Gene Transfection Of Cysteinyl Leukotriene Receptors And Signal Tranduction Pathway Of Cysteinyl Leukotriene Receptor 2

Cysteinyl leukotriens (CysLTs, including LTC4, LTD4 and LTE4) are important inflammatory mediators derived from arachidonic acid. CysLTs regulate various pathophysiological responses, such as inflammation, bronchial asthma, cerebral ischemia-reperfusion injury, wounds, and shock. CysLTs act mainly via CysLT1 and CysLT2 receptors as well as GPR17, a novel subtype of CysLT receptor.Currently, most of CysLT receptor antagonists are CysLT1 receptor selective antagonists, but the CysLT2 receptor selective antagonists are commercially not available. Therefore, the roles of CysLT1 receptor are well investigated, while those of CysLT2 receptor are little known. CysLT1 and CysLT2 receptors have been cloned and characterized. However, the GPR17 has not been investigated widely. Thus construction of eukaryotic GRR17 expression plasmid is necessary to provide a basis for studying its regulatory role.CysLT2 receptor closely relates to various diseases like pulmonary fibrosis, atherosclerotic disease and brain ischemia, so it is necessary to investigate the functional properties of CysLT2 receptor. Based on reported studies, CysLTs can change Egr-1 and IL-8 expression through the activation of CysLT2 receptor. However, how does it work? Whether the transcription factor Egr-1 is involved in regulation of IL-8 secretion remains unclear. Therefore, the regulation of IL-8 secretion by CysLT2 receptor activation through the ERK1/2-Egr-1 pathway needs investigation. Objective:To construct the eukaryotic expression plasmid of rat GPR17 (rGPR17) cDNA, and to identify the function of rGPR17 receptor in HEK293 cells.Methods:Total RNA was extracted from rat brain tissue; full-length GPR17 cDNA was prepared by RT-PCR, and cloned into pcDNA3.1(+) plasmid. The recombinant was converted into E. coli DH5α;the proper insert of rGPR17 cDNA was confirmed by PCR, double enzyme digestion analysis and sequencing. The recombinant plasmid pcDNA3.1(+)-rGPR17 was transiently transfected into HEK293 cells using Lipofectamin 2000. Expression of rGPR17 gene was confirmed with RT-PCR and immunofluorescence staining. The exogenous agonist LTD4-enhanced intracellular calcium was measured using Fluo-4.Results:RT-PCR, double enzyme digestion analysis and sequencing showed that the rGPR17 cDNA was cloned into pcDNA3.1(+) plasmid, and the recombinant rGPR17 receptor was expressed after transfection in HKE293 cells. LTD4 increased intracellular calcium release in the transfected HEK293 cells.Conclusion:The eukaryotic expression plasmid of rGPR17 cDNA has been constructed; rGPR17 receptor is functionally expressed in HEK293 cells.PartⅡGene transfection of CysLT receptors and the signal tranduction pathway of CysLT2 receptorObjective:To construct HEK293 cells stably expressed hCysLT1, hCysLT2, and rGPR17, and to determine the hCysLT2 signaling pathways after activation.Methods:The recombinant plasmids of pcDNA3.1(+)-hCysLT1, pcDNA3.1(+)-hCysLT2, PcDNA3.1-rGPR17 were transfected into HEK293 cells using Lipofectamin 2000. The transfected HEK293 cells were selected in 96 well plates by limiting dilution with 600μg/ml C418 for 8 weeks. IL-8 gene promoter activation was detected using dual luciferase reporter gene assay. Expression of EGR-1 was conformed by RT-PCR, Western blot and immunohistochemistry. Expression and secretion of IL-8 were measured by ELISA and RT-PCR. Activated signaling molecules were detected by Western blot analysis.Results:Using dual luciferase reporter gene assay, we found that LTD4 significantly induced IL-8 promoter activation in the HEK293 cells stably transfected with hCysLT2, but not in the cells stably expressing hCysLT1 and rGPR17. The effect of LTC4 was stronger than that of LTD4. In the HEK293 cells stably transfected with hCysLT2, LTC4 induced Egr-1 expression and stimulated IL-8 secretion. ERK1/2 inhibitor (U0126) inhibited Egr-1 and IL-8 expression. Egr-1 SiRNA inhibited the expression and secretion of IL-8.Conclusion:HEK293 cells stably expressing hCysLT1, hCysLT2 and rGPR17 receptor have been successfully constructed. In the HEK293 cells stably transfected with hCysLT2, leukotrienes (LTC4 and LTD4) may cause the secretion of IL-8 through the hCysLT2/ERKl/2-Egr-1 pathway.Summary1. The eukaryotic expression plasmid of rGPR17 cDNA has been constructed; GPR17 receptor is functionally expressed in HEK293 cells. This provides a basis for further research of the GPR17 receptor and its antagonists.2. HEK293 cell lines stably expressing hCysLT1, hCysLT2, rGPR17 have been successfully constructed.3. In the HEK293 cells stably transfected with hCysLT2, leukotrienes (LTD4, LTC4) may cause the secretion of IL-8 mainly through the hCysLT2/ERK1/2-Egr-1 pathway.