Publications

<p>Supplementary Figures: Figure S1. MBD2a and MBD2c exert opposing roles in BrCa cell migration. Figure S2. Hypoxia inhibits SRSF2-mediated MBD2 splicing. Figure S3. MBD2a and MBD2c inversely regulate EMT markers. Figure S4. DNA methylation controls MBD2-mediated FZD1 transcription. Figure S5. The effect of MBD2a and MBD2c on breast tumor growth in vivo. Supplementary Tables: Table S1, DAVID analysis of the co-regulated genes. Table S2, Metastasis-related Genes with altered expression according to RNA-seq data involved in cell migration. Table S3, DAVID analysis of the co-regulated and co-bound genes. Table S4, Primers used for quantification of mRNA and genome DNA. Table S5, Information of antibody, chemical reagent, cell line and software.</p>

Hypoxia-inducible factor-1 (HIF-1) regulates the transcription of genes whose products play critical roles in energy metabolism, erythropoiesis, angiogenesis, and cell survival. Limited information is available concerning its function in mammalian hematopoiesis. Previous studies have demonstrated that homozygosity for a targeted null mutation in the Hif1alpha gene, which encodes the hypoxia-responsive alpha subunit of HIF-1, causes cardiac, vascular, and neural malformations resulting in lethality by embryonic day 10.5 (E10.5). This study revealed reduced myeloid multilineage and committed erythroid progenitors in HIF-1alpha-deficient embryos, as well as decreased hemoglobin content in erythroid colonies from HIF-1alpha-deficient yolk sacs at E9.5. Dysregulation of erythropoietin (Epo) signaling was evident from a significant decrease in mRNA levels of Epo receptor (EpoR) in Hif1alpha-/- yolk sac as well as Epo and EpoR mRNA in Hif1alpha-/- embryos. The erythropoietic defects in HIF-1alpha-deficient erythroid colonies could not be corrected by cytokines, such as vascular endothelial growth factor and Epo, but were ameliorated by Fe-SIH, a compound delivering iron into cells independently of iron transport proteins. Consistent with profound defects in iron homeostasis, Hif1alpha-/- yolk sac and/or embryos demonstrated aberrant mRNA levels of hepcidin, Fpn1, Irp1, and frascati. We conclude that dysregulated expression of genes encoding Epo, EpoR, and iron regulatory proteins contributes to defective erythropoiesis in Hif1alpha-/- yolk sacs. These results identify a novel role for HIF-1 in the regulation of iron homeostasis and reveal unexpected regulatory differences in Epo/EpoR signaling in yolk sac and embryonic erythropoiesis.

HIF-1 alpha is required for IPC-induced mitochondrial ROS production and myocardial protection against ischaemia-reperfusion injury.

<p>Characterization of fibronectin-collagen gels.</p>

(Cell Reports 18, 1946–1957; February 21, 2017) In the originally published version of this article, the NANOG blot in Figure 5E was inadvertently included in Figure 5D. Likewise, the NANOG blot in Figure S5E was inadvertently included in Figure S5D. The corrected versions of the figures are provided here. Neither the initial errors nor the corrections alter the paper’s conclusions. The authors regret the errors.Figure S5Related to Figure 5View Large Image Figure ViewerDownload Hi-res image Download (PPT) Chemotherapy-Induced Ca2+ Release Stimulates Breast Cancer Stem Cell EnrichmentLu et al.Cell ReportsFebruary 21, 2017In BriefLu et al. report that exposure of breast cancer cells to chemotherapy induces HIF-dependent expression of GSTO1, which interacts with RYR1 to increase intracellular Ca2+ levels and trigger PYK2 → SRC → STAT3 signaling leading to breast cancer stem cell enrichment. GSTO1 knockdown blocks cancer stem cell enrichment, tumor initiation, and metastasis. Full-Text PDF Open Access

