Tissue specific expression of argininosuccinate synthase, a component of the citrulline‐NO cycle, is mediated through differential methylation and acetylation of the promoter

The enzymes argininosuccinate synthase (AS) and argininosuccinate lyase (AL) are components of the urea cycle and are thus highly expressed in the liver. However, these enzymes are found in low levels in many other tissues, including the endothelium. It was the discovery of nitric oxide (NO) that led to the understanding that AS and AL are components of the citrulline‐NO cycle. AS and AL effectively recycle citrulline to arginine, which is then used by endothelial nitric oxide synthase (eNOS) to produce NO and citrulline. AS, which is the rate‐limiting step in the conversion of citrulline to arginine, plays an important role in endothelial NO production and cell viability. This report provides evidence for a differential, tissue‐specific regulation of AS in the endothelium versus the liver. The AS promoter is GC rich (72%) and contains a large number of CpG repeats in the proximal promoter. Endothelial and liver cells were treated with 5‐Aza‐dC, a demethylating agent, and Trichostatin A, an HDAC inhibitor, to investigate the role of methylation and acetylation in the expression of AS in these tissues. Western blot analysis showed that both agents induce the expression of AS in the endothelium, while they had no effect in the liver. Reporter gene analysis demonstrated that methylation of the AS proximal promoter results in 90% suppression of promoter activity. Overall, the results of this study suggest that differential AS promoter methylation and acetylation play a role in tissue‐specific expression. (Supported by AHA Grant 0455228B)