Bone Abstracts (2016) 5 P425 | DOI: 10.1530/boneabs.5.P425

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

Akira Kurozumi, Kazuhisa Nakano, Kaoru Yamagata, Yosuke Okada, Shingo Nakayamada & Yoshiya Tanaka


First Department of Internal Medicine, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan.


Objective: Both inflammation and vascular calcification have been known as independent risk factors of cardiovascular events. However, the induction mechanism of vascular calcification by inflammation is still unclear. We here assessed the molecular effects of pro-inflammatory cytokines 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 (100 ng/ml), TNF-α (10 ng/ml) and IL-1β (2 ng/ml). Expression of mRNA and protein was determined by quantitative real-time PCR and WB, respectively. Cell calcification was evaluated by Alizarin Red S staining on day 21. Histone modification of RUNX2 promoter was determined by ChIP-PCR.

Results: Among pro-inflammatory cytokines, IL-6 caused the greatest induction in calcification of VSMCs. Stimulation with IL-6 for 3 days significantly increased mRNA expression of RUNX2, a master regulator for osteoblast differentiation, and ALP (3–5 and eightfold vs control, respectively) but decreased mRNA expression of SM-MHC (0.2-fold vs control), a SMC differentiation marker. IL-6 also increased phosphorylation of STAT3 and STAT3-binding to the Runx2 promoter region in VSMCs. STAT3-knockdown of VSMC 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 region after stimulation with or without IL-6 for 20 min. The level of H3K9ac, H3K14ac, H3K4me3 and H3K36me3 was similar in both groups, while the level of H3K9me3, a repressive mark, was strongly decreased in VSMCs treated with IL-6. In addition, IL-6-dependent H3K9me3 suppression was canceled by STAT3 siRNA.

Conclusion: Our findings indicate that IL-6 is a strong inducer of vascular calcification and that STAT3 binding and decreased H3K9me3 to Runx2 is associated with IL-6-dependent vascular calcification.

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