Abstract 607: Endothelin-1 Increases ß-Actin Expression During Osteochondrogenesis of Aortic Smooth Muscle Cells from Leptin-Deficient ob/ob Mice

Abstract

β-actin, a highly conserved gene/protein, which is usually not modulated by different stimuli, is routinely used to normalize target gene or protein expression in experimental biology, and so defined endogenous housekeeping gene. However, investigators showed that β-actin expression could change during cellular growth and differentiation. We hypothesized that endothelin 1 (ET-1), a cellular proliferative agonist, increases β-actin expression and stimulates calcification in vascular smooth muscle cells (SMC) from obob mice. Primary aortic SMC passage 4-6 isolated from obese leptin-deficient (obob) and from C57BL/6 (C57) mice were incubated without (Cont) or with ET-1 50nM (ET) for 0-72h. Cell lysates were assessed for β-actin and RUNX2 expression by western blotting and normalized by GAPDH. ObobET SMC increased β-actin expression after 24h, 48h and 72h (1.68±0.06, 1.66±0.05, 1.73±0.06 respectively) vs obobCont (1.01±0.07), p<.05 n=6. However, C57ET did not change β-actin expression (0.91±0.06, 1.04±0.03, 0.99±0.08) after 24h, 48h and 72h respectively vs. C57Cont (0.95 ± 0.04), p=NS n=6. Concomitantly, obobET SMC increased osteochondrogenic differentiation, by modulating RUNX2 (1.23±0.03 vs obobCont 1±0.01, p<.05 n=3) after 48h, which did not occur in C57 (C57ET 1.07±0.07 vs C57Cont 1.02±0.11), p=NS. Furthermore, obobET increased calcification vs obobCont (1.69±0.025 vs 1.03±0.03) after 14 days p<.05, n=3 and C57ET did not calcify (0.97±0.02 vs C57Cont 1±0.05), p=NS. We showed that ET-1 modulates β-actin expression and osteochodrogenenic differentiation only in obob SMC, thus increasing calcification. In conclusion, β-actin is not a consistent endogenous control in ET-1-stimulated SMC from obob mice during osteochondrogenic differentiation.