HIF- and Non-HIF-Regulated Hypoxic Responses Require the Estrogen-Related Receptor in Drosophila melanogaster

Low-oxygen tolerance is supported by an adaptive response that includes a coordinate shift in metabolism and the activation of a transcriptional program that is driven by the hypoxia-inducible factor (HIF) pathway. The precise contribution of HIF-1a in the adaptive response, however, has not been determined. Here, we investigate how HIF influences hypoxic adaptation throughout Drosophila melanogaster development. We find that hypoxic-induced transcriptional changes are comprised of HIF-dependent and HIF-independent pathways that are distinct and separable. We show that normoxic set-points of carbohydrate metabolites are significantly altered in sima mutants and that these animals are unable to mobilize glycogen in hypoxia. Furthermore, we find that the estrogen-related receptor (dERR), which is a global regulator of aerobic glycolysis in larvae, is required for a competent hypoxic response. dERR binds to dHIFa and participates in the HIF-dependent transcriptional program in hypoxia. In addition, dERR acts in the absence of dHIFa in hypoxia and a significant portion of HIF-independent transcriptional responses can be attributed to dERR actions, including upregulation of glycolytic transcripts. These results indicate that competent hypoxic responses arise from complex interactions between HIF-dependent and -independent mechanisms, and that dERR plays a central role in both of these programs

Hypertonic Stress Induces VEGF Production in Human Colon Cancer Cell Line Caco-2: Inhibitory Role of Autocrine PGE2

Vascular Endothelial Growth Factor (VEGF) is a major regulator of angiogenesis. VEGF expression is up regulated in response to micro-environmental cues related to poor blood supply such as hypoxia. However, regulation of VEGF expression in cancer cells is not limited to the stress response due to increased volume of the tumor mass. Lipid mediators in particular arachidonic acid-derived prostaglandin (PG)E2 are regulators of VEGF expression and angiogenesis in colon cancer. In addition, increased osmolarity that is generated during colonic water absorption and feces consolidation seems to activate colon cancer cells and promote PGE2 generation. Such physiological stimulation may provide signaling for cancer promotion. Here we investigated the effect of exposure to a hypertonic medium, to emulate colonic environment, on VEGF production by colon cancer cells. The role of concomitant PGE2 generation and MAPK activation was addressed by specific pharmacological inhibition. Human colon cancer cell line Caco-2 exposed to a hypertonic environment responded with marked VEGF and PGE2 production. VEGF production was inhibited by selective inhibitors of ERK 1/2 and p38 MAPK pathways. To address the regulatory role of PGE2 on VEGF production, Caco-2 cells were treated with cPLA2 (ATK) and COX-2 (NS-398) inhibitors, that completely block PGE2 generation. The Caco-2 cells were also treated with a non selective PGE2 receptor antagonist. Each treatment significantly increased the hypertonic stress-induced VEGF production. Moreover, addition of PGE2 or selective EP2 receptor agonist to activated Caco-2 cells inhibited VEGF production. The autocrine inhibitory role for PGE2 appears to be selective to hypertonic environment since VEGF production induced by exposure to CoCl2 was decreased by inhibition of concomitant PGE2 generation. Our results indicated that hypertonicity stimulates VEGF production in colon cancer cell lines. Also PGE2 plays an inhibitory role on VEGF production by Caco-2 cells exposed to hyperosmotic stress through EP2 activation

PGE₂ stimulates VEGF production by Caco-2 cells activated with CoCl₂.

<p>Caco-2 cells were stimulated with 0.1–1 mM of CoCl₂ during 24 h before PGE₂ and VEGF production (<b>A</b> and <b>D</b>, respectively) and COX-2 protein expression (<b>B</b>) analysis. PGE₂ and VEGF production by Caco-2 cells stimulated with 1 mM of CoCl₂ during 24 h after pre-treatment with inhibitor of COX-2, NS-398 (<b>C</b> and <b>E</b>, respectively). VEGF production by Caco-2 cells stimulated with 0.1–1 mM of CoCl₂ during 24 h (<b>D</b>). PGE₂ and VEGF production were determined by ELISA in supernatants of Caco-2 cells. COX-2 and GAPDH expression in cell pellets was analyzed by Western blotting. +, * <i>p<0.05</i>, when compared to non-stimulated cells or stimulated cells, respectively. Graph bars show means ± SEM from triplicate samples.</p

Role of MAPKs in VEGF production by Caco-2 cells.

