L. Eric Huang

Activation of Hypoxia-inducible Transcription Factor Depends Primarily upon Redox-sensitive Stabilization of Its α Subunit

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor that is critical for hypoxic induction of a number of physiologically important genes. We present evidence that regulation of HIF-1 activity is primarily determined by the stability of the HIF-1α protein. Both HIF-1α and HIF-1β mRNAs were constitutively expressed in HeLa and Hep3B cells with no significant induction by hypoxia. However, the HIF-1α protein was barely detectable in normoxic cells, even when HIF-1α was overexpressed, but was highly induced in hypoxic cells, whereas HIF-1β protein levels remained constant, regardless of pO2. Hypoxia-induced HIF-1 binding as well as the HIF-1α protein were rapidly and drastically decreased in vivo following an abrupt increase to normal oxygen tension. Moreover, short pre-exposure of cells to hydrogen peroxide selectively prevented hypoxia-induced HIF-1 binding via blocking accumulation of HIF-1α protein, whereas treatment of hypoxic cell extracts with H2O2 had no effect on HIF-1 binding. These observations suggest that an intact redox-dependent signaling pathway is required for destablization of the HIF-1α protein. In hypoxic cell extracts, HIF-1 DNA binding was reversibly abolished by sulfhydryl oxidation. Furthermore, the addition of reduced thioredoxin to cell extracts enhanced HIF-1 DNA binding. Consistent with these results, overexpression of thioredoxin and Ref-1 significantly potentiated hypoxia-induced expression of a reporter construct containing the wild-type HIF-1 binding site. These experiments indicate that activation of HIF-1 involves redox-dependent stabilization of HIF-1α protein.

HIF‐1α induces cell cycle arrest by functionally counteracting Myc

Hypoxia induces angiogenesis and glycolysis for cell growth and survival, and also leads to growth arrest and apoptosis. HIF‐1α, a basic helix–loop–helix PAS transcription factor, acts as a master regulator of oxygen homeostasis by upregulating various genes under low oxygen tension. Although genetic studies have indicated the requirement of HIF‐1α for hypoxia‐induced growth arrest and activation of p21cip1, a key cyclin‐dependent kinase inhibitor controlling cell cycle checkpoint, the mechanism underlying p21cip1 activation has been elusive. Here we demonstrate that HIF‐1α, even in the absence of hypoxic signal, induces cell cycle arrest by functionally counteracting Myc, thereby derepressing p21cip1. The HIF‐1α antagonism is mediated by displacing Myc binding from p21cip1 promoter. Neither HIF‐1α transcriptional activity nor its DNA binding is essential for cell cycle arrest, indicating a divergent role for HIF‐1α. In keeping with its antagonism of Myc, HIF‐1α also downregulates Myc‐activated genes such as hTERT and BRCA1. Hence, we propose that Myc is an integral part of a novel HIF‐1α pathway, which regulates a distinct group of Myc target genes in response to hypoxia.

Hypoxia facilitates Alzheimer's disease pathogenesis by up-regulating BACE1 gene expression

The molecular mechanism underlying the pathogenesis of the majority of cases of sporadic Alzheimers disease (AD) is unknown. A history of stroke was found to be associated with development of some AD cases, especially in the presence of vascular risk factors. Reduced cerebral perfusion is a common vascular component among AD risk factors, and hypoxia is a direct consequence of hypoperfusion. Previously we showed that expression of the β-site β-amyloid precursor protein (APP) cleavage enzyme 1 (BACE1) gene BACE1 is tightly controlled at both the transcriptional and translational levels and that increased BACE1 maturation contributes to the AD pathogenesis in Downs syndrome. Here we have identified a functional hypoxia-responsive element in the BACE1 gene promoter. Hypoxia up-regulated β-secretase cleavage of APP and amyloid-β protein (Aβ) production by increasing BACE1 gene transcription and expression both in vitro and in vivo. Hypoxia treatment markedly increased Aβ deposition and neuritic plaque formation and potentiated the memory deficit in Swedish mutant APP transgenic mice. Taken together, our results clearly demonstrate that hypoxia can facilitate AD pathogenesis, and they provide a molecular mechanism linking vascular factors to AD. Our study suggests that interventions to improve cerebral perfusion may benefit AD patients.

