Minerva T. Garcia-Barrio

Covalent Peroxisome Proliferator-activated Receptor γ Adduction by Nitro-fatty Acids SELECTIVE LIGAND ACTIVITY AND ANTI-DIABETIC SIGNALING ACTIONS

The peroxisome proliferator-activated receptor-γ (PPARγ) binds diverse ligands to transcriptionally regulate metabolism and inflammation. Activators of PPARγ include lipids and anti-hyperglycemic drugs such as thiazolidinediones (TZDs). Recently, TZDs have raised concern after being linked with increased risk of peripheral edema, weight gain, and adverse cardiovascular events. Most reported endogenous PPARγ ligands are intermediates of lipid metabolism and oxidation that bind PPARγ with very low affinity. In contrast, nitro derivatives of unsaturated fatty acids (NO2-FA) are endogenous products of nitric oxide (•NO) and nitrite (NO2−)-mediated redox reactions that activate PPARγ at nanomolar concentrations. We report that NO2-FA act as partial agonists of PPARγ and covalently bind PPARγ at Cys-285 via Michael addition. NO2-FA show selective PPARγ modulator characteristics by inducing coregulator protein interactions, PPARγ-dependent expression of key target genes, and lipid accumulation is distinctively different from responses induced by the TZD rosiglitazone. Administration of this class of signaling mediators to ob/ob mice revealed that NO2-FA lower insulin and glucose levels without inducing adverse side effects such as the increased weight gain induced by TZDs.The peroxisome proliferator-activated receptor-gamma (PPARgamma) binds diverse ligands to transcriptionally regulate metabolism and inflammation. Activators of PPARgamma include lipids and anti-hyperglycemic drugs such as thiazolidinediones (TZDs). Recently, TZDs have raised concern after being linked with increased risk of peripheral edema, weight gain, and adverse cardiovascular events. Most reported endogenous PPARgamma ligands are intermediates of lipid metabolism and oxidation that bind PPARgamma with very low affinity. In contrast, nitro derivatives of unsaturated fatty acids (NO(2)-FA) are endogenous products of nitric oxide ((*)NO) and nitrite (NO(2)(-))-mediated redox reactions that activate PPARgamma at nanomolar concentrations. We report that NO(2)-FA act as partial agonists of PPARgamma and covalently bind PPARgamma at Cys-285 via Michael addition. NO(2)-FA show selective PPARgamma modulator characteristics by inducing coregulator protein interactions, PPARgamma-dependent expression of key target genes, and lipid accumulation is distinctively different from responses induced by the TZD rosiglitazone. Administration of this class of signaling mediators to ob/ob mice revealed that NO(2)-FA lower insulin and glucose levels without inducing adverse side effects such as the increased weight gain induced by TZDs.

Extracellular Nef Protein Targets CD4+ T Cells for Apoptosis by Interacting with CXCR4 Surface Receptors

ABSTRACT The effects of soluble Nef protein on CD4+ T cells were examined. CD4+-T-cell cultures exposed to soluble Nef were analyzed for apoptosis by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling and hallmarks of apoptosis including cytoplasmic shrinkage, nuclear fragmentation, DNA laddering, and caspase activation. We observed dose- and time-dependent inductions of apoptosis. DNA laddering and activated caspase 3 were also evident. Cells treated with Nef/protein kinase inhibitor complexes were protected from Nef-induced apoptosis, suggesting possible roles for protein kinases in the apoptosis pathway. Similarly, cells treated with Nef/anti-Nef antibody complexes were protected from Nef-induced apoptosis. The cellular receptor responsible for Nef-induced apoptosis was identified through antibody- and ligand-blocking experiments as a receptor commonly involved in viral entry. CXCR4 antibodies, as well as the endogenous ligand SDF-1α, were effective in blocking Nef-induced apoptosis, while CCR5 and CD4 antibodies were ineffective. Moreover, a CXCR4-deficient cell line, MDA-MB-468, which was resistant to Nef-induced apoptosis, became sensitive upon transfection with a CXCR4-expressing vector. This study suggests that extracellular Nef protein could contribute to the decline of CD4 counts prior to and during the onset of AIDS in patients with human immunodeficiency virus type 1 infections.

