Mark A. Goldberg

Post-transcriptional Regulation of Vascular Endothelial Growth Factor by Hypoxia

The major control point for the hypoxic induction of the vascular endothelial growth factor (VEGF) gene is the regulation of the steady-state level of the mRNA. We previously demonstrated a discrepancy between the transcription rate and the steady-state mRNA level induced by hypoxia. This led us to examine the post-transcriptional regulation of VEGF expression. Actinomycin D experiments revealed that hypoxia increased VEGF mRNA half-life from 43 ± 6 min to 106 ± 9 min. Using an in vitro mRNA degradation assay, the half-life of VEGF mRNA 3′-untranslated region (UTR) transcripts were also found to be increased when incubated with hypoxic versus normoxic extracts. Both cis-regulatory elements involved in VEGF mRNA degradation under normoxic conditions and in increased stabilization under hypoxic conditions were mapped using this degradation assay. A hypoxia-induced protein(s) was found that bound to the sequences in the VEGF 3′-UTR which mediated increased stability in the degradation assay. Furthermore, genistein, a tyrosine kinase inhibitor, blocked the hypoxia-induced stabilization of VEGF 3′-UTR transcripts and inhibited hypoxia-induced protein binding to the VEGF 3′-UTR. These findings demonstrate a significant post-transcriptional component to the regulation of VEGF.

Inhibition of Hypoxia-inducible Factor 1 Activation by Carbon Monoxide and Nitric Oxide IMPLICATIONS FOR OXYGEN SENSING AND SIGNALING

It has been proposed that cells sense hypoxia by a heme protein, which transmits a signal that activates the heterodimeric transcription factor hypoxia-inducible factor 1 (HIF-1), thereby inducing a number of physiologically relevant genes such as erythropoietin (Epo). We have investigated the mechanism by which two heme-binding ligands, carbon monoxide and nitric oxide, affect oxygen sensing and signaling. Two concentrations of CO (10 and 80%) suppressed the activation of HIF-1 and induction of Epo mRNA by hypoxia in a dose-dependent manner. In contrast, CO had no effect on the induction of HIF-1 activity and Epo expression by either cobalt chloride or the iron chelator desferrioxamine. The affinity of CO for the putative sensor was much lower than that of oxygen (Haldane coefficient, ∼0.5). Parallel experiments were done with 100 μm sodium nitroprusside, a nitric oxide donor. Both NO and CO inhibited HIF-1 DNA binding by abrogating hypoxia-induced accumulation of HIF-1α protein. Moreover, both NO and CO specifically targeted the internal oxygen-dependent degradation domain of HIF-1α, and also repressed the C-terminal transactivation domain of HIF-1α. Thus, NO and CO act proximally, presumably as heme ligands binding to the oxygen sensor, whereas desferrioxamine and perhaps cobalt appear to act at a site downstream.

Hypoxia-inducible Protein Binding to Vascular Endothelial Growth Factor mRNA and Its Modulation by the von Hippel-Lindau Protein

Hypoxia induces an increase in the stability of the mRNA encoding vascular endothelial growth factor (VEGF). We have previously demonstrated that a 500-base region of the 3′-untranslated region of VEGF mRNA that is critical for stabilization of VEGF mRNA in an in vitro degradation assay forms a RNA-protein complex in a hypoxia-inducible fashion. We report here the identification of three adenylate-uridylate-rich RNA elements within this region that form an identical or closely related hypoxia-inducible RNA-protein complex. This complex is constitutively elevated in a tumor cell line lacking the wild type von Hippel-Lindau tumor suppressor gene and in which VEGF mRNA is constitutively stabilized. Furthermore, the glucose transporter-1 mRNA, which is also stabilized by hypoxia, forms a hypoxia-inducible RNA-protein complex with similar sequence and protein binding characteristics to that described for VEGF mRNA. Finally, RNA affinity purification and UV cross-linking were used to identify three proteins of 32, 28, and 17 kDa that are derived from this hypoxia-inducible RNA-protein complex.

