Jaime Caro

Hypoxia-inducible Factor 1α (HIF-1α) Protein Is Rapidly Degraded by the Ubiquitin-Proteasome System under Normoxic Conditions ITS STABILIZATION BY HYPOXIA DEPENDS ON REDOX-INDUCED CHANGES

The hypoxia-inducible factor 1 transcriptional activator complex (HIF-1) is involved in the activation of the erythropoietin and several other hypoxia-responsive genes. The HIF-1 complex is composed of two protein subunits: HIF-1β/ARNT (aryl hydrocarbon receptor nuclear translocator), which is constitutively expressed, and HIF-1α, which is not present in normal cells but induced under hypoxic conditions. The HIF-1α subunit is continuously synthesized and degraded under normoxic conditions, while it accumulates rapidly following exposure to low oxygen tensions. The involvement of the ubiquitin-proteasome system in the proteolytic destruction of HIF-1 in normoxia was studied by the use of specific inhibitors of the proteasome system. Lactacystin and MG-132 were found to protect the degradation of the HIF-1 complex in cells transferred from hypoxia to normoxia. The same inhibitors were able to induce HIF-1 complex formation when added to normoxic cells. Final confirmation of the involvement of the ubiquitin-proteasome system in the regulated degradation of HIF-1α was obtained by the use ofts20TG R cells, which contain a temperature-sensitive mutant of E1, the ubiquitin-activating enzyme. Exposure of ts20 cells, under normoxic conditions, to the non-permissive temperature induced a rapid and progressive accumulation of HIF-1. The effect of proteasome inhibitors on the normoxic induction of HIF-1 binding activity was mimicked by the thiol reducing agentN-(2-mercaptopropionyl)-glycine and by the oxygen radical scavenger 2-acetamidoacrylic acid. Furthermore,N-(2-mercaptopropionyl)-glycine induced gene expression as measured by the stimulation of a HIF-1-luciferase expression vector and by the induction of erythropoietin mRNA in normoxic Hep 3B cells. These last findings strongly suggest that the hypoxia induced changes in HIF-1α stability and subsequent gene activation are mediated by redox-induced changes.

Histone Deacetylase Inhibitors Induce VHL and Ubiquitin-Independent Proteasomal Degradation of Hypoxia-Inducible Factor 1α

ABSTRACT Adaptation to hypoxic microenvironment is critical for tumor survival and metastatic spread. Hypoxia-inducible factor 1α (HIF-1α) plays a key role in this adaptation by stimulating the production of proangiogenic factors and inducing enzymes necessary for anaerobic metabolism. Histone deacetylase inhibitors (HDACIs) produce a marked inhibition of HIF-1α expression and are currently in clinical trials partly based on their potent antiangiogenic effects. Although it has been postulated that HDACIs affect HIF-1α expression by enhancing its interactions with VHL (von Hippel Lindau), thus promoting its ubiquitination and degradation, the actual mechanisms by which HDACIs decrease HIF-1α levels are not clear. Here, we present data indicating that HDACIs induce the proteasomal degradation of HIF-1α by a mechanism that is independent of VHL and p53 and does not require the ubiquitin system. This degradation pathway involves the enhanced interaction of HIF-1α with HSP70 and is secondary to a disruption of the HSP70/HSP90 axis function that appears mediated by the activity of HDAC-6.

6-Phosphofructo-2-kinase (pfkfb3) gene promoter contains hypoxia-inducible factor-1 binding sites necessary for transactivation in response to hypoxia

