PGC-1α Downregulation in Steatotic Liver Enhances Ischemia-Reperfusion Injury and Impairs Ischemic Preconditioning.

AIMS: Liver steatosis is associated with mitochondrial dysfunction and elevated reactive oxygen species (ROS) levels together with enhanced sensitivity to ischemia-reperfusion (IR) injury and limited response to preconditioning protocols. Here, we sought to determine whether the downregulation in the steatotic liver of peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α), a master regulator of mitochondrial metabolism and ROS that is known to play a role in liver metabolic control, could be responsible for the sensitivity of the steatotic liver to ischemic damage. RESULTS: PGC-1α was induced in normal liver after exposure to an IR protocol, which was concomitant with an increase in the levels of antioxidant proteins. By contrast, its induction was severely blunted in the steatotic liver, resulting in a modest induction of antioxidant proteins. Livers of PGC-1α-/- mice on a chow diet were normal, but they exhibited an enhanced sensitivity to IR injury and also a lack of response to ischemic preconditioning (IPC), a phenotype that recapitulated the features of the steatotic liver in terms of liver damage, although the inflammatory response differed between both models. Utilizing an in vitro model of IPC, we found that PGC-1α expression was downregulated in hepatic cells cultured at 1% O2; whereas it was induced after reoxygenation (3% O2), and it was responsible for the recovery of antioxidant gene expression after the ischemic period. Innovation & Conclusion: PGC-1α plays an important role in the protection against IR injury in the liver, which is likely associated with its capacity to induce antioxidant gene expression. Antioxid. Redox Signal. 27, 1332-1346.

Single-cell analyses reveal an attenuated NF-κB response in the Salmonella-infected fibroblast.

The eukaryotic transcriptional regulator Nuclear Factor kappa B (NF-κB) plays a central role in the defense to pathogens. Despite this, few studies have analyzed NF-κB activity in single cells during infection. Here, we investigated at the single cell level how NF-κB nuclear localization - a proxy for NF-κB activity - oscillates in infected and uninfected fibroblasts co-existing in cultures exposed to Salmonella enterica serovar Typhimurium. Fibroblasts were used due to the capacity of S. Typhimurium to persist in this cell type. Real-time dynamics of NF-κB was examined in microfluidics, which prevents cytokine accumulation. In this condition, infected (ST+) cells translocate NF-κB to the nucleus at higher rate than the uninfected (ST-) cells. Surprisingly, in non-flow (static) culture conditions, ST- fibroblasts exhibited higher NF-κB nuclear translocation than the ST+ population, with these latter cells turning refractory to external stimuli such as TNF-α or a second infection. Sorting of ST+ and ST- cell populations confirmed enhanced expression of NF-κB target genes such as IL1B, NFKBIA, TNFAIP3, and TRAF1 in uninfected (ST-) fibroblasts. These observations proved that S. Typhimurium dampens the NF-κB response in the infected fibroblast. Higher expression of SOCS3, encoding a "suppressor of cytokine signaling," was also observed in the ST+ population. Intracellular S. Typhimurium subverts NF-κB activity using protein effectors translocated by the secretion systems encoded by pathogenicity islands 1 (T1) and 2 (T2). T1 is required for regulating expression of SOCS3 and all NF-κB target genes analyzed whereas T2 displayed no role in the control of SOCS3 and IL1B expression. Collectively, these data demonstrate that S. Typhimurium attenuates NF-κB signaling in fibroblasts, an effect only perceptible when ST+ and ST- populations are analyzed separately. This tune-down in a central host defense might be instrumental for S. Typhimurium to establish intracellular persistent infections.

Oxidative stress induces loss of pericyte coverage and vascular instability in PGC-1α-deficient mice.

Peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) is a regulator of mitochondrial oxidative metabolism and reactive oxygen species (ROS) homeostasis that is known to be inactivated in diabetic subjects. This study aimed to investigate the contribution of PGC-1α inactivation to the development of oxygen-induced retinopathy. We analyzed retinal vascular development in PGC-1α(-/-) mice. Retinal vasculature of PGC-1α(-/-) mice showed reduced pericyte coverage, a de-structured vascular plexus, and low perfusion. Exposure of PGC-1α(-/-) mice to hyperoxia during retinal vascular development exacerbated these vascular abnormalities, with extensive retinal hemorrhaging and highly unstructured areas as compared with wild-type mice. Structural analysis demonstrated a reduction in membrane-bound VE-cadherin, which was suggestive of defective intercellular junctions. Interestingly, PGC-1α(-/-) retinas showed a constitutive activation of the VEGF-A signaling pathway. This phenotype could be partially reversed by antioxidant administration, indicating that elevated production of ROS in the absence of PGC-1α could be a relevant factor in the alteration of the VEGF-A signaling pathway. Collectively, our findings suggest that PGC-1α control of ROS homeostasis plays an important role in the regulation of de novo angiogenesis and is required for vascular stability.

