Santiago Lamas

Presence of nitric oxide synthase activity in roots and nodules of Lupinus albus

NO is a widespread messenger molecule in physiology. We were interested in investigating whether an NO‐generating system could be present in plants. NO and l‐[14C]citrulline were synthesized by roots and nodules of Lupinus albus in an l‐arginine‐dependent manner. l‐[14C]Citrulline production was inhibited by NG‐monomethyl‐l‐arginine, a nitric oxide synthase antagonist, in a competitive way. NADPH‐diaphorase activity was localized in the vascular bundles in root and nodules, and also in the nodule infected zone. This staining was significantly reduced in the presence of NG‐monomethyl‐l‐arginine. These results indicate the presence of a putative nitric oxide synthase in plants.

Redox regulation of c-Jun DNA binding by reversible S-glutathiolation

Redox control of the transcription factor c‐Jun maps to a single cysteine in its DNA binding domain. However, the nature of the oxidized state of this cysteine and, thus, the potential molecular mechanisms accounting for the redox regulation of c‐Jun DNA binding remain unclear. To address this issue, we have analyzed the purified recombinant c‐Jun DNA binding domain for redox‐dependent thiol modifications and concomitant changes in DNA binding activity. We show that changes in the ratio of reduced to oxidized glutathione provide the potential to oxidize c‐Jun sulfhydryls by mechanisms that include both protein disulfide formation and S‐glutathiolation. We provide evidence that S‐glutathiolation, which is specifically targeted to the cysteine residue located in the DNA binding site of the protein, may account for the reversible redox regulation of c‐Jun DNA binding. Furthermore, based on a molecular model of the S‐glutathiolated protein, we discuss the structural elements facilitating S‐glutathiolation and how this modification interferes with DNA binding. Given the structural similarities between the positively charged cysteine‐containing DNA binding motif of c‐Jun and the DNA binding site of related oxidant‐sensitive transcriptional activators, the unprecedented phenomenon of redox‐triggered S‐thiolation of a transcription factor described in this report suggests a novel role for protein thiolation in the redox control of transcription.—Klatt, P., Molina, E. P., de Lacoba, M. G., Padilla, C. A., Martínez‐Galisteo, E., Bárcena, J. A., Lamas, S. Redox regulation of c‐Jun DNA binding by reversible S‐glutathiolation. FASEB J. 13, 1481–1490 (1999)

Nitric oxide signaling: classical, less classical, and nonclassical mechanisms.

Although nitric oxide (NO) was identified more than 150 years ago and its effects were clinically tested in the form of nitroglycerine, it was not until the decades of 1970-1990 that it was described as a gaseous signal transducer. Since then, a canonical pathway linked to cyclic GMP (cGMP) as its quintessential effector has been established, but other modes of action have emerged and are now part of the common body of knowledge within the field. Classical (or canonical) signaling involves the selective activation of soluble guanylate cyclase, the generation of cGMP, and the activation of specific kinases (cGMP-dependent protein kinases) by this cyclic nucleotide. Nonclassical signaling alludes to the formation of NO-induced posttranslational modifications (PTMs), especially S-nitrosylation, S-glutathionylation, and tyrosine nitration. These PTMs are governed by specific biochemical mechanisms as well as by enzymatic systems. In addition, a less classical but equally important pathway is related to the interaction between NO and mitochondrial cytochrome c oxidase, which might have important implications for cell respiration and intermediary metabolism. Cross talk trespassing these necessarily artificial conceptual boundaries is progressively being identified and hence an integrated systems biology approach to the comprehension of NO function will probably emerge in the near future.

Antioxidant responses and cellular adjustments to oxidative stress

Redox biological reactions are now accepted to bear the Janus faceted feature of promoting both physiological signaling responses and pathophysiological cues. Endogenous antioxidant molecules participate in both scenarios. This review focuses on the role of crucial cellular nucleophiles, such as glutathione, and their capacity to interact with oxidants and to establish networks with other critical enzymes such as peroxiredoxins. We discuss the importance of the Nrf2-Keap1 pathway as an example of a transcriptional antioxidant response and we summarize transcriptional routes related to redox activation. As examples of pathophysiological cellular and tissular settings where antioxidant responses are major players we highlight endoplasmic reticulum stress and ischemia reperfusion. Topologically confined redox-mediated post-translational modifications of thiols are considered important molecular mechanisms mediating many antioxidant responses, whereas redox-sensitive microRNAs have emerged as key players in the posttranscriptional regulation of redox-mediated gene expression. Understanding such mechanisms may provide the basis for antioxidant-based therapeutic interventions in redox-related diseases.

