Gieri Camenisch

Integration of Oxygen Signaling at the Consensus HRE

The hypoxia-inducible factor 1 (HIF-1) was initially identified as a transcription factor that regulated erythropoietin gene expression in response to a decrease in oxygen availability in kidney tissue. Subsequently, a family of oxygen-dependent protein hydroxylases was found to regulate the abundance and activity of three oxygen-sensitive HIFα subunits, which, as part of the HIF heterodimer, regulated the transcription of at least 70 different effector genes. In addition to responding to a decrease in tissue oxygenation, HIF is proactively induced, even under normoxic conditions, in response to stimuli that lead to cell growth, ultimately leading to higher oxygen consumption. The growing cell thus profits from an anticipatory increase in HIF-dependent target gene expression. Growth stimuli–activated signaling pathways that influence the abundance and activity of HIFs include pathways in which kinases are activated and pathways in which reactive oxygen species are liberated. These pathways signal to the HIF protein hydroxylases, as well as to HIF itself, by means of covalent or redox modifications and protein-protein interactions. The final point of integration of all of these pathways is the hypoxia-response element (HRE) of effector genes. Here, we provide comprehensive compilations of the known growth stimuli that promote increases in HIF abundance, of protein-protein interactions involving HIF, and of the known HIF effector genes. The consensus HRE derived from a comparison of the HREs of these HIF effectors will be useful for identification of novel HIF target genes, design of oxygen-regulated gene therapy, and prediction of effects of future drugs targeting the HIF system. Oxygen availability regulates many physiological and pathophysiological processes, including embryonic development, high-altitude adaptation, wound healing, and inflammation, as well as contributing to the pathophysiology of ischemic diseases and cancer. Central to our understanding of these processes is an elucidation of the molecular mechanisms by which cells react and adapt to insufficient oxygen supply (hypoxia). The last few years have brought a wealth of novel insights into these processes. Oxygen-sensing protein hydroxylases have been discovered that regulate the abundance and activity of three hypoxia-inducible transcription factors (HIFs) and thereby the activity of at least 70 effector genes involved in hypoxic adaptation. In addition to the increase in HIF abundance in response to a decrease in tissue oxygenation, it became evident that HIF abundance is also proactively increased, even under normoxic conditions, in response to stimuli that lead to cell growth and thus ultimately require higher oxygen consumption. The growing cell thus profits from an anticipatory increase in HIF-dependent target gene expression. Growth stimuli–activated signaling pathways that influence the abundance and activity of HIFs include pathways that involve the activation of kinases and liberation of reactive oxygen species. All of these pathways converge at the hypoxia-response elements (HREs) of effector genes, to which the HIFs bind, thereby enabling HIF-dependent induction of gene expression.

Increased prolyl 4-hydroxylase domain proteins compensate for decreased oxygen levels. Evidence for an autoregulatory oxygen-sensing system.

Prolyl 4-hydroxylase domain (PHD) proteins are oxygen-dependent enzymes that hydroxylate hypoxia-inducible transcription factor (HIF) α-subunits, leading to their subsequent ubiquitination and degradation. Paradoxically, the expression of two family members (PHD2 and PHD3) is induced in hypoxic cell culture despite the reduced availability of the oxygen co-substrate, and it has been suggested that they become functionally relevant following re-oxygenation to rapidly terminate the HIF response. Here we show that PHDs are also induced in hypoxic mice in vivo, albeit in a tissue-specific manner. As demonstrated under chronically hypoxic conditions in vitro, PHD2 and PHD3 show a transient maximum but remain up-regulated over more than 10 days, suggesting a feedback down-regulation of HIF-1α which then levels off at a novel set point. Indeed, hypoxic induction of PHD2 and PHD3 is paralleled by the attenuation of endogenous HIF-1α. Using an engineered oxygen-sensitive reporter gene in a cellular background lacking endogenous HIF-1α and hence inducible PHD expression, we could show that increased exogenous PHD levels can compensate for a wide range of hypoxic conditions. Similar data were obtained in a reconstituted cell-free system in vitro. In summary, these results suggest that due to their high O2 Km values, PHDs have optimal oxygen-sensing properties under all physiologically relevant oxygen concentrations; increased PHDs play a functional role even under oxygen-deprived conditions, allowing the HIF system to adapt to a novel oxygen threshold and to respond to another hypoxic insult. Furthermore, such an autoregulatory oxygen-sensing system would explain how a single mechanism works in a wide variety of differently oxygenated tissues.

