Influence of steroids and GnRH on biosynthesis and secretion of secretogranin II and chromogranin A in relation to LH release in LbetaT2 gonadotroph cells

Abstract

The granin proteins secretogranin II (SgII) and chromogranin A (CgA) are commonly found associated with LH and/or FSH within specialised secretory granules in gonadotroph cells, and it is possible that they play an important role in the differential secretion of the gonadotrophins. In this study we have examined the regulation of the biosynthesis and secretion of SgII and CgA, in relation to LH secretion, in the LbetaT2 mouse pituitary gonadotroph cell line. Three experiments were carried out to investigate the effects of oestradiol (E2) and dexamethasone (Dex) in the presence and absence of GnRH (experiment 1), differing GnRH concentrations (experiment 2) and alterations in GnRH pulse frequency (experiment 3). In experiment 1, exposure to E2, Dex or E2+Dex, either with or without GnRH treatment, resulted in increased LH secretion. Steroids alone had no effect on LHbeta mRNA levels, but in the presence of GnRH LHbeta mRNA levels were increased in Dex- and E2+Dex-treated cells. GnRH receptor (GnRH-R) mRNA levels were up-regulated by Dex and E2+Dex, but were unaffected by GnRH. There were no steroid-induced changes in SgII or CgA mRNA, but increased levels of CgA mRNA were observed after GnRH treatment in cells cultured in the presence of Dex. In experiment 2, increasing concentrations of GnRH resulted in increases in LH secretion that were inversely dose-dependent. No changes in LHbeta, GnRH-R or SgII mRNA levels were observed, but there were dose-dependent increases in CgA mRNA levels. In experiment 3, GnRH was given as either 1 pulse/day or 4 pulses/day for 3 days. Both pulse regimes resulted in increased LH, SgII and CgA secretion compared with controls during the first 15 min pulse on day 3. Exposure to GnRH at 4 pulses/day increased LH and SgII secretion compared with controls during all 4 pulses, but secretion of both proteins was reduced during pulses 2-4 compared with pulse 1. CgA secretion also increased due to GnRH in pulse 1, but was decreased by GnRH treatment during pulse 2, and unchanged by GnRH during pulses 3 and 4. Total daily secretion of LH and SgII from cells given 1 pulse/day of GnRH increased compared with controls on all three treatment days, while total CgA secretion increased in response to GnRH on days 2 and 3 only. Intracellular levels of SgII, but not LH, decreased after GnRH treatment. In contrast, intracellular CgA was increased, but only after 4 pulses/day of GnRH. Levels of LHbeta, but not SgII, mRNA were increased by both pulse regimes, while CgA mRNA levels increased after 1 pulse/day of GnRH. These results indicate that there is a close correlation between the GnRH-stimulated release of LH and SgII from LbetaT2 cells, suggesting that SgII may have an influential role in the regulated secretion of LH, possibly by inducing LH aggregation to facilitate trafficking into secretory granules. CgA secretion does not appear to be closely associated with that of LH, but CgA expression does appear to be regulated by GnRH, which may indicate involvement in the control of LH secretion, possib...

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

“…Interestingly, in cardiac tissues, ANP and BNP are colocalised with chromogranin A (CgA) in secretory granules (Tota et al 2008). As CgA and secretogranin II are implicated in gonadotrophin secretion (Nicol et al 2002(Nicol et al , 2004, it is tempting to speculate that CNP may well be cosecreted with gonadotrophins in response to GNRH.…”

Secretoneurin stimulates the production and release of luteinizing hormone in mouse LβT2 gonadotropin cells

“…We hypothesize that the well-conserved SN peptide also plays a role in regulating LH secretion in mammals. The mouse L␤T2 gonadotroph cell line was chosen because it is a highly amenable model system that exhibits the major functional characteristics consistent with those of normal mammalian pituitary gonadotrophs (38) and produces SgII (28,29), and intracellular signal transduction pathways are well described (14,19,20,32,40,43). Our results show that SN simulates intracellular signaling pathways similar to those regulated by GnRH.…”

“…The levels of LH release to the culture medium increased from 3 to 12 h in the control group as well as in the mGnRH-A and SN treatments. We adopted 10 nM of mGnRH-A as the positive control in this experiment (28). After 3 h of treatment, mGnRH-A induced a 3.1-fold increment of LH levels (P Ͻ 0.001) compared with the control (Fig.…”

“…The LH levels in the culture medium were assayed by a mouse LH-specific RIA, as described previously, and validated using the L␤T2 cell line (24,28,29). The intra-and interassay coefficients of variance were Ͻ10% for LH RIA.…”

“…Nicol et al (28) utilized radioimmunoassay (RIA) to investigate protein release from the mouse L␤T2 gonadotroph cell line. They observed that pulsatile GnRH treatments resulted in the marked increments of both LH and SgII release together at each time point, suggesting a close correlation between the secretions of these two proteins.…”