Rationale: Inositol polyphosphate multikinase (IPMK) and its major product inositol pentakisphosphate (IP5) regulate a variety of cellular functions, but their role in vascular biology remains unexplored. Objective: We have investigated the role of IPMK in regulating angiogenesis. Methods and Results: Deletion of IPMK in fibroblasts induces angiogenesis in both in vitro and in vivo models. IPMK deletion elicits a substantial increase of VEGF (vascular endothelial growth factor), which mediates the regulation of angiogenesis by IPMK. The regulation of VEGF by IPMK requires its catalytic activity. IPMK is predominantly nuclear and regulates gene transcription. However, IPMK does not apparently serve as a transcription factor for VEGF. HIF (hypoxia-inducible factor)-1α is a major determinant of angiogenesis and induces VEGF transcription. IPMK deletion elicits a major enrichment of HIF-1α protein and thus VEGF. HIF-1α is constitutively ubiquitinated by pVHL (von Hippel–Lindau protein) followed by proteasomal degradation under normal conditions. However, HIF-1α is not recognized and ubiquitinated by pVHL in IPMK KO (knockout) cells. IP5 reinstates the interaction of HIF-1α and pVHL. HIF-1α prolyl hydroxylation, which is prerequisite for pVHL recognition, is interrupted in IPMK-deleted cells. IP5 promotes HIF-1α prolyl hydroxylation and thus pVHL-dependent degradation of HIF-1α. Deletion of IPMK in mouse brain increases HIF-1α/VEGF levels and vascularization. The increased VEGF in IPMK KO disrupts blood–brain barrier and enhances brain blood vessel permeability. Conclusions: IPMK, via its product IP5, negatively regulates angiogenesis by inhibiting VEGF expression. IP5 acts by enhancing HIF-1α hydroxylation and thus pVHL-dependent degradation of HIF-1α.

Exposure of rats to hypoxia (7% O2) markedly increased the level of heme oxygenase-1 (HO-1) mRNA in several tissues. Accumulation of HO-1 transcripts was also observed after exposure of rat aortic vascular smooth muscle (VSM) cells to 1% O2, and this induction was dependent on gene transcription. Activation of the mouse HO-1 gene by all agents thus far tested is mediated by two 5'-enhancer sequences, SX2 and AB1, but neither fragment was responsive to hypoxia in VSM cells. Hypoxia-dependent induction of the chloramphenicol acetyltransferase (CAT) reporter gene was mediated by a 163-bp fragment located approximately 9.5 kilobases upstream of the transcription start site. This fragment contains two potential binding sites for hypoxia-inducible factor 1 (HIF-1). A role for HIF-1 in HO-1 gene regulation was established by the following observations: 1) HIF-1 specifically bound to an oligonucleotide spanning these sequences, 2) mutation of these sequences abolished HIF-1 binding and hypoxia-dependent gene activation in VSM cells, 3) hypoxia increased HIF-1alpha and HIF-1beta protein levels in VSM cells, and 4) hypoxia-dependent HO-1 mRNA accumulation was not observed in mutant hepatoma cells lacking HIF-1 DNA-binding activity. Taken together, these data demonstrate that hypoxia induces HO-1 expression in animal tissues and cell cultures and implicate HIF-1 in this response.

Chronic hypoxia is an inciting factor for the development of pulmonary arterial hypertension. The mechanisms involved in the development of hypoxic pulmonary hypertension (HPH) include hypoxia-inducible factor 1 (HIF-1)–dependent transactivation of genes controlling pulmonary arterial smooth muscle cell (PASMC) intracellular calcium concentration ([Ca 2+ ] i ) and pH. Recently, digoxin was shown to inhibit HIF-1 transcriptional activity. In this study, we tested the hypothesis that digoxin could prevent and reverse the development of HPH. Mice were injected daily with saline or digoxin and exposed to room air or ambient hypoxia for 3 wk. Treatment with digoxin attenuated the development of right ventricle (RV) hypertrophy and prevented the pulmonary vascular remodeling and increases in PASMC [Ca 2+ ] i , pH, and RV pressure that occur in mice exposed to chronic hypoxia. When started after pulmonary hypertension was established, digoxin attenuated the hypoxia-induced increases in RV pressure and PASMC pH and [Ca 2+ ] i . These preclinical data support a role for HIF-1 inhibitors in the treatment of HPH.

