Intermedin Functions as a Pituitary Paracrine Factor Regulating Prolactin Release

Chia Lin Chang, Jaesook Roh, Jae-Il Park, Cynthia Klein, Nicole Cushman, Rainer V. Haberberger and Sheau Yu Teddy Hsu

Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, California 94305-5317

Address all correspondence and requests for reprints to: Sheau Yu Teddy Hsu, Stanford University School of Medicine, Department of Obstetrics and Gynecology, 300 Pasteur Drive, Room A344E, Stanford, California 94305-5317. E-mail: teddyhsu{at}stanford.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Calcitonin, {alpha}- and ß-calcitonin gene-related peptides, amylin, and adrenomedullin belong to a unique group of peptide hormones important for homeostasis maintenance. We recently identified intermedin (IMD) as a novel member of the calcitonin/calcitonin gene-related peptide family expressed in the pituitary, digestive tract, and other organs of vertebrates. Real-time PCR and immunohistochemical analysis of pituitaries from rats at different stages of development showed that IMD is expressed in the intermediate lobe and select adrenocorticotrophs in the anterior lobe, suggesting that IMD could function as a paracrine factor regulating anterior pituitary hormone secretion. In support of a paracrine role for IMD in the pituitary, quantitative and in situ hybridization analyses showed the expression of IMD receptor transcripts including the calcitonin receptor-like receptor and receptor activity-modifying proteins in the pituitary. Treatment with IMD leads to a dose-dependent increase of prolactin release in cultured rat pituitary cells. In contrast, IMD treatment has negligible effects on the release of GH, FSH, or ACTH. Likewise, in vivo treatment with IMD leads to an elevation of plasma prolactin levels in conscious rats. Based on these functional characteristics, we hypothesized that IMD could represent one of the intermediate lobe-derived prolactin-releasing factors important for prolactin regulation during reproduction. In support of this hypothesis, studies of IMD expression in lactating and ovariectomized rats showed that pituitary IMD transcripts in lactating animals increased to more than 2-fold over nonlactating controls whereas ovariectomy leads to a 90% reduction of IMD expression in the pituitary. Of importance, subsequent treatment with 17ß-estradiol or diethylstilbestrol increased pituitary IMD expression in ovariectomized rats. In addition, analysis of the proximate region of the IMD gene promoter showed that the IMD gene promoter contains consensus estrogen response element sequences, and estrogen treatments up-regulate the promoter reporter activity in transfected pituitary cells. Collectively, the present study indicates that IMD represents a novel estrogen-dependent intermediate lobe-derived prolactin-releasing factor and could play important roles in the regulation of prolactin release during reproduction in females.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CALCITONIN/CALCITONIN gene-related peptide (CGRP) family peptides include calcitonin, {alpha}- and ß-calcitonin gene-related peptides ({alpha}CGRP and ßCGRP), amylin, adrenomedullin, and intermedin (IMD) (1, 2, 3, 4, 5, 6, 7). Among these peptides, adrenomedullin and CGRPs are important endocrine and neurocrine regulators in the vascular and respiratory systems, whereas amylin and calcitonin are essential for optimal glucose metabolism and calcium homeostasis, respectively. Utilizing genome searches, we recently identified a novel peptide hormone gene, IMD, that shares sequence similarity with the calcitonin/CGRP family peptides (1). Independent of our study, another laboratory also reported the identification of IMD as an adrenomedullin paralog and named it as adrenomedullin 2 (8, 9). Of interest, Northern and immunohistochemical analyses showed that IMD is expressed in intermediate and anterior lobes of the pituitary (1), suggesting that IMD could function as an endocrine factor or a paracrine factor regulating pituitary hormone secretion.

CGRP and adrenomedullin signaling are mediated by receptor complexes consisting of the calcitonin receptor-like receptor (CRLR) and one of the three receptor activity-modifying proteins (RAMPs) (10, 11). Among the three receptor complexes, the CGRPs and adrenomedullin primarily interact with CRLR/RAMP1, and CRLR/RAMP2 or 3, respectively, thereby allowing the specificity of their signaling (10). Using recombinant CRLR and different RAMPs, we have shown that IMD peptides from mammals and pufferfish are capable of activating CRLR coexpressed with any one of the three RAMP coreceptors, but exhibit a greater potency upon the activation of CRLR/RAMP1 and CRLR/RAMP3 in transfected human embryonic kidney 293T cells (1, 12). In addition, in vivo studies demonstrated that peripherally administered IMD exhibits a potent hypotensive effect and has suppressive effects on gastric emptying activities, food intake, and water intake (1, 8, 9, 12, 13, 14). In contrast, administration of IMD into the lateral cerebroventricle of rats caused an elevation in arterial pressure and heart rate (14). These data established that IMD is a novel ligand specific for CRLR/RAMP1 and CRLR/RAMP3 receptor complexes and could be important for the regulation of diverse physiological processes that have been attributed to signaling by adrenomedullin or CGRPs (1, 10).

