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Hypoxia occurs in pathological conditions, such as cancer, as a result of the imbalance between oxygen supply and consumption by proliferating cells. HIFs are critical molecular mediators of the physiological response to hypoxia but also regulate multiple steps of carcinogenesis including tumor progression and metastasis. Recent data support that sumoylation, the covalent attachment of the Small Ubiquitin-related MOdifier (SUMO) to proteins, is involved in the activation of the hypoxic response and the ensuing signaling cascade. To gain insights into differences of the SUMO1 and SUMO2/3 proteome of HeLa cells under normoxia and cells grown for 48 h under hypoxic conditions, we employed endogenous SUMO-immunoprecipitation in combination with quantitative mass spectrometry (SILAC). The group of proteins whose abundance was increased both in the total proteome and in the SUMO IPs from hypoxic conditions was enriched in enzymes linked to the hypoxic response. In contrast, proteins whose SUMOylation status changed without concommitant change in abundance were predominantly transcriptions factors or transcription regulators. Particularly interesting was transcription factor TFAP2a (Activating enhancer binding Protein 2 alpha), whose sumoylation decreased upon hypoxia. TFAP2a is known to interact with HIF-1 and we provide evidence that deSUMOylation of TFAP2a enhances the transcriptional activity of HIF-1 under hypoxic conditions. Overall, these results support the notion that SUMO-regulated signaling pathways contribute at many distinct levels to the cellular response to low oxygen.

Hypoxia-inducible factors (HIF) are master regulators of the response to hypoxia. Although several kinases are known to modify their oxygen sensitive HIF-α subunits or affect indirectly their function, little is known about the role of phosphatases in HIF control. To address this issue, a library containing siRNAs for the 25 known catalytic subunits of human phosphatases was used to screen for their effect on HIF transcriptional activity in HeLa cells. Serine-threonine phosphatase PPP3CA (calcineurin A, isoform a) was identified as the strongest candidate for a negative regulator of HIF activity. Indeed, independent silencing of PPP3CA expression stimulated HIF transcriptional activity under hypoxia, without increasing the protein levels of HIF-1α or HIF-2α. Overexpression of a constitutively active PPP3CA form, but not its catalytically inactive counterpart, inhibited HIF activity and expression of HIF target genes but did not affect HIF-1α or HIF-2α expression. These results were phenocopied by treatment with the ionophore ionomycin, that activates endogenous PPP3CA. The effect of ionomycin was mediated by PPP3CA as it was largely abolished by PPP3CA silencing. Furthermore, ionomycin enhanced the down-regulation of HIF activity by wild-type PPP3CA overexpression. Overall, our results suggest the involvement of PPP3CA in fine-tuning the HIF-dependent transcriptional response to hypoxia.

HIF-1alpha is the inducible subunit of the dimeric transcription factor HIF-1 (Hypoxia Inducible Factor 1). It is induced by hypoxia and hypoxia-mimetics in most cell types, as well as non-hypoxic signals such as growth factors, cytokines and oncogenes, often in a cell specific manner. HIF-1 is present in virtually all cells of higher eukaryotes and its function is of great biomedical relevance since it is highly involved in development, tumor progression and tissue ischemia. Intracellular signaling to HIF-1alpha, as well as its further action, involves its participation in numerous protein complexes. Using the yeast two-hybrid system we have identified MgcRacGAP (male germ cell Rac GTPase Activating Protein) as a HIF-1alpha interacting protein. The MgcRacGAP protein is a regulator of Rho proteins, which are principally involved in cytoskeletal organization. We have verified specific binding of HIF-1alpha and MgcRacGAP in vitro and in vivo in mammalian cells. We have additionally shown that MgcRacGAP overexpression inhibits HIF-1alpha transcriptional activity, without lowering HIF-1alpha protein levels, or altering its subcellular localization. Moreover, this inhibition is dependent on the MgcRacGAP domain that interacts with HIF-1alpha. In conclusion, our findings demonstrate that HIF-1alpha function is negatively affected by its interaction with MgcRacGAP.

Quercetin, a flavonoid with anti-oxidant, metal chelating, kinase modulating and anti-proliferative properties, can induce hypoxia-inducible factor-1alpha (HIF-1alpha) in normoxia, but its mechanism of action has not been determined. In this study we characterized the induction of HIF-1alpha and the inhibition of cell proliferation caused by quercetin in HeLa and ASM (airway smooth muscle) cells and examined the effect of iron on these processes. Furthermore, we investigated the relevance of the intracellular levels of quercetin to HIF-1alpha expression and cell proliferation. Our data demonstrate that quercetin depletes intracellular calcein-chelatable iron and that supplying additional iron from extracellular or intracellular pools abrogates the induction of HIF-1alpha by quercetin. Moreover, addition of iron reverses the quercetin-induced inhibition of DNA synthesis, cell proliferation and cycle progression, but to different extents, depending on cell type. We propose that quercetin stabilises HIF-1alpha and inhibits cell proliferation predominantly by decreasing the concentration of intracellular iron through chelation.

