Ischemia Regulated Transcription Factors: Hypoxia Inducible Factor 1 and Activating Transcription Factor 4

Introduction

Loss of blood flow during stroke creates significant cellular stress via a host of stresses including hypoxia, glucose deprivation, and oxidative stress. Preconditioning studies teach us that homeostatic programs to cell stress are rapidly engaged via activation of preexisting proteins and sustained via adaptive gene expression . Whether activated prior or after a stroke, if these adaptive mechanisms fail to restore homeostasis at a single cell level, then cell death mechanisms are engaged. For postmitotic neurons, deciding to die is a critical, irreversible decision, so transcription of death proteins adds another layer of regulation. Reversing hypoxia, glucose deprivation, or oxidative stress individually via small molecules has not been effective clinically. Failure is likely because each stress creates parallel and overlapping pathways to trigger cell death. Accordingly, a successful therapeutic will target all three pathways by restoring homeostasis. Modulating transcriptional programs that affect a large cassette of genes at cellular, local, and systemic levels provides a strategy for targeting many parallel and interacting cell stress pathways. A number of studies have focused on identifying downstream transcriptional changes that occur before neurons under stress commit to a cell death pathway . In this chapter, we discuss two known stroke-activated transcription factors, hypoxia inducible factor (HIF) and activating transcription factor 4 (ATF4) and potential therapeutic strategies targeting these transcription factors to prevent neuronal death.

Hypoxia-Mediated Transcription Changes During Stroke

Ischemic stroke leads to a decrease in essential cellular fuels, such as oxygen (hypoxia) and glucose (hypoglycemia), required for cell metabolism and survival. Because of the high-energy demands of synaptic activity, neurons critically depend on oxygen and have evolved mechanisms to detect and deal with hypoxia. These adaptive mechanisms involve changes in gene expression that would favor nonaerobic energy generation and increase vascularly derived oxygen delivery, thus counteracting the deleterious effects of hypoxia on energy production. To facilitate recovery of damaged neurons and prevent neuronal death, many have attempted to identify adaptive transcriptional programs and determine how these can be augmented for therapeutic advantage. The HIF transcriptional system has emerged as a key regulatory system that responds to hypoxia at both a local and systemic level and aspects of hypoxia signaling have emerged as key targets for therapeutic intervention.

Hypoxia Regulates Hypoxia-Inducible Factor System

Reductions in oxygen levels below a critical threshold can trigger a series of elegantly evolved biochemical events that lead to the stabilization of the transcription factor HIF-1α and its isoforms (HIF-2α, HIF-3α). Hypoxia-stabilized HIF-1α translocates into the nucleus, forms a heterodimer transcription factor complex with constitutively synthesized HIF-1β, and recruits p300/CBP for transactivation of target genes . This HIF transcriptional factor complex binds specifically to a conserved promoter sequence known as the hypoxia response element (HRE; 5′-RCGTG-3′) in the 3′ enhancer region of HIF target genes . A number of genes enhanced during hypoxia by HIF-1α are known to have protective and reparative properties [e.g., erythropoietin (EPO), vascular endothelial growth factor, and glycolytic enzymes], whereas others trigger cell death or mitophagy [e.g., BCL2/adenovirus E1B 19 kDa protein-interacting protein 3(BNIP3)] . The structure of HIF transcriptional proteins is divided into four domains: an N-terminus with a basic helix-loop-helix structure for DNA binding, a central domain with a per-ARNT-sim domain for heterodimerization or ligand activation, an oxygen-dependent (ODD) domain that confers oxygen-dependent stability, and C and N terminal transactivation domains that regulate the basal transcriptional machinery.

Preconditioning with a sublethal dose of hypoxia in neonatal rats increases HIF-1α and HIF-2α protein expression and upregulates mRNA for HIF target genes . Pretreatment of neonatal animals with known “hypoxia mimics,” such as cobalt chloride and deferoxamine (DFO) provided neuroprotection during hypoxia . These studies initially suggested that pharmacological induction of the hypoxic adaptive response via small molecules could be neuroprotective.