Evolutionarily conserved role of calcineurin in phosphodegron-dependent degradation of phosphodiesterase 4D.

Calcineurin is a widely expressed and highly conserved Ser/Thr phosphatase. Calcineurin is inhibited by the immunosuppressant drug cyclosporine A (CsA) or tacrolimus (FK506). The critical role of CsA/FK506 as an immunosuppressant following transplantation surgery provides a strong incentive to understand the phosphatase calcineurin. Here we uncover a novel regulatory pathway for cyclic AMP (cAMP) signaling by the phosphatase calcineurin which is also evolutionarily conserved in Caenorhabditis elegans. We found that calcineurin binds directly to and inhibits the proteosomal degradation of cAMP-hydrolyzing phosphodiesterase 4D (PDE4D). We show that ubiquitin conjugation and proteosomal degradation of PDE4D are controlled by a cullin 1-containing E(3) ubiquitin ligase complex upon dual phosphorylation by casein kinase 1 (CK1) and glycogen synthase kinase 3beta (GSK3beta) in a phosphodegron motif. Our findings identify a novel signaling process governing G-protein-coupled cAMP signal transduction-opposing actions of the phosphatase calcineurin and the CK1/GSK3beta protein kinases on the phosphodegron-dependent degradation of PDE4D. This novel signaling system also provides unique functional insights into the complications elicited by CsA in transplant patients.

Hydrogen sulfide attenuates hepatic ischemia-reperfusion injury: role of antioxidant and antiapoptotic signaling.

Hydrogen sulfide (H(2)S) is an endogenously produced gaseous signaling molecule with diverse physiological activity. The potential protective effects of H(2)S have not been evaluated in the liver. The purpose of the current study was to investigate if H(2)S could afford hepatoprotection in a murine model of hepatic ischemia-reperfusion (I/R) injury. Hepatic injury was achieved by subjecting mice to 60 min of ischemia followed by 5 h of reperfusion. H(2)S donor (IK1001) or vehicle were administered 5 min before reperfusion. H(2)S attenuated the elevation in serum alanine aminotransferase (ALT) by 68.6% and aspartate aminotransferase (AST) by 70.8% compared with vehicle group. H(2)S-mediated cytoprotection was associated with an improved balance between reduced glutathione (GSH) vs. oxidized glutathione (GSSG), an attenuated formation of lipid hydroperoxides, and an increased expression of thioredoxin-1 (Trx-1). Furthermore, H(2)S inhibited the progression of apoptosis after I/R injury by increasing the protein expression of heat shock protein (HSP-90) and Bcl-2. These results indicate that H(2)S protects the murine liver against I/R injury through an upregulation of intracellular antioxidant and antiapoptotic signaling pathways.

Genetic overexpression of eNOS attenuates hepatic ischemia-reperfusion injury.

Previous studies have shown that endothelial nitric oxide (NO) synthase (eNOS)-derived NO is an important signaling molecule in ischemia-reperfusion (I-R) injury. Deficiency of eNOS-derived NO has been shown to exacerbate injury in hepatic and myocardial models of I-R. We hypothesized that transgenic overexpression of eNOS (eNOS-TG) would reduce hepatic I-R injury. We subjected two strains of eNOS-TG mice to 45 min of hepatic ischemia and 5 h of reperfusion. Both strains were protected from hepatic I-R injury compared with wild-type littermates. Because the mechanism for this protection is still unclear, additional studies were performed by using inhibitors and activators of both soluble guanylyl cyclase (sGC) and heme oxygenase-1 (HO-1) enzymes. Blocking sGC with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and HO-1 with zinc (III) deuteroporphyrin IX-2,4-bisethyleneglycol (ZnDPBG) in wild-type mice increased hepatic I-R injury, whereas pharmacologically activating these enzymes significantly attenuated I-R injury in wild-type mice. Interestingly, ODQ abolished the protective effects of eNOS overexpression, whereas ZnDPBG had no effect. These results suggest that hepatic protection in eNOS-TG mice may be mediated in part by NO signaling via the sGC-cGMP pathway and is independent of HO-1 signal transduction pathways.

eNOS gene therapy exacerbates hepatic ischemia-reperfusion injury in diabetes: a role for eNOS uncoupling.

Previous studies indicate that endothelial nitric oxide synthase (eNOS) function is impaired in diabetes as a result of increased vascular generation of reactive oxygen species. We hypothesized that eNOS gene therapy would augment NO. bioavailability and protect against hepatic ischemia-reperfusion (I-R) injury in type 2 diabetes mellitus. We developed a transgenic (Tg) diabetic mouse in which eNOS is systemically overexpressed. We also examined the effects of hepatic eNOS adenovirus therapy in diabetic mice. Diabetic (db/db) and nondiabetic mice were subjected to hepatic I-R injury. In nondiabetic mice, genetic overexpression of eNOS (both eNOS-Tg and eNOS adenovirus) resulted in hepatoprotection. In contrast, hepatic I-R injury was significantly increased in the db/db eNOS-Tg mouse, as serum alanine aminotransaminase (ALT) levels were increased by 3.3-fold compared with diabetic controls. Similarly, eNOS adenovirus treatment resulted in a 3.2-fold increase in serum ALT levels as compared with diabetic controls. We determined that hepatic eNOS was dysfunctional in the db/db mouse and increased genetic expression of eNOS resulted in greater production of peroxynitrite. Treatment with the eNOS cofactor tetrahydrobiopterin (BH4) or the BH4 precursor sepiapterin resulted in a significant decrease in serum ALT levels following I-R injury. We present clear examples of the protective and injurious nature of NO. therapy in I-R. Our data indicate that eNOS exists in an "uncoupled" state in the setting of diabetes and that "recoupling" of the eNOS enzyme with cofactor therapy is beneficial.

Cytoprotective effects of nitrite during in vivo ischemia-reperfusion of the heart and liver.

Nitrite represents a circulating and tissue storage form of NO whose bioactivation is mediated by the enzymatic action of xanthine oxidoreductase, nonenzymatic disproportionation, and reduction by deoxyhemoglobin, myoglobin, and tissue heme proteins. Because the rate of NO generation from nitrite is linearly dependent on reductions in oxygen and pH levels, we hypothesized that nitrite would be reduced to NO in ischemic tissue and exert NO-dependent protective effects. Solutions of sodium nitrite were administered in the setting of hepatic and cardiac ischemia-reperfusion (I/R) injury in mice. In hepatic I/R, nitrite exerted profound dose-dependent protective effects on cellular necrosis and apoptosis, with highly significant protective effects observed at near-physiological nitrite concentrations. In myocardial I/R injury, nitrite reduced cardiac infarct size by 67%. Consistent with hypoxia-dependent nitrite bioactivation, nitrite was reduced to NO, S-nitrosothiols, N-nitros-amines, and iron-nitrosylated heme proteins within 1-30 minutes of reperfusion. Nitrite-mediated protection of both the liver and the heart was dependent on NO generation and independent of eNOS and heme oxygenase-1 enzyme activities. These results suggest that nitrite is a biological storage reserve of NO subserving a critical function in tissue protection from ischemic injury. These studies reveal an unexpected and novel therapy for diseases such as myocardial infarction, organ preservation and transplantation, and shock states.