© 2000 American Heart Association, Inc.
Vascular Biology |
Inducible Expression of Manganese Superoxide Dismutase by Phorbol 12-Myristate 13-Acetate Is Mediated by Sp1 in Endothelial Cells
From the Second Department of Internal Medicine, Gunma University School of Medicine, Maebashi, Gunma, Japan.
Correspondence to Masahiko Kurabayashi, MD, Second Department of Internal Medicine, Gunma University School of Medicine, 3-39-15, Showa-machi, Maebashi, Gunma, 371-8511, Japan. E-mail mkuraba{at}news.sb.gunma-u.ac.jp
Abstract—The expression of manganese superoxide dismutase (Mn-SOD), an important component of the cellular defense system against oxidative stress, is induced in response to a variety of stimuli, including cytokines and phorbol esters, in endothelial cells. To define the molecular mechanisms regulating the expression of Mn-SOD, we have characterized the promoter of the human Mn-SOD gene. In calf pulmonary artery endothelial cells, phorbol 12-myristate 13-acetate (PMA) gradually increased Mn-SOD mRNA levels, with a peak at 6 to 12 hours after stimulation. The increase in Mn-SOD mRNA was significantly inhibited by a protein kinase C (PKC) inhibitor (calphostin C) but not by a mitogen-activated protein kinase kinase-1 inhibitor (PD98059) or a p38 mitogen-activated protein kinase inhibitor (SB203580). By reporter gene transfection experiments of a series of promoter deletions and site-directed mutation constructs, we found 2 consensus Sp1 binding sequences located at -97 and at -77 to play an important role in PMA-induced Mn-SOD transcription. Electrophoretic gel mobility shift assays have indicated that this sequence serves as an Sp1 binding site. Northern and Western blot analysis has revealed that PMA-induced promoter activity of Mn-SOD correlates with an increased expression of Sp1. Nuclear proteins from PMA-treated calf pulmonary artery endothelial cells displayed an increased DNA binding to the Sp1 site. Furthermore, the Mn-SOD promoter was activated either by overexpression of Sp1 or the constitutively activated form of PKCß in an Sp1 site–dependent manner. These results suggest that PMA stimulates transcription of the Mn-SOD gene through an increase in Sp1 expression and thus implicate Sp1 as an effector mediating the PKC-signaling pathway elicited by extracellular signals.
Key Words: manganese superoxide dismutase • protein kinase C • Sp1 • endothelial cells
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