Expression of AGPAT2, an enzyme involved in the glycerophospholipid/triacylglycerol biosynthesis pathway, is directly regulated by HIF-1 and promotes survival and etoposide resistance of cancer cells under hypoxia.

Hypoxia inducible factor-1 (HIF-1) supports survival of normal cells under low oxygen concentration and cancer cells in the hypoxic tumor microenvironment. This involves metabolic reprogramming via upregulation of glycolysis, downregulation of oxidative phosphorylation and, less well documented, effects on lipid metabolism. To investigate the latter, we examined expression of relevant enzymes in cancer cells grown under hypoxia. We show that expression of acylglycerol-3-phosphate acyltransferase 2 (AGPAT2), also known as lysophosphatidic acid acyltransferase ß (LPAATß), was upregulated under hypoxia and this was impaired by siRNA-mediated knockdown of HIF-1α. Moreover, a sequence of the AGPAT2 gene promoter region, containing 6 putative Hypoxia Response Elements (HREs), activated transcription of a reporter gene under hypoxic conditions or in normoxic cells over-expressing HIF-1α. Chromatin immunoprecipitation experiments confirmed binding of HIF-1α to one of these HREs, mutation of which abolished hypoxic activation of the AGPAT2 promoter. Knockdown of AGPAT2 by siRNA reduced lipid droplet accumulation and cell viability under hypoxia and increased cancer cell sensitivity to the chemotherapeutic etoposide. In conclusion, our findings demonstrate that AGPAT2, which is mutated in patients with congenital generalized lipodystrophy and over-expressed in different types of cancer, is a direct transcriptional target of HIF-1, suggesting that upregulation of lipid storage by HIF-1 plays an important role in adaptation and survival of cancer cells under low oxygen conditions.

MgcRacGAP, a cytoskeleton regulator, inhibits HIF-1 transcriptional activity by blocking its dimerization.

Hypoxia inducible factor-1 (HIF-1), a dimeric transcription factor of the bHLH-PAS family, is comprised of HIF-1α, which is inducible by hypoxia and ARNT or HIF-1ß, which is constitutively expressed. HIF-1 is involved in cellular homeostasis under hypoxia, in development and in several diseases affected by oxygen availability, particularly cancer. Since its expression is positively correlated with poor outcome prognosis for cancer patients, HIF-1 is a target for pharmaceutical therapy. We have previously shown that male germ cell Rac GTPase activating protein (MgcRacGAP), a regulator of Rho proteins which are principally involved in cytoskeletal organization, binds to HIF-1α and inhibits its transcriptional activity. In this work, we have explored the mechanism of the MgcRacGAP-mediated HIF-1 inactivation. We show that the Myo domain of MgcRacGAP, which is both necessary and sufficient for HIF-1 repression, binds to the PAS-B domain of HIF-1α. Furthermore MgcRacGAP competes with ARNT for binding to the HIF-1α PAS-B domain, as shown by in vitro binding pull down assays. In mammalian cells, ARNT overexpression can overcome the MgcRacGAP-mediated inhibition and MgcRacGAP binding to HIF-1α in vivo inhibits its dimerization with ARNT. We additionally present results indicating that MgcRacGAP binding to HIF-1α is specific, since it does not affect the transcriptional activity of HIF-2, a close evolutionary relative of HIF-1 also involved in hypoxia regulation and cancer. Our results reveal a new mechanism for HIF-1 transcriptional activity regulation, suggest a novel hypoxia-cytoskeleton link and provide new tools for selective HIF-1 inhibition.

Short-term hypoxia triggers ROS and SAFB mediated nuclear matrix and mRNA splicing remodeling.