Involvement of Rho GTPases in the Transcriptional Inhibition of Preproendothelin-1 Gene Expression by Simvastatin in Vascular Endothelial Cells

Endothelial dysfunction is characterized by an impaired vasodilatory response to endothelial agonists as well as by alterations in adhesion and coagulation processes. 3-Hydroxy-3-methylglutaryl-CoA reductase inhibitors (statins) have been shown to be useful in the reversal of endothelial dysfunction, an effect that may be independent of the reduction in cholesterol levels. Both the l-arginine–nitric oxide–cGMP and endothelin pathways are involved in the regulation of vascular tone. Here, we show that the basal transcription rate of the preproendothelin-1 gene was decreased by simvastatin (10 &mgr;mol/L) in bovine aortic endothelial cells. Transfection studies with the preproendothelin-1 gene promoter showed that mevalonate (100 &mgr;mol/L) was able to prevent the inhibitory effect mediated by simvastatin. Protein geranylgeranylation, but not farnesylation, proved to be crucial for a correct expression of the preproendothelin-1 gene. The C3 exotoxin from Clostridium botulinum that selectively inactivates Rho GTPases, the processing of which involves geranylgeranylation, reproduced the inhibitory effect of simvastatin on the expression of preproendothelin-1. Overexpression of dominant-negative mutants of RhoA and RhoB led to a significant reduction in the preproendothelin-1 promoter activity, whereas the expression of wild-type and constitutively active forms of these proteins resulted in an increase, in support that Rho proteins are required for the basal expression of the preproendothelin-1 gene. Finally, we show that the Rho-dependent activation of the preproendothelin-1 gene transcription was inhibited by simvastatin. Thus, the control of vascular tone and proliferative response mediated by endothelin-1 is regulated at multiple levels, among which the Rho proteins play an essential role.

Nitric oxide inhibits c-Jun DNA binding by specifically targeted S-glutathionylation.

This study addresses potential molecular mechanisms underlying the inhibition of the transcription factor c-Jun by nitric oxide. We show that in the presence of the physiological sulfhydryl glutathione nitric oxide modifies the two cysteine residues contained in the DNA binding module of c-Jun in a selective and distinct way. Although nitric oxide induced the formation of an intermolecular disulfide bridge between cysteine residues in the leucine zipper site of c-Jun monomers, this same radical directed the covalent incorporation of stoichiometric amounts of glutathione to a single conserved cysteine residue in the DNA-binding site of the protein. We found that covalent dimerization of c-Jun apparently did not affect its DNA binding activity, whereas the formation of a mixed disulfide with glutathione correlated well with the inhibition of transcription factor binding to DNA. Furthermore, we provide experimental evidence that nitric oxide-inducedS-glutathionylation and inhibition of c-Jun involves the formation of S-nitrosoglutathione. In conclusion, our results support the reversible formation of a mixed disulfide between glutathione and c-Jun as a potential mechanism by which nitrosative stress may be transduced into a functional response at the level of transcription.

Mutual Dependence of Foxo3a and PGC-1α in the Induction of Oxidative Stress Genes

Oxidative stress is a hallmark of metabolism-related diseases and a risk factor for atherosclerosis. FoxO factors have been shown to play a key role in vascular endothelial development and homeostasis. Foxo3a can protect quiescent cells from oxidative stress through the regulation of detoxification genes such as sod2 and catalase. Here we show that Foxo3a is a direct transcriptional regulator of a group of oxidative stress protection genes in vascular endothelial cells. Importantly, Foxo3a activity requires the transcriptional co-activator PGC-1α, because it is severely curtailed in PGC-1α-deficient endothelial cells. Foxo3a and PGC-1α appear to interact directly, as shown by co-immunoprecipitation and in vitro interaction assays, and are recruited to the same promoter regions. The notion that Foxo3a and PGC-1α interact directly to regulate oxidative stress protection genes in the vascular endothelium is supported by the observation that PGC-1α transcriptional activity at the sod2 (manganese superoxide dismutase) promoter requires a functional FoxO site. We also demonstrate that Foxo3a is a direct transcriptional regulator of PGC-1α, suggesting that an auto-regulatory cycle regulates Foxo3a/PGC-1α control of the oxidative stress response.

