【Objective】Vascular medial calcification is often complicated in CKD, diabetes and have been known as an independent risk factor of cardiovascular events. It was reported that concentration of IL-6 rise as renal function worsen and the group of high IL-6 concentration have more cardiovascular death. However, the induction mechanism of vascular calcification by inflammation is still unclear. We assessed the molecular effects of IL-6 on vascular smooth muscle cell (VSMC) calcification.
【Methods】VSMCs from human aorta cultured in osteoblast induction medium (OIM) and stimulated by pro-inflammatory cytokines, such as IL-6/sIL-6R (100ng/ml), TNF-α (10ng/ml) and IL-1β (2ng/ml). Expression of mRNA and protein was determined by qPCR and WB, respectively. Cell calcification was evaluated by Alizarin Red S staining. Histone modification of RUNX2 promoter was determined by ChIP-PCR.
【Results】IL-6 caused the greatest induction in calcification of VSMCs. Stimulation with IL-6 for 72h increased mRNA expression of RUNX2 and ALP but decreased mRNA expression of SM-MHC. IL-6 also increased p-STAT3 in VSMCs. STAT3-knockdown with siRNA inhibited both IL-6-induced calcification and RUNX2 expression. To elucidate the relationship between histone modification and STAT3-dependent transcription of RUNX2, we next examined the level of histone modification on the RUNX2 promoter after stimulation with IL-6 for 20 minutes. The level of H3K9ac, H3K14ac and H3K4me3 were similar, while the level of H3K9me3, a repressive mark, strongly decreased in VSMCs treated with IL-6. Hisotone demethylase of H3K9me3, JMJD 2A, 2B, 2C and 2D were present in VSMCs by qPCR, but only JMJD2B increased on the RUNX2 promoter in VSMCs treated with IL-6.
【Conclusion】Our findings indicate that IL-6 was a strong inducer of vascular calcification and that p-STAT3 was essential for IL-6-induced calcification and demethylation of H3K9me3 by JMJD2B at RUNX2 promoter was important for IL-6-induced activation of RUNX2.