Ana M. Cervera

A targeted antioxidant reveals the importance of mitochondrial reactive oxygen species in the hypoxic signaling of HIF‐1α

Exposure to limiting oxygen in cells and tissues induce the stabilization and transcriptional activation of the hypoxia‐inducible factor 1 alpha (HIF‐1α) protein, a key regulator of the hypoxic response. Reactive oxygen species (ROS) generation has been implicated in the stabilization of HIF‐1α during this response, but this is still a matter of some debate. In this study we utilize a mitochondria‐targeted antioxidant, mitoubiquinone (MitoQ), and examine its effects on the hypoxic stabilization of HIF‐1α. Our results show that under conditions of reduced oxygen (3% O2), MitoQ ablated the hypoxic induction of ROS generation and destabilized HIF‐1α protein. This in turn led to an abrogation of HIF‐1 transcriptional activity. Normoxic stabilization of HIF‐1α, on the other hand, was unchanged in the presence of MitoQ suggesting that ROS were not involved. This study strongly suggests that mitochondrial ROS contribute to the hypoxic stabilization of HIF‐1α.

Inhibition of succinate dehydrogenase dysregulates histone modification in mammalian cells

Remodelling of mitochondrial metabolism is a hallmark of cancer. Mutations in the genes encoding succinate dehydrogenase (SDH), a key Krebs cycle component, are associated with hereditary predisposition to pheochromocytoma and paraganglioma, through mechanisms which are largely unknown. Recently, the jumonji-domain histone demethylases have emerged as a novel family of 2-oxoglutarate-dependent chromatin modifiers with credible functions in tumourigenesis. Using pharmacological and siRNA methodologies we show that increased methylation of histone H3 is a general consequence of SDH loss-of-function in cultured mammalian cells and can be reversed by overexpression of the JMJD3 histone demethylase. ChIP analysis revealed that the core promoter of IGFBP7, which encodes a secreted protein upregulated after loss of SDHB, showed decreased occupancy by H3K27me3 in the absence of SDH. Finally, we provide the first evidence that the chief (type I) cell is the major methylated histone-immunoreactive constituent of paraganglioma. These results support the notion that loss of mitochondrial function alters epigenetic processes and might provide a signature methylation mark for paraganglioma.

Mitochondrial Reactive Oxygen Species Mediate Cardiomyocyte Formation from Embryonic Stem Cells in High Glucose

Accumulating evidence points to reactive oxygen species (ROS) as important signaling molecules for cardiomyocyte differentiation in embryonic stem (ES) cells. Given that ES cells are normally maintained and differentiated in medium containing supraphysiological levels of glucose (25 mM), a condition which is known to result in enhanced cellular ROS formation, we questioned whether this high glucose concentration was necessary for cardiomyocyte lineage potential. We show here that ES cells cultured in physiological glucose (5 mM), maintained their general stemness qualities but displayed an altered mitochondrial metabolism, which resulted in decreased ROS production. Furthermore, ES and induced pluripotent stem (iPS) cells differentiated in lower glucose concentrations failed to generate cardiomyocyte structures; an effect mimicked with antioxidant treatments using catalase, N‐acetyl cysteine and mitoubiquinone, under high glucose conditions in ES cells. Molecular analysis revealed that ES cells differentiated in 5 mM glucose had reduced expression of the pro‐cardiac NOX4 gene and diminished phosphorylation of p38 mitogen‐activated protein kinase (MAPK), together with specific changes in the cardiac transcriptional network. These outcomes could be reversed by supplementation of low glucose cultures with ascorbic acid, paradoxically acting as a pro‐oxidant. Furthermore, forced expression of an upstream p38 MAPK kinase (MKK6) could bypass the requirement for ROS during differentiation to cardiomyocytes under low glucose conditions, illustrating a key role for p38 in the cardiac differentiation program. Together these data demonstrate that endogenous ROS control is important for cardiomyocyte formation from ES cells, and furthermore that supraphysiological glucose, by supplying ROS, is absolutely required. STEM CELLS 2010;28:1132–1142

Cells Silenced for SDHB Expression Display Characteristic Features of the Tumor Phenotype

