Molecular characterisation and functional interrogation of a local natriuretic peptide system in rodent pituitaries, αT3-1 and LβT2 gonadotroph cells

Abstract

In the pituitary, C-type natriuretic peptide (CNP) has been implicated as a gonadotroph-specific factor, yet expression of the CNP gene (Nppc) and CNP activity in gonadotrophs is poorly defined. Here, we examine the molecular expression and putative function of a local gonadotroph natriuretic peptide system. Nppc, along with all three natriuretic peptide receptors (Npr1, Npr2 and Npr3), was expressed in both aT3-1 and LbT2 cells and primary mouse pituitary tissue, yet the genes for atrial-(ANP) and B-type natriuretic peptides (Nppa and Nppb) were much less abundant. Putative processing enzymes of CNP were also expressed in aT3-1 cells and primary mouse pituitaries. Transcriptional analyses revealed that the proximal 50 bp of the murine Nppc promoter were sufficient for GNRH responsiveness, in an apparent protein kinase C and calcium-dependent manner. Electrophoretic mobility shift assays showed Sp1/Sp3 proteins form major complexes within this region of the Nppc promoter. CNP protein was detectable in rat anterior pituitaries, and electron microscopy detected CNP immunoreactivity in secretory granules of gonadotroph cells. Pharmacological analyses of natriuretic peptide receptor activity clearly showed ANP and CNP are potent activators of cGMP production. However, functional studies failed to reveal a role for CNP in regulating cell proliferation or LH secretion. Surprisingly, CNP potently stimulated the human glycoprotein hormone a-subunit promoter in LbT2 cells but not in aT3-1 cells. Collectively, these findings support a role for CNP as the major natriuretic peptide of the anterior pituitary, and for gonadotroph cells as the major source of CNP expression and site of action.

Expression of guanylyl cyclase-B (GC-B/NPR2) receptors in normal human fetal pituitaries and human pituitary adenomas implicates a role for C-type natriuretic peptide

“…Cell culture aT3-1, LbT2, GH3, and AtT-20 cells were cultured as described previously , Thompson et al 2009) using DMEM (Sigma-Aldrich) containing 10% FCS, penicillin, and streptomycin.…”

“…Pituitaries were removed from 8-week-old random cycling female C57/BL6 mice (20 per experiment) and subjected to enzymatic dispersion as described previously (McArdle & Poch 1992, Thompson et al 2009. Dispersed cells were then cultured overnight in 24-well plates before transfection with the indicated NPR2 reporter gene constructs.…”

“…Tissue was collected immediately at the postoperative stage, snap frozen, and stored at K80 8C before extracting total RNA using the Trizol method followed by RNA cleanup and DNase treatment, as described previously (Thompson et al 2009). Normal human fetal pituitary RNA samples were obtained from fetuses collected following medical termination of pregnancy (between 14 and 18 weeks of gestation), subject to local ethical approval (by the Lothian Paediatrics/Reproductive Medicine Research Ethics Subcommittee) as described previously (Pope et al 2006).…”

“…Our previous molecular and pharmacological studies performed on well-established rodent anterior pituitary cell lines (Fowkes et al 1999, Thompson et al 2009) have shown that GC-B signaling is the major pathway used by CNP in most anterior pituitary cell types. Despite this, there is a paucity of data describing the expression of GC-B in human pituitaries and pituitary adenomas.…”

Regulation of C-type natriuretic peptide expression

“…On the contrary, Thompson et al [88] have very recently shown evidence using electrophoretic mobility shift assays (EMSA) that both Sp-1 and Sp-3 bind as a complex to the putative GC-rich regulatory site in the proximal Nppc promoter. It might be worth noting that although both studies employed nuclear extract from pituitary cell lines or the pituitary proper, the CNP gene examined in the Ohta et al study was human, but the gene in the Thompson et al study was from the mouse [88].…”