Abstract
Abstract: :
Purpose: Endothelins (ETs) have inotropic, chemotactic and mitogenic properties. In particular, ET–1 is a potent mitogen for many cells especially when ET–1 levels are elevated under pathophysiological conditions. However, the signal transduction pathway utilized by ET–1 in astrocyte proliferation is not clear. In the present study, the signaling pathways involved in ET–1–mediated astrocyte proliferation were determined. Methods: Specifically, we focused on the involvement of the MAPK, PKC and PI3 kinase signal pathways in cell proliferation after treatment with ET–1 in both U373MG astrocytoma and human optic nerve head astrocytes (hONAs) in culture. A formazan MTT assay was used for quantifying cell proliferation. Phosphorylation of ERK1/2, PKC, and Akt was detected by western blot analysis. A kinase assay was employed to detect the activities and translocation of PKC in membrane and cytosolic fractions. Changes in [Ca2+]i levels were determined by Fura–2 calcium imaging. Results: ET–1 stimulated cell proliferation both in U373MG and in hONA cells in a MAPK and PKC–dependent manner. ET–1 caused a rapid phosphorylation of ERK1/2, which could be blocked by treatment with PD98059 and U0126 (a MEK inhibitor), in both cell types. While PKC inhibitor chelerythrine attenuated ET–1–induced cell proliferation, it was unable to block ET–1–induced ERK phosphorylation. In U373MG cells, ET–1 did not activate PKCs (c– and n–PKCs) and did not elevate [Ca2+]i. U73122 (a phospholipase C inhibitor) also had no effect on ET–1–induced ERK1/2 phosphorylation. FTI–277, a Ras inhibitor, and genistein, a protein tyrosine kinase inhibitor, did not abolish the ERK1/2 phosphorylation. LY294002, a PI3K inhibitor, completely blocked the phosphorylation of Akt and cell proliferation, but did not block the phosphorylation of ERK1/2. Conclusions: ET–1 activates phosphorylation of ERK1/2, which plays an important role in astroglial proliferation for hONAs and U373MG astrocytoma cells. Conventional and novel PKCs appear not to be involved in astrocyte cell proliferation in U373MG cells. The PI3 kinase pathway is involved in signal transduction induced by ET–1, but it does not appear to participate in crosstalk with the MAPK pathway. The mitogenic effects of ET–1 may provide insight into ET’s role in astrogliosis leading to optic nerve damage as seen in glaucoma.
Keywords: astrocytes: optic nerve head • proliferation • signal transduction