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 Peptidyl Prolyl cis/trans Isomerase FKBP38 Determines Hypoxia-Inducible Transcription Factor Prolyl-4-Hydroxylase PHD2 Protein Stability

ABSTRACT The heterodimeric hypoxia-inducible transcription factors (HIFs) are central regulators of the response to low oxygenation. HIF-α subunits are constitutively expressed but rapidly degraded under normoxic conditions. Oxygen-dependent hydroxylation of two conserved prolyl residues by prolyl-4-hydroxylase domain-containing enzymes (PHDs) targets HIF-α for proteasomal destruction. We identified the peptidyl prolyl cis/trans isomerase FK506-binding protein 38 (FKBP38) as a novel interactor of PHD2. Yeast two-hybrid, glutathione S-transferase pull-down, coimmunoprecipitation, colocalization, and mammalian two-hybrid studies confirmed specific FKBP38 interaction with PHD2, but not with PHD1 or PHD3. PHD2 and FKBP38 associated with their N-terminal regions, which contain no known interaction motifs. Neither FKBP38 mRNA nor protein levels were regulated under hypoxic conditions or after PHD inhibition, suggesting that FKBP38 is not a HIF/PHD target. Stable RNA interference-mediated depletion of FKBP38 resulted in increased PHD hydroxylation activity and decreased HIF protein levels and transcriptional activity. Reconstitution of FKBP38 expression abolished these effects, which were independent of the peptidyl prolyl cis/trans isomerase activity. Downregulation of FKBP38 did not affect PHD2 mRNA levels but prolonged PHD2 protein stability, suggesting that FKBP38 is involved in PHD2 protein regulation.

Hypoxic pulmonary artery fibroblasts trigger proliferation of vascular smooth muscle cells: role of hypoxia-inducible transcription factors

Chronic lung hypoxia causes vascular remodeling with pulmonary artery smooth muscle cell (SMCPA) hyperplasia, resulting in pulmonary hypertension and cor pulmonale. We investigated SMCPA and pulmonary artery adventitial fibroblasts (FBPA) for their proliferative response to hypoxia. Strong SMCPA growth occurred under hypoxic conditions in SMCPA/FBPA co‐cultures, but not in SMCPA monocultures. SMCPA growth was fully reproduced by transferring serum‐free supernatant from hypoxic cultured FBPA to normoxic SMCPA. Hypoxia‐inducible‐transcriptionfactor subtypes (HIF‐1α, HIF‐2α, HIF‐3α) and its dependent target genes, carrying the hypoxiaresponsive‐element as regulatory component, were strongly activated in both hypoxic FBPA and SMCPA. HIF‐transcription‐factor decoy technique, employed to FBPA during hypoxic culturing, blocked the mitogenic activity of FBPA conditioned medium on SMCPA. The data suggest that hypoxia‐driven gene regulation in pulmonary artery fibroblasts results in a mitogenic stimulus on adjacent pulmonary artery smooth muscle cells, and HIF‐transcription‐decoy may offer a new therapeutic approach to suppress these events.

Attenuation of HIF-1 DNA-binding activity limits hypoxia-inducible endothelin-1 expression.

