Dolores Pérez-Sala

15-Deoxy-Delta 12,14-prostaglandin J2 inhibition of NF-kappaB-DNA binding through covalent modification of the p50 subunit.

Cyclopentenone prostaglandins display anti-inflammatory activities and interfere with the signaling pathway that leads to activation of transcription factor NF-κB. Here we explore the possibility that the NF-κB subunit p50 may be a target for the cyclopentenone 15-deoxy-Δ12,14-prostaglandin J2(15d-PGJ2). This prostaglandin inhibited the DNA binding ability of recombinant p50 in a dose-dependent manner. The inhibition required the cyclopentenone moiety and could be prevented but not reverted by glutathione and dithiothreitol. Moreover, a p50 mutant with a C62S mutation was resistant to inhibition, indicating that the effect of 15d-PGJ2 was probably due to its interaction with cysteine 62 in p50. The covalent modification of p50 by 15d-PGJ2 was demonstrated by reverse-phase high pressure liquid chromatography and mass spectrometry analysis that showed an increase in retention time and in the molecular mass of 15d-PGJ2-treated p50, respectively. The interaction between p50 and 15d-PGJ2 was relevant in intact cells. 15d-PGJ2 effectively inhibited cytokine-elicited NF-κB activity in HeLa without reducing IκBα degradation or nuclear translocation of NF-κB subunits. 15d-PGJ2 reduced NF-κB DNA binding activity in isolated nuclear extracts, suggesting a direct effect on NF-κB proteins. Finally, treatment of HeLa with biotinylated-15d-PGJ2 resulted in the formation of a 15d-PGJ2-p50 adduct as demonstrated by neutravidin binding and immunoprecipitation. These results clearly show that p50 is a target for covalent modification by 15d-PGJ2 that results in inhibition of DNA binding.

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.

Molecular basis for the direct inhibition of AP-1 DNA binding by 15-deoxy-Δ12,14-prostaglandin J2

Cyclopentenone prostaglandins may interfere with cellular functions by multiple mechanisms. The cyclopentenone 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) has been reported to inhibit the activity of the transcription factor AP-1 in several experimental settings. We have explored the possibility of a direct interaction of 15d-PGJ2 with AP-1 proteins. Here we show that 15d-PGJ2 covalently modifies c-Jun and directly inhibits the DNA binding activity of AP-1. The modification of c-Jun occurs both in vitro and in intact cells as detected by labeling with biotinylated 15d-PGJ2 and mass spectrometry analysis. Attachment of the cyclopentenone prostaglandin occurs at cysteine 269, which is located in the c-Jun DNA binding domain. In addition, 15d-PGJ2 can promote the oligomerization of a fraction of c-Jun through the formation of intermolecular disulfide bonds or 15d-PGJ2-bonded dimers. Our results identify a novel site of interaction of 15d-PGJ2 with the AP-1 activation pathway that may contribute to the complex effects of cyclopentenone prostaglandins on the cellular response to pro-inflammatory agents. They also show the first evidence for the induction of protein cross-linking by 15d-PGJ2.

The cyclopentenone 15-deoxy-delta 12,14-prostaglandin J2 binds to and activates H-Ras

The cyclopentenone 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) induces cell proliferation and mitogen-activated protein kinase activation. Here, we describe that these effects are mediated by 15d-PGJ2-elicited H-Ras activation. We demonstrate that this pathway is specific for H-Ras through the formation of a covalent adduct of 15d-PGJ2 with Cys-184 of H-Ras, but not with N-Ras or K-Ras. Mutation of C184 inhibited H-Ras modification and activation by 15d-PGJ2, whereas serum-elicited stimulation was not affected. These results describe a mechanism for the activation of the Ras signaling pathway, which results from the chemical modification of H-Ras by formation of a covalent adduct with cyclopentenone prostaglandins.

Novel application of S-nitrosoglutathione-Sepharose to identify proteins that are potential targets for S-nitrosoglutathione-induced mixed-disulphide formation

