Investigation of the activation mechanism of the hypoxia inducible factor HIF-1a in vitro and in primary cultures of airway smooth muscle cells

The present study deals with the investigation of the molecular mechanisms involved in the regulation and activation of the hypoxia inducible factor 1α (HIF-1α). At first, we examined the induction mechanisms of HIF-1α in primary cultures of airway smooth muscle (ASM) cells. The use of specific inhibitors revealed that ΗΙF- 1α, in ASM cells that are cultured in the absence of fetal bovine serum and thus obtain their “differentiated” contractile apparatus, is induced by two distinct mechanisms. The first mechanism that is induced by cobalt is the stabilization of the protein due to a possible inhibition of prolyl-hydroxylases and the second one that is induced by serum, involves enhanced synthesis of HIF-1α through transcription, translation and involvement of the PI-3K pathway. On the contrary, cobalt induces HIF-1α mainly through his protein synthesis, in “synthetic” ASM cells, that are cultured in the presence of serum, by using the activated PI-3K pathway. In order to further study the molecular properties of HIF-1α in vitro we used recombinant full length HIF-1α that was produced for the first time in bacteria. This unmodified form of HIF-1α was able to form a stable heterodimer with the second subunit of HIF-1 (ARNT) when both proteins were co-expressed in E. coli. The reconstituted heterodimer exhibited specific DNA binding activity. The amino-terminal regions of the two subunits were bound to DNA but this activity was affected by the carboxy-terminal regions of the two proteins. Furthermore, during the study of interaction of recombinant HIF-1α with p42-MAPK, the exact phosphorylation positions of HIF-1α were revealed. Finally, we studied the unknown nuclear transport mechanism of HIF-1α and we showed that importins 4 and 7 are responsible for HIF-1α import and thus define two distinct import pathways for the protein.Στην παρούσα διδακτορική διατριβή μελετήθηκαν οι μοριακοί μηχανισμοί ρύθμισης και ενεργοποίησης του παράγοντα που επάγεται από την υποξία ΗΙF-1α. Αρχικά διερευνήθηκε ο μηχανισμός επαγωγής του HIF-1α σε λεία μυϊκά κύτταρα αεραγωγών (ΛΜΚΑ). Η χρήση αναστολέων κυτταρικών μονοπατιών αποκάλυψε πως η επαγωγή του HIF-1α σε ΛΜΚΑ που καλλιεργούνται απουσία ορού και παρουσιάζουν τον επιμήκη «διαφοροποιημένο» φαινότυπο μπορεί να επιτευχθεί μέσω δύο τουλάχιστον μηχανισμών. Ο πρώτος μηχανισμός που ενεργοποιείται από το κοβάλτιο οδηγεί στη σταθεροποίηση του HIF-1α πιθανά μέσω αναστολής των πρόλυλο-υδροξυλασών ενώ ο δεύτερος που ενεργοποιείται από τον ορό ενέχει την αύξηση της σύνθεσης του μέσω μεταγραφής και μετάφρασης ενώ φαίνεται να εμπλέκει και το μονοπάτι της PI-3K. Αντίθετα σε «συνθετικά» ΛΜΚΑ που καλλιεργούνται συνεχώς παρουσία ορού το κοβάλτιο επάγει τον HIF-1α κυρίως μέσω αύξησης της μετάφρασης του mRNA του χρησιμοποιώντας το μονοπάτι της PI-3K που είναι ήδη ενεργοποιημένο από τον ορό. Για να διερευνηθεί περαιτέρω ο μηχανισμός ενεργοποίησης του HIF-1α in vitro χρησιμοποιήθηκε ανασυνδυασμένος HIF-1α πλήρους μεγέθους που παράχθηκε για πρώτη φορά, σε βακτήρια. Δείχθηκε πως ο ανασυνδυασμένος HIF-1α έχει τη δυνατότητα να συνδέεται σταθερά με τον συμπαράγοντα του ARNT μόνο όταν οι δυο πρωτεΐνες συνεκφραστούν στα ίδια E.coli κύτταρα. Επιπλέον, το σύμπλοκο που προκύπτει είναι ικανό για ειδική σύνδεση σε αλληλουχία του DNA που περιέχει τα στοιχεία απόκρισης στην υποξία (HRE). H σύνδεση γίνεται μέσω των αμινοτελικών άκρων των δύο υπομονάδων αλλά επηρεάζεται και από τα καρβοξυτελικά άκρα τους. Ο μη τροποποιημένος ανασυνδυασμένος HIF-1α χρησιμοποιήθηκε επίσης στη μελέτη της φωσφορυλίωσης του από την p42 MAP κινάση. Με αυτόν τον τρόπο αποκαλύφθηκαν για πρώτη φορά οι ακριβείς θέσεις φωσφορυλίωσης του από τη συγκεκριμένη κινάση. Τέλος μελετήθηκε ο άγνωστος μέχρι στιγμής μηχανισμός μεταφοράς του HIF-1α στον πυρήνα και δείχθηκε ότι οι ιμπορτίνες 4 και 7 είναι υπεύθυνες για την είσοδο του στον πυρήνα υποδεικνύοντας έτσι την ύπαρξη δύο διαφορετικών μονοπατιών εισόδου

