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 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 PKCbeta 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.
Tanaka
T
T
Second Department of Internal Medicine, Gunma University School of Medicine, Maebashi, Gunma, Japan.
Kurabayashi
M
M
Aihara
Y
Y
Ohyama
Y
Y
Nagai
R
R
D000818:Animals; D001483:Base Sequence / Q000235:genetics; D002417:Cattle; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology*; D004789:Enzyme Activation / Q000502:physiology; D004790:Enzyme Induction; D006801:Humans; D009154:Mutation / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000235:genetics; D011493:Protein Kinase C / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects
D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D013482:Superoxide Dismutase; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate