Role of interleukin 6 in osteogenic transition and calcification of human umbilical artery smooth muscle cells in vitro and the possible cell signal transduction way / 中华肾脏病杂志

ABSTRACT

Objective To investigate the role of recombinant human interleukin 6 (rhlL-6) in calcification and osteogenic transition of cultured human umbilical artery smooth muscle cells (HUASMC), and the possible cell signal transduction way. Methods HUASMCs were isolated by the explant method. HUASMCs were treated with (treatment groups) or without (control group) rhIL-6. Alizarin Red S stain was applied for calcium deposition in extracellular matrix of control ceils and the cells treated with rhIL-6 50 μg/L at day 12. Calcium concentration in cell layer of control group and treatment group (treated with rhIL-6 10 μg/L and 50 μg/L, respectively) was determined calorimetrically by the o-cresolphthalein complexone method at day 3, 6, 9 and 12, and corrected by total cell proteins. The mRNA expressions of bone-specific alkaline phosphatase (BAP), osteopontin (OPN), bone morphogenetic protein-2 (BMP2) and osteoprotegerin (OPG) were estimated by real-time PCR in 12, 24 and 72 hours. OPN, BMP2 and OPG expressions were assessed by Western blotting and the BAP concentration at the same time was checked by fluorometry method . Electrophoretie mobility shift assays (EMSA) was used to detect the binding activity of transcription factor Cbfα1 with or without inhibitors of p38-MAPK (SB203580) and PKC (DHC) after 6 hours stimulation by rhIL-6 10 μg/L. Results rhIL-6 induced a positive Alizarin Red S stain and a time-dose-dependent increasing of cell layer calcium deposition.Compared with control group, rhIL-6 10 μg/L enhanced gene expression and protein levels of BAP and BMP2 at the early time (12 and 24 hours), and of OPN and OPG at later hours (24 and 72 hours). RhIL-6 still induced an increasing of binding activity of Cbfα1, which could be partially blocked by DHC but not SB203580. Conclusions rhIL-6 induces HUASMCs calcification and osteogenie transition in vitro, which may be one of the mechanism involved in IL-6 associated vascular calcification as observed in clinical studies. The role of IL-6 in HUASMCs may partially achieved through the PKC cell signal transduction way.