Increased expression of CD47 has been reported to enable cancer cells to evade phagocytosis by macrophages and to promote the cancer stem cell phenotype, but the molecular mechanisms regulating CD47 expression have not been determined. Here we report that hypoxia-inducible factor 1 (HIF-1) directly activates transcription of the CD47 gene in hypoxic breast cancer cells. Knockdown of HIF activity or CD47 expression increased the phagocytosis of breast cancer cells by bone marrow-derived macrophages. CD47 expression was increased in mammosphere cultures, which are enriched for cancer stem cells, and CD47 deficiency led to cancer stem cell depletion. Analysis of datasets derived from thousands of patients with breast cancer revealed that CD47 expression was correlated with HIF target gene expression and with patient mortality. Thus, CD47 expression contributes to the lethal breast cancer phenotype that is mediated by HIF-1.

Intermittent hypoxia (IH) increases HIF‐1α expression and HRE activation in PC12 cells ( Yuan et al. J. Biol. Chem. 2005). Reactive oxygen species (ROS)‐mediated signaling plays a critical for IH‐evoked cellular and systemic responses. In the present study we examined whether ROS play a role in HIF‐1α accumulation by IH, and if so to assess the source of ROS generation. Experiments were performed on PC12 cells exposed to either 20% O 2 (normoxic control) or 60 cycles of IH (30 sec at 1.5% O 2 followed by 5 min at 20% O 2 ). ROS scavengers, MnTMPyP, and PEG‐catalase prevented IH‐induced HIF‐1α accumulation. IH increased ROS generation as evidenced by decreases in aconitase enzyme activity in both cytosolic and mitochondrial fractions. Apocynin, an inhibitor of NADPH oxidase prevented IH‐induced HIF‐1α accumulation and ROS generation. IH upregulated NADPH oxidase subunits including gp91phox, p47phox, p22phox, and p67phox and increased NADPH oxidase enzyme activity. These observations suggest that ROS generated by NADPH oxidase plays an important role in IH‐induced up‐regulation of HIF‐1α. (Supported by NIH‐HL‐25830).

HIF-1 is a heterodimeric transcription factor that mediates adaptive responses to hypoxia and plays critical roles in cancer progression. Using a cell-based screening assay we have identified acriflavine as a drug that binds directly to HIF-1α and HIF-2α and inhibits HIF-1 dimerization and transcriptional activity. Pretreatment of mice bearing prostate cancer xenografts with acriflavine prevented tumor growth and treatment of mice bearing established tumors resulted in growth arrest. Acriflavine treatment inhibited intratumoral expression of angiogenic cytokines, mobilization of angiogenic cells into peripheral blood, and tumor vascularization. These results provide proof of principle that small molecules can inhibit dimerization of HIF-1 and have potent inhibitory effects on tumor growth and vascularization.

Many patients with diabetic eye disease respond inadequately to anti-VEGF therapies, implicating additional vasoactive mediators in its pathogenesis. We demonstrate that levels of angiogenic proteins regulated by HIF-1 and -2 remain elevated in the eyes of people with diabetes despite treatment with anti-VEGF therapy. Conversely, by inhibiting HIFs, we normalized the expression of multiple vasoactive mediators in mouse models of diabetic eye disease. Accumulation of HIFs and HIF-regulated vasoactive mediators in hyperglycemic animals was observed in the absence of tissue hypoxia, suggesting that targeting HIFs may be an effective early treatment for diabetic retinopathy. However, while the HIF inhibitor acriflavine prevented retinal vascular hyperpermeability in diabetic mice for several months following a single intraocular injection, accumulation of acriflavine in the retina resulted in retinal toxicity over time, raising concerns for its use in patients. Conversely, 32-134D, a recently developed HIF inhibitor structurally unrelated to acriflavine, was not toxic to the retina, yet effectively inhibited HIF accumulation and normalized HIF-regulated gene expression in mice and in human retinal organoids. Intraocular administration of 32-134D prevented retinal neovascularization and vascular hyperpermeability in mice. These results provide the foundation for clinical studies assessing 32-134D for the treatment of patients with diabetic eye disease.