SFE2006 Oral Communications Pituitary, ovary and steroids (8 abstracts)
Royal Veterinary College, London, United Kingdom.
In target tissues, CNP acts via the cell surface guanylyl cyclase B (GC-B) receptor to stimulate cGMP accumulation. Transgenic mice studies deleting either CNP or the GC-B receptor suggest a role for CNP in regulating bone formation and pituitary growth hormone secretion. Our previous studies have implicated a role for CNP in inhibiting calcium transients in alphaT3-1 pituitary gonadotroph cells; however the biological consequence of cGMP accumulation remains unclear. Therefore, we investigated novel signalling pathways activated by CNP in rat GH3 somatotroph and alphaT3-1 gonadotroph cell lines. CNP potently stimulated cGMP accumulation in both GH3 and alphaT3-1 cells (assessed by EIA), demonstrating the presence of active GC-B receptors. Western blotting was used to examine the effect of CNP pre-treatment (30 min) on acute TRH and GnRH (both 100 nM, 15 min) induced MAPK phosphorylation (P) in GH3 and alphaT3-1 cells respectively. CNP failed to alter the magnitude of either TRH-stimulated or GnRH-stimulated P-ERK, P-JNK or P-p38MAPK in GH3 and alphaT3-1 cells respectively. However, in GH3 cells, basal MAPK phosphorylation in CNP pre-treated cells was dramatically enhanced. Time-course studies (090 min) in GH3 cells using 100 nM CNP alone showed a time-dependent enhancement of ERK and JNK phosphorylation after 5 min, an enhancement of p38MAPK phosphorylation after 15 min, and a concomitant increase in MKP-1 expression. P-ERK and P-JNK levels remained elevated for 60 min, and P-p38MAPK level remained elevated at 90 min. Immunofluorescent confocal microscopy demonstrated enhanced nuclear localisation of P-ERK and P-p38MAPK following CNP treatment (up to 60 min). Pharmacological inhibition of PKC, PKA or calcium influx failed to inhibit the CNP-mediated ERK and p38MAPK phosphorylation, but MEK (UO126) and p38MAPK (SB203580) inhibition abrogated these effects. This study is the first to report a role for CNP in the positive regulation of MAPK family proteins in any cell type.