Calcium-sensing receptor stimulates secretion of an interferon-gamma-induced monokine (CXCL10) and monocyte chemoattractant protein-3 in immortalized GnRH neurons.
Biology of GnRH neurons is critically dependent on extracellular Ca(2+) (Ca(2+) (o)). We evaluated differences in gene expression patterns with low and high Ca(2+) (o) in an immortalized GnRH neuron line, GT1-7 cells. Mouse global oligonucleotide microarray was used to evaluate transcriptional differences among the genes regulated by elevated Ca(2+) (o). Our result identified two interferon-gamma (IFNgamma)-inducible chemokines, CXCL9 and CXCL10, and a beta chemokine, monocyte chemoattractant protein-3 (MCP-3/CCL7), being up-regulated in GT1-7 cells treated with high Ca(2+) (o) (3.0 mM) compared with low Ca(2+) (o) (0.5 mM). Up-regulation of these mRNAs by elevated Ca(2+) (o) was confirmed by quantitative PCR. Elevated Ca(2+) (o) stimulated secretion of CXCL10 and MCP-3 but not CXCL9 in GT1-7 cells, and this effect was mediated by an extracellular calcium-sensing receptor (CaR) as the dominant negative CaR attenuated secretion of CXCL10 and MCP-3. CXCL10 and MCP-3 were localized in mouse GnRH neurons in the preoptic hypothalamus. Suppression of K(+) channels (BK channels) with 25 nM charybdotoxin inhibited high-Ca(2+) (o)-stimulated CXCL10 release. Accordingly, CaR activation by a specific CaR agonist, NPS-467, resulted in the activation of a Ca(2+)-activated K(+) channel in these cells. CaR-mediated MCP-3 secretion involves the PI3 kinase pathway in GT1-7 cells. MCP-3 stimulated chemotaxis of astrocytes treated with transforming growth factor-beta (TGFbeta). With TGFbeta-treated astrocytes, we next observed that conditioned medium from GT1-7 cells treated with high Ca(2+) promoted chemotaxis of astrocytes, and this effect was attenuated by a neutralizing antibody to MCP-3. These results implicate CaR as an important regulator of GnRH neuron function in vivo by stimulating secretion of heretofore unsuspected cytokines, i.e., CXCL10 and MCP-3.
Bandyopadhyay
Sanghamitra
S
Neurochemistry Laboratory, Department of Psychiatry and Genetics and Aging Research Unit, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02115, USA.
Jeong
Kyeong-Hoon
KH
Hansen
Jacob Tfelt
JT
Vassilev
Peter M
PM
Brown
Edward M
EM
Chattopadhyay
Naibedya
N
D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects; D002118:Calcium / Q000494:pharmacology; D002478:Cells, Cultured; D018999:Charybdotoxin / Q000494:pharmacology; D054410:Chemokine CCL7; D054357:Chemokine CXCL10; D019743:Chemokines, CXC / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D020869:Gene Expression Profiling / Q000379:methods; D007031:Hypothalamus / Q000166:cytology; D051379:Mice; D008810:Mice, Inbred C57BL; D018945:Monocyte Chemoattractant Proteins / Q000378:metabolism* / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D009498:Neurotoxins / Q000494:pharmacology; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D044169:Receptors, Calcium-Sensing / Q000502:physiology*; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology*
C515451:Ccl7 protein, mouse; D054410:Chemokine CCL7; D054357:Chemokine CXCL10; D019743:Chemokines, CXC; D018945:Monocyte Chemoattractant Proteins; D009498:Neurotoxins; D044169:Receptors, Calcium-Sensing; D016212:Transforming Growth Factor beta; D018999:Charybdotoxin; D002118:Calcium