Control of endothelial function and angiogenesis by PGC-1α relies on ROS control of vascular stability.

Peroxisome proliferator activated receptor g co-activator 1alpha (PGC-1α) is a regulator of oxidative metabolism and reactive oxygen species (ROS) homeostasis that has been show to play a relevant role in angiogenesis. PGC-1α KO mice show reduced vascular density in the retinas and KO primary vascular endothelial cells (ECs) migrate faster than the wild type, an effect that can be rescued by antioxidants, suggesting that excessive ROS levels might be relevant in PGC-1 α role in angiogenesis. This study aims to investigate the role of ROS homeostasis on the regulation by PGC-1 α of angiogenesis. We found that endothelial cells (ECs) from mice deleted for PGC-1 α display attenuated adhesion to the extracellular matrix, together with slower spreading, reduced formation of cellular junctions, a disorganized cytoskeleton and random motility, and a enhanced tip phenotype. Aditionally, PGC-1 α -deleted ECs exhibit an altered response to vascular endothelial growth factor-A (VEGF-A). In vivo, deletion of PGC-1 α results in addition to reduced retinal vascular density, sparse pericyte coverage. Exposure of PGC-1 α deleted mice to hyperoxia during retinal vascular development exacerbates these vascular abnormalities and mice show extensive retinal hemorrhaging, with highly unstructured areas and very poor perfusion, compared with wild-type mice. Structural analysis demonstrates a reduction of endothelial VE-cadherin, suggesting defective inter-cellular junctions. Interestingly, this hyperoxia-induced phenotype is partially reversed by antioxidant administration, indicating that elevated production of mitochondrial reactive oxygen species (ROS) in the absence of PGC-1 α is functionally important. Finally, in vitro studies show that antioxidant treatment improves VEGF-A signaling, suggesting that toxic effect of ROS may be caused by the alteration of the VEGF-A signaling pathway. In summary, our findings indicate that PGC-1 α control of ROS homeostasis plays an important role in the control of de novo angiogenesis, and is required for vascular stability.

Genome expression analysis of nonproliferating intracellular Salmonella enterica serovar Typhimurium unravels an acid pH-dependent PhoP-PhoQ response essential for dormancy.

Genome-wide expression analyses have provided clues on how Salmonella proliferates inside cultured macrophages and epithelial cells. However, in vivo studies show that Salmonella does not replicate massively within host cells, leaving the underlying mechanisms of such growth control largely undefined. In vitro infection models based on fibroblasts or dendritic cells reveal limited proliferation of the pathogen, but it is presently unknown whether these phenomena reflect events occurring in vivo. Fibroblasts are distinctive, since they represent a nonphagocytic cell type in which S. enterica serovar Typhimurium actively attenuates intracellular growth. Here, we show in the mouse model that S. Typhimurium restrains intracellular growth within nonphagocytic cells positioned in the intestinal lamina propria. This response requires a functional PhoP-PhoQ system and is reproduced in primary fibroblasts isolated from the mouse intestine. The fibroblast infection model was exploited to generate transcriptome data, which revealed that ∼2% (98 genes) of the S. Typhimurium genome is differentially expressed in nongrowing intracellular bacteria. Changes include metabolic reprogramming to microaerophilic conditions, induction of virulence plasmid genes, upregulation of the pathogenicity islands SPI-1 and SPI-2, and shutdown of flagella production and chemotaxis. Comparison of relative protein levels of several PhoP-PhoQ-regulated functions (PagN, PagP, and VirK) in nongrowing intracellular bacteria and extracellular bacteria exposed to diverse PhoP-PhoQ-inducing signals denoted a regulation responding to acidic pH. These data demonstrate that S. Typhimurium restrains intracellular growth in vivo and support a model in which dormant intracellular bacteria could sense vacuolar acidification to stimulate the PhoP-PhoQ system for preventing intracellular overgrowth.

Induction of PGC-1α expression can be detected in blood samples of patients with ST-segment elevation acute myocardial infarction.