<p>Inhibitors of JNK, SP600125; p38, SB202190; and MEK 1/2, U0126 were added before stimulation with hypertonic stress (100 mM NaCl) (<b>A</b>) or 1 mM CoCl₂ (<b>B</b>) for 24 h. Caco-2 cells were pretreated with 1 µM of NS-398 to prevent endogenous PGE₂ production in all samples. VEGF production was determined by ELISA in supernatants of Caco-2 cells. +, * <i>p<0.05</i>, when compared to non-stimulated cells or stimulated cells, respectively. Graph bars show means ± SEM from triplicate samples.</p

EP₂ receptor plays a role in endogenous PGE₂ regulation of VEGF production by Caco-2 stimulated with hypertonic stress.

<p>(<b>A</b>) Caco-2 cells were stimulated by hypertonic stress (100 mM NaCl) during 24 h after pre-treatment with EP and DP receptors antagonist, AH 6809 (<b>A</b>); with inhibitor of cPLA₂, ATK (10 µM); PGE₂; EP receptors agonist, 16,16-dimethyl Prostaglandin E₂; EP1 and EP3 receptors agonist, 17-phenyl trinor Prostaglandin E₂; EP2 receptor agonist, butaprost and EP3 receptor agonist, sulprostone (<b>B</b>); or with PKA inhibitor, H-89. PGE₂ and its analogs were used at 0.1 µM. VEGF production was determined by ELISA in supernatants of Caco-2 cells. +, * <i>p<0.05</i>, when compared to non-stimulated cells or stimulated cells, respectively. ** <i>p<0.05</i>, when compared to ATK-treated cells. Graph bars show means ± SEM from triplicate samples.</p

The influence of dERR and dHIFa on hypoxic transcripts.

<p>(A) HIF-independent (HI), HIF-dependent (HD), ERR-dependent (ED), and ERR&HIF-dependent (DM) gene sets identified by microarray schemes outlined in Figures S1 and S2. Circles are scaled to size by number of transcripts in each set. (B) A Venn diagram demonstrating the overlap of the HI/HD/ED H-genes sets. Note, HI and HD genes sets are, by definition, mutually exclusive. The asterisks indicate that the overlap is significant (<i>p</i>-value<0.05), as determined by hypergeometric probability. (C) A Venn diagram demonstrating the overlap of the HD/ED/DM H-genes sets. qRT-PCR analysis of hypoxia-regulated genes falling into specific Venn overlaps, as indicated by arrows. (D) The top ten affected transcripts, as assessed by the H-responses measured in the control background, for each of the seven Venn categories shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003230#pgen-1003230-g007" target="_blank">Figure 7C</a>. Hypoxic expression for each transcript in the different mutant backgrounds (compared to <i>w¹¹¹⁸</i>) is reported as fold-change difference. Additionally shown is the N/H ratio obtained for <i>w¹¹¹⁸</i> animals. (E) Normoxic and hypoxic expression of each of the six genes (<i>Pfk, fatiga, spermine oxidase, NMNAT, LDH, ALAS</i>) was determined using RNA collected from animals of the indicated genotypes at late-L3. Samples were collected in triplicate and are independent from those used in the microarrays. Values are normalized to <i>rp49</i> expression and are reported relative to the value obtained for <i>w¹¹¹⁸</i> in normoxia.</p

List of 20 top transcripts whose expression changes in response to hypoxic challenge in a dHIF-dependent or -independent fashion.

<p>For HIF-dependent genes (taken from the top 20 down-regulated transcripts in the HD H-genes set), transcripts are sorted according to normalized microarray values obtained when comparing <i>w¹¹¹⁸</i> hypoxia samples with <i>sima</i> hypoxia samples. For the HIF-independent genes (taken from the top 20 up-regulated HI H-genes set), transcripts are sorted according to the normalized microarray values obtained when comparing <i>sima</i> normoxia with <i>sima</i> hypoxia samples. For comparative purposes, the respective hypoxic changes observed in <i>w¹¹¹⁸</i> control animals are reported in the last column. Additionally, the first four columns show the Affymetrix probe set ID, CG number, gene title, and the putative process/function of the encoded protein.</p

Temporal-dependent hypoxic responses.

<p>(A) Hypoxic treatment regimen of <i>w¹¹¹⁸</i> animals that were allowed to develop in normoxia (N) until they reached one of three developmental stages, at which point they were treated for 6 hours in N or hypoxia (H) (4% O₂). (B–D) qRT-PCR analysis was performed to assess the expression of <i>fatiga</i>, <i>LDH</i>, and <i>Pfk</i> at 18–24 hr AEL, mid-L2, or partial clear-gut larvae in late-L3. All experiments were performed in triplicate from pools of biological replicates. Values are normalized to <i>rp49</i> expression and are reported as the relative fold-change of H/N. Error bars are the SEM. * = <i>p</i>-value<0.05.</p