Abstract
Stromal cell-derived factor alpha (SDF1alpha) and its cognate receptor CXCR4 play an important role in neuronal development in the hippocampus, but the genes directly regulated by SDF1alpha/CXCR4 signaling are unknown. To study the role of CXCR4 targeted genes in neuronal development, we used neuronal cultures established from embryonic day 18 rats. Hippocampal neurons express CXCR4 receptor proteins and are stimulated by SDF1alpha resulting in activation of extracellular signal-regulated kinase (ERK)1/2 and the transcription factor cAMP-response element-binding protein. SDF1alpha rapidly induces the expression of the early growth response gene Egr1, a transcription factor involved in activity-dependent neuronal responses, in a concentration-dependent manner. Gel-shift analysis showed that SDF1alpha enhances DNA binding activity to the Egr1-containing promoter for GAD67. Chromatin immunoprecipitation analysis using an Egr1 antibody indicated that SDF1alpha stimulation increases binding of Egr1 to a GAD67 promoter DNA sequence. SDF1alpha stimulation increases the expression of GAD67 at both the mRNA and protein levels, and increases the amount and neurite localization of gamma-aminobutyric acid (GABA) in neurons already expressing GABA. SDF1alpha-induced Egr1/GAD67 expression is mediated by the G protein-coupled CXCR4 receptor and activation of the ERK pathway. Reduction of Egr1 gene expression using small interfering RNA technology lowers the level of GAD67 transcripts and inhibits SDF1alpha-induced GABA production. Inhibition of CXCR4 activation in the developing mouse brain in utero greatly reduced Egr1 and GAD67 mRNA levels and GAD67 protein levels, suggesting a pivotal role for CXCR4 signaling in the development of GABAergic neurons in vivo. Our data suggest that SDF1alpha/CXCR4/G protein/ERK signaling induces the expression of the GAD67 system via Egr1 activation, a mechanism that may promote the maturation of GABAergic neurons during development.
Highlights
Chemokines are a family of small secreted proteins with diverse immune and neural functions, including control of leukocyte trafficking, organization of the hematopoietic/lymphopoietic system, and angiogenesis [1]
Our results suggest that SDF1␣/CXCR4 signaling enhances GAD67 enzyme expression and subsequent GABA production through early growth response gene 1 (Egr1) activation in rat embryonic hippocampal neurons
5) SDF1␣-induced GAD67 expression is inhibited by pretreatment of the neurons with AMD3100, pertussis toxin V (PTX), and by PD98059. 6) SDF1␣ enhances endogenous GAD67 promoter binding activity
Summary
SDF1␣, stromal cell-derived factor ␣; ERK, extracellular signal-regulated kinase; GABA, ␥-aminobutyric acid; CREB, cAMPresponse element-binding protein; PTX, pertussis toxin; DIV, day in vitro; EMSA, electrophoretic mobility shift assay; RT, reverse transcription; PBS, phosphate-buffered saline; GDP, giant depolarizing potential; ChIP, chromatin immunoprecipitation; GAD, glutamic acid decarboxylase; ERE, Egrresponse element; siRNA, small interfering RNA; GAPDH, glyceraldehyde3-phosphate dehydrogenase. We previously reported that SDF1␣/CXCR4 is up-regulated as neuronal stem cells from embryonic spinal cord differentiate into more restricted precursors [24]. In further characterizing SDF1␣/CXCR4 signaling in neuronal precursors from embryonic spinal cord, we found that SDF1␣ activates the ERK pathway, cross-talks with Wnt signaling, and regulates early growth response gene 1 (Egr1) expression using both microarray and RT-PCR analysis [25, 26]. Because the promoter of GAD67 contains an ERE consensus binding site [29, 30], we tested the hypothesis that SDF1␣/CXCR4 signaling regulates the GAD67/GABA system via an Egr1-mediated mechanism in developing hippocampal neurons
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