease in activity of Agxt2 core promoter after disruption of the HNF4α binding site. Direct binding of HNF4α to Agxt2 promoter was confirmed by chromatin immunoprecipitation assay. siRNA-mediated knockdown of Hnf4a led to an almost 50% reduction in Agxt2 mRNA levels in Hepa 1–6 cells. Liver-specific Hnf4a knockout mice exhibited a 90% decrease in liver Agxt2 expression and activity, and elevated plasma levels of ADMA, SDMA and BAIB, compared to wild-type littermates. Thus we identified HNF4α as a major regulator of Agxt2 expression. Considering a strong association between human HNF4A polymorphisms and increased risk of type 2 diabetes our current findings suggest that downregulation of AGXT2 and subsequent impairment in metabolism of dimethylarginines and BAIB caused by HNF4α deficiency might contribute to development of cardiovascular complications in diabetic patients.
Highlights
Endogenous methylated derivatives of L-arginine, such as asymmetric (ADMA) and symmetric (SDMA) dimethylarginines, have been widely studied as markers and potential mediators of cardiovascular diseases[1,2]
The only highly conserved region found in mammals within the predicted AGXT2 core promoter region was located −89 bp to −77 bp upstream relative to the murine Agxt[2] gene translation start site
Analysis of the publicly accessible microarray data obtained from 22 C57BL/6 wild type mouse tissues (Gene Expression Omnibus database, dataset GDS314235,36 demonstrated a strong correlation between Hnf4a and Agxt[2] expression levels (Pearson’s R = 0.736)
Summary
Endogenous methylated derivatives of L-arginine, such as asymmetric (ADMA) and symmetric (SDMA) dimethylarginines, have been widely studied as markers and potential mediators of cardiovascular diseases[1,2]. Agxt[2] knockout mice have elevated plasma levels of both ADMA and SDMA and develop hypertension[14]. AGXT2 polymorphisms lead to increased BAIB levels in urine (hyper-β-aminoisobutyric aciduria), presumably the most common autosomal recessive metabolic trait in humans[17] (OMIM 210100). Despite the growing evidence that AGXT2 and its substrates may play an important role in the pathogenesis of cardiovascular and metabolic diseases, the mechanisms of regulation of AGXT2 expression and activity are still unknown. Battle and coauthors demonstrated downregulation of Agxt[2] in the liver of Hnf4a conditional knockout mice, which suggest an important role of HNF4a in regulation of AGXT2 expression[23]. The goal of the current study was to test the hypothesis that HNF4αis a major regulator of AGXT2 expression in vivo
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