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-induced genetic instability : a calculated mechanism underlying tumor progression

The cause of human cancers is imputed to the genetic alterations at nucleotide and chromosomal levels of ill-fated cells. It has long been recognized that genetic instability—the hallmark of human cancers—is responsible for the cellular changes that confer progressive transformation on cancerous cells. How cancer cells acquire genetic instability, however, is unclear. We propose that tumor development is a result of expansion and progression—two complementary aspects that collaborate with the tumor microenvironment—hypoxia in particular, on genetic alterations through the induction of genetic instability. In this article, we review the recent literature regarding how hypoxia functionally impairs various DNA repair pathways resulting in genetic instability and discuss the biomedical implications in cancer biology and treatment.

The phosphorylation status of PAS‐B distinguishes HIF‐1α from HIF‐2α in NBS1 repression

Hypoxia promotes genetic instability for tumor progression. Recent evidence indicates that the transcription factor HIF‐1α impairs DNA mismatch repair, yet the role of HIF‐1α isoform, HIF‐2α, in tumor progression remains obscure. In pursuit of the involvement of HIF‐α in chromosomal instability, we report here that HIF‐1α, specifically its PAS‐B, induces DNA double‐strand breaks at least in part by repressing the expression of NBS1, a crucial DNA repair gene constituting the MRE11A–RAD50–NBS1 complex. Despite strong similarities between the two isoforms, HIF‐2α fails to do so. We demonstrate that this functional distinction stems from phosphorylation of HIF‐2α Thr‐324 by protein kinase D1, which discriminates between subtle differences of the two PAS‐B in amino‐acid sequence, thereby precluding NBS1 repression. Hence, our findings delineate a molecular pathway that functionally distinguishes HIF‐1α from HIF‐2α, and arguing a unique role for HIF‐1α in tumor progression by promoting genomic instability.

Suppression of Hypoxia-inducible Factor 1α (HIF-1α) Transcriptional Activity by the HIF Prolyl Hydroxylase EGLN1

The cellular response to hypoxia is, at least in part, mediated by the transcriptional regulation of hypoxia-responsive genes involved in balancing the intracellular ATP production and consumption. Recent evidence suggests that the transcription factor, HIF-1α, functions as a master regulator of oxygen homeostasis by controlling a broad range of cellular events in hypoxia. In normoxia, HIF-1α is targeted for destruction via prolyl hydroxylation, an oxygen-dependent modification that signals for recognition by the ubiquitin ligase complex containing the von Hippel-Lindau tumor suppressor. Three HIF prolyl hydroxylases (EGLN1, EGLN2, and EGLN3) have been identified in mammals, among which EGLN1 and EGLN3 are hypoxia-inducible at their mRNA levels in an HIF-1α-dependent manner. In this study, we demonstrated that apart from promoting HIF-1α proteolysis in normoxia, EGLN1 specifically represses HIF-1α transcriptional activity in hypoxia. Ectopic expression of EGLN1 inhibited HIF-1α transcriptional activity without altering its protein levels in a von Hippel-Lindau-deficient cell line, indicating a discrete activity of EGLN1 in transcriptional repression. Conversely, silencing of EGLN1 expression augmented HIF-1α transcriptional activity and its target gene expression in hypoxia. Thus, we proposed that the accumulated EGLN1 in hypoxia acts as a negative-feedback mechanism to modulate HIF-1α target gene expression. Our finding also provided new insight into the pharmacological manipulation of the HIF prolyl hydroxylase for ischemic diseases.