Hypoxia-inducible factors (HIFs) locate to HIF-binding sites (HBSs) within the hypoxia-response elements (HREs) of oxygen-regulated genes. Whereas HIF-1α is expressed ubiquitously, HIF-2α is found primarily in the endothelium, similar to endothelin-1 (ET-1) and fms-like tyrosine kinase 1 (Flt-1), the expression of which is controlled by HREs. We identified an unique sequence alteration in both ET-1 and Flt-1 HBSs not found in other HIF-1 target genes, implying that these HBSs might cause binding of HIF-2 rather than HIF-1. However, electrophoretic mobility shift assays showed HIF-1 and HIF-2 DNA complex formation with the unique ET-1 HBS to be about equal. Both DNA-binding and hypoxic activation of reporter genes using the ET-1 HBS was decreased compared with transferrin and erythropoietin HBSs. The Flt-1 HBS was non-functional when assayed in isolation, suggesting that additional factors are required for hypoxic up-regulation via the reported Flt-1 HRE. Interestingly, HIF-1 activity could be restored fully by point-mutating the ET-1 (but not the Flt-1) HBS, suggesting that the wild-type ET-1 HBS attenuated the full hypoxic response known from other oxygen-regulated genes. Such a mechanism might serve to limit the expression of this potent vasoconstrictor in hypoxia.

Hypoxia-inducible Factor Prolyl-4-hydroxylase PHD2 Protein Abundance Depends on Integral Membrane Anchoring of FKBP38

Prolyl-4-hydroxylase domain (PHD) proteins are 2-oxoglutarate and dioxygen-dependent enzymes that mediate the rapid destruction of hypoxia-inducible factor α subunits. Whereas PHD1 and PHD3 proteolysis has been shown to be regulated by Siah2 ubiquitin E3 ligase-mediated polyubiquitylation and proteasomal destruction, protein regulation of the main oxygen sensor responsible for hypoxia-inducible factor α regulation, PHD2, remained unknown. We recently reported that the FK506-binding protein (FKBP) 38 specifically interacts with PHD2 and determines PHD2 protein stability in a peptidyl-prolyl cis-trans isomerase-independent manner. Using peptide array binding assays, fluorescence spectroscopy, and fluorescence resonance energy transfer analysis, we defined a minimal linear glutamate-rich PHD2 binding domain in the N-terminal part of FKBP38 and showed that this domain forms a high affinity complex with PHD2. Vice versa, PHD2 interacted with a non-linear N-terminal motif containing the MYND (myeloid, Nervy, and DEAF-1)-type Zn2+ finger domain with FKBP38. Biochemical fractionation and immunofluorescence analysis demonstrated that PHD2 subcellular localization overlapped with FKBP38 in the endoplasmic reticulum and mitochondria. An additional fraction of PHD2 was found in the cytoplasm. In cellulo PHD2/FKBP38 association, as well as regulation of PHD2 protein abundance by FKBP38, is dependent on membrane- anchored FKBP38 localization mediated by the C-terminal transmembrane domain. Mechanistically our data indicate that PHD2 protein stability is regulated by a ubiquitin-independent proteasomal pathway involving FKBP38 as adaptor protein that mediates proteasomal interaction. We hypothesize that FKBP38-bound PHD2 is constantly degraded whereas cytosolic PHD2 is stable and able to function as an active prolyl-4-hydroxylase.

Prolyl-4-hydroxylase PHD2- and hypoxia-inducible factor 2-dependent regulation of amphiregulin contributes to breast tumorigenesis

Hypoxia-elicited adaptations of tumor cells are essential for tumor growth and cancer progression. Although ample evidence exists for a positive correlation between hypoxia-inducible factors (HIFs) and tumor formation, metastasis and bad prognosis, the function of the HIF-α protein stability regulating prolyl-4-hydroxylase domain enzyme PHD2 in carcinogenesis is less well understood. In this study, we demonstrate that downregulation of PHD2 leads to increased tumor growth in a hormone-dependent mammary carcinoma mouse model. Tissue microarray analysis of PHD2 protein expression in 281 clinical samples of human breast cancer showed significantly shorter survival times of patients with low-level PHD2 tumors over a period of 10 years. An angiogenesis-related antibody array identified, amongst others, amphiregulin to be increased in the absence of PHD2 and normalized after PHD2 reconstitution. Cultivation of endothelial cells in conditioned media derived from PHD2-downregulated cells resulted in enhanced tube formation that was blocked by the addition of neutralizing anti-amphiregulin antibodies. Functionally, amphiregulin was regulated on the transcriptional level specifically by HIF-2 but not HIF-1. Our data suggest that PHD2/HIF-2/amphiregulin signaling has a critical role in the regulation of breast tumor progression and propose PHD2 as a potential tumor suppressor in breast cancer.

