20‐HETE Inhibited DNA synthesis in Vascular Smooth Muscle Cells. Role of Transforming Growth Factor β 1

Abstract

20-hydroxyeicosatetraenoic acid (20-HETE), an arachidonate metabolite of CYP4A, has been reported to be a mitogen in vascular smooth muscle cells (VSMC). However, we found that 20-HETE inhibited DNA synthesis in R22D cells, a clonal VSMC from neonatal rat. Because long chain polyunsaturated fatty acid could modulate cell growth by releasing transforming growth factor β1 (TGF-£]1), we asked whether TGF-β1 mediates the effect of 20-HETE on DNA synthesis in R22D cells. Incubation of cultured R22D cells with 20-HETE for 24 hr inhibited the [3H]-thymidine incorporation in a concentration-dependent manner without altering cell morphology. At 5 μM, 20-HETE reduced [3H]-thymidine incorporation by 30–40%; anti-TGF-β neutralizing antibody, but not nonspecific IgG, completely reversed the effect of 20-HETE on [3H]-thymidine incorporation. Incubation of 20-HETE for 12 and 24 hr increased TGF-β1 release by 141 ± 27 and 218 ± 64 pg/ml, respectively, as measured by ELISA. However, 20-HETE exhibited no effect on steady state mRNA level of TGF-β1. Exogenous addition of authentic TGF-β1 (200 pg/ml) reduced [3H]-thymidine incorporation by 36%. In addition, western blot analysis indicated that expression of cyclin D1, a downstream effector of TGF-β1, was attenuated by 20-HETE after incubation for 24 hr. These results suggested that inhibitory effect of 20-HETE on DNA synthesis may be mediated by TGF-β1 in R22D cells.