In support of the hypothesis that IMD functions as a paracrine factor regulating pituitary hormone release, we demonstrated the expression of CRLR and RAMP transcripts in rat pituitaries at different stages of development. Importantly, IMD treatment selectively stimulated prolactin release, but not the release of FSH, ACTH, or GH from cultured anterior pituitary cells in vitro. Likewise, IMD treatment in vivo led to a rise in plasma prolactin levels in conscious rats.

Earlier studies on the regulation of pituitary prolactin secretion have shown that the intermediate lobe of the pituitary contains potent prolactin-releasing peptides that are critical for the suckling- and estradiol-induced rise in prolactin release (15, 16, 17, 18, 19, 20), and the activity of these uncharacterized prolactin-releasing factors cannot be accounted for by oxytocin, TRH, vasopressin, or angiotensin II (15, 20, 21). To support the notion that IMD could represent one of the intermediate lobe-derived prolactin-releasing factors, we investigated the expression of IMD in the pituitary of lactating and ovariectomized rats as well as the effect of 17ß-estradiol on IMD gene promoter activity in transfected pituitary cells. Because the expression of IMD in pituitary was elevated in lactating animals whereas estrogen treatments increased IMD expression in the pituitary, intermediate and anterior lobe-derived IMD could play important roles in the regulation of prolactin release during different reproductive states in females in addition to potential roles in the cardiovascular, gastrointestinal, and renal systems.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
IMD Is Expressed in the Pituitary of Rats at Different Developmental Stages
Rat IMD (GenBank accession no. AY590103) cDNA encodes a 146-amino-acid open reading frame sharing 64% amino acid sequence identity with the human IMD precursor (Fig. 1AGo) (1). Although the putative preproregion of rat IMD shares less than 52% identity with the preproregion of human IMD, putative 47-amino acid mature rat IMD differs from the mature human IMD peptide by only six amino acids (Fig. 1BGo).



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Fig. 1. Subcloning of Rat IMD cDNA

A, Rat IMD cDNA encodes a 146-amino acid open reading frame. The open reading frame starts with a 25-amino acid signal peptide for secretion and contains a predicted 47-amino acid mature peptide. Nucleotide sequences are numbered on the right and a dark background highlights the signal peptide for secretion. The N-terminal dibasic cleavage residues and the C-terminal amidation donor residue are in bold italicized letters. B, Alignment of rat and human IMD orthologs. Residues shared by IMD orthologs are shown in between. h, Human; r, rat. The putative mature regions of human and rat IMD are underlined. Amino acid sequences are numbered on the right.

 
To investigate the expression pattern of the IMD transcript in pituitary during development, pituitary tissues were obtained from rats at 6, 12, 18, 24, 30, or 36 d of age as well as 8-wk-old adult rats. Quantitative real-time PCR analysis of total RNA showed that low levels of IMD transcripts could be detected as early as 6 d of age in both males and females (Fig. 2AGo). In males, the expression increased after 6 d of age and decreased after 18 d of age (Fig. 2AGo, upper panel). Likewise, the expression of IMD transcript in females decreased before puberty (Fig. 2AGo, lower panel). However, a significant increase of expression was observed in 8-wk-old adult females (Fig. 2AGo, lower panel). To study the expression of IMD polypeptides, specific rabbit anti-IMD antibodies were generated using synthetic peptides corresponding to residues 28–47 of human IMD. In immunoblot analysis, the antihuman IMD antibody showed negligible cross-reaction with paralogous peptides but cross-reacted with orthologous peptides including that from pufferfish (1). In support of real-time PCR analysis, immunohistochemical analysis showed that immunoreactive IMD is detected in the anterior and intermediate lobes of pituitary from rats at all studied stages of development (Fig. 2Go, B–H). Although IMD was detected in the majority of cells in the intermediate lobe, only select cells in the anterior pituitary lobe showed positive staining. In contrast, no specific signal was observed in the posterior lobe.



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Fig. 2. Expression of IMD in the Pituitary of Rats at Different Ages

A, Real-time PCR analysis of rat IMD transcript in the pituitary. For analysis of pituitary IMD mRNAs during development, male (upper panel) and female (lower panel) rats at 6, 12, 18, 24, 30, and 36 d of age as well as 8-wk-old adult rats were killed and the pituitary was collected for total RNA extraction. First-strand cDNAs reverse-transcribed from 50 ng of total RNA were analyzed for IMD transcripts using TaqMan real-time PCR. Expression of the GAPDH transcripts in different cDNA templates was also analyzed to assess the quality of cDNA templates. Expression of the IMD transcript was normalized by the expression of the GAPDH transcript (mean ± SEM, n = 3). Similar results were observed in at least three different experiments. B–H, Detection of immunoreactive IMD in pituitary sections of female rats at 6 (B), 12 (C), 18 (D), 24 (E), 30 (F), or 36 (G) d of age, as well as 8 wk of age (H) using paraffin-fixed tissues and a specific anti-IMD antibody (x160) (1 ). Similar results were observed in at least five different individuals. AL, Anterior lobe; IL, intermediate lobe; PL, posterior lobe; d, days of age; W, weeks of age.

