Development of an ERK1/2 activation assay to determine relaxin-3/RXFP3 activation

OBJECTIVE To develop ERK1/2 activation assays to detect RXFP3 activation or inhibition by its agonists or antagonists.METHODS Plated HEK-RXFP3,CHO-RXFP3,HEK293 Tand CHO-K1 cells in poly-L-lysine coated well plates.The cells were serum starved and treated with either human relaxin-3(H3relaxin)(10nmol·L-1),R3B1-22R(10μmol·L-1)and pertussis toxin(PTX,100ng·mL-1).The cells were lysed and the ERK1/2 activation was determined by SDS-PAGE followed by immunoblotting for phosphorylated ERK1/2(pERK1/2)and total ERK1/2(tERK1/2)for the lysates.RESULTSThe quantification of the data revealed that the peak of ERK1/2activation can be detected precisely at 10 min post stimulation with 10nmol·L-1 H3 relaxin in both HEK-RXFP3 and CHO-RXFP3 cell lines in all three trials compared to the cells treated with vehicle(P<0.05).However,HEK-RXFP3 cells demonstrated a transient activation of ERK1/2and CHORXFP3 cells demonstrated a continuous activation of ERK1/2which was inhibited by the Gi inhibitor,PTX.Activation of ERK1/2was significantly inhibited by pre-treating the cells with RXFP3 antagonist R3B1-22 Rin HEK-RXFP3 cells.ERK1/2 activation was observed neither in wild type HEK293 Tnor in CHO-K1 cells.CONCLUSION The developed assay can detect RXFP3 activation or inhibition by agonists and antagonists via the detection of pERK1/2 in multiple cell lines.This assay will also be useful to detect signaling pathways upstream to ERK1/2 activation mediated by RXFP3.Activation of ERK1/2 in CHO-RXFP3 cells was mediated by Gi proteins at 10 min as well as at 25-35 min time points.

Determining relaxin-3/RXFP3 activation by inhibition of forskolin induced cAMP assay

OBJECTIVE Relaxin-3 is a novel neuropeptide of the relaxin insulin family of peptides.So far,many studies have reported the role of relaxin-3/RXFP3 system in regulating feeding,stress response and cognition.In previous studies using RXFP3 stable cell lines,inhibition of adenylate cyclase by the activation of Gi/o proteins have been reported.Based on this signaling event,we have developed inhibition of forskolin induced cAMP assay to detect RXFP3 activation or inhibition by its agonists or antagonists.METHODS To detect inhibition of adenylate cyclase up on RXFP3 activation by human relaxin-3(H3relaxin),HEK-RXFP3,CHO-RXFP3,SN56 and GT1-7cells were plated in poly-L-lysine coated 24 well plates.The following day,the cells were starved in serum free cell culture media for 6h.Next,cells were treated in triplicate with serum free media(control)and H3 relaxin for 5-15 min.Then,the cells were treated with serum free media with DMSO(control)or forskolin(5-100μmol·L-1)for 15 min at 37℃ with 5%CO2.At the end of the incubation,cell culture media was discarded and the cells were lysed with 0.1mol·L-1 HCl.The cAMP concentration in each lysate was detected by ELISA(Cayman Chemicals).The data from three experiments were analysed using one way ANOVA followed by Bonferroni post hoc test or Dunnett′s post hoc test.RESULTS In CHO-RXFP3 and HEK-RXFP3 cells,10nmol·L-1 of H3 relaxin was able to significantly inhibit the forskolin(5μmol·L-1)induced cAMP levels(P<0.05).In SN56 neuronal like cell line endogenously expressing RXFP3,100nmol·L-1 H3 relaxin was able to significantly reduce forskolin(3μmol·L-1)induced cAMP(P<0.05).However,in wild type HEK293 Tand CHO-K1 cells,10n mol·L-1 H3 relaxin was not able to significantly reduce the forskolin 22(5μmol·L-1)induced cAMP levels.In GT1-7 mouse hypothalamic cells endogenously expressing RXFP3,100nmol·L-1 H3 relaxin and 5or 3μmol·L-1 forskolin,was able toa significantly increase cAMP levels(P<0.05).CONCLUSION Inhibition of forskolin induced cAMP assay can be used to detect Gi/o mediated cAMP inhibition related signaling events due to RXFP3 activation by its agonists in CHO-RXFP3,HEK-RXFP3 and SN56 cell lines.