Hypoxia enhances CXCR4 expression favoring microglia migration via HIF-1a activation

Migration toward pathological area is the first critical step in microglia engagement during the central nervous system (CNS) injury, although the molecular mechanisms underlying microglia mobilization have not been fully understood. Here, we report that hypoxia promotes stromal cell-derived factor-1a (SDF-1a) induced microglia migration by inducing the CXC chemokine receptor 4 (CXCR4) expression. Exposure to hypoxia significantly enhanced CXCR4 expression levels in N9 microglia cell. Then, cell migration induced by SDF-1, a CXCR4-specific ligand, was observed accelerated. Blockade of hypoxia inducible factor-1a (HIF-1a) activation by inhibitors of phosphoinositide-3-kinase (PI3K)/Akt signaling pathway abrogated both of hypoxia-induced CXCR4 up-regulation and cell-migration acceleration. These results point to a crucial role of Hypoxia-HIF-1a-CXCR4 pathway during microglia migration. 2008 Elsevier Inc. All rights reserved. Hypoxia is one of the important physiological stimuli that are often associated with a variety of pathological states such as ischemia, respiratory disease, and tumorigenesis. In central nervous system (CNS), hypoxia not only causes neuronal cell injury, but also induces pathological microglial activation [1,2]. Microglia is believed to play a crucial role in the development and regeneration of the CNS. During CNS injury, microglia participate in inflammation and wound healing by migrating into damaged tissue, where they proliferate and act as scavengers [3,4]. Thus, the migration of microglia in response to pathological conditions including hypoxia is a critical step to carry out microglia functions, but the mechanisms are not fully understood. The chemokines are a large family of small, structurally related cytokines which possess chemotactic activities. Chemokine receptors are classified according to which group of chemokines they bind and are designated as CXCR1-CXCR6, CCR1-CCR6, CX3CR1, and XCR1 [5]. There is growing evidence for the role of chemokines in the regulation of CNS disease. Elevated levels of chemokines have been observed in several brain diseases, suggesting that these molecules function as regulators of brain inflammation. [6,7]. Only recently, the chemokine stromal cell-derived factor-1a (SDF-1a) and its receptor CXC chemokine receptor-4 (CXCR4) have been recognized to control the migration of microglia [8,9]. After exposed to hypoxia, many kinds of cells increase their synthesis of a protein named HIF, which in turn binds to and activates many genes [10]. Hypoxia inducible factor-1 (HIF-1) is a heterodimer composed of a HIF-1b subunit that is constitutively expressed and a HIF-1a subunit that is rapidly degraded by ubiquitination via the proteasomal pathway, a process that is inhibited under hypoxic conditions [11]. A role of HIF-1a in the regulation of CXCR4 mRNA expression is suggested by several previous findings [12,13]. CXCR4 promoter includes four potential hypoxia-response elements (HREs) located within 2.6 kb upstream of the transcriptional start site and one at position 1.3 kb within the intron [14]. This implies that CXCR4 is a hypoxia response gene. Therefore, hypoxia may affect migroglia migration process by altering the expression of CXCR4 via activation of HIF-1a. To test this hypothesis, we employed N9 microglia cell line to evaluate the effect of hypoxia on microglia. Here, we report that hypoxia increased mRNA and protein expression levels of CXCR4 in N9 cells and enhanced microglia migration. In addition, we also demonstrated that the induction of CXCR4 expression by hypoxia was possibly through the activation of HIF-1a. We believe this knowledge may lead to a potentially important therapy for regulating microglia function targeting CXCR4. Materials and methods Cell culture and hypoxia. The N9 murine microglia cell line (kindly donated by Dr. Yun BAI, Department of genetics, the Third Military Medical University, China) was maintained in Dulbecco’s modified Eagle’s medium (DMEM; HyClone, Logan, UT) 0006-291X/$ see front matter 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.04.055 * Corresponding author. Fax: +86 23 68752290. E-mail addresses: [email protected] (Z. Yu), [email protected] (Z. Yu). Biochemical and Biophysical Research Communications 371 (2008) 283–288