 
IMD Colocalizes with ACTH in the Anterior Pituitary
Because IMD is found in the intermediate lobe and its expression overlaps with that of {alpha}-MSH (1), it is likely that IMD could be coexpressed with other proopiomelanocortin gene products such as ACTH in the anterior lobe. Studies using the anti-IMD antibody and a rhodamine-conjugated secondary antibody showed that IMD is expressed in the intermediate lobe and in select anterior pituitary cells in immature rats (Fig. 3AGo). In contrast, studies using an fluorescein isothiocyanate-conjugated secondary antibody and an ACTH-specific monoclonal antibody showed that immunoreactive ACTH is found primarily in the adrenocorticotrophs in the anterior lobe with minimal staining in the intermediate lobe (Fig. 3BGo). Of importance, the staining for IMD and ACTH in the anterior lobe was observed in the same population of cells (Fig. 3Go, C–E). Sections stained with both antibodies showed an overlay of the signals in an almost identical cell population (Fig. 3EGo). Similar to immature rats, studies of pituitaries from mature female rats showed that immunoreactive {alpha}-MSH (Fig. 3FGo) and ACTH (Fig. 3GGo) were detected largely in the intermediate and anterior lobe of pituitaries, respectively, whereas IMD was present in the intermediate lobe and the adrenocorticotrophs of the anterior lobe (Fig. 3HGo). In contrast, immunoreactive prolactin was detected exclusively in the anterior lobe (Fig. 3IGo), and its expression was more prevalent as compared with IMD or ACTH.



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Fig. 3. Coexpression of IMD and ACTH in the Anterior Lobe of Pituitary

Immunofluorescent detection of immunoreactive IMD (red signal, upper panel) (A) and ACTH (green signal, lower panel) (B) in the pituitary of a 12-d-old female rat. White tracing indicates the contour of the intermediate lobe. C–E, Colocalization of immunoreactive IMD (upper left panel) (C) and ACTH (upper right panel) (D) in anterior pituitary cells. Orange signals in the lower panel show the overlay signal of ACTH and IMD (E) as observed using a 51004v2 F/R filter (Chroma Technology Corp., Rockingham, VT). F–I, Localization of immunoreactive {alpha}-MSH (F), ACTH (G), IMD (H), and prolactin (I) in the pituitary of 10-wk-old mature females. Similar results were observed in at least four different individuals. For the detection of {alpha}-MSH, IMD, and prolactin, a rhodamine-conjugated secondary antibody was used whereas the signal for ACTH was detected with an fluorescein isothiocyanate-conjugated secondary antibody. AL, Anterior lobe; IL, intermediate lobe; PL, posterior lobe.

 
Expression of IMD Receptor Transcripts, CRLR, RAMP1, and RAMP3, in the Pituitary
Earlier studies demonstrated that IMD signals through receptor complexes formed by the CRLR and one of the three RAMPs (1). Of the three receptor complexes, IMD had a greater effect on the CRLR/RAMP1 and CRLR/RAMP3 receptor complexes. To investigate whether IMD could function as a paracrine factor in the pituitary, we performed quantitative real-time PCR with rat gene-specific primers to study the expression of CRLR, RAMP1, and RAMP3 in the pituitary from male and female rats at different ages. As shown in Fig. 4Go, A and B, all three transcripts were detected in the pituitary. RAMP1 showed a greater level of expression in both males and females as compared with RAMP3 and CRLR. Whereas comparable levels of RAMP1 and CRLR were detected at different stages of development, the expression of RAMP3 exhibited a pattern similar to that of IMD in both male and female animals (Figs. 2AGo and 4Go, A, and B). The highest levels of RAMP3 expression in males and females were observed at 18 d and 8 wk of age, respectively (Fig. 4Go, A and B). Furthermore, to demonstrate that CRLR/RAMP receptor complexes are expressed in pituitary cells, we performed competitive receptor-binding assays using radiolabeled CGRP. Specific binding of the CGRP tracer to anterior pituitary cells was competed in a dose-dependent manner by increasing concentrations of nonlabeled IMD or CGRP (Fig. 4CGo).



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Fig. 4. Expression of CRLR, RAMP1, and RAMP3 Transcripts in the Pituitary of Rats at Different Stages of Development

Expression of CRLR, RAMP1, and RAMP3 transcripts in the pituitary of male (A) and female (B) rats were studied by real-time PCR analysis. Pituitary samples include rats at 6, 12, 18, 24, 30, or 36 d of age as well as 8-wk-old adult rats. The expression of CRLR and RAMP transcripts was normalized by the expression of the GAPDH transcript in different cDNA templates. d, Days of age; W, weeks of age. Data are means ± SEM (n = 4). Similar results were observed in at least three separate independent studies. C, Competitive displacement of [125I]CGRP binding to anterior pituitary cells by nonlabeled IMD and CGRP. Data are means ± SEM (n = 4).