Site-specific S-glutathionylation is emerging as a novel mechanism by which S-nitrosoglutathione (GSNO) may modify functionally important protein thiols. Here, we show that GSNO-Sepharose mimicks site-specific S-glutathionylation of the transcription factors c-Jun and p50 by free GSNO in vitro. Both c-Jun and p50 were found to bind to immobilized GSNO through the formation of a mixed disulphide, involving a conserved cysteine residue located in the DNA-binding domains of these transcription factors. Furthermore, we show that c-Jun, p50, glycogen phosphorylase b, glyceraldehyde-3-phosphate dehydrogenase, creatine kinase, glutaredoxin and caspase-3 can be precipitated from a mixture of purified thiol-containing proteins by the formation of a mixed-disulphide bond with GSNO-Sepharose. With few exceptions, protein binding to this matrix correlated well with the susceptibility of the investigated proteins to undergo GSNO- but not diamide-induced mixed-disulphide formation in vitro. Finally, it is shown that covalent GSNO-Sepharose chromatography of HeLa cell nuclear extracts results in the enrichment of proteins which incorporate glutathione in response to GSNO treatment. As suggested by DNA-binding assays, this group of nuclear proteins include the transcription factors activator protein-1, nuclear factor-kappaB and cAMP-response-element-binding protein. In conclusion, we introduce GSNO-Sepharose as a probe for site-specific S-glutathionylation and as a novel and potentially useful tool to isolate and identify proteins which are candidate targets for GSNO-induced mixed-disulphide formation.

Identification of Novel Protein Targets for Modification by 15-Deoxy-Δ12,14-Prostaglandin J2 in Mesangial Cells Reveals Multiple Interactions with the Cytoskeleton

The cyclopentenone prostaglandin 15-deoxy-Delta12,14-PGJ2 (15d-PGJ2) has been shown to display protective effects against renal injury or inflammation. In cultured mesangial cells (MC), 15d-PGJ2 inhibits the expression of proinflammatory genes and modulates cell proliferation. Therefore, cyclopentenone prostaglandins (cyPG) have been envisaged as a promise in the treatment of renal disease. The effects of 15d-PGJ2 may be dependent on or independent from its role as a peroxisome proliferator-activated receptor agonist. It was shown recently that an important determinant for the peroxisome proliferator-activated receptor-independent effects of 15d-PGJ2 is the capacity to modify proteins covalently and alter their function. However, a limited number of protein targets have been identified to date. Herein is shown that a biotinylated derivative of 15d-PGJ2 recapitulates the effects of 15d-PGJ2 on the stress response and inhibition of inducible nitric oxide synthase levels and forms stable adducts with proteins in intact MC. Biotinylated 15d-PGJ2 was then used to identify proteins that potentially are involved in cyPG biologic effects. Extracts from biotinylated 15d-PGJ2-treated MC were separated by two-dimensional electrophoresis, and the spots of interest were analyzed by mass spectrometry. Identified targets include proteins that are regulated by oxidative stress, such as heat-shock protein 90 and nucleoside diphosphate kinase, as well as proteins that are involved in cytoskeletal organization, such as actin, tubulin, vimentin, and tropomyosin. Biotinylated 15d-PGJ2 binding to several targets was confirmed by avidin pull-down. Consistent with these findings, 15d-PGJ2 induced early reorganization of vimentin and tubulin in MC. The cyclopentenone moiety and the presence of cysteine were important for vimentin rearrangement. These studies may contribute to the understanding of the mechanism of action and therapeutic potential of cyPG.

Bcl-2 differentially targets K-, N-, and H-Ras to mitochondria in IL-2 supplemented or deprived cells : Implications in prevention of apoptosis

IL-2 deprivation triggers apoptosis in the murine T cell line TS1αβ, a process that can be blocked by overexpression of Bcl-2. Here we show that Bcl-2 and Ras proteins interact in mitochondria from TS1αβ cells in the presence or absence of IL-2, as evidenced by co-immunoprecipitation. All three Ras proteins, K-, N- and H-Ras, interact with Bcl-2; however, their mitochondrial localization is differentially regulated in IL-2-supplemented or -deprived cells. K-Ras is found in mitochondria only in IL-2-supplemented cells, whereas H-Ras is observed in mitochondria only after IL-2 withdrawal. N-Ras is detected in mitochondria under both experimental conditions. Bcl-2 transfection partially restored K- and N-Ras association with mitochondria in IL-2-deprived cells and rendered H-Ras association independent of IL-2 withdrawal. Inhibitors of Ras posttranslational processing did not alter the IL-2-induced differential pattern of mitochondrial localization. The processed forms of K- and N-Ras associated with mitochondria, although unprocessed H-Ras was also detected in mitochondria from mevastatin-treated cells. These results evidence a distinct behavior among the three Ras proteins in TS1αβ cells, depending on IL-2 supply, and suggest homologue-specific roles for Ras proteins in IL-2-dependent events.

