Poster Presentation Annual Meetings of the Endocrine Society of Australia and Society for Reproductive Biology and Australia and New Zealand Bone and Mineral Society 2016

IL-6/STAT3 pathway is critically involved in vascular calcification via histone modification of the RUNX2 promoter in human vascular smooth muscle cells (#319)

Akira Kurozumi 1 , Kazuhisa Nakano 1 , Kaoru Yamagata 1 , Yosuke Okada 1 , Shingo Nakayamada 1 , Yoshiya Tanaka 1
  1. First Department of Internal Medicine, School of Medicine, University of Occupational and Environmental Health, Japan, Kitakyushu, FUKUOKA, Japan

【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.