Oxygen tension modulates beta-globin switching in embryoid bodies.

Little is known about the factors influencing the hemoglobin switch in vertebrates during development. Inasmuch as the mammalian conceptus is exposed to changing oxygen tensions in utero, we examined the effect of different oxygen concentrations on β‐globin switching. We used an in vitro model of mouse embryogenesis based on the differentiation of blastocyst‐derived embryonic stem cells to embryoid bodies (EBs). Cultivation of EBs at increasing oxygen concentrations (starting at 1% O2) did not influence the temporal expression pattern of embryonic (βH1) globin compared to the normoxic controls (20% O2). In contrast, when compared to normoxically grown EBs, expression of fetal/adult (βmaj) globin in EBs cultured at varying oxygen concentrations was delayed by about 2 days and persisted throughout differentiation. Quantitation of hemoglobin in EBs using a 2,7‐diaminofluorene‐based colorimetric assay revealed the appearence of hemoglobin in two waves, an early and a late one. This observation was verified by spectrophotometric analysis of hemoglobin within single EBs. These two waves might reflect the switch of erythropoiesis from yolk sac to fetal liver. Reduced oxygenation is known to activate the hypoxia‐inducible factor‐1 (HIF‐1), which in turn specifically induces expression of a variety of genes among them erythropoietin (EPO). Although EBs increased EPO expression upon hypoxic exposure, the altered β‐globin appearance was not related to EPO levels as determined in EBs overexpressing EPO. Since mRNA from both mouse HIF‐1α isoforms was detected in all EBs tested at different differentiation stages, we propose that HIF‐1 modulates β‐globin expression during development.—Bichet, S., Wenger, R. H., Camenisch, G., Rolfs, A., Ehleben, W., Porwol, T., Acker, H., Fandrey, J., Bauer, C., Gassmann, M. Oxygen tension modulates β‐globin switching in embryoid bodies. FASEB J. 13, 285–295 (1999)

A Dominant-Negative Isoform of Hypoxia-Inducible Factor-1α Specifically Expressed in Human Testis

Abstract Spermatogenesis in the seminiferous tubuli of the testis occurs under a high proliferation rate, suggesting considerable oxygen consumption. Because of the lack of blood vessels, the oxygen partial pressure in the lumen of these tubuli is very low. We previously identified a testis isoform of the hypoxia-inducible factor (HIF)-1α in the mouse, termed mHIF-1αI.1. Here, we demonstrate that expression of mHIF-1αI.1 increases during puberty, further demonstrating its gene induction in postmeiotic germ cells. Using 5′-rapid amplification of cDNA ends, we identified a novel HIF-1α isoform in the human testis, called hHIF-1αTe. Like mHIF-1αI.1, hHIF-1αTe mRNA is derived from an alternative promoter-first exon combination, but with a different genomic organization and a different nucleotide sequence. Reverse transcription-polymerase chain reaction analysis confirmed that hHIF-1αTe is exclusively expressed in the testis. As determined by immunofluorescence of ejaculated sperm cells, HIF-1α protein is mainly localized in the postacrosomal head and in the midpiece of spermatozoa. Though overlapping with mitochondrial localization in human and mouse spermatozoa, neither hHIF-1αTe nor hHIF-1α associated with mitochondria. In contrast with the ubiquitously expressed HIF-1α protein and the mouse testis-specific mHIF-1αI.1 isoform, the hHIF-1αTe mRNA sequence predicts a protein with an N-terminal truncation of the DNA-binding domain. As shown by yeast two-hybrid assays, hHIF-1αTe still formed heterodimeric complexes with HIF-1β. However, hHIF-1αTe was incapable of forming a DNA-binding HIF-1 complex. Overexpression of exogenous hHIF-1αTe resulted in the inhibition of the endogenous HIF-1 transcriptional activity, demonstrating that the testis-specific hHIF-1αTe isoform is a dominant-negative regulator of normal HIF-1 activity.