Regulation of gene expression by hypoxia: Integration of the HIF-transduced hypoxic signal at the hypoxia-responsive element

Abstract

Cells experiencing lowered O₂ levels (hypoxia) undergo a variety of biological responses in order to adapt to these unfavorable conditions. The master switch, orchestrating the cellular response to low O₂ levels, is the transcription factor, termed hypoxia-inducible factor (HIF). The α subunits of HIF are regulated by 2-oxoglutarate-dependent oxygenases that, in the presence of O₂, hydroxylate specific prolyl and asparaginyl residues of HIF-α, inducing its proteasome-dependent degradation and repression of transcriptional activity, respectively. Hypoxia inhibits oxygenases, stabilized HIF-α translocates to the nucleus, dimerizes with HIF-β, recruits the coactivators p300/CBP, and induces expression of its transcriptional targets via binding to hypoxia-responsive elements (HREs). HREs are composite regulatory elements, comprising a conserved HIF-binding sequence and a highly variable flanking sequence that modulates the transcriptional response. In summary, the transcriptional response of a cell is the end product of two major functions. The first (trans-acting) is the level of activation of the HIF pathway that depends on regulation of stability and transcriptional activity of the HIF-α. The second (cis-acting) comprises the characteristics of endogenous HREs that are determined by the availability of transcription factors cooperating with HIF and/or individual HIF-α isoforms.

Introduction

Oxygen (O₂) is essential for the survival of all aerobic organisms. O₂ is required for aerobic metabolism that maintains intracellular energy balance. Aerobic energy metabolism is dependent on oxidative phosphorylation, in which the oxido-reduction energy of the mitochondrial electron transport is converted into the high-energy phosphate bond of ATP. In this process, O₂ serves as the final electron acceptor. Depending largely on the distance from the nearest functional blood vessel, cells in mammalian tissues typically experience O₂ concentrations in the 40–60 mm Hg range [1]. Hypoxia, defined as a state of reduced O₂ level below normal values, occurs under various physiological (embryonic development, adaptation to high altitudes, wound healing) as well as pathological (ischemic diseases, cancer) conditions [1]. In order to cope with hypoxia, organisms undergo a variety of systemic and local changes to restore O₂ homeostasis and limit the effect of low O₂ [2]. Systemic adjustments include enhanced O₂ delivery by the bloodstream, whereas angiogenesis features prominently among the local adjustments [3]. At the cellular level, the most noticeable response to hypoxia is reduction in oxidative phosphorylation, accompanied by increased glycolysis to compensate for lower ATP production [4].

Although hypoxia generally inhibits mRNA synthesis, transcription of subsets of genes increases dramatically. At the molecular level, the master switch orchestrating the cellular response to low O₂ tension is generally considered to be the transcription factor hypoxia-inducible factor (HIF) [5], [6]. The importance of the HIF pathway can be inferred from the fact that it is present in virtually all cell types and all higher eukaryotes [7]. HIF is a heterodimer that consists of one of the regulatable HIF-α subunits and the constitutively expressed HIF-1β (also known as aryl hydrocarbon receptor nuclear translocator or ARNT). Both α and β subunits belong to the family of the basic helix–loop–helix (bHLH) and PER-ARNT-SIM (PAS) domain-containing transcription factors [8]. bHLH and PAS domains mediate DNA binding and dimerization; the other domains in the α subunits include a unique O₂-dependent degradation domain (ODDD) and two transactivation domains: the N-terminal activation domain (NAD) and C-terminal activation domain (CAD) [7] (Fig. 1). Three structurally closely related α subunits (HIF-1α, HIF-2α, and HIF-3α) have been identified to date [7]. In addition to these three isoforms, their splicing variants also have been described. HIF-1α variants lacking exon 14 or exons 11 and 12 display severely compromised transcriptional activity [9], [10]. Inhibitory PAS protein is a splice variant of HIF-3α that preferentially dimerizes with HIF-1α and thus precludes formation of active HIF-1α/ HIF-1β heterodimers [11].

Hypoxia activates the HIF pathway by a sophisticated mechanism that regulates post-translational modifications of the α subunits. In this mechanism, two independent but co-regulated molecular switches can be recognized: the first switch controls the abundance of HIF-α whereas the second regulates its transcriptional activity. Upon activation by the hypoxic signal, HIF-α translocates to the nucleus, dimerizes with HIF-β [12], recruits p300/CBP, and induces the expression of its transcriptional targets via binding to hypoxia-responsive elements (HRE) [5], [6] (Fig. 2).