 
Stimulation of Prolactin Release from Cultured Anterior Pituitary Cells by IMD
Previous studies have shown that the intermediate lobe of pituitary secretes potent prolactin-releasing peptides that remain to be characterized (15, 17, 18, 19). Because IMD showed predominant expression in the intermediate lobe, we tested whether IMD could represent one of the prolactin-releasing peptides in the intermediate lobe using in vitro cultured rat anterior pituitary cells. Earlier studies indicated that the IMD precursor may be processed to generate two isoforms (IMDL, IMD long; and IMDS, IMD short) through alternative cleavages at two conserved cleavage sites and that both isoforms are bioactive (1). Treatment of cultured pituitary cells from male rats with a known prolactin-releasing factor, TRH, a recently isolated hypothalamic prolactin-releasing peptide (22), or RFamide peptide 1 (22) resulted in an increase of prolactin release in vitro (Fig. 5AGo). Likewise, treatment with two different IMD isoforms, IMDL or IMDS, stimulated prolactin release by cultured pituitary cells in a dose-dependent manner (Fig. 5AGo). Similarly, IMDL or IMDS treatments increased prolactin release from pituitary cells prepared from female rats (Fig. 5BGo). In contrast, no significant change was observed in the secretion of GH, ACTH, or FSH (data not shown). To verify that IMD action is mediated by the CRLR/RAMP receptor complex-induced adenylyl cyclase activation, we analyzed the effect of an IMD receptor antagonist and a specific protein kinase A (PKA) inhibitor, H89 (23), on IMD-induced prolactin release. As expected, pretreatment with an IMD receptor antagonist, IMD17–47, reduced the IMD-induced prolactin release significantly whereas treatment with a shorter IMD peptide fragment, IMD28–47, had negligible effect (Fig. 5CGo). Likewise, H89 treatment caused a significant reduction of IMD-induced prolactin release from cultured pituitary cells (Fig. 5DGo). In contrast, treatment with a MEK kinase-specific inhibitor, PD98059, had minimal effect on IMD-induced prolactin release (24). These data suggest that IMD increases prolactin release through the CRLR/RAMP receptor complexes and the PKA-dependent pathway.



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Fig. 5. IMD Stimulates Prolactin Release by Cultured Anterior Pituitary Cells

Prolactin contents in culture media of pituitary cells treated with IMD (47-amino acid IMD, IMDL; 40-amino acid IMD, IMDS) (1 ), TRH, a hypothalamus RFamide peptide (RFRP1) (22 ), prolactin releasing peptide (PrRP) (43 ), or adrenomedullin (ADM) from male (A) or female (B) rats. Data are mean ± SEM (n = 3). Similar results were observed in at least three separate independent studies. C, Suppression of the IMD-induced prolactin release by an IMD receptor antagonist, IMD17–47. The IMD17–47 peptide design was based on the putative receptor-binding domain of IMD and functions as a competitive receptor-binding antagonist (1 ). Cultured pituitary cells from 8-wk-old male rats were preincubated with IMD17–47 (300 nM) or a truncated IMD fragment, IMD28–47, (300 nM) for 1 h before treatment with IMD (100 nM). Data are mean ± SEM (n = 8). D, Inhibition of the IMD-induced prolactin release by a specific PKA inhibitor, H89. Cultured pituitary cells from 8-wk-old male rats were preincubated with H89 (10 µM) or a MEK kinase inhibitor, PD98059 (10 µM) for 1 h before treatment with IMD (100 nM). Data are mean ± SEM (n = 4). Conditioned media were harvested for hormone assays 30 min after IMD treatment. *, Significantly different from controls (P < 0.05).

 
To further substantiate the hypothesis that IMD acts as a paracrine factor regulating prolactin release in the pituitary, we performed in situ hybridization to localize the site of expression for CRLR and the more abundant RAMP, RAMP1, in the pituitary. As expected, CRLR and RAMP1 transcripts were detected in the anterior lobe of the pituitary (Fig. 6Go, A–F), overlapping with that of prolactin (Fig. 6Go, G and H). CRLR and RAMP1 transcripts also were detected in the intermediate lobe, which showed a negligible signal for the prolactin transcript. The combined results indicate that IMD, CRLR/RAMP receptor complexes, and prolactin form a paracrine regulatory pathway in the pituitary (Fig. 6IGo).



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Fig. 6. In Situ Hybridization Localization of CRLR, RAMP1, and Prolactin Transcripts in the Rat Pituitary

Positive signal for CRLR (panel A, bright field; panel B, dark field) and RAMP1 (panel D, bright field; panel E, dark field) transcripts were detected in both anterior and intermediate lobes of the pituitary (x100). Negative controls hybridized with a sense riboprobe for CRLR or RAMP1 are shown in panel C and F, respectively (dark field, x100). Sequential sections were also hybridized with an antisense prolactin riboprobe to demonstrate the specificity of in situ hybridization (panel G, bright field; panel H, dark field). I, A schematic representation of the intrapituitary regulatory circuit in which IMD acts as a paracrine factor regulating prolactin secretion via the CRLR/RAMP receptor complexes in lactotrophs. AL, Anterior lobe; IL, intermediate lobe; PL, posterior lobe.

