Background and Aims: Various metabolites of tryptophan participate in diverse physiological functions. We have shown that tryptophan metabolite 5-methoxytryptophan (5-MTP) reduces inflammatory cytokine-induced p38 MAPK activation in vascular smooth muscle cells (VSMCs) and protects against stenosis and restenosis in animal injury models. As such, it is important to understand the synthetic enzymes for 5-MTP. Hydroxyindole O-methyltransferase (HIOMT) is likely to be a key enzyme for 5-MTP production. Our goal is to investigate the role of HIOMT in 5-MTP synthesis and function in VSMCs. Methods: Human HIOMT has 3 isoforms, of 373, 345, and 298 amino acids, respectively. Mouse HIOMT has one isoform of 387 amino acids; however, there are two mutations, R78G and R242C, in C57BL/6 strain compared with that of C3H strain. To clarify functions of these isoforms, we generated expression plasmids of various isoforms, transfected into VSMCs, stimulated with inflammatory cytokines, and examined p38 MAPK phosphorylation levels. Results: Transfection experiments revealed that different inflammatory cytokines activated p38 phosphorylation in VSMCs. Interestingly, compared with vector control, hHIOMT373 but not hHIOMT298 isoform reduced p38 phosphorylation. Conditioned medium from hHIOMT373-transfected VSMCs also showed a similar effect. Intriguingly, mHIOMT-C3H but not mHIOMT-C57 repressed p38 activation to a similar degree as hHIOMT373 and 5-MTP, suggesting HIOMT might be defective in C57BL/6 strain. Double immunofluorescence staining showed colocalized staining of 5-MTP and hHIOMT in hHIOMT373-transfected VSMCs. Conclusions: Our results indicate that hHIOMT373 and mHIOMT-C3H rather than hHIOMT298 or mHIOMT-C57 are responsible for 5-MTP production and could functionally substitute 5-MTP for exerting anti-inflammatory effect and vascular protection in VSMCs.
