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-1 (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-1 alpha and HIF-1 beta 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-1 beta in astrocytes is mediated by HIF-1a. In this study, we demonstrate the role of HIF-1 alpha 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-1 alpha(+/+) and HIF-1 alpha(+/-) mice were subjected to hypoxia (< 2% oxygen) or 125 mu M CoCl2 for 4 h and 6 h, respectively. The expression of HIF-1 alpha, MCP-1 and MCP-5 was determined by semi-quantitative RT-PCR, western blot or ELISA. The interaction of HIF-1 alpha 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-1 alpha 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-1 alpha inducer, CoCl2, strongly up-regulated HIF-1 alpha expression in astrocytes. Mouse HIF-1 alpha(+/-) astrocytes had lower basal levels of HIF-1 alpha and MCP-5 expression. The up- regulation of MCP-5 by hypoxia or CoCl2 in HIF-1a(+/+) and HIF-1a(+/-) astrocytes was correlated with the levels of HIF-1 alpha in cells. Both hypoxia and CoCl2 also up- regulated HIF-1 alpha 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. Conclusion: These findings suggest that both MCP-1 and MCP-5 are HIF- 1 target genes and that HIF-1 alpha is involved in transcriptional induction of these two chemokines in astrocytes by hypoxia.