Abstract-Cyclic GMP, produced in response to nitric oxide and natriuretic peptides, is a key regulator of vascular smooth muscle cell contractility, growth, and differentiation, and is implicated in opposing the pathophysiology of hypertension, cardiac hypertrophy, atherosclerosis, and vascular injury/restenosis. cGMP regulates gene expression both positively and negatively at transcriptional as well as at posttranscriptional levels. cGMP-regulated transcription factors include the cAMP-response element binding protein CREB, the serum response factor SRF, and the nuclear factor of activated T cells NF/AT. cGMP can regulate CREB directly, through phosphorylation by cGMP-dependent protein kinase, or indirectly, through activation of mitogen-activated protein kinase pathways; regulation of SRF and NF/AT by cGMP is indirect, through modulation of RhoA and calcineurin signaling, respectively. Downregulation of the RNA-binding protein HuR by cGMP leads to destabilization of guanylate cyclase mRNA, but this posttranscriptional mechanism may affect many more cGMP-regulated genes. In this review, we discuss the role of cGMP-regulated gene expression in (patho)physiological processes most relevant to the cardiovascular system, such as regulation of vascular tone, cardiac hypertrophy, phenotypic modulation of vascular smooth muscle cells, and regulation of cell proliferation and apoptosis.

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Hydrogen sulfide is a highly toxic gas—second only to carbon monoxide as a cause of inhalational deaths. Its mechanism of toxicity is only partially known, and no specific therapy exists for sulfide poisoning. We show in several cell types, including human inducible pluripotent stem cell (hiPSC)-derived neurons, that sulfide inhibited complex IV of the mitochondrial respiratory chain and induced apoptosis. Sulfide increased hydroxyl radical production in isolated mouse heart mitochondria and F2-isoprostanes in brains and hearts of mice. The vitamin B12 analog cobinamide reversed the cellular toxicity of sulfide, and rescued Drosophila melanogaster and mice from lethal exposures of hydrogen sulfide gas. Cobinamide worked through two distinct mechanisms: direct reversal of complex IV inhibition and neutralization of sulfide-generated reactive oxygen species. We conclude that sulfide produces a high degree of oxidative stress in cells and tissues, and that cobinamide has promise as a first specific treatment for sulfide poisoning.

We recently demonstrated that cyclic GMP (cGMP)-dependent protein kinase (G-kinase) activates the human fos promoter in a strictly cGMP-dependent manner (T. Gudi et al., J. Biol. Chem. 271: [4597][4598][4599][4600] 1996). Here, we demonstrate that G-kinase translocates to the nucleus by an active transport mechanism which requires a nuclear localization signal (NLS) and is regulated by cGMP. Immunofluorescent staining of G-kinase was predominantly cytoplasmic in untreated cells, but intense nuclear staining appeared in 8-bromo (Br)-cGMP-treated cells. We identified a putative NLS in the G-kinase ATP binding domain which resembles the NLS of the interleukin-1␣ precursor. Fusion of the G-kinase NLS to the N terminus of ␤-galactosidase produced a chimeric protein which localized to the nucleus. Mutation of a single amino acid residue (K 407 3E) within the G-kinase NLS produced an enzyme with normal cGMP-dependent activity in vitro which did not translocate to the nucleus and did not transactivate the fos promoter in the presence of 8-Br-cGMP in vivo. In contrast, N-terminally truncated versions of G-kinase with constitutive, cGMP-independent activity in vitro localized to the nucleus and transactivated the fos promoter in the absence of 8-Br-cGMP. These results indicate that nuclear localization of G-kinase is required for transcriptional activation of the fos promoter and suggest that a conformational change of the kinase, induced by cGMP binding or by removal of the N-terminal autoinhibitory domain, functionally activates an otherwise cryptic NLS.The NO/cyclic GMP (cGMP) signal transduction pathway is present in many mammalian cells and involved in the regulation of important physiological functions such as neurotransmission, cell differentiation and proliferation, changes in vascular smooth muscle tone, endothelial cell permeability, and platelet aggregation (13,36,37,39,40,44,45). Whereas the cyclic AMP (cAMP) signal transduction pathway is well known to regulate gene transcription, regulation of gene expression by the NO/cGMP signal transduction pathway has been demonstrated only recently in different cell types (2,5,16,18,19,24,38,41,47). In rat embryo fibroblasts and thyroid epithelial cells, we showed that NO-releasing agents and membranepermeable cGMP analogs increase c-fos and junB mRNA expression, AP-1 DNA binding, and the transcriptional activity of promoters containing phorbol ester response elements (41). We subsequently demonstrated that transfection of G-kinase I␤ into G-kinase-deficient baby hamster kidney (BHK) cells causes induction of endogenous c-fos mRNA and activation of a cotransfected human fos promoter construct in a strictly cGMP-dependent manner (18, 18a). The effect of G-kinase is mediated by several sequence elements in the fos promoter, most notably the cAMP response element, the AP-1 binding site, and the serum response element with adjacent C/EBP-␤ binding site (18). The magnitude of G-kinase transactivation of the fos promoter was similar to that of cAMP-dependent protein kinase (A...

Nitric oxide (NO) increases cytosolic guanylate cyclase activity and thereby activates the cGMP signal transduction pathway. The cAMP and Ca2+/phospholipid signal transduction pathways activate transcription factors that bind to the cAMP response element (CRE) and phorbol ester response element (TRE), respectively. Little is known about transcriptional regulation of gene expression by NO/cGMP. In transient and stable transfection experiments and in microinjection studies we found that three different NO-releasing agents and two membrane-permeable cGMP analogs activated TRE-regulated but not CRE-regulated reporter genes in rodent fibroblast and epithelial cell lines. Activation of TRE-regulated genes by NO-releasing agents and cGMP analogs appeared to be mediated by the AP-1 (Jun/Fos) transcription factor complex because we observed increased DNA binding of AP-1 and increased junB and c-fos mRNA in cells treated with these agents. The mechanism of gene activation by NO/cGMP was distinct from that used by phorbol esters and cAMP because it was not associated with c-jun mRNA induction and was not observed with CRE-containing promoters.