Recently, enzymes of the tricarboxylic acid (TCA) cycle have emerged as novel tumor suppressors. In particular, mutations in the nuclear-encoded subunits of succinate dehydrogenase (SDHB, SDHC, and SDHD) cause paragangliomas and pheochromocytomas. Although the mechanism(s) by which disruption of mitochondrial metabolism leads to neoplasia is largely unknown, increasing evidence points to an activation of pseudohypoxia. In this study, we have shown that silencing of SDHB using DNA-based small interfering RNA resulted in major impairments in cellular proliferation, respiration, and a corresponding shift to glycolysis. The levels of reactive oxygen species, however, were unchanged. As expected, hypoxia-inducible factor-1 alpha (HIF-1 alpha) and HIF-2alpha were up-regulated in chronically silenced cells, suggesting that a pseudohypoxic state was attained. In addition, the c-Jun amino-terminal kinase and p38 kinase stress signaling proteins were hyperphosphorylated in SDHB-silenced cells. Microarray analysis showed that >400 genes were influenced (6-fold or more up-regulation or down-regulation) by silencing of SDHB, confirming the importance of the TCA cycle in cellular metabolism. Examples of dysregulated genes included those involved in proliferation, adhesion, and the hypoxia pathway. Of interest, SDHB-silenced cells had a greater capacity to adhere to extracellular matrix components, including fibronectin and laminin, than control cells, thus suggesting a possible mechanism of tumor initiation. Although transient silencing of the HIF-1 alpha transcription factor in SDHB-silenced cells had little effect on the expression of a subset of up-regulated genes, it partially reversed the adhesion phenotype to fibronectin, pointing to a potentially important role for HIF-1 in this process.

ICOS controls Foxp3+ regulatory T‐cell expansion, maintenance and IL‐10 production during helminth infection

Foxp3+ regulatory T (Treg) cells are key immune regulators during helminth infections, and identifying the mechanisms governing their induction is of principal importance for the design of treatments for helminth infections, allergies and autoimmunity. Little is yet known regarding the co‐stimulatory environment that favours the development of Foxp3+ Treg‐cell responses during helminth infections. As recent evidence implicates the co‐stimulatory receptor ICOS in defining Foxp3+ Treg‐cell functions, we investigated the role of ICOS in helminth‐induced Foxp3+ Treg‐cell responses. Infection of ICOS−/− mice with Heligmosomoides polygyrus or Schistosoma mansoni led to a reduced expansion and maintenance of Foxp3+ Treg cells. Moreover, during H. polygyrus infection, ICOS deficiency resulted in increased Foxp3+ Treg‐cell apoptosis, a Foxp3+ Treg‐cell specific impairment in IL‐10 production, and a failure to mount putatively adaptive Helios−Foxp3+ Treg‐cell responses within the intestinal lamina propria. Impaired lamina propria Foxp3+ Treg‐cell responses were associated with increased production of IL‐4 and IL‐13 by CD4+ T cells, demonstrating that ICOS dominantly downregulates Type 2 responses at the infection site, sharply contrasting with its Type 2‐promoting effects within lymphoid tissue. Thus, ICOS regulates Type 2 immunity in a tissue‐specific manner, and plays a key role in driving Foxp3+ Treg‐cell expansion and function during helminth infections.

The role of ICOS in the development of CD4 T cell help and the reactivation of memory T cells

We have addressed the role of the inducible costimulator (ICOS) in the development of T cell help for B cells and in the generation, survival and reactivation of memory CD4 T cells and B cells. We find that while T cell help for all antibody isotypes (including IgG2c) is impaired in ICOS knockout (ICOS‐KO) mice, the IFN‐γ response is little affected, indicating a defect in helper function that is unrelated to cytokine production. In addition, the ICOS‐negative T cells do not accumulate in B cell follicles. Secondary (memory), but not primary, clonal proliferation of antigen‐specific B cells is impaired in ICOS‐KO mice, as is the generation of secondary antibody‐secreting cells. Analysis of endogenous CD4 memory cells in ICOS‐KO mice, using MHC class II tetramers, reveals normal primary clonal expansion, formation of memory clones and long‐term (10 wk) survival of memory cells, but defective expansion upon reactivation in vivo. The data point to a role of ICOS in supporting secondary, memory and effector T cell responses, possibly by influencing cell survival. The data also highlight differences in ICOS dependency of endogenous T cell proliferation in vivo compared to that of adoptively transferred TCR‐transgenic T cells.

