Hypoxia-inducible factor-1 (HIF-1) is involved in the regulation of hypoxia-stimulated expression of monocyte chemoattractant protein-1 (MCP-1/CCL2) and MCP-5 (Ccl12) in astrocytes.
BACKGROUND
Neuroinflammation 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-1alpha and HIF-1beta 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-1beta in astrocytes is mediated by HIF-1alpha. In this study, we demonstrate the role of HIF-1alpha 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.


METHODS
Primary fetal human astrocytes or mouse astrocytes generated from HIF-1alpha+/+ and HIF-1alpha+/- mice were subjected to hypoxia (<2% oxygen) or 125 muM CoCl2 for 4 h and 6 h, respectively. The expression of HIF-1alpha, MCP-1 and MCP-5 was determined by semi-quantitative RT-PCR, western blot or ELISA. The interaction of HIF-1alpha 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-1alpha was analyzed by reporter gene assay.


RESULTS
Sequence analyses identified HIF-1-binding sites in the promoters of MCP-1 and MCP-5 genes. Both hypoxia and HIF-1alpha inducer, CoCl2, strongly up-regulated HIF-1alpha expression in astrocytes. Mouse HIF-1alpha+/- astrocytes had lower basal levels of HIF-1alpha and MCP-5 expression. The up-regulation of MCP-5 by hypoxia or CoCl2 in HIF-1alpha+/+ and HIF-1alpha+/- astrocytes was correlated with the levels of HIF-1alpha in cells. Both hypoxia and CoCl2 also up-regulated HIF-1alpha 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.


CONCLUSIONS
These findings suggest that both MCP-1 and MCP-5 are HIF-1 target genes and that HIF-1alpha is involved in transcriptional induction of these two chemokines in astrocytes by hypoxia.
Mojsilovic-Petrovic
Jelena
J
Neurobiology Program, Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada. PETROVIC@email.chop.edu <PETROVIC@email.chop.edu>
Callaghan
Debbie
D
Cui
Hong
H
Dean
Clare
C
Stanimirovic
Danica B
DB
Zhang
Wandong
W
D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D015687:Cell Hypoxia / Q000235:genetics / Q000502:physiology*; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000096:biosynthesis* / Q000235:genetics; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D051793:Hypoxia-Inducible Factor 1 / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008822:Mice, Transgenic; D018945:Monocyte Chemoattractant Proteins / Q000096:biosynthesis* / Q000235:genetics
C491224:Ccl12 protein, mouse; D018932:Chemokine CCL2; D051793:Hypoxia-Inducible Factor 1; D018945:Monocyte Chemoattractant Proteins