Regulation of Vascular Endothelial Growth Factor in Cardiac Myocytes

Collateral blood vessels supplement normal coronary blood flow and coronary blood flow compromised by coronary artery disease, thereby protecting the myocardium from ischemia. Collateral vessel formation is the result of angiogenesis. Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), is a secreted mitogen specific for endothelial cells and an extremely potent angiogenic factor. In the present study, VPF/VEGF mRNA and protein were demonstrated to be markedly stimulated in primary rat cardiac myocytes in vitro in response to reduction of the oxygen tension to 1% or inhibition of the electron transport chain. Four isoforms of VPF/VEGF were coordinately regulated by hypoxia, including a novel isoform not previously described. Phorbol ester and the depolarizing agent veratridine, stimulators of protein kinase C and calcium influx, respectively, were found to markedly increase VPF/VEGF mRNA expression in cardiac myocytes. Forskolin, a potent stimulator of adenylate cyclase, produced a small but significant increase in VPF/VEGF mRNA expression in the cardiac myocytes. However, only H7, an inhibitor of protein kinase C, inhibited the hypoxic induction of VPF/VEGF mRNA; inhibitors of calcium influx and the calcium-calmodulin-dependent protein kinase II as well as inhibition of protein kinase A did not block the hypoxic induction of VPF/VEGF mRNA. This suggests that more than one signal transduction pathway is involved in regulating VPF/VEGF expression. The sensor that regulates the expression of hypoxia-responsive genes has been proposed to be a heme protein. Consistent with this model, transition metals initiate a genetic program similar to hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of Vascular Endothelial Growth Factor Expression by Insulin-Like Growth Factor I

Insulin-like growth factor I (IGF-I) and vascular endothelial growth factor (VEGF) levels are correlated with retinal ischemia-associated intraocular neovascu-larization in humans. Since VEGF is required for iris and retinal neovascularization in animal models of retinal ischemia, we tested whether IGF-I could act as an indirect angiogenic factor by increasing VEGF gene expression. IGF-I increased retinal pigment epithelial (RPE) cell VEGF mRNA in a concentration-dependent manner with an EC50 of 7 nmol/l (53.6 ng/ml). RPE and bovine smooth muscle cells exposed to 50 nmol/1 (383 ng/ml) IGF-I achieved peak VEGF mRNA expression within 2 h. IGF-I-treated RPE cells increased VEGF protein levels in conditioned media and stimulated capillary endothelial cell proliferation. Blockade of the IGF-I receptor with a neutralizing antibody abrogated the VEGF increases in RPE cells. Further, hypoxia-mediated and IGF-I-mediated increases in VEGF mRNA and protein levels were additive in RPE cells, and the hypoxia-induced VEGF increases were independent of endogenous IGF-I. VEGF promoter activity was enhanced by IGF-I in RPE cells, but VEGF transcript half-life was unaltered. In summary, the supplementation of RPE and smooth muscle cell cultures with IGF-I at 5-100 nmol/1 increased VEGF mRNA and secreted protein levels. The VEGF increases in RPE cells occurred primarily through enhanced transcription of the VEGF gene and via the IGF-I receptor. Elevated IGF-I levels may promote neovascularization through increased retinal VEGF gene expression.

Identification and Characterization of a Low Oxygen Response Element Involved in the Hypoxic Induction of a Family ofSaccharomyces cerevisiaeGenes: IMPLICATIONS FOR THE CONSERVATION OF OXYGEN SENSING IN EUKARYOTES

An organisms ability to respond to changes in oxygen tension depends in large part on alterations in gene expression. The oxygen sensing and signaling mechanisms in eukaryotic cells are not fully understood. To further define these processes, we have studied the Δ9 fatty acid desaturase gene OLE1 inSaccharomyces cerevisiae. We have confirmed previous data showing that the expression of OLE1 mRNA is increased in hypoxia and in the presence of certain transition metals.OLE1 expression was also increased in the presence of the iron chelator 1,10-phenanthroline. A 142-base pair (bp) region 3′ to the previously identified fatty acid response element was identified as critical for the induction of OLE1 in response to these stimuli using OLE1 promoter-lacZ reporter constructs. Electromobility shift assays confirmed the presence of an inducible band shift in response to hypoxia and cobalt. Mutational analysis defined the nonameric sequence ACTCAACAA as necessary for transactivation. A 20-base pair oligonucleotide containing this nonamer confers up-regulation by hypoxia and inhibition by unsaturated fatty acids when placed upstream of a heterologous promoter in alacZ reporter construct. Additional yeast genes were identified which respond to hypoxia and cobalt in a manner similar toOLE1. A number of mammalian genes are also up-regulated by hypoxia, cobalt, nickel, and iron chelators. Hence, the identification of a family of yeast genes regulated in a similar manner has implications for understanding oxygen sensing and signaling in eukaryotes.