Regulation of cell adhesion to collagen via β1 integrins is dependent on interactions of filamin A with vimentin and protein kinase C epsilon

Cell adhesion and spreading on collagen, which are essential processes for development and wound healing in mammals, are mediated by beta1 integrins and the actin and intermediate filament cytoskeletons. The mechanisms by which these separate cytoskeletal systems interact to regulate beta1 integrins and cell spreading are poorly defined. We previously reported that the actin cross-linking protein filamin A binds the intermediate filament protein vimentin and that these two proteins co-regulate cell spreading. Here we used deletional mutants of filamin A to define filamin A-vimentin interactions and the subsequent phosphorylation and re-distribution of vimentin during cell spreading on collagen. Imaging of fixed and live cell preparations showed that phosphorylated vimentin is translocated to the cell membrane during spreading. Knockdown of filamin A inhibited cell spreading and the phosphorylation and re-distribution of vimentin. Knockdown of filamin A and/or vimentin reduced the cell surface expression and activation of beta1 integrins, as indicated by immunoblotting of plasma membrane-associated proteins and shear force assays. In vitro pull-down assays using filamin A mutants showed that both vimentin and protein kinase Cvarepsilon bind to repeats 1-8 of filamin A. Reconstitution of filamin-A-deficient cells with full-length filamin A or filamin A repeats 1-8 restored cell spreading, vimentin phosphorylation, and the cell surface expression of beta1 integrins. We conclude that the binding of filamin A to vimentin and protein kinase Cepsilon is an essential regulatory step for the trafficking and activation of beta1 integrins and cell spreading on collagen.

PPAR agonists amplify iNOS expression while inhibiting NF-κB: Implications for mesangial cell activation by cytokines

In acute inflammation, the transcription factor NF-kappaB is activated and increases the expression of multiple pro-inflammatory genes. Agonists of peroxisome proliferator activated receptors (PPAR) have been reported to exert antiinflammatory effects in various systems. In keeping with such an antiinflammatory role, it was found that several PPAR agonists, including Wy14,643, clofibrate, carbaprostacyclin, and ciglitazone inhibited NF-kappaB activity and increased IkappaBalpha levels in cytokine-stimulated mesangial cells (MC). Activation of NF-kappaB has been found to be crucial to the cytokine-elicited expression of inducible nitric oxide synthase (iNOS). Despite the inhibitory effect of PPAR agonists on NF-kappaB activity, this study provides experimental data demonstrating that these agonists amplify cytokine-elicited NO generation in MC, potentiating iNOS protein expression approximately threefold. The upregulation of iNOS expression occurred at the mRNA level and apparently did not result from iNOS mRNA stabilization. Clofibrate and ciglitazone amplified the cytokine-elicited stimulation of a 16-Kb human iNOS promoter construct in stably transfected MC, suggesting that PPAR agonists potentiate iNOS induction through transcriptional mechanisms. MC express all three PPAR proteins. However, iNOS potentiation did not correlate with increased PPAR activity. In addition, Wy14,643-induced amplification of cytokine-elicited iNOS levels also occurred in RAW264.7 macrophages and in human epithelial Caco-2 and HT-29 cells. The observation that these epithelial cell lines express an inactive, truncated PPARalpha variant suggests that a classical PPARalpha agonist, such as Wy14,643, may act through PPARalpha-independent mechanisms. In conclusion, these results show that, despite reducing NF-kappaB activity, PPAR agonists may amplify the expression of certain NF-kappaB-dependent genes that are relevant to the inflammatory process, like iNOS.

Isoprenylation of RhoB is necessary for its degradation. A novel determinant in the complex regulation of RhoB expression by the mevalonate pathway

Statins improve vascular functions by mechanisms independent from their cholesterol-lowering effect. Rho GTPases are emerging as key targets for the vascular effects of statins. RhoB is a short-lived, early-response inducible protein involved in receptor endocytosis, apoptosis, and gene expression. Here we show that statins regulate RhoB expression by acting at multiple levels. Simvastatin increased RhoB protein levels by 8- to 10-fold. This effect was related to a depletion of isoprenoid intermediates, as deduced from the observation that several metabolites of the cholesterol biosynthetic pathway, namely, mevalonate and geranylgeranyl-pyrophosphate, attenuated simvastatin-induced RhoB up-regulation. Moreover, prenyltransferase inhibitors mimicked simvastatin effect. Cholesterol supplementation did not prevent simvastatin-elicited up-regulation but increased RhoB levels per se. Simvastatin moderately augmented RhoB transcript levels, but markedly impaired the degradation of RhoB protein, which accumulated in the cytosol in its non-isoprenylated form. Inhibition of RhoB isoprenylation was apparently required for simvastatin-induced up-regulation, because levels of an isoprenylation-deficient RhoB mutant were not affected by simvastatin. Moreover, this mutant was found to be markedly more stable than the wild-type protein. These results show that RhoB isoprenylation is necessary for rapid turnover of this protein and identify a novel link between the cholesterol biosynthetic pathway and the regulation of G-protein expression.