Inducible expression of manganese superoxide dismutase by phorbol 12-myristate 13-acetate is mediated by Sp1 in endothelial cells

The expression of manganese superoxide dismutase (Mn-SOD), an important com ponent 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 e xpression 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 1 2 hours after stimulation. The increase in Mn-SOD mRNA was significantly in hibited by a protein kinase C (PKC) inhibitor (calphostin C) but not by a m itogren-activated protein kinase kinase-1 inhibitor (PD98059) or a p38 mito gen-activated protein kinase inhibitor (SB203580). By reporter gene transfe ction experiments of a series of promoter deletions and site-directed mutat ion constructs, we found 2 consensus Sp1 binding sequences located at -97 a nd at -77 to play an important role in PMA-induced Mn-SOD transcription. El ectrophoretic gel mobility shift assays have indicated that this sequence s erves as an Sp1 binding site. Northern and Western blot analysis has reveal ed that PMA-induced promoter activity of Mn-SOD correlates with an increase d expression of Sp1. Nuclear proteins from PMA-treated calf pulmonary arter y endothelial cells displayed an increased DNA binding to the Sp1 site. Fur thermore, the Mn-SOD promoter was activated either by overexpression of Sp1 or the constitutively activated form of PKC beta in an Spl site-dependent manner. These results suggest that PMA stimulates transcription of the Mn-S OD gene through an increase in Sp1 expression and thus implicate Sp1 as an effector mediating the PKC-signaling pathway elicited by extracellular sign als.