MGA2 Is Involved in the Low-Oxygen Response Element-Dependent Hypoxic Induction of Genes in Saccharomyces cerevisiae

ABSTRACT Eukaryotes have the ability to respond to changes in oxygen tension by alterations in gene expression. For example,OLE1 expression in Saccharomyces cerevisiae is upregulated under hypoxic conditions. Previous studies have suggested that the pathway regulating OLE1expression by unsaturated fatty acids may involve Mga2p and Spt23p, two structurally and functionally related proteins. To define the possible roles of each of these genes on hypoxia-inducedOLE1 expression, we examined OLE1expression under normoxia, hypoxia, and cobalt treatment conditions in Δmga2 or Δspt23 deletion strains. The results of OLE1promoter-lacZ reporter gene and Northern blot analyses showed that hypoxia- and cobalt-induced OLE1 expression was dramatically decreased in a Δmga2 strain but not in a Δspt23 strain. Further analyses using low-oxygen response element (LORE)-CYC1-lacZ fusion reporter assays and electrophoretic mobility shift assays (EMSAs) demonstrated that MGA2 significantly affects the LORE-dependent hypoxic induction pathway of gene expression. When MGA2 was supplied by a plasmid, the LORE-dependent hypoxia-inducible reporter expression was recovered, as was the hypoxia-inducible complex in EMSAs in the S. cerevisiae Δmga2 strain. Supershift analysis of EMSAs using crude extracts containing mycMga2p indicated that Mga2p is a component of the LORE-binding complex. Another LORE-dependent, hypoxia-inducible gene, ATF1, was similarly affected in the Δmga2 strain. These results indicate thatMGA2 is required for the LORE-dependent hypoxic gene induction in S. cerevisiae.

Erythropoiesis, erythropoietin, and iron metabolism in elective surgery: Preoperative strategies for avoiding allogeneic blood exposure

Preoperative autologous donation (PAD) of blood and administration of recombinant human erythropoietin (Epoetin alfa) are two strategies for increasing red blood cell (RBC) mass preoperatively. The success of PAD depends primarily on the patients ability to manufacture new RBCs before surgery to replace those removed during PAD. Red blood cell manufacture depends in turn on adequate supplies of iron and the increased renal production of endogenous erythropoietin following PAD. Successful PAD also requires adequate time for regeneration of predonated RBCs. Parenteral administration of Epoetin alfa causes a dose-dependent stimulation of RBC production. Its use has been studied as an adjunct to PAD and as a method to enhance endogenous erythropoiesis without PAD. Several studies suggest that administration of Epoetin alfa, begun several days before surgery, may stimulate erythropoiesis and help decrease the number of RBC transfusions required postoperatively. The precise role of Epoetin alfa in the surgical setting is not yet established, and optimal dosage regimens have not been determined.

Clinical validation of an RIA for natural and recombinant erythropoietin in serun and plasma

A sensitive radioimmunoassay (RIA) for the detection of erythropoietin (EPO) was developed using antibody directed against EPO from human urine. With 100 microL of sample, the assay is sensitive to 7 U/L, well below the mean EPO level in normal males (15.1 +/- 3.5 U/L) or females (15.4 +/- 4.8 U/L). Dilutions of a variety of human serum samples show a parallel relationship with the standard EPO. Clinical validation of the RIA was confirmed by appropriate increases or decreases of EPO levels in various types of anemia and polycythemia. Serum EPO levels were also measured in volunteers participating in an autologous blood donation study. The RIA proved to be quite sensitive, detecting small increases even after a single unit phlebotomy. This RIA of human EPO meets all the requirements of a routine clinical assay in terms of specificity and clinical sensitivity and can be easily conducted in routine clinical laboratories.