Cajal bodies are nuclear structures that are involved in biogenesis of snRNPs and snoRNPs, maintenance of telomeres and processing of histone mRNA. Recently, the SUMO isopeptidase USPL1 was identified as a component of Cajal bodies that is essential for cellular growth and Cajal body integrity. However, a cellular function for USPL1 is so far unknown. Here, we use RNAi-mediated knockdown in human cells in combination with biochemical and fluorescence microscopy approaches to investigate the function of USPL1 and its link to Cajal bodies. We demonstrate that levels of snRNAs transcribed by RNA polymerase (RNAP) II are reduced upon knockdown of USPL1 and that downstream processes such as snRNP assembly and pre-mRNA splicing are compromised. Importantly, we find that USPL1 associates directly with U snRNA loci and that it interacts and colocalises with components of the Little Elongation Complex, which is involved in RNAPII-mediated snRNA transcription. Thus, our data indicate that USPL1 plays a key role in RNAPII-mediated snRNA transcription.

BACKGROUND: Epstein-Barr virus (EBV) encodes six nuclear transformation-associated proteins that induce extensive changes in cellular gene expression and signaling and induce B-cell transformation. The role of HIF1A in EBV-induced B-cell immortalization has not been previously studied. METHODS AND FINDINGS: Using Western blotting and Q-PCR, we found that HIF1A protein is stabilized in EBV-transformed lymphoblastoid cells. Western blotting, GST pulldown assays, and immunoprecipitation showed that EBV-encoded nuclear antigens EBNA-5 and EBNA-3 bind to prolylhydroxylases 1 and 2, respectively, thus inhibiting HIF1A hydroxylation and degradation. Immunostaining and Q-PCR showed that the stabilized HIF1A translocates to the nucleus, forms a heterodimer with ARNT, and transactivates several genes involved in aerobic glycolysis. Using biochemical assays and Q-PCR, we also found that lymphoblastoid cells produce high levels of lactate, lactate dehydrogenase and pyruvate. CONCLUSIONS: Our data suggest that activation of the aerobic glycolytic pathway, corresponding to the Warburg effect, occurs in EBV-transformed lymphoblastoid cells, in contrast to mitogen-activated B-cells.

Airway smooth muscle (ASM) cells are characterized by phenotypic plasticity and can switch between differentiated and proliferative phenotypes. In rabbit tracheal ASM cells that had been differentiated in vitro by serum starvation, readdition of FBS caused initiation of proliferation and induction of nuclear and transcriptionally active hypoxia-inducible factor (HIF)-1alpha. In addition, FBS stimulated the induction of HIF-1alpha by the hypoxia mimetic cobalt. Treatment with actinomycin D, cycloheximide, the phosphatidylinositol 3-kinase inhibitors LY-294002 and wortmannin or the reactive oxygen species scavenger diphenyleneiodonium inhibited the FBS-dependent induction of HIF-1alpha. These data indicate that, in differentiated ASM cells, FBS upregulates HIF-1alpha by a transcription-, translation-, phosphatidylinositol 3-kinase-, and reactive oxygen species-dependent mechanism. Interestingly, addition of FBS and cobalt also induced HIF-1alpha in organ cultures of rabbit trachea strips and synergistically increased their contractile response to ACh, suggesting that HIF-1alpha might be implicated in airway hypercontractility.

Hypoxia-inducible factor 1 (HIF-1) controls the expression of most genes induced by hypoxic conditions. Regulation of expression and activity of its inducible subunit, HIF-1alpha, involves several post-translational modifications. To study HIF-1alpha phosphorylation, we have used human full-length recombinant HIF-1alpha as a substrate in kinase assays. We show that at least two different nuclear protein kinases, one of them identified as p42/p44 MAPK, can modify HIF-1alpha. Analysis of in vitro phosphorylated HIF-1alpha by mass spectroscopy revealed residues Ser-641 and Ser-643 as possible MAPK phosphorylation sites. Site-directed mutagenesis of these residues reduced significantly the phosphorylation of HIF-1alpha. When these mutant forms of HIF-1alpha were expressed in HeLa cells, they exhibited much lower transcriptional activity than the wild-type form. However, expression of the same mutants in yeast revealed that their capacity to stimulate transcription was not significantly compromised. Localization of the green fluorescent protein-tagged HIF-1alpha mutants in HeLa cells showed their exclusion from the nucleus in contrast to wild-type HIF-1alpha. Treatment of the cells with leptomycin B, an inhibitor of the major exportin CRM1, reversed this exclusion and led to nuclear accumulation and partial recovery of the activity of the HIF-1alpha mutants. Moreover, inhibition of the MAPK pathway by PD98059 impaired the phosphorylation, nuclear accumulation, and activity of wild-type GFP-HIF-1alpha. Overall, these data suggest that phosphorylation of Ser-641/643 by MAPK promotes the nuclear accumulation and transcriptional activity of HIF-1alpha by blocking its CRM1-dependent nuclear export.

Cobalt can mimic hypoxia and has been implicated as a cause of lung defects. However, the effect of cobalt on airway smooth muscle (ASM) cells has not been analyzed in detail. In this article, we use primary cultures of ASM cells from rabbit trachea and show that exposure to cobalt chloride causes a rapid increase of the intracellular levels of hypoxia-inducible factor-1alpha, which is detected predominantly inside the nucleus. With the use of specific inhibitors, we demonstrate that induction of hypoxia-inducible factor-1alpha by cobalt depends on active protein synthesis but not transcription. Furthermore, wortmannin, LY294002, and N-acetyl-L-cysteine inhibit the effect of cobalt, suggesting that it involves the phosphatidylinositol 3 kinase pathway and production of reactive oxygen species. Interestingly, cobalt chloride attenuates the contractile response of rabbit airways induced by potassium chloride, but not by acetylcholine, suggesting a link between the cellular response to hypoxic stimuli and the contractile properties of ASM cells.