The up-regulation of glycolysis to enhance the production of energy under reduced pO2 is a hallmark of the hypoxic response. A key regulator of glycolytic flux is fructose-2,6-bisphosphate, and its steady state concentration is regulated by the action of different isozymes product of four genes (pfkfb1–4). pfkfb3 has been found in proliferating cells and tumors, being induced by hypoxia. To understand the organization of cis-acting sequences that are responsible for the oxygen-regulated pfkfb3 gene, we have studied its 5′-flanking region. Extensive analysis of the 5′ pfkfb3 promoter sequence revealed the presence of putative consensus binding sites for various transcription factors that could play an important role in pfkfb3 gene regulation. These DNA consensus sequences included estrogen receptor, hypoxia response element (HRE), early growth response, and specific protein 1 putative binding sites. Promoter deletion analysis as well as putative HREs sequences (wild type and mutated) fused to a c-fos minimal promoter unit constructs demonstrate that the sequence located from -1269 to -1297 relative to the start site is required for hypoxia-inducible factor 1 (HIF-1) induction. The effective binding of HIF-1 transcription factor to the HREs at -1279 and -1288 was corroborated by electrophoretic mobility shift assay and biotinylated oligonucleotide pull-down. In addition, HIF-1α null mouse embryo fibroblasts transfected with a full-length pfkfb3 promoter-luciferase reporter construct further demonstrated that HIF-1 protein was critically involved for hypoxia transactivation of this gene. Altogether, these results demonstrate that pfkfb3 is a hypoxia-inducible gene that is stimulated through HIF interaction with the consensus HRE site in its promoter region.

Hypoxic regulation of the 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase gene family (PFKFB‐1–4) expression in vivo

When oxygen becomes limiting, cells shift primarily to a glycolytic mode for generation of energy. A key regulator of glycolytic flux is fructose‐2,6‐bisphosphate (F‐2,6‐BP), a potent allosteric regulator of 6‐phosphofructo‐1‐kinase (PFK‐1). The levels of F‐2,6‐BP are maintained by a family of bifunctional enzymes, 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase (PFKFB or PFK‐2), which have both kinase and phosphatase activities. Each member of the enzyme family is characterized by their phosphatase:kinase activity ratio (K:B) and their tissue‐specific expression. Previous work demonstrated that one of the PFK‐2 isozyme genes, PFKFB‐3, was induced by hypoxia through the hypoxia‐inducible factor‐1 (HIF‐1) pathway. In this study we examined the basal and hypoxic expression of three members of this family in different organs of mice. Our findings indicate that all four isozymes (PFKFB‐1–4) are responsive to hypoxia in vivo. However, their basal level of expression and hypoxia responsiveness varies in the different organs studied. Particularly, PFKFB‐1 is highly expressed in liver, heart and skeletal muscle, with the highest response to hypoxia found in the testis. PFKFB‐2 is mainly expressed in the lungs, brain and heart. However, the highest hypoxia responses are found only in liver and testis. PFKFB‐3 has a variable low basal level of expression in all organs, except skeletal muscle, where it is highly expressed. Most importantly, its hypoxia responsiveness is the most ample of all three genes, being strongly induced in the lungs, liver, kidney, brain, heart and testis. Further studies showed that PFKFB‐1 and PFKFB‐2 were highly responsive to hypoxia mimics such as transition metals, iron chelators and inhibitors of HIF hydroxylases, suggesting that the hypoxia responsiveness of these genes is also regulated by HIF proteins. In summary, our data demonstrate that PFK‐2 genes are responsive to hypoxia in vivo, indicating a physiological role in the adaptation of the organism to environmental or localized hypoxia/ischemia.

Carboxyl-Terminal Transactivation Activity of Hypoxia-Inducible Factor 1α Is Governed by a von Hippel-Lindau Protein-Independent, Hydroxylation-Regulated Association with p300/CBP

ABSTRACT Hypoxia-inducible factor 1 complex (HIF-1) plays a pivotal role in oxygen homeostasis and adaptation to hypoxia. Its function is controlled by both the protein stability and the transactivation activity of its alpha subunit, HIF-1α. Hydroxylation of at least two prolyl residues in the oxygen-dependent degradation domain of HIF-1α regulates its interaction with the von Hippel-Lindau protein (VHL) that targets HIF-1α for ubiquitination and proteasomal degradation. Several prolyl hydroxylases have been found to specifically hydroxylate HIF-1α. In this report, we investigated possible roles of VHL and hydroxylases in the regulation of the transactivation activity of the C-terminal activating domain (CAD) of HIF-1α. We demonstrate that regulation of the transactivation activity of HIF-1α CAD also involves hydroxylase activity but does not require functional VHL. In addition, stimulation of the CAD activity by a hydoxylase inhibitor, hypoxia, and desferrioxamine was severely blocked by the adenoviral oncoprotein E1A but not by an E1A mutant defective in targeting p300/CBP. We further demonstrate that a hydroxylase inhibitor, hypoxia, and desferrioxamine promote the functional and physical interaction between HIF-1α CAD and p300/CBP in vivo. Taken together, our data provide evidence that hypoxia-regulated stabilization and transcriptional stimulation of HIF-1α function are regulated through partially overlapping but distinguishable pathways.

