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

Jelena Mojsilovic-Petrovic 0 2 Debbie Callaghan 2 Hong Cui 2 3 Clare Dean 2 Danica B Stanimirovic 1 2 Wandong Zhang 1 2 0 Children's Hospital of Philadelphia, Department of Neurology , ARC-814, Philadelphia, PA 19104 , USA 1 Faculty of Medicine, University of Ottawa , Ottawa , Canada 2 Neurobiology Program, Institute for Biological Sciences, National Research Council of Canada , 1200 Montreal Road, Ottawa, Ontario, K1A0R6 , Canada 3 Visiting Scholar from the Beijing Friendship Hospital affiliated to the Capital University of Medical Sciences , Beijing , China 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 and HIF-1 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 in astrocytes is mediated by HIF-1. In this study, we demonstrate the role of HIF-1 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+/+ and HIF-1+/- mice were subjected to hypoxia (<2% oxygen) or 125 M CoCl2 for 4 h and 6 h, respectively. The expression of HIF-1, MCP-1 and MCP-5 was determined by semi-quantitative RT-PCR, western blot or ELISA. The interaction of HIF-1 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 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 HIF1 inducer, CoCl2, strongly up-regulated HIF-1 expression in astrocytes. Mouse HIF-1+/- astrocytes had lower basal levels of HIF-1 and MCP-5 expression. The up-regulation of MCP-5 by hypoxia or CoCl2 in HIF-1+/+ and HIF-1+/- astrocytes was correlated with the levels of HIF-1 in cells. Both hypoxia and CoCl2 also up-regulated HIF-1 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 MCP1 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 is involved in transcriptional induction of these two chemokines in astrocytes by hypoxia. - Background Ischemic brain damage, including that caused by stroke and trauma, elicits inflammation in the injured areas [13]. A number of inflammatory mediators are expressed in the brain in response to ischemia and hypoxia [1-4]. Hypoxia or ischemia stimulates the expression of inflammatory cytokines (IL-1, TNF-), 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 not only in the initiation and propagation of ischemica/ hypoxia-evoked neuroinflammation but also in the resolution of brain damage [1-4]. However, triggers of inflammatory chemokine up-regulation by hypoxia/ischemia in these cells are poorly understood. We have recently shown that hypoxia-stimulated IL-1 expression in astrocytes is mediated by hypoxia-inducible factor-1 (HIF-1) [11]. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that plays a central role in cellular and systemic homeostatic responses to hypoxia [12-14]. HIF-1 is a heterodimeric protein complex consisting of two subunits, the redox-sensitive HIF-1 (120130 kD), which is unique to HIF-1, and the constitutively expressed HIF-1 (9194 kD), a common partner for many other transcription factors [12-14]. Both subunits are necessary for DNA binding and activation of HIF-1 target genes [15,16]. Several HIF-1 isoforms have been found, including HIF-2 and HIF-3, both of which have significant homologies to HIF-1 [13,14,17]. Although these HIF-1 isoforms may also contribute to the response to hypoxia, HIF-1 is considered the major regulator of O2-tension sensitive genes in cells [12,13]. Decrease in cellular O2 tension or the presence of CoCl2 or desferroxamine leads to elevation of HIF-1 expression, whereas carbon monoxide and nitric oxide inhibit HIF-1 activation [18-20]. HIF-1 is cytosolic and degraded by ubiquitin-proteasome pathway [21,22] via binding of von Hippel-Lindau tumor suppressor protein to the oxygen-dependent degradation domain [23]. Hypoxia induces HIF-1 expression in tissues and cultured cells [12,13,24]. The length of hypoxic stress determines HIF-1 half-life upon reoxygenation. During hypoxia, HIF-1 is stabilized and dimerized with HIF-1, and the complex is translocated into nucleus where it binds to hypoxia-responsive elements in the promoters or enhancers of the target genes, such as the genes encoding erythropoetin (EPO), glucose transporters, glycolytic enzymes, heme oxygenase-1, inducible nitric oxide synthase, transferin, and vascular endothelial growth factor (VEGF) [12-14,25,26]. The consensus DNA sequence for HIF-1 binding in the hypoxia-response element is 5'-[A/ G]CGTG-3' flanked with or without a second consensus site 5'-[A/C]ACAG-3' [12]. Mutations of the consensus sequences result in loss of HIF-1 binding and transcriptional response of the genes to hypoxia [12]. In vitro exposure to CoCl2 or iron chelator deferoxamine under normoxic conditions produces a hypoxia-mimetic effect with up-regulation of HIF-1 and target gene expression [12-14,26]. 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. Astroglial cells are the most abundant cells in the brain and serve as an important source of inflammatory mediators during the course of neuroinflammation [1-3]. Astrocytes subjected to in vitro ischemia/hypoxia produce a large amount of chemoattractant MCP-1 which is 30-time higher than that secreted by human brain endothelial cells subjected to the same treatment [6]. MCP-1 is a potent chemokine and directs the transmigration of blood-borne monocytes/macrophages across the bloodbrain barrier (BBB) into the inflammatory sites in the brain [1-3]. Mouse monocyte chemoattractant protein-5 (MCP-5), known as chemokine (C-C motif) ligand 12 (Ccl12) or sm (...truncated)