 
Stimulation of Prolactin Release by IMD in Vivo
To analyze IMD action in vivo, we studied plasma levels of prolactin and GH in adult male rats after iv injection of IMD. At 30 min after iv injection of a 100-nmol dose of IMD, plasma prolactin levels rose to approximately 3-fold of those present in saline-injected controls (Fig. 7Go). In contrast to the stimulatory effect on plasma prolactin levels, IMD treatment led to a decline of GH levels, suggesting that the stimulatory effect of IMD on lactotrophs is specific.



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Fig. 7. IMD Stimulates Prolactin, but not GH, Release in Vivo

Analysis of prolactin and GH in the plasma of conscious rats. Adult male rats (12 wk of age) were anesthetized and IMD was administered via the femoral vein. At 30 min after treatment, plasma samples were collected and frozen at –20 C before hormone assays. Data are mean ± SEM (n = 5). Similar results were observed in two separate independent studies. *, Significantly different from control animals injected with saline alone (P < 0.05).

 
Increased Expression of Pituitary IMD in Lactating Rats
Based on the finding that IMD increases pituitary prolactin release and earlier studies showing that uncharacterized intermediate lobe-derived prolactin-releasing factors are critical for mediating suckling-induced prolactin release (15, 17, 19, 20), we hypothesized that the expression of IMD in the pituitary could be elevated during nursing in female rats (17, 18, 19). To test this hypothesis, we analyzed the expression of IMD and its receptors including CRLR, RAMP1, and RAMP3 in pituitaries of lactating and nonlactating 10-wk-old female rats using real-time PCR. The expression of IMD and RAMP3 transcripts in the pituitary of lactating animals at 15 d postpartum was more than 2-fold that of nonlactating control animals (Fig. 8Go). In contrast, levels of CRLR and RAMP1 transcripts in the pituitary were not altered in lactating animals (Fig. 8Go).



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Fig. 8. Elevation of IMD Transcript Expression in the Pituitary of Lactating Rats

Analysis of IMD, RAMP1, CRLR, and RAMP3 transcripts in the pituitary of lactating females and nonlactating controls. Pituitaries were collected from 10-wk-old lactating female rats at 15 d postpartum and age-matched nonlactating females and analyzed for the expression of IMD, RAMP1, CRLR, and RAMP3 transcripts separately using real-time PCR. Data are mean ± SEM (n = 4). Similar results were observed in three separate independent studies. *, Significantly different from control animals (P < 0.05).

 
Stimulation of Pituitary IMD Expression by Estrogens
In addition to being associated with prolactin release during lactation, the uncharacterized prolactin-releasing factors from the intermediate lobe play roles in mediating estrogen-induced prolactin release in vivo. To further support the hypothesis that IMD represents one of the uncharacterized intermediate lobe-derived prolactin-releasing factors, we analyzed the expression of IMD in the pituitary of 9-wk-old ovariectomized rats. As compared with age-matched intact rats, the level of pituitary IMD transcripts was reduced by more than 90% in ovariectomized rats (Fig. 9Go, A and B). Of interest, daily injection of 17ß-estradiol (10 µg/kg body weight) or implantation of a diethylstilbestrol pellet for 4 d one week after ovariectomy increased IMD expression in the pituitary of ovariectomized rats by more than 6- and 9-fold, respectively (Fig. 9Go, A and B). Likewise, immunohistochemical analysis showed that immunoreactive IMD in both intermediate and anterior lobes decreased drastically after ovariectomy as compared with control animals (Fig. 9Go, C and D). In support of real-time PCR analysis, estrogen treatment also effectively increased the staining of immunoreactive IMD in the pituitary of ovariectomized rats (Fig. 9EGo).



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Fig. 9. Regulation of Pituitary IMD Expression by Estrogens

Real-time PCR analysis of IMD transcripts in the pituitary of 9-wk-old intact female rats, ovariectomized rats, and ovariectomized rats receiving 17ß-estradiol (A) or diethylstilbestrol (DES) implant (B) treatment. Ovaries were surgically removed from 9-wk-old female rats. One week after surgery, groups of ovariectomized rats were injected daily with 17ß-estradiol for 4 d or implanted with a DES capsule for 4 d. On d 11, ovariectomized rats with or without estrogen treatment together with age-matched intact female rats were killed for analysis of pituitary IMD expression using real-time PCR and immunohistochemistry. Data are mean ± SEM (n = 6). *, Significantly different from control animals (P < 0.05). C–E, Analysis of immunoreactive IMD in pituitary sections from control intact rats (C), ovariectomized rats (D), and ovariectomized rats receiving a DES implant for 4 d (E). For the detection of IMD, a fluorescein isothiocyanate-conjugated secondary antibody was used. White tracing indicates the contour of the intermediate lobe. For the comparison of IMD expression in histological sections, batches of sections were processed at the same time and images captured with a fixed exposure time using a Leica DFC300 FX digital camera and Image-Pro software. AL, Anterior lobe; IL, intermediate lobe; PL, posterior lobe.