General applicability of chicken egg yolk antibodies: the performance of IgY immunoglobulins raised against the hypoxia-inducible factor 1alpha.

Avian embryos and neonates acquire passive immunity by transferring maternal immunoglobulins from serum to egg yolk. Despite being a convenient source of antibodies, egg yolk immunoglobulins (IgY) from immunized hens have so far received scant attention in research. Here we report the generation and rapid isolation of IgY from the egg yolk of hens immunized against the α subunit of the human hypoxia‐inducible factor 1 (HIF‐1α). Anti‐HIF‐1α IgY antibodies were affinity purified and tested for their performance in various applications. Abundant HIF‐1α protein was detected by Western blot analysis in nuclear extracts derived from hypoxic cells of human, mouse, monkey, swine, and dog origin whereas in hypoxic quail and frog cells, the HIF‐1α signal was weak or absent, respectively. In electro‐phoretic mobility shift assays, affinity‐purified IgY antibody was shown to recognize the native HIF‐1 (but not the related HIF‐2) complex that specifically binds an oligonucleotide containing the HIF‐1 DNAbinding site. Furthermore, IgY antibody immunoprecipitated HIF‐1α from hypoxic cell extracts. Immunofluorescence experiments using IgY antibody allowed the detection of HIF‐1α in the nucleus of hypoxic COS‐7 cells. For comparison, the application of a mouse monoclonal antibody raised against the identical HIF‐1 α fragment was more restricted. Because chicken housing is inexpensive, egg collection is noninvasive, isolation and affinity purification of IgY antibodies are fast and simple, and the applicability of IgY is widespread, immunization of hens represents an excellent alternative for the generation of polyclonal antibodies. —Camenisch, G., Tini, M., Chilov, D., Kvietikova, I., Srinivas, V., Caro, J., Spielmann, P., Wenger, R. H., Gassmann, M. General applicability of chicken egg yolk antibodies: the performance of IgY immunoglobulins raised against the hypoxia‐inducible factor 1α. FASEB J. 13, 81–88 (1999)

The autophagic tumor stroma model of cancer or "battery-operated tumor growth": A simple solution to the autophagy paradox.

The role of autophagy in tumorigenesis is controversial. Both autophagy inhibitors (chloroquine) and autophagy promoters (rapamycin) block tumorigenesis by unknown mechanism(s). This is called the “Autophagy Paradox”. We have recently reported a simple solution to this paradox. We demonstrated that epithelial cancer cells use oxidative stress to induce autophagy in the tumor microenvironment. As a consequence, the autophagic tumor stroma generates recycled nutrients that can then be used as chemical building blocks by anabolic epithelial cancer cells. This model results in a net energy transfer from the tumor stroma to epithelial cancer cells (an energy imbalance), thereby promoting tumor growth. This net energy transfer is both unilateral and vectorial, from the tumor stroma to the epithelial cancer cells, representing a true host-parasite relationship. We have termed this new paradigm “The Autophagic Tumor Stroma Model of Cancer Cell Metabolism” or “Battery-Operated Tumor Growth”. In this sense, autophagy in the tumor stroma serves as a “battery” to fuel tumor growth, progression, and metastasis, independently of angiogenesis. Using this model, the systemic induction of autophagy will prevent epithelial cancer cells from using recycled nutrients, while the systemic inhibiton of autophagy will prevent stromal cells from producing recycled nutrients—both effectively “starving” cancer cells. We discuss the idea that tumor cells could become resistant to the systemic induction of autophagy, by the up-regulation of natural endogenous autophagy inhibitors in cancer cells. Alternatively, tumor cells could also become resistant to the systemic induction of autophagy, by the genetic silencing/deletion of pro-autophagic molecules, such as Beclin1. If autophagy resistance develops in cancer cells, then the systemic inhibition of autophagy would provide a therapeutic solution to this type of drug resistance, as it would still target autophagy in the tumor stroma. As such, an anti-cancer therapy that combines the alternating use of both autophagy promoters and autophagy inhibitors would be expected to prevent the onset of drug resistance. We also discuss why anti-angiogenic therapy has been found to promote tumor recurrence, progression, and metastasis. More specifically, anti-angiogenic therapy would induce autophagy in the tumor stroma via the induction of stromal hypoxia, thereby converting a non-aggressive tumor type to a “lethal” aggressive tumor phenotype. Thus, uncoupling the metabolic parasitic relationship between cancer cells and an autophagic tumor stroma may hold great promise for anti-cancer therapy. Finally, we believe that autophagy in the tumor stroma is the local microscopic counterpart of systemic wasting (cancer-associated cachexia), which is associated with advanced and metastatic cancers. Cachexia in cancer patients is not due to decreased energy intake, but instead involves an increased basal metabolic rate and increased energy expenditures, resulting in a negative energy balance. Importantly, when tumors were surgically excised, this increased metabolic rate returned to normal levels. This view of cachexia, resulting in energy transfer to the tumor, is consistent with our hypothesis. So, cancer-associated cachexia may start locally as stromal autophagy, and then spread systemically. As such, stromal autophagy may be the requisite precursor of systemic cancer-associated cachexia.