The cellular response to hypoxia, in addition to HIF-dependent transcriptional reprogramming, also involves less characterized transcription-independent processes, such as alternative splicing of the VEGFA transcript leading to the production of the proangiogenic VEGF form. We now show that this event depends on reorganization of the splicing machinery, triggered after short-term hypoxia by ROS production and intranuclear redistribution of the nucleoskeletal proteins SAFB1/2. Exposure to low oxygen causes fast dissociation of SAFB1/2 from the nuclear matrix, which is reversible, inhibited by antioxidant treatment, and also observed under normoxia when the mitochondrial electron transport chain is blocked. This is accompanied by altered interactions between SAFB1/2 and the splicing machinery, translocation of kinase SRPK1 to the cytoplasm, and dephosphorylation of RS-splicing factors. Depletion of SAFB1/2 under normoxia phenocopies the hypoxic and ROS-mediated switch in VEGF mRNA splicing. These data suggest that ROS-dependent remodeling of the nuclear architecture can promote production of splicing variants that facilitate adaptation to hypoxia.

Enhancer of rudimentary homologue interacts with scaffold attachment factor B at the nuclear matrix to regulate SR protein phosphorylation.

Scaffold attachment factor B1 (SAFB1) is an integral component of the nuclear matrix of vertebrate cells. It binds to DNA on scaffold/matrix attachment region elements, as well as to RNA and a multitude of different proteins, affecting basic cellular activities such as transcription, splicing and DNA damage repair. In the present study, we show that enhancer of rudimentary homologue (ERH) is a new molecular partner of SAFB1 and its 70% homologous paralogue, scaffold attachment factor B2 (SAFB2). ERH interacts directly in the nucleus with the C-terminal Arg-Gly-rich region of SAFB1/2 and co-localizes with it in the insoluble nuclear fraction. ERH, a small ubiquitous protein with striking homology among species and a unique structure, has also been implicated in fundamental cellular mechanisms. Our functional analyses suggest that the SAFB/ERH interaction does not affect SAFB1/2 function in transcription (e.g. as oestrogen receptor α co-repressors), although it reverses the inhibition exerted by SAFB1/2 on the splicing kinase SR protein kinase 1 (SRPK1), which also binds on the C-terminus of SAFB1/2. Accordingly, ERH silencing decreases lamin B receptor and SR protein phosphorylation, which are major SRPK1 substrates, further substantiating the role of SAFB1 and SAFB2 in the co-ordination of nuclear function.

An association study between hypoxia inducible factor-1alpha (HIF-1α) polymorphisms and osteonecrosis.

Bone hypoxia resulting from impaired blood flow is the final pathway for the development of osteonecrosis (ON). The aim of this study was to evaluate if HIF-1α, the major transcription factor triggered by hypoxia, is genetically implicated in susceptibility to ON. For this we analyzed frequencies of three known HIF-1α polymorphisms: one in exon 2 (C111A) and two in exon 12 (C1772T and G1790A) and their association with ON in a Greek population. Genotype analysis was performed using PCR-RFLP and rare alleles were further confirmed with sequencing. We found that genotype and allele frequency of C1772T and G1790A SNP of HIF-1α (SNPs found in our cohort) were not significantly different in ON patients compared to control patients. Furthermore these SNPs could not be associated with the different subgroups of ON. At the protein level we observed that the corresponding mutations (P582S and A588T, respectively) are not significant for protein function since the activity, expression and localization of the mutant proteins is practically indistinguishable from wt in HEK293 and Saos-2 cells. These results suggest that these missense mutations in the HIF-1α gene are not important for the risk of developing ON.

Serine-arginine protein kinases: a small protein kinase family with a large cellular presence.

Serine-arginine protein kinases (SPRKs) constitute a relatively novel subfamily of serine-threonine kinases that specifically phosphorylate serine residues residing in serine-arginine/arginine-serine dipeptide motifs. Fifteen years of research subsequent to the purification and cloning of human SRPK1 as a SR splicing factor-phosphorylating protein have lead to the accumulation of information on the function and regulation of the different members of this family, as well as on the genomic organization of SRPK genes in several organisms. Originally considered to be devoted to constitutive and alternative mRNA splicing, SRPKs are now known to expand their influence to additional steps of mRNA maturation, as well as to other cellular activities, such as chromatin reorganization in somatic and sperm cells, cell cycle and p53 regulation, and metabolic signalling. Similarly, SRPKs were considered to be constitutively active kinases, although several modes of regulation of their function have been demonstrated, implying an elaborate cellular control of their activity. Finally, SRPK gene sequence information from bioinformatics data reveals that SRPK gene homologs exist either in single or multiple copies in every single eukaryotic organism tested, emphasizing the importance of SRPK protein function for cellular life.

