Investigaton Of The Effect And Mechanisms Of Urotensin Ii On Promotion Of Osteopontin Expression In Aortic Adventitial Fibroblasts Of Rats

Background Urotensin II (UII) as a new vasoactive peptide, has a wide range of biological functions, such as vasoconstriction, promoting cell proliferation. Recently, we have reported that UII induced migration of adventitial fibroblasts, and found that Osteopontin (OPN) may be involved in the process.Objective To explore the effect of UII in promoting OPN expression of rat aortic adventitial fibroblasts(AFs) , and the possible intracellular signal transduction might be activated.Methods The adventitia from male Sprague–Dawley rats thoracic aorta was isolated and cultured in vitro with DMEM medium containing 20% fetal calf serum to obtain the adventitial fibroblasts(AFs) , and passage 3–5 AFs were used for experiments after incubation with serum-free medium for 24h. Cultured cells were separated into (1)Control group: Cells were cultured in serum-free DMEM and absence of UII; (2)UII group: different concentration(10-10, 10-9, 10-8 and 10-7mol/L) and hours(1h, 3h, 6h, 12h, 24h) of UII stimulation; (3)UII + inhibitors group: Cells were pretreated in DMEM for 0.5 h with UII receptor antagonist SB710411(10-6M), mitogen activated protein kinase(MAPK) inhibitor PD98059 (10-5M), calcineurin inhibitor Cyclosporine A(CSA) (10-5M), Ca2+ channel blocker nicardipine (10-5M), protein kinase C (PKC) inhibitor H7 (10-5M), or Rho protein kinase inhibitor Y-27632 (10-5M) followed by UII (10-8 mol/L). The cells and medium were collected respectively after incubation. The mRNA and protein expression of OPN in rat AFs induced by UII were examined by semi-quantitative reverse transcriptase polymerase chain reaction(RT-PCR) and enzyme-linked immunosorbent assay(ELISA), respectively.Results UII induced OPN expression in rat aortic adventitial fibroblasts. UII incduced a dose-dependent increase in OPN mRNA expression and protein secretion, compare with control group, AFs OPN mRNA was increased by 20.1%(P>0.05), 59.8%(P<0.05), 88.7%(P<0.01) and 68.3%(P<0.05) in 10-10, 10-9, 10-8 and 10-7mol/L UII group, respectively. The OPN protein secretion was increased by 25.6%(P>0.05), 49.8%(P<0.05), 74.9%(P< 0.01) and 24.6%(P>0.05) in 10-10, 10-9, 10-8 and 10-7mol/L UII group, respectively. UII(10-8 mol/L) incduced a time-dependent increase in OPN mRNA expression and protein secretion, compare with control group, AFs OPN mRNA was increased by 111.2%(P<0.01), 253.2%(P<0.01), 119.1%(P<0.01) and 4.9%(P>0.05) in 1h, 3h, 6h, 12h group, respectively. The OPN protein secretion was increased by was increased by 59.4% (P<0.05), 74.9% (P<0.05) and 42.7% (P> 0.05) in 6h, 12h, 24h group , respectively. This effect of UII was inhibited by SB710411, nicardipine, H7, CSA, Y-27632 and PD98059. The expression of OPN mRNA was inhibited by 25.2%, 23.7%, 20.4%, 43.1%, 15.6%, 29.0% (P<0.01), and the OPN protein secretion was inhibited by 29.3%(P<0.05), 52.7%(P<0.01), 24.7%(P<0.05), 46.7%(P<0.01), 38.7%(P<0.01) and 39.7%(P<0.01)compare with control group, respectively .Conclusion UII induced a dose-dependent and time-dependent increase in mRNA and protein expression of OPN in rat AFs. This effect could be inhibited by UII receptor antagonist SB710411, MAPK inhibitor PD98059, calcineurin inhibitor CSA, Ca2+ channel blocker nicardipine, PKC inhibitor H7, or Rho protein kinase inhibitor Y-27632.