Figure 5.

CRH-R subtypes and PKA involved in regulation of IL-1β-induced PGHS2 expression in human term nonlaboring myometrial cells. Primary cells were isolated from myometrial tissues obtained from pregnant women at term before the onset of labor. Subsequently, cells were pretreated with specific CRH-R antagonists for 1–2 hours (A), specific CRH-R1 siRNA oligonucleotide probes as described in Materials and Methods (B), or the specific PKA inhibitor peptide 14-22 amide (myristoylated) (1 μM for 1 hour). Subsequently, cells were treated with IL-1β (1 ng/mL) alone or with 100 nM CRH for the indicated time period, and PGHS2 mRNA and protein levels were determined by real-time quantitative RT-PCR or immunoblotting. Relative mRNA expression levels were normalized against β-actin mRNA and GAPDH was used as a protein loading control. Data are expressed as mean values ± SEM of 3 estimations from at least 4 patients and are presented as a percentage change of maximum CRH effect on IL-1β-induced PGHS2 mRNA (inhibitory at 2 hours or potentiating at 18 hours)(A) or a percentage change of maximum IL-1β effect on PGHS2 protein (B) or mRNA (C). *, P < 0.05 compared with control (no inhibitor) (A) or IL-1β alone (B and C) values; +, P < .05 compared with IL-1β alone.