Role of reactive oxygen species in the signalling cascade of cyclosporine A-mediated up-regulation of eNOS in vascular endothelial cells

Cyclosporine A (CsA) increases eNOS mRNA expression in bovine cultured aortic endothelial cells (BAEC). As some effects of CsA may be mediated by reactive oxygen species (ROS), present experiments were devoted to test the hypothesis that the CsA‐induced eNOS up‐regulation could be dependent on an increased synthesis of ROS. CsA induced a dose‐dependent increase of ROS synthesis, with the two fluorescent probes used, DHR123 (CsA 1 μM: 305±7% over control) and H2DCFDA (CsA 1 μM: 178±6% over control). Two ROS generating systems, xanthine plus xanthine oxidase (XXO) and glucose oxidase (GO), increased the expression of eNOS mRNA in BAEC, an effect which was maximal after 8 h of incubation (XXO: 168±21% of control values. GO: 208±18% of control values). The ROS‐dependent increased eNOS mRNA expression was followed by an increase in eNOS activity. The effect of CsA on eNOS mRNA expression was abrogated by catalase, and superoxide dismutase (SOD). In contrast, the antioxidant PDTC augmented eNOS mRNA expression, both in basal conditions and in the presence of CsA. The potential participation of the transcription factor AP‐1 was explored. Electrophoretic mobility shift assays were consistent with an increase in AP‐1 DNA‐binding activity in BAEC treated with CsA or glucose oxidase. The present results support a role for ROS, particularly superoxide anion and hydrogen peroxide, as mediators of the CsA‐induced eNOS mRNA up‐regulation. Furthermore, they situate ROS as potential regulators of gene expression in endothelial cells, both in physiological and pathophysiological situations.

Constitutive ALK5-Independent c-Jun N-Terminal Kinase Activation Contributes to Endothelin-1 Overexpression in Pulmonary Fibrosis: Evidence of an Autocrine Endothelin Loop Operating through the Endothelin A and B Receptors

ABSTRACT The signal transduction mechanisms generating pathological fibrosis are almost wholly unknown. Endothelin-1 (ET-1), which is up-regulated during tissue repair and fibrosis, induces lung fibroblasts to produce and contract extracellular matrix. Lung fibroblasts isolated from scleroderma patients with chronic pulmonary fibrosis produce elevated levels of ET-1, which contribute to the persistent fibrotic phenotype of these cells. Transforming growth factor β (TGF-β) induces fibroblasts to produce and contract matrix. In this report, we show that TGF-β induces ET-1 in normal and fibrotic lung fibroblasts in a Smad-independent ALK5/c-Jun N-terminal kinase (JNK)/Ap-1-dependent fashion. ET-1 induces JNK through TAK1. Fibrotic lung fibroblasts display constitutive JNK activation, which was reduced by the dual ETA/ETB receptor inhibitor, bosentan, providing evidence of an autocrine endothelin loop. Thus, ET-1 and TGF-β are likely to cooperate in the pathogenesis of pulmonary fibrosis. As elevated JNK activation in fibrotic lung fibroblasts contributes to the persistence of the myofibroblast phenotype in pulmonary fibrosis by promoting an autocrine ET-1 loop, targeting the ETA and ETB receptors or constitutive JNK activation by fibrotic lung fibroblasts is likely to be of benefit in combating chronic pulmonary fibrosis.

Nitric oxide regulates mitochondrial oxidative stress protection via the transcriptional coactivator PGC-1α

Nitric oxide (NO) has both prooxidant and antioxidant activities in the endothelium; however, the molecular mechanisms involved are still a matter of controversy. PGC‐1α [peroxisome proliferators‐activated receptor (PPAR) γ coactivator 1‐α] induces the expression of several members of the mitochondrial reactive oxygen species (ROS) detoxification system. Here, we show that NO regulates this system through the modulation of PGC‐1α expression. Short‐term (<12 h) treatment of endothelial cells with NO donors down‐regulates PGC‐1α expression, whereas long‐term (>24 h) treatment up‐regulates it. Treatment with the NOS inhibitor L‐NAME has the opposite effect. Down‐regulation of PGC‐1α by NO is mediated by protein kinase G (PKG). It is blocked by the soluble guanylate cyclase (sGC) inhibitor ODQ and the PKG inhibitor KT5823, and mimicked by the cGMP analog 8‐Br‐cGMP. Changes in PGC‐1α expression are in all cases paralleled by corresponding variations in the mitochondrial ROS detoxification system. Cells that transiently overexpress PGC‐1α from the cytomeglovirus (CMV) promoter respond poorly to NO donors. Analysis of tissues from eNOS−/− mice showed reduced levels of PGC‐1α and the mitochondrial ROS detoxification system. These data suggest that NO can regulate the mitochondrial ROS detoxification system both positively and negatively through PGC‐1α. —Borniquel, S., Valle, I., Cadenas, S., Lamas, S., and Monsalve, M. Nitric oxide regulates mitochondrial oxidative stress protection via the transcriptional coactivator PGC‐1α. FASEB J. 20, E1216–E1227 (2006)