Lipid Metabolism in Cancer: The Role of Acylglycerolphosphate Acyltransferases (AGPATs)

Altered lipid metabolism is an emerging hallmark of aggressive tumors, as rapidly proliferating cancer cells reprogram fatty acid (FA) uptake, synthesis, storage, and usage to meet their increased energy demands. Central to these adaptive changes, is the conversion of excess FA to neutral triacylglycerides (TAG) and their storage in lipid droplets (LDs). Acylglycerolphosphate acyltransferases (AGPATs), also known as lysophosphatidic acid acyltransferases (LPAATs), are a family of five enzymes that catalyze the conversion of lysophosphatidic acid (LPA) to phosphatidic acid (PA), the second step of the TAG biosynthesis pathway. PA, apart from its role as an intermediate in TAG synthesis, is also a precursor of glycerophospholipids and a cell signaling molecule. Although the different AGPAT isoforms catalyze the same reaction, they appear to have unique non-overlapping roles possibly determined by their distinct tissue expression and substrate specificity. This is best exemplified by the role of AGPAT2 in the development of type 1 congenital generalized lipodystrophy (CGL) and is also manifested by recent studies highlighting the involvement of AGPATs in the physiology and pathology of various tissues and organs. Importantly, AGPAT isoform expression has been shown to enhance proliferation and chemoresistance of cancer cells and correlates with increased risk of tumor development or aggressive phenotypes of several types of tumors

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

Epstein-Barr Virus Immortalization of Human B-Cells Leads to Stabilization of Hypoxia-Induced Factor 1 Alpha, Congruent with the Warburg Effect

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

Schematic view on the role of HIF1A in EBV-infected B-cell.

<p><b>A</b> – HIF1A is hydroxylated by the PHDs under normoxic conditions. The hydroxylated HIF1A is recognized by the VHL, E3 ubiquitin ligase, and HIF1A is degraded on proteasomes in activated B-cells. <b>B –</b> Upon EBV infection, EBNA-3 and EBNA-5 bind to PHD2 and PHD1, respectively, and inhibit HIF1A hydroxylation and degradation. The stabilized HIF1A translocates to the nucleus, forms a heterodimer with ARNT and transactivates genes such as <i>GLUT1</i>, <i>PDK1</i> and <i>LDHA</i>. This results in conversion of pyruvate to lactate, i.e., aerobic glycolysis.</p

Analysis of HIF-1α polymorhisms using RFLP.

<p>(A) Analysis of HIF-1α gene C1772T polymorphism with HphI enzyme, shown on 2% agarose electrophoresis. CT heterozygous genotype yielded three bands 467, 251 and 216 bp; C allele wt yielded two bands (251 and 216 bp). T allele remained uncut and yielded one fragment at 467 bp. Molecular weight standards are shown on the right. (B) Chromatograms of DNA sequence analysis of HIF-1α exon12 fragment showing the corresponding C, CT, T allelic variations at position 1772. (C) Analysis of HIF-1α gene G1790A polymorphism with AciI enzyme, shown on 2% agarose electrophoresis. G allele wt yielded two bands (269 and 198 bp). A allele would remained uncut and yielded one fragment at 467 bp, as represented. Molecular weight standards are shown on the right. (D) Analysis of HIF-1α gene C111A polymorphism with BglII enzyme, shown on 2% agarose electrophoresis. C allele wt yielded two bands (143 and 44 bp). A allele would remained uncut and yielded one fragment at 187 bp. Molecular weight standards are shown on the right.</p

HIF1A is localized to the nucleus and cytoplasm in LCLs, in contrast to mitogen-activated B-cells.

<p><b>A –</b> Western blotting of cellular subfractions. The membrane was probed with mouse anti-HIF1A and anti-actin antibodies (left panel). Nuclear HIF1A was detected in the lysates of LCL; however, no nuclear signal for HIF1A was detected in mitogen-activated cells. The HIF1A/actin ratio for all the probes was calculated (right panel). <b>B –</b> Immunostaining of LCLs and mitogen-activated cells with mouse anti-HIF1A antibody (green signal). Nuclear DNA is stained in blue. Notice the presence of nuclear HIF1A signal in LCL (panels <b>a</b> and <b>b</b>) and its absence in the mitogen-activated cells (<b>c</b> and <b>d</b>).</p

Expression of HIF1A-responsive genes in EBV-infected and mitogen-activated B-cells and LCLs.

<p>Expression of HIF1A-responsive genes, assessed by Q-PCR. CD40+IL4-activated (ABC) and freshly EBV-infected (EBC) B-cells were compared with LCLs. Untreated and cells treated with NiCl₂ were compared. Notice that many genes are upregulated in LCL and freshly EBV-infected cells, compared with CD40+IL4-activated B-cells.</p