Following acute myocardial infarction (MI), cardiomyocyte survival depends on its mitochondrial oxidative capacity. Cell death is normally followed by activation of the immune system. Peroxisome proliferator activated receptor γ-coactivator 1α (PGC-1α) is a transcriptional coactivator and a master regulator of cardiac oxidative metabolism. PGC-1α is induced by hypoxia and facilitates the recovery of the contractile capacity of the cardiac muscle following an artery ligation procedure. We hypothesized that PGC-1α activity could serve as a good molecular marker of cardiac recovery after a coronary event. The objective of the present study was to monitor the levels of PGC-1α following an ST-segment elevation acute myocardial infarction (STEMI) episode in blood samples of the affected patients. Analysis of blood mononuclear cells from human patients following an STEMI showed that PGC-1α expression was increased and the level of induction correlated with the infarct size. Infarct size was determined by LGE-CMR (late gadolinium enhancement on cardiac magnetic resonance), used to estimate the percentage of necrotic area. Cardiac markers, maximum creatine kinase (CK-MB) and Troponin I (TnI) levels, left ventricular ejection function (LVEF) and regional wall motion abnormalities (RWMA) as determined by echocardiography were also used to monitor cardiac injury. We also found that PGC-1α is present and active in mouse lymphocytes where its expression is induced upon activation and can be detected in the nuclear fraction of blood samples. These results support the notion that induction of PGC-1α expression can be part of the recovery response to an STEMI and could serve as a prognosis factor of cardiac recovery.

PGC-1α regulates translocated in liposarcoma activity: role in oxidative stress gene expression.

Translocated in liposarcoma (TLS) is a poorly characterized multifunctional protein involved in the genotoxic response. TLS regulates gene expression at several steps, including splicing and mRNA transport, possibly connecting transcriptional and posttranscriptional events. AIMS: In this study we aimed to idenfity molecular targets and regulatory partners of TLS. RESULTS AND INNOVATION: Here we report that TLS transcriptionally regulates the expression of oxidative stress protection genes. This regulation requires interaction with the transcriptional coactivator peroxisome proliferator activated receptor γ-coactivator 1α (PGC-1α), a master regulator of mitochondrial function that coordinately induces the expression of genes involved in detoxification of mitochondrial reactive oxygen species (ROS). Microarray gene expression analysis showed that TLS transcriptional activity is impaired in the absence of PGC-1α, and is thus largely dependent on PGC-1α. CONCLUSION: These results suggest the existence of a regulatory circuit linking the control of ROS detoxification to the coordinated cross-talk between oxidative metabolism and the cellular response to genomic DNA damage.

Mutual dependence of Foxo3a and PGC-1alpha 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-1alpha, because it is severely curtailed in PGC-1alpha-deficient endothelial cells. Foxo3a and PGC-1alpha 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-1alpha interact directly to regulate oxidative stress protection genes in the vascular endothelium is supported by the observation that PGC-1alpha 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-1alpha, suggesting that an auto-regulatory cycle regulates Foxo3a/PGC-1alpha control of the oxidative stress response.

Salmonella enterica serovar Choleraesuis derivatives harbouring deletions in rpoS and phoP regulatory genes are attenuated in pigs, and survive and multiply in porcine intestinal macrophages and fibroblasts, respectively.

Live attenuated Salmonella enterica strains have been extensively studied as potential vectors for the oral delivery of heterologous antigens. Due to its ability to target immune cells, its specific mechanism for crossing the intestinal barrier, and its swine-restricted tropism, S. enterica subspecies enterica serovar Choleraesuis (S. Choleraesuis) has attracted a great deal of interest for the production of bacterial-based oral carriers specifically adapted to swine. In this study, two mutants of S. Choleraesuis were constructed and their attenuation and intracellular fate analysed with the purpose of engineering new attenuated live strains with improved properties as oral vaccine carriers. Those strains harboured a specific deletion either within the phoP or rpoS genes, which encode virulence-related regulators in S. Typhimurium. In comparison to the wild-type parental S. Choleraesuis, the mutant strains, especially DeltaphoP, were extremely low in virulence in the murine model and in the natural host, the pig. Moreover, when compared with a commercial live vaccine strain, SC-54, the two mutants showed a higher level of attenuation in mice and DeltaphoP also in pigs. In addition, DeltarpoS and DeltaphoP presented a proliferation and survival phenotype within swine intestinal primary fibroblast and macrophage cell cultures, respectively. Collectively, the present results indicate that the DeltarpoS and DeltaphoP strains of S. Choleraesuis gather adequate features to be potential candidates for vaccine vectors for the specific delivery of heterologous antigens adapted to pigs.

