No abstract

Redox regulation of DNA-binding proteins through the reversible oxidation of key cysteine sulfhydryl groups has been demonstrated to occur in vitro for a range of transcription factors. The direct redox regulation of DNA binding has not been described in vivo, possibly because most protein thiol groups are strongly buffered against oxidation by the highly reduced intracellular environment mediated by glutathione, thioredoxin, and associated pathways. For this reason, only accessible protein thiol groups with high thiol-disulfide oxidation potentials are likely to be responsive to intracellular redox changes. In this article, we demonstrate that zinc finger DNA-binding proteins, in particular members of the Sp-1 family, appear to contain such redox-sensitive -SH groups. These proteins displayed a higher sensitivity to redox regulation than other redox-responsive factors both in vitro and in vivo. This effect was reflected in the hyperoxidative repression of transcription from promoters with essential Sp-1 binding sites, including the simian virus 40 early region, glycolytic enzyme, and dihydrofolate reductase genes. Promoter analyses implicated the Sp-1 sites in this repression. Non-Sp-1-dependent redox-regulated genes including metallothionein and heme oxygenase were induced by the same hyperoxic stress. The studies demonstrate that cellular redox changes can directly regulate gene expression in vivo by determining the level of occupancy of strategically positioned GC-binding sites.There is compelling evidence for both direct and indirect pathways for the regulation of gene expression by changes in cellular redox state. Hypoxic and hyperoxic stresses can activate or repress the transcription of certain genes by pathways that probably involve protein kinases (5,16,18,24,28,52,68,70,82,84). The response to severe oxidative stress may involve an additional effect in which redox-sensitive factors can be directly activated or inactivated through the oxidation of sulfhydryl residues. The binding of factors AP-1, Sp-1, Egr-1, NF-B, v-rel, c-myb, E2, IRE-BP, p53, and USF to nucleic acid is reduced or lost when critical cysteine residues are oxidized or alkylated (1, 3, 4, 33-35, 37, 48, 53, 60, 66, 78, 89). HoxB5, a member of the mammalian homeodomain gene family, is an example of a factor that is activated by oxidation (30). In the case of AP-1, a cellular DNA-repair protein that may regulate the redox equilibrium has been described previously (90). Oxidative inactivation of USF has been shown to correlate directly with transcriptional activity in an in vitro assay (60). It has been proposed that the reactive cysteines may constitute redox-sulfhydryl switches which directly regulate gene expression (35,60). In support of this, factor Sp-1 in rat liver appears to become progressively oxidized during aging, resulting in the reversible loss of binding activity by an in vitro assay (3). To date, there have been no reports to demonstrate that oxidizing agents or redox stresses can directly mediate transcription factor bindi...

The transcription rates of glycolytic enzyme genes are coordinately induced when cells are exposed to low oxygen tension. This effect has been described in many cell types and is not restricted to species or phyla. In mammalian cells, there are 11 distinct glycolytic enzymes, at least 9 of which are induced by hypoxia. Recent reports described a role for the hypoxia-inducible factor-1 (HIF-1) in the transcriptional activation of lactate dehydrogenase A, aldolase-A, phosphoglycerate kinase, and enolase-1 genes. It is not known whether the HIF-1 factor acts exclusively to regulate these genes during hypoxia, or how the other genes of the pathway are regulated. In this paper, we describe analyses of the musclespecific pyruvate kinase-M and ␤-enolase promoters that implicate additional mechanisms for the regulation of glycolytic enzyme gene transcription by hypoxia. Transient transcription of a reporter gene directed by either promoter was activated when transfected muscle cells were exposed to hypoxia. Neither of these promoters contain HIF-1 binding sites. Instead, the hypoxia response was localized to a conserved GC-rich element positioned immediately upstream of a GATAA site in the proximal promoter regions of both genes. The GC element was essential for both basal and hypoxia-induced expression and bound the transcription factors Sp1 and Sp3. Hypoxia caused the progressive depletion of Sp3 determined by DNA binding studies and Western analyses, whereas Sp1 protein levels remained unchanged. Overexpression of Sp3 repressed expression of ␤-enolase promoters. It is concluded that hypoxia activates these glycolytic enzyme gene promoters by down-regulating Sp3, thereby removing the associated transcriptional repression.

Abstract-Hearts of wild-type and insulin-like growth factor-1 overexpressing (Igf-1 ϩ/Ϫ ) transgenic mice were subjected to Langendorff perfusions and progressive periods of ischemia followed by reperfusion. Apoptosis was measured by DNA nucleosomal cleavage and a hairpin probe labeling assay to detect single-base overhang. Transgenic hearts subjected to 20 minutes of ischemia and 4 hours of reperfusion (I/R) sustained a rate of apoptosis of 1.8Ϯ0.3% compared with 4.6Ϯ1.1% for wild-type controls (nϭ4; PϽ0.03). Phosphorylation of the protein kinase Akt/protein kinase B was elevated 6.2-fold in transgenic hearts at baseline and increased another 4.4-fold within 10 minutes of reperfusion, remaining elevated for up to 2 hours. I/R activated Akt in wild-type hearts but to a lesser extent (1.6Ϯ0.3-fold). Pretreatment of transgenic hearts with wortmannin immediately before and during ischemia eliminated reperfusionmediated activation of Akt and neutralized the resistance to apoptosis. The stress-activated kinase p38 was also activated during ischemia and reperfusion in both wild-type and transgenic hearts. Perfusion with the p38 inhibitor SB203580 (10 mol/L) blocked both p38 activation and phosphorylation of Akt and differentially modulated apoptosis in wild-type and transgenic hearts. Pretreatment with SB203580 reduced apoptosis in wild-type hearts but increased apoptosis in transgenic hearts. These results demonstrate that Akt phosphorylation during I/R is modulated by IGF-1 and prevents apoptosis in hearts that overexpress the IGF-1 transgene. (Circ Res. 2001;88:609-614.) Key Words: ischemia Ⅲ hypoxia Ⅲ phosphoinositol-3Ј-kinase Ⅲ p38 mitogen-activated protein kinase Ⅲ SB203580 P rotective and antiapoptotic properties of insulin-like growth factor-1 (IGF-1) have been demonstrated in different models of myocardial ischemia and infarction 1-4 as well as in isolated cardiac myocytes subjected to ischemic or oxidative stress. 5,6 IGF-1 stimulates the phosphoinositol-3Ј (PI3)-kinase pathway, producing phosphoinositides that promote activation of the kinase Akt. 7,8 Activated Akt kinase plays a central role in suppressing apoptosis by modulating the activities of Bcl-2 family proteins, 9 caspase 9, 10 and Fas ligand. 11 Transfer of mutationally activated PI3-kinase and Akt genes has been shown to prevent apoptosis of cardiac myocytes in vitro, 6 and activated Akt delivered by an adenovirus vector reduced apoptosis in the intact heart subjected to ischemia and reperfusion (I/R). 4 We previously reported that Igf-1 ϩ/Ϫ transgenic mice overexpressing IGF-1 had reduced rates of necrosis and apoptosis after myocardial infarction caused by coronary artery ligation. 3 The same hearts were also resistant to necrosis but not apoptosis caused by nonocclusive coronary artery constriction. 12 Other work has shown that the endogenous PI3-kinase pathway is activated in the postinfarcted myocardium, where it may contribute to cell survival and hypertrophy. [13][14][15] Regulation of the endogenous PI3-kinase pathway during I/R has not b...