Figure 2.

Activation of the reporter genes AP-1 (A), NF-κB (B) and SRE (C) and inhibition of the forskolin-stimulated cAMP production (D) in CHO-RXFP3 or Flp-In CHO cells by H3 relaxin, H2 relaxin or R3(BΔ23–27)R/I5. CHO-RXFP3 or Flp-In CHO cells (negative control) were transiently co-transfected with either AP-1-SEAP, NF-κB-SEAP, or SRE-SEAP reporter genes and a constitutively active β-galactosidase reporter. Activation was determined by increased SEAP in the culture medium 24 h after stimulation. H3 relaxin activated AP-1, NF-κB and SRE (A, B, C), but only the AP-1 reporter was blocked by R3(BΔ23–27)R/I5 (A). H2 relaxin stimulated AP-1 and SRE and R3(BΔ23–27)R/I5 had no inhibitory effect (A, C). SRE activation was also detected following R3(BΔ23–27)R/I5 (C). FBS (10%) was used as a positive control in all experiments to demonstrate functional reporter genes. Data are expressed as percentage β-galactosidase activation and normalized to the FBS response. Data are mean ± SEM of six to eight independent experiments, conducted in triplicate. In the cAMP assay (D), forskolin-stimulated cAMP accumulation (30 μM) was inhibited in CHO-RXFP3 cells by H3 relaxin and to a lesser extent by H2 relaxin and R3(BΔ23–27)R/I5. R3(BΔ23–27)R/I5 blocked H3 relaxin, but not H2 relaxin-induced cAMP inhibition. Data are expressed as % response to forskolin. Vehicle represents signalling in the absence of forskolin and ligands. Data are mean ± SEM of three independent experiments, conducted in duplicate. Controls for CHO-RXFP3 cells were parallel treatments of the parent cell line Flp-In CHO. Data were analysed by one-way anova with Dunnett's post hoc test. *P ≤ 0.05 and ***P ≤ 0.001.