SAFB1 interacts with and suppresses the transcriptional activity of p53.

A significant amount of nuclear p53 is found associated with the nuclear matrix in cells that were exposed to genotoxic stress. In this study we identified Scaffold attachment factor B1 (SAFB1), a nuclear matrix-associated protein that binds the scaffold or matrix attachment regions (S/MARs) of genomic DNA, as a novel p53-interacting protein. SAFB1 was able to associate with p53 through its C-terminal domain, while significant co-localization of the two proteins was observed in cells treated with 5-fluorouracil or mithramycin. Binding of p53 to SAFB1 had a significant functional outcome, since SAFB1 was shown to suppress p53-mediated reporter gene expression. These data suggest that nuclear matrix-associated proteins may play a critical role in regulating p53 localization and activity.

The enzymatic activity of SR protein kinases 1 and 1a is negatively affected by interaction with scaffold attachment factors B1 and 2.

SR protein kinases (SRPKs) phosphorylate Ser/Arg dipeptide-containing proteins that play crucial roles in a broad spectrum of basic cellular processes. Phosphorylation by SRPKs constitutes a major way of regulating such cellular mechanisms. In the past, we have shown that SRPK1a interacts with the nuclear matrix protein scaffold attachment factor B1 (SAFB1) via its unique N-terminal domain, which differentiates it from SRPK1. In this study, we show that SAFB1 inhibits the activity of both SRPK1a and SRPK1 in vitro and that its RE-rich region is redundant for the observed inhibition. We demonstrate that kinase activity inhibition is caused by direct binding of SAFB1 to SRPK1a and SRPK1, and we also present evidence for the in vitro binding of SAFB2 to the two kinases, albeit with different affinity. Moreover, we show that both SR protein kinases can form complexes with both scaffold attachment factors B in living cells and that this interaction is capable of inhibiting their activity, depending on the tenacity of the complex formed. Finally, we present data demonstrating that SRPK/SAFB complexes are present in the nucleus of HeLa cells and that the enzymatic activity of the nuclear matrixlocalized SRPK1 is repressed. These results suggest a new role for SAFB proteins as regulators of SRPK activity and underline the importance of the assembly of transient intranuclear complexes in cellular regulation.

MgcRacGAP interacts with HIF-1alpha and regulates its transcriptional activity.

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.

Identification of MAPK phosphorylation sites and their role in the localization and activity of hypoxia-inducible factor-1alpha.

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.

Bacterially produced human HIF-1alpha is competent for heterodimerization and specific DNA-binding.

Hypoxia-inducible factor 1alpha (HIF-1alpha) is the regulatory subunit of HIF-1, the transcriptional activator and key mediator of the cellular response to hypoxia. Regulation of HIF-1alpha occurs at multiple levels and involves several different post-translational modifications. In order to examine the importance of these modifications for the basic function of HIF-1alpha, we have produced in bacteria recombinant full-length human HIF-1alpha using different expression systems. We show that this unmodified form of HIF-1alpha is able to form a stable heterodimer with the second subunit of HIF-1 (HIF-1beta or ARNT) when both proteins are co-expressed in Escherichia coli. Furthermore, this bacterially reconstituted heterodimer exhibits specific DNA-binding activity. These data indicate that post-translational modification of HIF-1alpha is not essential for its interaction with ARNT and DNA, and provide an in vitro system for the characterization of the molecular properties of HIF-1alpha.