 
To investigate the molecular mechanism underlying the regulation of pituitary IMD gene expression by estrogens, we retrieved the IMD gene promoter sequences of human, mouse, and rat from the GenBank genome database and searched for the presence of putative estrogen response element (ERE) and binding sites for other transcription factors (25, 26). The ERE is a palindrome of GGTCA motifs separated by 3 bp (Fig. 10AGo) (27). Consistent with the finding that pituitary IMD expression is regulated by ovarian estrogens, the proximate region of the IMD gene promoter from all three species contain one consensus ERE important for the regulation of gene expression by estrogen receptors (Fig. 10AGo) (25, 26). ERE in the IMD gene promoter differs from the consensus sequence by only one or two nucleotides. Because EREs of many typical estrogen-responsive genes showed great divergence from the consensus sequence (25, 26), estrogen receptors have a high probability of interacting with the highly conserved ERE in the IMD gene promoter. Based on this finding, we subcloned a 2.7-kb and a 1.1-kb mouse IMD gene promoter fragments into the pGL2-luciferase reporter vector and tested the ability of 17ß-estradiol to stimulate the promoter reporter activity in transfected pituitary cells (Fig. 10AGo). In addition to sites for activator protein 2, HIF1, progesterone receptor, HFH8, and GATA1, the 2.7-kb proximate region of the mouse IMD gene promoter contained one consensus ERE site (–1362 ~ –1374). Pituitary cells obtained from 8-wk-old female rats were cultured for 2 d before transfection. As shown in Fig. 10BGo, treatment with 17ß-estradiol, but not testosterone, significantly increased the luciferase activity in cells transfected with the ERE-containing 2.7-kb promoter reporter construct. In contrast, 17ß-estradiol treatment had negligible effect on the reporter activity in cells transfected with the construct containing the 1.1-kb proximate promoter region devoid of the consensus ERE sequence.



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Fig. 10. Estradiol Stimulates the IMD Gene Promoter Reporter Activity in Pituitary Cells

A, Schematic representation of the 2.7-kb (pGL2–2.7kbIMD-luc) and 1.1-kb (pGL2–1.1kbIMD-luc) mouse IMD gene promoter-luciferase reporter constructs. Putative ERE sequences in the promoter of human, mouse, and rat IMD genes are shown in parallel with the consensus ERE sequence. Nucleotides that differ from the consensus sequence are italicized. B, Increases of luciferase activity in pituitary cells transfected with the pGL2–2.7kbIMD-luc construct, but not the pGL2–1.1kbIMD-luc construct, by 17ß-estradiol. Cultured rat pituitary cells were cotransfected with a pCMV-ß-galactosidase expression plasmid to monitor the transfection efficiency, followed by treatment with ethanol, 30 nM 17ß-estradiol, 300 nM 17ß-estradiol, or 300 nM testosterone as indicated. The results are presented as the ratio of luciferase reporter and ß-galactosidase activities in each treatment. Values are mean ± SEM (n = 4). Similar results were observed in two separate independent studies. *, Significantly different from controls treated with ethanol (P < 0.05).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the present study, we demonstrate that IMD receptor transcripts are expressed in the pituitary of rats at different stages of development and that IMD treatment leads to the select release of prolactin from pituitary cells in vitro and in vivo. Studies of female rats showed that the expression of IMD transcript in the pituitary is reduced by more than 9-fold after ovariectomy whereas subsequent estrogen treatments increased pituitary IMD expression in vivo. In addition, we demonstrated that estradiol treatment increases IMD gene promoter reporter activity in transfected pituitary cells. Together with the finding that pituitary IMD expression is increased in postpartum lactating animals, the results suggest that IMD could represent one of the uncharacterized intermediate lobe-derived prolactin-releasing factors important for prolactin regulation during lactation and other stages of reproduction by acting on neighboring lactotrophs (17, 18, 19).

