Natriuretic Peptide Expression and Function in GH3 Somatolactotropes and Feline Somatotrope Pituitary Tumours

Abstract

Patients harbouring mutations in genes encoding C-type natriuretic peptide (CNP; NPPC) or its receptor guanylyl cyclase B (GC-B, NPR2) suffer from severe growth phenotypes; loss-of-function mutations cause achondroplasia, whereas gain-of-function mutations cause skeletal overgrowth. Although most of the effects of CNP/GC-B on growth are mediated directly on bone, evidence suggests the natriuretic peptides may also affect anterior pituitary control of growth. Our previous studies described the expression of NPPC and NPR2 in a range of human pituitary tumours, normal human pituitary, and normal fetal human pituitary. However, the natriuretic peptide system in somatotropes has not been extensively explored. Here, we examine the expression and function of the CNP/GC-B system in rat GH3 somatolactotrope cell line and pituitary tumours from a cohort of feline hypersomatotropism (HST; acromegaly) patients. Using multiplex RT-qPCR, all three natriuretic peptides and their receptors were detected in GH3 cells. The expression of Nppc was significantly enhanced following treatment with either 100 nM TRH or 10 µM forskolin, yet only Npr1 expression was sensitive to forskolin stimulation; the effects of forskolin and TRH on Nppc expression were PKA- and MAPK-dependent, respectively. CNP stimulation of GH3 somatolactotropes significantly inhibited Esr1, Insr and Lepr expression, but dramatically enhanced cFos expression at the same time point. Oestrogen treatment significantly enhanced expression of Nppa, Nppc, Npr1, and Npr2 in GH3 somatolactotropes, but inhibited CNP-stimulated cGMP accumulation. Finally, transcripts for all three natriuretic peptides and receptors were expressed in feline pituitary tumours from patients with HST. NPPC expression was negatively correlated with pituitary tumour volume and SSTR5 expression, but positively correlated with D2R and GHR expression. Collectively, these data provide mechanisms that control expression and function of CNP in somatolactotrope cells, and identify putative transcriptional targets for CNP action in somatotropes.

Keywords: CNP; acromegaly; feline; multiplex RT-qPCR; pituitary; somatotrope.

Figure 1

Molecular characterisation and regulation of the natriuretic peptide system in GH3 somatolactotropes. (A) Expression of natriuretic peptide and natriuretic peptide receptor genes in GH3 cells. Data shown are means ± SEM of relative gene expression (normalised to Actb) from 3 to 8 individual RNA extractions. (B–E) Effect of forskolin (FSK) and TRH on natriuretic peptide and natriuretic peptide receptor gene expression in GH3 cells. Cells were treated for up to 24 h with either 0, 10 µM FSK (C,E), or 100 nM TRH (D) before extraction of RNA, prior to multiplex RT-qPCR assay. Data shown are means ± SEM normalised as fold changes compared to 0 time-point, from 3 to 5 individual RNA extractions (**** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, significantly different from 0 time-point).

Figure 2

Role of PKA and MEK–ERK signalling pathways in the regulation of Nppc expression. Effect of (A) PKA inhibitor (H89) on FSK-stimulated, or (B) MEK inhibitor (PD98059) on TRH-stimulated, Nppc gene expression in GH3 cells. Cells were pre-treated for 30 min with either 0, 10 µM H89 or 1 µM PD98059 before stimulation with either 0, 10 µM FSK or 100 nM TRH for 24 h, followed by extraction of RNA and subsequent multiplex RT-qPCR assay. Data shown are means ± SEM normalised as fold changes compared to control, from 3 to 6 individual RNA extractions. (** p < 0.01, * p < 0.05, significantly different from control).

Figure 3

Effect of C-type natriuretic peptide (CNP) on somatolactotrope-enriched gene expression in GH3 cells. (A) Heatmap of relative gene expression in rat pituitary and GH3 somatolactotropes (normalised to Actb). (B–L) GH3 cells were cultured in the presence of 0 or 100 nM CNP for up to 24 h, before extracting RNA and performing multiplex RT-qPCR to examine alterations in gene expression profiling of somatolactotrope-enriched transcripts (Insr, Lepr, Esr1, Trhr, Sstr1, Sstr2, Sstr3, Sstr4, Sstr5, cFos, Egr1, Gh1, Prl, Aip, Pou1f1, Fos). Data shown are means ± SEM (n = 3 to 5 individual RNA extractions) of relative gene expression (normalized to ActB); (**** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, significantly different from basal.

Figure 4

Effect of 17β-oestradiol on natriuretic peptide expression and function in GH3 somatolactotropes and rat anterior pituitaries. (A,B) GH3 cells were cultured in the presence of 0 or 100 nM 17β-oestradiol for up to 24 h. RNA was extracted and analysed by multiplex RT-qPCR to examine alterations in gene expression profiling of natriuretic peptide system transcripts (Nppa, Nppc, Npr1, Npr2). Data shown are means ± SEM (n = 3 to 5 individual RNA extractions) of relative gene expression (normalized to ActB); * p < 0.05, ** p < 0.01, significantly different from basal. (C,D) GH3 cells were cultured in the presence of 0 or 100 nM 17β-oestradiol for 24 h and 72 h, followed by exposure to 0 or 100 nM CNP in the presence of 1 mM IBMX for 1 h. Total cGMP concentration were determined using a commercially available enzyme immunoassay kit. Data shown are means ± SEM of 5 independent experiments, each performed in triplicates; **** p < 0.0001, *** p < 0.001, * p < 0.05, significantly different from control, or in the presence of E2 (as indicated). (E) Effect of oestrous cycle on pituitary CNP content. Vaginal smears were performed on random cycling Sprague–Dawley rats (8–10 weeks old); many neutrophils were present at diestrous, with small nucleated epithelial cells present at proestrous, and predominantly keratinized epithelial cells at estrous and metestrous. Pituitary CNP and LH content, and plasma LH were measured by RIAs. Data shown are means ± SEM from 3 to 12 animals, normalised as fold changes compared to diestrous (** p < 0.01, * p < 0.05, significantly different from diestrous).

Figure 5

Molecular characterisation of the natriuretic peptide system in feline somatotrope tumours. (A) Optimisation of the multiplex RT-qPCR assay to detect natriuretic peptide transcripts and somatotrope-enriched genes from feline pituitary tissue, showing relative expression heatmap of transcripts, normalised to RPL18. (B–G) Total RNA was extracted from feline pituitary samples taken from patients with hypersomatotropism (HST) (n = 19) and healthy controls (n = 10), and analysed by multiplex RT-qPCR to examine alterations in gene expression profiling of the natriuretic peptide system (NPPA, NPPB, NPPC, NPR1, NPR2, NPR3). Violin plots show median and quartiles; *** p < 0.0001, significantly different compared to control.

Figure 6

Correlation relationships between NPPC and somatotrope-enriched genes in feline somatotrope tumours. (A–F) Dot plots showing significant correlations between NPPC and (A) NPPA, (B) NPR2, (C) NPR3, (D) D2R, (E) SSTR5, and (F) GHR. (G) Representative transverse CT scan of feline pituitary tumour, from which dorsoventral mass size was measured (in mm), and correlated with NPPC expression. Data shown are from 19 feline HST patients.