Mitochondria Determine the Differentiation Potential of Cardiac Mesoangioblasts

An understanding of cardiac progenitor cell biology would facilitate their therapeutic potential for cardiomyocyte restoration and functional heart repair. Our previous studies identified cardiac mesoangioblasts as precommitted progenitor cells from the postnatal heart, which can be expanded in vitro and efficiently differentiated in vitro and in vivo to contribute new myocardium after injury. Based on their proliferation potential in culture, we show here that two populations of mesoangioblasts can be isolated from explant cultures of mouse and human heart. Although both populations express similar surface markers, together with a panel of instructive cardiac transcription factors, they differ significantly in their cellular content of mitochondria. Slow dividing (SD) cells, containing many mitochondria, can be efficiently differentiated with 5‐azacytidine (5‐aza) to generate cardiomyocytes expressing mature structural markers. In contrast, fast dividing (FD) mesoangioblasts, which contain decreased quantities of mitochondria, do not respond to 5‐aza treatment. Notably, increasing mitochondrial numbers using pharmacological nitric oxide (NO) donors reverses the differentiation block in FD mesoangioblasts and leads to a progressive maturation to cardiomyocytes; conversely decreasing mitochondrial content, using respiratory chain inhibitors and chloramphenicol, perturbs cardiomyocyte differentiation in SD populations. Furthermore, isolated cardiac mesoangioblasts from aged mouse and human hearts are found to be almost exclusively mitochondria‐low FD populations, which are permissive for cardiomyocyte differentiation only after NO treatment. Taken together, this study illustrates a key role for mitochondria in cardiac mesoangioblast differentiation and raises the interesting possibility that treatments, which increase cellular mitochondrial content, may have utility for cardiac stem cell therapy. STEM CELLS 2011;29:1064–1074

Targeted Disruption of the SUCNR1 Metabolic Receptor Leads to Dichotomous Effects on Obesity

A number of metabolites have signaling properties by acting through G-protein–coupled receptors. Succinate, a Krebs cycle intermediate, increases after dysregulated energy metabolism and can bind to its cognate receptor succinate receptor 1 (Sucnr1, or GPR91) to activate downstream signaling pathways. We show that Sucnr1 is highly expressed in the white adipose tissue (WAT) compartment of mice and regulates adipose mass and glucose homeostasis. Sucnr1−/− mice were generated, and weight gain was monitored under basal and nutritional stress (high-fat diet [HFD]) conditions. On chow diet, Sucnr1−/− mice had increased energy expenditure, were lean with a smaller WAT compartment, and had improved glucose buffering. Lipolysis measurements revealed that Sucnr1−/− mice were released from succinate-induced inhibition of lipolysis, demonstrating a function of Sucnr1 in adipose tissue. Sucnr1 deletion also protected mice from obesity on HFD, but only during the initial period; at later stages, body weight of HFD-fed Sucnr1−/− mice was almost comparable with wild-type (WT) mice, but WAT content was greater. Also, these mice became progressively hyperglycemic and failed to secrete insulin, although pancreas architecture was similar to WT mice. These findings suggest that Sucnr1 is a sensor for dietary energy and raise the interesting possibility that protocols to modulate Sucnr1 might have therapeutic utility in the setting of obesity.

Molecular cloning and characterization of a Candida albicans gene coding for cytochrome c haem lyase and a cell wall-related protein

Immunoscreening of a Candida albicans cDNA library with a monoclonal antibody (mAb 4C12) recognizing an epitope present in high‐molecular‐weight mannoprotein (HMWM) components specific for the mycelial cell walls (a 180 kDa component and a polydispersed 260 kDa species) resulted in the isolation of the gene CaCYC3 encoding for cytochrome c haem lyase (CCHL). The CaCYC3 gene was transcribed preferentially in mycelial cells in which two mRNA transcripts of 0.8 and 1 kb were found. The nucleotide and the deduced amino acid sequences of this gene displayed 45% homology and 46% identity, respectively, to the Saccharomyces cerevisiae CYC3 gene and shared common features with other reported genes encoding for CCHL. The CaCYC3 gene restored the respiratory activity when transformed in a S. cerevisiae cyc3 − mutant strain. A C. albicans CYC3 null mutant was constructed after sequential disruption using the hisG ::URA3 ::hisG (‘ura‐blaster’) cassette. Null mutant cells were unable to use lactate as a sole carbon source and had a reduced ability to form germ tubes. Western immunoblotting analysis of subcellular fractions from wild‐type and null mutant strains demonstrated the presence of two gene products, a 33 kDa mitochondrial protein and a 40 kDa cell wall‐associated moiety reacting with antibodies against CCHL, in both yeast cells and germ tubes. mAb 4C12 still reacted with the CaCYC3 null mutant (by immunofluorescence and immunoblotting) but showed an altered pattern of immunoreactivity against cell wall HMWM species, indicating a relationship between these moieties and the CaCYC3 gene products. The results suggest that the CaCYC3 gene encodes two proteins, one targeted to the mitochondria and the other to the cell wall.

Molecular Cloning of a Candida albicans Gene (SSB1) Coding for a Protein Related to the Hsp70 Family

We have cloned and sequenced a Candida albicans gene (SSB1) encoding a potential member of the heat‐shock protein seventy (hsp70) family. The protein encoded by this gene contains 613 amino acids and shows a high degree (85%) of sequence identity to the ssb subfamily (ssb1 and ssb2) of the Saccharomyces cerevisiae hsp70 family. The transcribed mRNA (2·1 kb) is present in similar amounts both in yeast and germ tube cells of C. albicans. The sequence data has been deposited in the GenBank data library under the Accession Number X97723.