Based on genomic analysis, we and others identified IMD as a novel paralog of CGRPs and adrenomedullin in all vertebrates (1, 8, 12, 13). Earlier studies have established that CGRPs are important neuropeptides for the modulation of sympathetic activity and inflammatory reactions (2, 3, 4, 28, 29, 30, 31) whereas adrenomedullin is a hypotensive hormone indispensable for vascular morphogenesis during embryonic development (5, 7, 32, 33, 34). Together with IMD, these peptide hormones share the same G protein-coupled receptor, CRLR, for signaling. However, they exhibit distinct signaling characteristics through differential interactions with three CRLR/RAMP receptor complexes (10, 11). Among the three CRLR/RAMP receptor complexes, CGRPs and adrenomedullin primarily interact with CRLR/RAMP1, and CRLR/RAMP2 or -3, respectively. In contrast, IMD from mammals and teleosts exhibits a greater effect on the activation of CRLR/RAMP1 and CRLR/RAMP3 receptor complexes (1, 12). Therefore, IMD could regulate diverse physiological functions that have been attributed previously to adrenomedullin or CGRPs. We and others have shown that IMD exhibits potent effects on cardiovascular, gastrointestinal, and renal systems reminiscent of that of CGRP or adrenomedullin (1, 8, 12, 13, 14); however, its physiological roles remain to be investigated. Based on the unique expression pattern of IMD, we hypothesized that the physiological activities of IMD could include the regulation of pituitary hormone release. Earlier studies have shown that administration of exogenous adrenomedullin in patients with pulmonary hypertension leads to increases in the circulation of prolactin, but not ACTH, TSH, FSH, LH, or cortisol (35). Although immunoreactive adrenomedullin has been reported to localize in the paraventricular and supraoptic nuclei of the hypothalamus (36), the exact role of adrenomedullin in pituitary prolactin secretion has not been studied specifically. Because adrenomedullin is capable of activating all three CRLR/RAMP receptor complexes, exogenous adrenomedullin could stimulate prolactin release through pituitary CRLR/RAMP receptors that are normally acted upon by endogenous IMD. Based on the finding that IMD is expressed mainly in the intermediate lobe and adrenocorticotrophs, the select stimulation of prolactin release by IMD in vitro and in vivo is likely mediated by a direct action on lactotrophs and not due to a secondary stimulation of other prolactin-releasing factor(s) from the posterior lobe or hypothalamus. This hypothesis is supported by observations that 1) components of IMD receptors, CRLR, RAMP1, and RAMP3, are expressed in pituitary cells; and 2) the IMD-induced prolactin release is suppressed by cotreatment with an IMD receptor antagonist, IMD17–47.

Prolactin is secreted in a pulsatile fashion and the serum prolactin level displays a circadian rhythm. During development, plasma prolactin in rats was low between d 5 and 15 and increased thereafter (37). During pregnancy and lactation, prolactin levels rose to maintain a physiological hyperprolactinemia. Diverse hypothalamic releasing peptides are important in the specific regulation of the secretion of different anterior pituitary hormones such as GH, ACTH, TSH, LH, and FSH (38, 39, 40, 41). However, regulation of prolactin release from the pituitary involves stimulatory factors originating from both the hypothalamus and the intermediate lobe of pituitary as well as the tonic inhibition by dopamine (19, 42). In addition to TRH, several newly identified G protein-coupled receptor ligands including prolactin-releasing peptide (43), RFamide peptides, and neuropeptide W have been proposed to function as prolactin-releasing factors from the hypothalamus (22, 44, 45, 46). Although in vivo evidence is incomplete, these peptides could stimulate prolactin release through the central nervous system or through the portal vessel (46).

In contrast to the uncertain role of hypothalamic prolactin-releasing factors, the intermediate lobe-derived prolactin-releasing factors are important for mediating suckling-induced prolactin release in lactating animals and for stimulation of prolactin release by estradiol in normal animals (15, 16, 17, 18, 19, 20, 21). Although these factors have been partially purified and shown to include small peptide ligands, the chemical identity of these factors has not been determined (15). The specific stimulation of prolactin release, but not ACTH or FSH release, by IMD is consistent with earlier studies on the specificity of the uncharacterized intermediate lobe-derived prolactin-releasing factors (19). Importantly, studies of ovariectomized rats showed that pituitary IMD expression is regulated by ovarian estrogens and is increased during the lactation period. Therefore, in addition to sharing similar chemical properties and functional characteristics, the findings that pituitary IMD expression is increased in lactating animals and is stimulated by estrogens are consistent with the proposed role for IMD as one of the uncharacterized intermediate lobe-derived prolactin-releasing factors essential for the regulation of estrogen- and suckling-induced prolactin release (15, 17, 18, 19, 21).

Suppression of IMD-induced prolactin release by a specific PKA inhibitor, H89, and an IMD receptor antagonist, IMD17–47, supports the hypothesis that IMD stimulates pituitary prolactin secretion through CRLR/RAMP receptor complexes and the cAMP-dependent pathway, consistent with the observation that IMD activates adenylyl cyclase via the CRLR/RAMP receptors in transfected cells (1, 12). Studies on the expression of IMD and its receptors in the pituitary show that IMD is expressed in both anterior and intermediate lobes and is present from the neonatal stage to the adulthood. These data indicate that IMD may play a role in regulating the tonic release of prolactin in concert with or independent of the hypothalamic factors. In addition, we observed a parallel increase of IMD and RAMP3 transcripts in the pituitary during development and in lactating animals. Because IMD exhibits a potent effect on the activation of the CRLR/RAMP3 receptor complex (1, 12), the regulation of RAMP3 expression in the pituitary may play a role in the regulation of IMD-induced prolactin secretion.

