Female-specific induction of rat pituitary dentin matrix protein-1 by GnRH

Abstract

Hypothalamic GnRH is the primary regulator of reproduction in vertebrates, acting via the G protein-coupled GnRH receptor (GnRHR) in pituitary gonadotrophs to control synthesis and release of gonadotropins. To identify elements of the GnRHR-coupled gene network, GnRH was applied in a pulsatile manner for 6 hours to a mixed population of perifused pituitary cells from cycling females, mRNA was extracted, and RNA sequencing analysis was performed. This revealed 83 candidate-regulated genes, including a large number coding for secreted proteins. Most notably, GnRH induces a greater than 600-fold increase in expression of dentin matrix protein-1 (Dmp1), one of five members of the small integrin-binding ligand N-linked glycoprotein gene family. The Dmp1 response is mediated by the GnRHR, not elicited by other hypothalamic releasing factors, and is approximately 20-fold smaller in adult male pituitary cells. The sex-dependent Dmp1 response is established during the peripubertal period and independent of the developmental pattern of Gnrhr expression. In vitro, GnRH-induced expression of this gene is coupled with release of DMP1 in extracellular medium through the regulated secretory pathway. In vivo, pituitary Dmp1 expression in identified gonadotrophs is elevated after ovulation. Cell signaling studies revealed that the GnRH induction of Dmp1 is mediated by the protein kinase C signaling pathway and reflects opposing roles of ERK1/2 and p38 MAPK; in addition, the response is facilitated by progesterone. These results establish that DMP1 is a novel secretory protein of female rat gonadotrophs, the synthesis and release of which are controlled by the hypothalamus through the GnRHR signaling pathway. This advance raises intriguing questions about the intrapituitary and downstream effects of this new player in GnRH signaling.

Figure 1.

Effects of GnRH on gene expression in perifused female rat pituitary cells. To systematically investigate the transcriptional activity of GnRHR, pituitary cells from 7-week-old female rats were attached on cytodex 1 microcarrier beads and stimulated 12 times with 10 nM GnRH for 10 minutes, with a 20-minute washout period in between the pulses. Samples were collected every 5 minutes to determine LH and PRL content. A, Basal LH release was low before GnRH application and high and fluctuating during GnRH application. The 5-minute collection periods precluded detection of the dynamics in LH release. B, Measurements of PRL content from the same samples revealed high and fluctuating basal release. C, Transcriptome profiling: At the end of experiments, mRNA was extracted from lysed cells and sequenced. Dmp1 transcript abundance increased more than 600-fold in response to 10 nM GnRH treatment (indicated). D–F, qRT-PCR analysis of the pituitary gonadotroph-specific genes in perifused pituitary cells with a range of patterns of GnRH application: Cga (left panels), Lhb (middle left panels), Fshb (middle right panels), and Gnrhr (right panels). Cells were stimulated with the indicated GnRH concentrations (indicated below the bars) once for 5 min/h during 6 hours (D), twice for 5 min/h during 6 hours (E), or twice for 5 min/h during 6 hours vs continuous application of 10 nM GnRH for 6 hours (F). The data presented come from two experiments, each performed with 12 million cells per chamber. Differences in the values from the two experiments were less than 10%.

Figure 2.

GnRH induces Dmp1 expression in cultured female rat anterior pituitary cells by qRT-PCR analysis. All experiments were done with pituitary cells from 7-week-old female rats; culture periods were 1–2 days. A, Concentration-dependent effect of GnRH on Dmp1 transcripts in perifused pituitary cells under different patterns of GnRH application (indicated above bars). Pulse duration was 5 minutes (first three panels) or 10 minutes (the last panel). The data presented in panel A are from two experiments, each performed with 12 million cells per chamber. B–H, Effects of GnRH on Dmp1 expression in static cultures. B and C, GnRH induced Dmp1-mRNA expression in a concentration- (B) and time (C)-dependent manner. D, Degradation of GnRH in culture medium does not account for transient Dmp1 response. Medium containing 10 nM GnRH was applied once (black), twice (gray), and three times (white). E, Decay in Dmp1 expression is facilitated by the removal of GnRH. Cells were stimulated with 10 nM GnRH for 4 hours, followed by the removal of GnRH (−GnRH) in some cases. F-H, Dmp1 expression in cultured pituitary cells is specific for gonadotrophs. F, Inhibition of GnRH-induced Dmp1 expression in cultured pituitary cells by [d-pGlu1,d-Phe2,d-Trp3,6]GnRH (DpGlu), a GnRHR-specific antagonist. G, The lack of several Ca²⁺-mobilizing G protein-coupled receptors native to gonadotrophs to initiate Dmp1 expression. ET, endothelin-1; OXT, oxytocin; PACAP, pituitary adenylate cyclase-activating peptide (all were applied in 100 nM concentration). H, The lack of induction of Dmp1 with 100 nM GHRH, which acts on somatotrophs; 100 nM TRH, which acts on lactotrophs and thyrotrophs; or 100 nM CRH, which acts on corticotrophs. Data are presented as means from two (D) and means ± SEM from four (E, F, G, and H) or 12–18 (B and C) determinations per time or dose point (B and C). In this and the following figures, when SEM error bars are not visible, they fall within the area covered by the symbols.

Figure 3.