Nitro-Oleic Acid Inhibits Angiotensin II–Induced Hypertension

Rationale Nitro-oleic acid (OA-NO2) is a bioactive, nitric-oxide derived fatty acid with physiologically relevant vasculoprotective properties in vivo. OA-NO2 exerts cell signaling actions as a result of its strong electrophilic nature and mediates pleiotropic cell responses in the vasculature. Objective The present study sought to investigate the protective role of OA-NO2 in angiotensin (Ang) II–induced hypertension. Methods and Results We show that systemic administration of OA-NO2 results in a sustained reduction of Ang II–induced hypertension in mice and exerts a significant blood pressure lowering effect on preexisting hypertension established by Ang II infusion. OA-NO2 significantly inhibits Ang II contractile response as compared to oleic acid (OA) in mesenteric vessels. The improved vasoconstriction is specific for the Ang II type 1 receptor (AT1R)-mediated signaling because vascular contraction by other G-protein–coupled receptors is not altered in response to OA-NO2 treatment. From the mechanistic viewpoint, OA-NO2 lowers Ang II–induced hypertension independently of peroxisome proliferation-activated receptor (PPAR)&ggr; activation. Rather, OA-NO2, but not OA, specifically binds to the AT1R, reduces heterotrimeric G-protein coupling, and inhibits IP3 (inositol-1,4,5-trisphosphate) and calcium mobilization, without inhibiting Ang II binding to the receptor. Conclusions These results demonstrate that OA-NO2 diminishes the pressor response to Ang II and inhibits AT1R-dependent vasoconstriction, revealing OA-NO2 as a novel antagonist of Ang II–induced hypertension.

Vascular Smooth Muscle Cell–Selective Peroxisome Proliferator–Activated Receptor-γ Deletion Leads to Hypotension

Background— Peroxisome proliferator–activated receptor-γ (PPARγ) agonists are commonly used to treat diabetes, although their PPARγ-dependent effects transcend their role as insulin sensitizers. Thiazolidinediones lower blood pressure (BP) in diabetic patients, whereas results from conventional/tissue-specific PPARγ experimental models suggest an important pleiotropic role for PPARγ in BP control. Little evidence is available on the molecular mechanisms underlying the role of vascular smooth muscle cell–specific PPARγ in basal vascular tone. Methods and Results— We show that vascular smooth muscle cell–selective deletion of PPARγ impairs vasoactivity with an overall reduction in BP. Aortic contraction in response to norepinephrine is reduced and vasorelaxation is enhanced in response to β-adrenergic receptor (β-AdR) agonists in vitro. Similarly, vascular smooth muscle cell–selective PPARγ knockout mice display a biphasic response to norepinephrine in BP, reversible on administration of β-AdR blocker, and enhanced BP reduction on treatment with β-AdR agonists. Consistent with enhanced β2-AdR responsiveness, we found that the absence of PPARγ in vascular smooth muscle cells increased β2-AdR expression, possibly leading to the hypotensive phenotype during the rest phase. Conclusion— These data uncovered the β2-AdR as a novel target of PPARγ transcriptional repression in vascular smooth muscle cells and indicate that PPARγ regulation of β2-adrenergic signaling is important in the modulation of BP.

Expression profiling of nuclear receptors in human and mouse embryonic stem cells

Nuclear receptors (NRs) regulate gene expression in essential biological processes including differentiation and development. Here we report the systematic profiling of NRs in human and mouse embryonic stem cell (ESC) lines and during their early differentiation into embryoid bodies. Expression of the 48 human and mouse NRs was assessed by quantitative real-time PCR. In general, expression of NRs between the two human cell lines was highly concordant, whereas in contrast, expression of NRs between human and mouse ESCs differed significantly. In particular, a number of NRs that have been implicated previously as crucial regulators of mouse ESC biology, including ERRbeta, DAX-1, and LRH-1, exhibited diametric patterns of expression, suggesting they may have distinct species-specific functions. Taken together, these results highlight the complexity of the transcriptional hierarchy that exists between species and governs early development. These data should provide a unique resource for further exploration of the species-specific roles of NRs in ESC self-renewal and differentiation.

Apoptotic effects in primary human umbilical vein endothelial cell cultures caused by exposure to virion-associated and cell membrane-associated HIV-1 gp120

Summary: During the course of HIV‐1 infection, free virus, infected cells, and free HIV‐1 proteins circulate within the host, exposing the host endothelium to these viral factors. We have previously presented evidence showing that soluble HIV‐1 gp120 protein interacts with chemokine receptors on primary human endothelium and (through those interactions) induces apoptosis as well as other intracellular effects. The current study examines the effect of exposure of vascular endothelium to gp120 IIIb expressed on the surface of Jurkat cells and in the context of viral particles. Apoptosis was observed in human umbilical vein endothelial cell (HUVEC) cultures exposed to gp160‐transfected Jurkat cells as well as to virion particles with gp120 on their surface. Additional experiments show that this apoptotic effect was caused by gp120 protein acting through chemokine receptors on the HUVEC surface, primarily the CXCR4 receptor. At higher concentrations of gp120, this lymphotrophic variant, which has been shown to interact predominantly with CXCR4, seems to interact with and induce apoptosis through the CCR5 receptor. Finally, this apoptotic effect in HUVEC cultures occurs at low levels of the inducing agent, gp120, on cell membranes or on virion particles. These results demonstrate that HIV‐1 gp120 is capable of interacting with and killing vascular endothelial cells in multiple in vivo contexts.

