Bone morphogenetic protein-4 interacts with activin and GnRH to modulate gonadotrophin secretion in LbetaT2 gonadotrophs

Abstract

We have shown previously that, in sheep primary pituitary cells, bone morphogenetic proteins (BMP)-4 inhibits FSHbeta mRNA expression and FSH release. In contrast, in mouse LbetaT2 gonadotrophs, others have shown a stimulatory effect of BMPs on basal or activin-stimulated FSHbeta promoter-driven transcription. As a species comparison with our previous results, we used LbetaT2 cells to investigate the effects of BMP-4 on gonadotrophin mRNA and secretion modulated by activin and GnRH. BMP-4 alone had no effect on FSH production, but enhanced the activin+GnRH-induced stimulation of FSHbeta mRNA and FSH secretion, without any effect on follistatin mRNA. BMP-4 reduced LHbeta mRNA up-regulation in response to GnRH (+/-activin) and decreased GnRH receptor expression, which would favour FSH, rather than LH, synthesis and secretion. In contrast to sheep pituitary gonadotrophs, which express only BMP receptor types IA (BMPRIA) and II (BMPRII), LbetaT2 cells also express BMPRIB. Smad1/5 phosphorylation induced by BMP-4, indicating activation of BMP signalling, was the same whether BMP-4 was used alone or combined with activin+/-GnRH. We hypothesized that activin and/or GnRH pathways may be modulated by BMP-4, but neither the activin-stimulated phosphorylation of Smad2/3 nor the GnRH-induced ERK1/2 or cAMP response element-binding phosphorylation were modified. However, the GnRH-induced activation of p38 MAPK was decreased by BMP-4. This was associated with increased FSHbeta mRNA levels and FSH secretion, but decreased LHbeta mRNA levels. These results confirm 1. BMPs as important modulators of activin and/or GnRH-stimulated gonadotrophin synthesis and release and 2. important species differences in these effects, which could relate to differences in BMP receptor expression in gonadotrophs.

Figure 1

Expression of BMP receptors. (A) mRNAs for BMP receptors IA, IB and II were detected in untreated LβT2 cells and adult mouse pituitary by RT-PCR at 35 cycles. (B) BMP receptor proteins IA, IB and II were detected in untreated LβT2 cells by immunofluorescence. Bar represents 10 μm.

Figure 2

Effects of BMP-4 on gonadotrophin secretion and FSHβ and LHβ mRNA expression. (A) FSH and (C) LH secreted on day 3 from LβT2 cells cultured with 0 or 50 ng/ml BMP-4 in the presence and absence of 50 ng/ml activin A±a daily 1 h pulse of 10 nM GnRH for 3 days were measured by RIA. Results represent total hormone secreted during the 1 h GnRH treatment and the subsequent overnight incubation; n=5. (B) FSHβ and (D) LHβ mRNA levels on day 4 in LβT2 cells, cultured as described, were measured by Taqman quantitative RT-PCR; n=3. Different letters indicate significant differences between treatment groups. Values represent means±s.e.m. from one representative experiment. Five experiments were performed with similar results.

Figure 3

Effects of BMP-4 on GnRH-R and follistatin mRNA expression. LβT2 cells were cultured with 0 or 50 ng/ml BMP-4 in the presence and absence of 50 ng/ml activin A±a daily 1-h pulse of 10 nM GnRH for 3 days. On day 4, total RNA was prepared and (A) GnRH receptor and (B) follistatin mRNA levels were measured by Taqman quantitative RT-PCR. Different letters indicate significant differences between treatment groups. Values represent means±s.e.m. from one representative experiment; n=3. Five experiments were performed with similar results.

Figure 4

Activation of Smad signalling pathways. LβT2 cells were cultured with 0 or 50 ng/ml BMP-4 in the presence and absence of 50 ng/ml activin A±a daily 1 h pulse of 10 nM GnRH for 3 days. On day 4, the cells were given a further 1-h GnRH treatment and total protein extracts prepared. Levels of (A) phosphorylated Smad1/5 and (B) phosphorylated Smad2/3 were measured by western blotting and expressed relative to the respective total Smad proteins. Different letters indicate significant differences between treatment groups. Values represent means±s.e.m. from one representative experiment; n=2.

Figure 5

Activation of ERK, p38 MAPK and CREB signalling. The LβT2 cells were cultured with 0 or 50 ng/ml BMP-4 in the presence and absence of 50 ng/ml activin A±a daily 1-h pulse of 10 nM GnRH for 3 days. On day 4, the cells were given a final GnRH pulse for 10 or 20 min and levels of (A) phosphorylated ERK1/2 expressed relative to total ERK1/2, (B) phosphorylated p38 expressed relative to α-tubulin and (C) phosphorylated CREB expressed relative to α-tubulin were measured by western blotting. Different letters indicate significant differences between treatment groups. Data shown for ERK1/2 and CREB are from 20-min GnRH treatment groups and for p38 from the 10-min GnRH treatment. Values represent means±s.e.m. from one representative experiment; n=2.