GnRH-induced Dmp1 expression is age and sex dependent. qRT-PCR analysis was done using mRNA from pituitary glands obtained immediately after euthanasia (in vivo, top and middle panels) and from cultured pituitary cells from male (M) and female (F) rats (in vitro, bottom panels). Numbers on top indicate the age of the animals. The status of cycling females was determined by vaginal smear, and animals were grouped as proestrus (FP) vs estrus + diestrus I + diestrus II (top and middle panels). Time-course studies of 10 nM GnRH-induced Dmp1 expression in pituitary cells were done 24–48 hours after dispersion (bottom panels). Data shown are mean ± SEM values from 5–20 animals per age group (top panels) and quadruplicate determinations (bottom panels). *, Significant differences between FP and M/F (top), M and F/FP (middle), and between pairs (bottom). Basal Dmp1 expression ranged between 0.005 and 0.09, relative to Gapdh, and was not subtracted (bottom panel).

Figure 4.

Dependence of Dmp1 expression in the rat pituitary cells on physiological conditions. A, Dmp1 expression is down-regulated during pregnancy but not lactation. Experiments were done with pituitary cells from 5-month-old animals 24 hours after dispersion. Cells were stimulated with 10 nM GnRH for the indicated times. Data shown are means ± SEM values from quadruplicate determinations. B, Effects of steroid hormones on 10 nM GnRH-induced Dmp1 expression in 2-month-old female and male pituitary cells. Pituitary cells were exposed to dihydrotestosterone (DHT), estradiol (E2), and progesterone (Pr) in the presence of 10 nM GnRH for 6 hours. Concentrations of steroid hormones are indicated below the bars. Data shown are means ± SEM values from quadruplicate determinations. *, P > .05 vs control (0).

Figure 5.

GnRH stimulates Dmp1 expression in the female rat pituitary through the PKC-MAPK signaling pathway. A, Elevation of cytosolic Ca²⁺ concentration by 25 mM KCl (K⁺), 1 μM BayK 8644 (BayK), or 100 nM thapsigargin (TG) did not elevate Dmp1 expression. B, Forskolin (FSK; 1 μM), an adenylyl cyclase activator, did not induce Dmp1 expression and H89 (10 μM), an inhibitor of cAMP-dependent protein kinase, did not affect basal and GnRH-stimulated Dmp1 expression. C, PKC activation by phorbol 12-myristate 13-acetate (PMA; 100 nM) elevated Dmp1 expression. D and E, PKC inhibition by 10 μM staurosporine (D) and Go6983 (E) down-regulated GnRH induction of Dmp1. F, GnRH-stimulated Dmp1 expression was reduced after PKC depletion by treatment with PMA (100 nM, 18 h). G and H, Inhibition of ERK1/2 with PD98059 (G) or UO126 (H) inhibited GnRH-induction of Dmp1; lack of effect of inhibition of JNK and big ERKs with 1 μM SP600125 and Bix022189, respectively. I, p38 MAPK inhibition by SB203580 enhanced GnRH-induced Dmp1 expression; SB203580 enhancement was abolished by Go6983 treatment. Pituitary cells from 7-week-old female rats were treated for 6 hours. *, Significant differences between PMA and GnRH-stimulated cells (C) and vs GnRH-stimulated cells (other panels). In all experiments GnRH concentration was 10 nM. J, Schematic representation of signaling pathways controlling Dmp1 expression in pituitary gonadotrophs.

Figure 6.

Coronal sections of female (estrus) and male pituitaries double labeled for DMP1 and FSHβ. DMP1 is expressed by female gonadotrophs, as indicated by overlapping of green and red fluorescence. DMP1 immunoreactivity was localized in the cytoplasm and was more intense in the perimembrane space (arrow), similar to FSH localization. In males, DMP1 immunoreactivity could be seen only occasionally. This weak immunofluorescence was localized in FSHβ-positive gonadotrophs (arrows). Scale bar applies to all images, 20 μm. See Materials and Methods for further details.

Figure 7.

DMP1 expression by gonadotrophs in pituitary of a female rat in estrus. Sections were double labeled for DMP1 and FSHβ. The two signals are almost completely overlapping. Arrows indicate that DMP1 localizes in the same subcellular compartments, as does FSH. Scale bar, 5 μm. See Materials and Methods for further details.

Figure 8.

Expression of DMP1 protein in pituitary cells. Western blot analysis of culture medium (A) and cell lysate (B and C) of adult female and male rat pituitary cells is shown. A specific signal for DMP1 was not detected in the medium and cell lysates of GnRH-untreated cells (A and B, lanes 1 and 5). DMP1 signal was detected in the medium of female cells treated with 10 nM GnRH for 4 and 8 hours (A, lanes 2 and 3), whereas no signal was detected in the lysate of these cells (B, lanes 2 and 3). BFA treatment prevented the secretion of newly synthetized DMP1 from cells treated with GnRH, resulting in no visible DMP1 signal in the culture medium (A, lane 4) and the appearance of strong DMP1 signal in the cell lysate (B, lane 4). The DMP1 protein signal was not detected in the culture medium and the cell lysates from GnRH-treated male pituitary cells with or without BFA treatment (A and B, lanes 6, 7, and 8). C, Anti-GAPDH antibody was used to detect GAPDH as a loading control protein.

Figure 9.

Intracellular distribution of DMP1 in GH₃ and COS-7 cells. A, pEGFP-N3-DMP1 expression in GH₃ cells in the absence (left) and presence (right) of BFA. The ER localization of EGFP-DMP protein is suggested because perinuclear localization of the fluorescent signal was observed in the absence of BFA (left, arrows). Furthermore, EGFP-DMP1-expressing cells could be observed after BFA treatment, which caused an accumulation of EGFP-DMP1 protein in the ER (right, arrow). Scale bar, 10 μm. B, Expression of pEGFP-N3-DMP1 in COS-7 cells showed intense EGFP intracellular labeling, suggestive of both ER and Golgi localization (arrows), even in the absence of BFA. Note the absence of DMP1 in the nucleus. Scale bar, 10 μm (left); 5 μm (right).