Mitochondrial Dysfunction and Adipogenic Reduction by Prohibitin Silencing in 3T3-L1 Cells

Increase in mitochondrial biogenesis has been shown to accompany brown and white adipose cell differentiation. Prohibitins (PHBs), comprised of two evolutionarily conserved proteins, prohibitin-1 (PHB1) and prohibitin-2 (PHB2), are present in a high molecular-weight complex in the inner membrane of mitochondria. However, little is known about the effect of mitochondrial PHBs in adipogenesis. In the present study, we demonstrate that the levels of both PHB1 and PHB2 are significantly increased during adipogenesis of 3T3-L1 preadipocytes, especially in mitochondria. Knockdown of PHB1 or PHB2 by oligonucleotide siRNA significantly reduced the expression of adipogenic markers, the accumulation of lipids and the phosphorylation of extracellular signal-regulated kinases. In addition, fragmentation of mitochondrial reticulum, loss of mitochondrial cristae, reduction of mitochondrial content, impairment of mitochondrial complex I activity and excessive production of ROS were observed upon PHB-silencing in 3T3-L1 cells. Our results suggest that PHBs are critical mediators in promoting 3T3-L1 adipocyte differentiation and may be the potential targets for obesity therapies.

Restoration of wild-type infectivity to human immunodeficiency virus type 1 strains lacking nef by intravirion reverse transcription

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Nef protein exerts several effects, both on infected cells and as a virion protein, which work together to enhance viral replication. One of these activities is the ability to enhance infectivity and the formation of proviral DNA. The mechanism of this enhancement remains incompletely understood. We show that virions with nef deleted can be restored to wild-type infectivity by stimulating intravirion reverse transcription. Particle composition and measures of reverse transcriptase activity remain the same for Nef+ and Nef− virions both before and after natural endogenous reverse transcription (NERT) treatment. The effect of NERT treatment on virions pseudotyped with murine leukemia virus envelope protein was similar to that on particles pseudotyped with HIV-1 envelope protein. However, virions pseudotyped with vesicular stomatitis virus G envelope protein showed no influence of Nef on NERT enhancement of infectivity. These observations suggest that Nef may function at a level prior to reverse transcription. Since NERT treatment results in partial disassembly of the viral core, we speculate that Nef may function at the level of core particle disassembly.

Perhexiline activates KLF14 and reduces atherosclerosis by modulating ApoA-I production

Recent genome-wide association studies have revealed that variations near the gene locus encoding the transcription factor Krüppel-like factor 14 (KLF14) are strongly associated with HDL cholesterol (HDL-C) levels, metabolic syndrome, and coronary heart disease. However, the precise mechanisms by which KLF14 regulates lipid metabolism and affects atherosclerosis remain largely unexplored. Here, we report that KLF14 is dysregulated in the liver of 2 dyslipidemia mouse models. We evaluated the effects of both KLF14 overexpression and genetic inactivation and determined that KLF14 regulates plasma HDL-C levels and cholesterol efflux capacity by modulating hepatic ApoA-I production. Hepatic-specific Klf14 deletion in mice resulted in decreased circulating HDL-C levels. In an attempt to pharmacologically target KLF14 as an experimental therapeutic approach, we identified perhexiline, an approved therapeutic small molecule presently in clinical use to treat angina and heart failure, as a KLF14 activator. Indeed, in WT mice, treatment with perhexiline increased HDL-C levels and cholesterol efflux capacity via KLF14-mediated upregulation of ApoA-I expression. Moreover, perhexiline administration reduced atherosclerotic lesion development in apolipoprotein E-deficient mice. Together, these results provide comprehensive insight into the KLF14-dependent regulation of HDL-C and subsequent atherosclerosis and indicate that interventions that target the KLF14 pathway should be further explored for the treatment of atherosclerosis.

The emerging roles of prohibitins in folliculogenesis.

Prohibitins are members of a highly conserved eukaryotic protein family containing the stomatin/prohibitin/flotillin/HflK/C (SPFH) domain (also known as the prohibitin (PHB) domain) found in divergent species from prokaryotes to eukaryotes. Prohibitins are found in unicellular eukaryotes, fungi, plants, animals and humans. Prohibitins are ubiquitously expressed and present in multiple cellular compartments including the mitochondria, nucleus, and the plasma membrane, and shuttles between the mitochondria, cytosol and nucleus. Multiple functions have been attributed to the mitochondrial and nuclear prohibitins, including cellular differentiation, anti-proliferation, and morphogenesis. In the present review, we focus on the recent developments in prohibitins research related to folliculogenesis. Based on current research findings, the data suggest that these molecules play important roles in modulating specific responses of granulose cells to follicle stimulating hormone (FSH) by acting at multiple levels of the FSH signal transduction pathway. Understanding the molecular mechanisms by which the intracellular signaling pathways utilize prohibitins in governing folliculogenesis is likely to result in development of strategies to overcome fertility disorders and suppress ovarian cancer growth.