Tumor suppressor p53 represses transcription of RECQ4 helicase

RECQ4 is a member of the RecQ helicase family, which has been implicated in the regulation of DNA replication, recombination and repair. p53 modulates the functions of RecQ helicases including BLM and WRN. In this study, we demonstrate that p53 can regulate the transcription of RECQ4. Using nontransformed, immortalized normal human fibroblasts, we show that p53-dependent downregulation of RECQ4 expression occurred in G1-arrested cells, both in the absence or presence of exogenous DNA damage. Wild-type p53 (but not the tumor-derived mutant forms) repressed RECQ4 promoter activity. The camptothecin or etoposide-dependent p53-mediated repression was attenuated by trichostatin A (TSA), an inhibitor of histone deacetylases (HDACs). Repression of the RECQ4 promoter was accompanied with an increased accumulation of HDAC1, and the loss of SP1 and p53 binding to the promoter. The simultaneous formation of a camptothecin-dependent p53-SP1 complex indicated its occurrence outside of the RECQ4 promoter. These data suggest that p53-mediated repression of RECQ4 transcription during DNA damage results from the modulation of the promoter occupancy of transcription activators and repressors.

Leu-574 of human HIF-1α is a molecular determinant of prolyl hydroxylation

Hypoxia‐inducible factor (HIF)‐1α, a master regulator of oxygen homeostasis, regulates genes crucial for cell growth and survival. In normoxia, HIF‐1α is constantly degraded via the ubiquitin‐proteasome pathway. The von Hippel‐Lindau (VHL) E3 ubiquitin ligase binds HIF‐1α through specific recognition of hydroxylated Pro‐402 or Pro‐564, both of which are modified by the oxygen‐dependent HIF prolyl hydroxylases (PHDs/HPHs). Despite the identification of a conserved Leu‐X‐X‐Leu‐Ala‐Pro motif, the molecular requirement of HIF‐1α for PHDs/HPHs binding remains elusive. Recently, we demonstrated that Leu‐574 of human HIF‐1α—10 residues downstream of Pro‐564—is essential for VHL recognition. We show here that the role of Leu‐574 is to recruit PHD2/HPH2 for Pro‐564 hydroxylation. An antibody specific for hydroxylated Pro‐564 has been used to determine the hydroxylation status; mutation or deletion of Leu‐574 results in a significant decrease in the ratio of the hydroxylated HIF‐1α to the total amount. The nine‐residue spacing between Pro‐564 and Leu‐574 is not obligatory for prolyl hydroxylation. Furthermore, mutation of Leu‐574 disrupts the binding of PHD2/HPH2, a key prolyl hydroxylase for oxygen‐dependent proteolysis of HIF‐1α. Hence, our findings indicate that Leu‐574 is essential for recruiting PHD2/HPH2, thereby providing a molecular basis for modulating HIF‐1α activity.

Hypoxic Suppression of the Cell Cycle Gene CDC25A in Tumor Cells

Hypoxia, a key microenvironmental factor for tumor development, not only stimulates angiogenesis and glycolysis for tumor expansion, but also induces cell cycle arrest and genetic instability for tumor progression. Several independent studies have shown hypoxic blockade of cell cycle progression at the G1/S transition, arising from the inactivation of S-phase promoting cyclin E–CDK2 kinase complex. Despite these findings, the biochemical pathways leading to the cell-cycle arrest remain poorly defined. We recently showed that hypoxic activates the expression of CDNK1A encoding the CDK2 inhibitor p21Cip1 through a novel HIF-1α–Myc pathway that involves Myc displacement from the CDNK1A promoter by the hypoxia-inducible transcription factor HIF-1α. In pursuit of further understanding of the hypoxic effects on cell cycle in tumor cells, here we report that hypoxia inhibits the expression of CDC25A, another cell cycle gene encoding a tyrosine phosphatase that maintains CDK2 activity. In accordance with the HIF-1α–Myc pathway, hypoxia requires HIF-1α for CDC25A repression, resulting in a selective displacement of an activating Myc from the CDC25A promoter that lacks a canonical E-box without affecting Myc binding in the intron. Intriguingly, HIF-1α alone fails to recapitulate the hypoxic effect, indicating that HIF-1α is necessary but insufficient for the hypoxic repression. Taken together, our studies support that hypoxia inhibits cell cycle progression by controlling the expression of various cell cycle genes.