Histone Deacetylase Inhibitors Repress the Transactivation Potential of Hypoxia-inducible Factors Independently of Direct Acetylation of HIF-α

Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors regulating the oxygen supply, glucose metabolism, and angiogenesis. HIF function requires the recruitment of p300/CREB-binding protein, two coactivators with histone acetyltransferase activity, by the C-terminal transactivation domain of HIF-α (HIF-αCAD). Histone deacetylase inhibitors (HDAIs) induce differentiation or apoptosis and repress tumor growth and angiogenesis, hence being explored intensively as anti-cancer agents. Using combined pharmacological, biochemical, and genetic approaches, here we show that HDAIs repress the transactivation potential of HIF-αCAD. This repression is independent of the function of tumor suppressors von Hippel-Lindau or p53 or the degradation of HIF-α. We also demonstrate the sufficiency of low concentrations of HDAIs in repression of HIF target genes in tumor cells. We further show that HDAIs induce hyperacetylation of p300 and repress the HIF-1α·p300 complex in vivo. In vitro acetylation analysis reveals that the p300CH1 region, but not HIF-αCAD, is susceptible to acetylation. Taken together, our data demonstrate that a deacetylase activity is indispensable for the transactivation potential of HIF-αCAD and support a model that acetylation regulates HIF function by targeting HIF-α·p300 complex, not by direct acetylating HIF-α. The demonstration that HDAIs repress both HIF-1α and HIF-2α transactivation potential independently of von Hippel-Lindau tumor suppressor and p53 function indicates that HDAIs may have biological effects in a broad range of tissues in addition to tumors.

Circulating erythropoietin in patients with myelodysplastic syndromes

Summary. Serum concentration of erythropoietin (Epo) has been measured by radioimmunoassay in 46 patients with myelodysplastic syndromes. There is an overall inverse relationship between the level of Epo and the degree of anaemia but a wide range of Epo response between patients with similar haemoglobin concentrations. No differences were found between the different FAB groups, but the highest Epo levels were found in those patients with erythroid hypoplasia in the bone marrow. It is suggested that the intensity of erythroid activity in the marrow, as well as the degree of anaemia, may be a factor determining serum Epo concentration.

Renal and Extrarenal Erythropoietin Production in Anaemic Rats

Summary. Rats were rendered anaemic by a single bleeding or by a single injection of phenylhydrazine. At various times after the onset of anaemia they were nephrec‐tomized and challenged with a 6 h exposure to hypoxia. The erythropoietin titre observed at the end of this hypoxic period was corrected for renal erythropoietin induced by the anaemia alone, and the resulting extrarenal component was compared to total erythropoietin production of nephric rats in response to anaemia plus 6 h hypoxia. Extrarenal erythropoietin production was found to increase from 10.3% in normal rats to 12.5% in moderately anaemic rats to 15.1% in rats with severe bleeding anaemia. In phenylhydrazine‐treated rats this extrarenal component was found to be 18.3% possibly due to stimulation of extrarenal erythropoietin by haemolysed red cells. Chronic phenylhydrazine administration resulted in splenomegaly and Kupffer cell hyperactivity but not in any further stimulation of extrarenal erythropoietin production.