New concepts in Salmonella virulence: the importance of reducing the intracellular growth rate in the host.

The literature refers to Salmonella enterica as an intracellular bacterial pathogen that proliferates within vacuoles of mammalian cells. However, recent in vivo studies have revealed that the vast majority of infected cells contain very few intracellular bacteria (three to four organisms). Salmonella intracellular growth is also limited in cultured dendritic cells and fibroblasts, two cell types abundant in tissues located underneath the intestinal epithelium. Recently, a Salmonella factor previously known for its role as a negative regulator of intracellular growth has been shown to tightly repress certain pathogen functions upon host colonization and to be critical for virulence. The connection between virulence and the negative control of intracellular growth is further sustained by the fact that some attenuated mutants overgrow in non-phagocytic cells located in the intestinal lamina propria. These findings are changing our classical view of Salmonella as a fast growing intracellular pathogen and suggest that this pathogen may trigger responses directed to reduce the growth rate within the infected cell. These responses could play a critical role in modulating the delicate balance between disease and persistence.

The Salmonella membrane protein IgaA modulates the activity of the RcsC-YojN-RcsB and PhoP-PhoQ regulons.

The Salmonella enterica serovar Typhimurium membrane protein IgaA and the PhoP-PhoQ two-component system are used by this pathogen to attenuate the intracellular growth rate within fibroblasts. IgaA has also recently been shown to contribute to virulence by exerting tight repression of the RcsC-YojN-RcsB phosphorelay in host tissues. Here we show that loss of repression of the RcsC-YojN-RcsB system, linked to an R188H mutation in the IgaA protein (igaA1 allele), is accompanied by altered expression of PhoP-PhoQ-activated (pag) genes. The changes in gene expression were different depending on the specific pag gene analyzed. Thus, transcription of ugd, which is required for lipopolysaccharide modification and colanic acid capsule synthesis, was enhanced in the igaA1 mutant. RcsB and its coregulator RcsA promoted this alteration in a PhoP-PmrA-independent manner. Unlike ugd, activation of the RcsC-YojN-RcsB phosphorelay negatively affected the expression of all other pag genes tested. In this case, RcsB alone was responsible for this effect. We also found that PhoP, but not PmrA, negatively modulates the expression of gmm, a gene required for colanic acid synthesis that is regulated positively by RcsC-YojN-RcsB. Finally, it was observed that the fine regulation of pag genes exerted by RcsB requires the RpoS protein and that an active RcsB, but not RcsA, diminishes expression of the phoP gene. These data support the hypothesis that in Salmonella there is an intimate regulatory circuit between the PhoP-PhoQ and RcsC-YojN-RcsB phosphorelays, which is revealed only when the RcsC-YojN-RcsB signaling route is derepressed. Consistent with the phenotypes observed in fibroblast cells, IgaA is predicted to favor expression of the entire PhoP-PhoQ regulon based on its repression of the RcsC-YojN-RcsB phosphorelay.

Regulation of capsule synthesis and cell motility in Salmonella enterica by the essential gene igaA.

Mutants of Salmonella enterica carrying the igaA1 allele, selected as able to overgrow within fibroblast cells in culture, are mucoid and show reduced motility. Mucoidy is caused by derepression of wca genes (necessary for capsule synthesis); these genes are regulated by the RcsC/YojN/RcsB phosphorelay system and by the RcsA coregulator. The induction of wca expression in an igaA1 mutant is suppressed by mutations in rcsA and rcsC. Reduced motility is caused by lowered expression of the flagellar master operon, flhDC, and is suppressed by mutations in rcsB or rcsC, suggesting that mutations in the igaA gene reduce motility by activating the RcsB/C system. A null igaA allele can be maintained only in an igaA(+)/igaA merodiploid, indicating that igaA is an essential gene. Lethality is suppressed by mutations in rcsB, rcsC, and yojN, but not in rcsA, suggesting that the viability defect of an igaA null mutant is mediated by the RcsB/RcsC system, independently of RcsA (and therefore of the wca genes). Because all the defects associated with igaA mutations are suppressed by mutations that block the RcsB/RcsC system, we propose a functional interaction between the igaA gene product and either the Rcs regulatory network or one of its regulated products.