Abstract
BackgroundNeuroinflammation has been implicated in various brain pathologies characterized by hypoxia and ischemia. Astroglia play an important role in the initiation and propagation of hypoxia/ischemia-induced inflammation by secreting inflammatory chemokines that attract neutrophils and monocytes into the brain. However, triggers of chemokine up-regulation by hypoxia/ischemia in these cells are poorly understood. Hypoxia-inducible factor-1 (HIF-1) is a dimeric transcriptional factor consisting of HIF-1α and HIF-1β subunits. HIF-1 binds to HIF-1-binding sites in the target genes and activates their transcription. We have recently shown that hypoxia-induced expression of IL-1β in astrocytes is mediated by HIF-1α. In this study, we demonstrate the role of HIF-1α in hypoxia-induced up-regulation of inflammatory chemokines, human monocyte chemoattractant protein-1 (MCP-1/CCL2) and mouse MCP-5 (Ccl12), in human and mouse astrocytes, respectively.MethodsPrimary fetal human astrocytes or mouse astrocytes generated from HIF-1α+/+ and HIF-1α+/- mice were subjected to hypoxia (<2% oxygen) or 125 μM CoCl2 for 4 h and 6 h, respectively. The expression of HIF-1α, MCP-1 and MCP-5 was determined by semi-quantitative RT-PCR, western blot or ELISA. The interaction of HIF-1α with a HIF-1-binding DNA sequence was examined by EMSA and supershift assay. HIF-1-binding sequence in the promoter of MCP-1 gene was cloned and transcriptional activation of MCP-1 by HIF-1α was analyzed by reporter gene assay.ResultsSequence analyses identified HIF-1-binding sites in the promoters of MCP-1 and MCP-5 genes. Both hypoxia and HIF-1α inducer, CoCl2, strongly up-regulated HIF-1α expression in astrocytes. Mouse HIF-1α+/- astrocytes had lower basal levels of HIF-1α and MCP-5 expression. The up-regulation of MCP-5 by hypoxia or CoCl2 in HIF-1α+/+ and HIF-1α+/- astrocytes was correlated with the levels of HIF-1α in cells. Both hypoxia and CoCl2 also up-regulated HIF-1α and MCP-1 expression in human astrocytes. EMSA assay demonstrated that HIF-1 activated by either hypoxia or CoCl2 binds to wild-type HIF-1-binding DNA sequence, but not the mutant sequence. Furthermore, reporter gene assay demonstrated that hypoxia markedly activated MCP-1 transcription but not the mutated MCP-1 promoter in transfected astrocytes.ConclusionThese findings suggest that both MCP-1 and MCP-5 are HIF-1 target genes and that HIF-1α is involved in transcriptional induction of these two chemokines in astrocytes by hypoxia.
Highlights
Neuroinflammation has been implicated in various brain pathologies characterized by hypoxia and ischemia
Hypoxia-inducible factor-1 (HIF-1)-binding regions in MCP-1 and monocyte chemoattractant protein-5 (MCP-5) genes Our recent work demonstrated that transcriptional activation of IL-1β in human and mouse astrocytes during hypoxia is mediated by hypoxia-inducible factor-1α (HIF-1α) [11]
MCP-5 in mouse astrycotes To study the role of HIF-1α in transcriptional regulation of monocyte chemokine MCP-5, primary astrocyte cultures with HIF-1α+/- or HIF-1α+/+ genotype were generated from HIF-1α+/- heterozygous and wild-type mice, respectively [30]
Summary
Neuroinflammation has been implicated in various brain pathologies characterized by hypoxia and ischemia. Hypoxia or ischemia stimulates the expression of inflammatory cytokines (IL-1β, TNF-α), chemokines (IL-8, MCP1/CCL2) and adhesion molecules (ICAM-1) in the brain and in cultured astrocytes and brain endothelial cells [510] These inflammatory mediators play a critical role in the initiation and propagation of ischemica/ hypoxia-evoked neuroinflammation and in the resolution of brain damage [1,2,3,4]. HIF-1 is a heterodimeric protein complex consisting of two subunits, the redox-sensitive HIF-1α (120–130 kD), which is unique to HIF-1, and the constitutively expressed HIF-1β (91–94 kD), a common partner for many other transcription factors [12,13,14] Both subunits are necessary for DNA binding and activation of HIF-1 target genes [15,16]. Cobalt chloride (CoCl2) increases erythropoetin (EPO) production in vitro [27] and in vivo [28] under normoxic conditions and was once given to human patients to treat anemia
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