Based on studies of ovariectomized rats and IMD gene promoter reporters, we demonstrated that ovarian estrogens could regulate IMD gene expression by directly acting on the IMD gene promoter. This hypothesis is corroborated by earlier studies showing that estrogen receptor {alpha} and ß are present in both anterior and intermediate lobes of the pituitary and that the expression of these receptors in the pituitary is dynamically regulated during development and reproduction (47, 48, 49, 50). In addition, because IMD has potent effects on the cardiovascular, gastrointestinal, and renal systems, the pivotal role played by estrogens on IMD expression indicated that IMD could participate in the regulation of a variety of sex-specific physiological responses in these systems. Further studies on the regulation of IMD gene promoter activity in pituitary cells by different neuroendocrine and steroid hormones would lead to a better understanding of the physiological role of IMD in the pituitary and other peripheral tissues.

Whereas it is evident that the regulation of prolactin release by IMD is mediated by a paracrine mechanism, the mechanism by which IMD reduces circulating GH in vivo is not clear. Because GHRH stimulates GH release via the cAMP-dependent pathway (51), the lack of effect of IMD on GH secretion in cultured pituitary cells indicated that the CRLR/RAMP signaling is specific to lactotrophs. Earlier studies on the neuronal regulation of pituitary hormone release in rodents have shown that treatment with various neuropeptides in the brain leads to the elevation of prolactin and a concomitant decrease of the circulating GH level (52, 53). However, the physiological importance and the underlying mechanisms for these observations are not clear. Future studies on the mechanism by which peripherally administered IMD decreases circulating GH could lead to a better understanding of the discordant regulation of prolactin and GH in rodents under various pharmacological conditions. During the preparation of this manuscript, it was reported that intracerebroventricular administration of IMD leads to the secretion of corticosterone, ACTH, prolactin, oxytocin, and vasopressin in conscious rats and the stimulatory effect of centrally administered IMD on stress hormone release could be mediated by the activation of CRH receptors (54). Thus, independent of the paracrine regulation mediated by pituitary IMD, the neuroendocrine regulation of pituitary prolactin secretion involves CRLR/RAMP receptor signaling in the brain.

Immunohistochemical studies revealed that IMD colocalizes with the proopiomelanocortin gene products at various stages of development; IMD and {alpha}-MSH are coexpressed in most cells of the intermediate lobe whereas IMD and ACTH are found in adrenocorticotrophs of the anterior lobe. It is well known that pituitary prolactin release is associated with stress induction under diverse conditions (55). Thus, it is possible that IMD is cosecreted with ACTH from the anterior lobe and contributes to the elevation of prolactin in circulation under stress conditions. Preliminary studies of the IMD gene showed that the IMD gene promoter contains putative glucocorticoid response element-like sequences (data not shown). Therefore, the secretion of IMD could be regulated by stress hormones such as glucocorticoids and CRH.

In conclusion, this study demonstrates that the newly identified IMD peptide functions to regulate pituitary prolactin release in vitro and in vivo. In addition to playing a regulatory role in the vasculature, gastrointestinal, and renal systems as demonstrated earlier (1, 8, 9), endogenous IMD may represent one of the long-sought prolactin-releasing factors from the intermediate lobe and could participate in the coordinated regulation of prolactin secretion with hypothalamic factors under various physiological conditions.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of IMD in the Pituitary
Rat IMD cDNA sequences were identified from an EST BQ192607 and the rat genomic sequence in GenBank. The predicted open reading frame (ORF) of rat IMD was verified by PCR amplification using a rat ovarian cDNA library. For quantitative analysis of IMD transcripts in rat pituitaries, IMD transcript-specific standards were produced by subcloning a PCR-amplified rat IMD cDNA fragment. Amplification of cDNA templates was performed in a 45-µl reaction volume consisting of 15 mM Tris-HCl, pH 8.0, containing 50 mM KCl, 2.5 mM MgCl2, 200 µM deoxynucleotide triphosphates, 0.5 µM primers, serial dilutions of cDNA template, and 0.3 U AmpliTaq Gold DNA polymerase (Applied Biosystems, Norwalk, CT) with the following cycling parameters: 1) initial denaturation, 94 C, 10 min; 2) denaturation, 94 C, 30 sec; annealing and extension, 72 C, 4 min (five cycles); 3) denaturation, 94 C, 30 sec; annealing and extension, 70 C, 4 min (five cycles); 4) denaturation, 94 C, 30 sec; annealing and extension, 68 C, 4 min (25 cycles); and 5) final extension, 72 C, 10 min.

For the quantitative analysis of transcript expression, total RNA from rat tissue was isolated using RNeasy Mini kit (QIAGEN, Valencia, CA). Aliquots of RNA were transcribed into first-strand cDNAs using Omniscript Reverse Transcriptase (QIAGEN). Real-time PCR was conducted using a Smart Cycler (Cepheid, Sunnyvale, CA) in a 25-µl Smart Cycler reaction tube. TaqMan primers used for amplification of the rat IMD cDNAs are forward primer rIMDFW (5'-ATCACAGACCACAGAGTACTCGA-3') and reverse primer rIMDRW (5'-TCCTGCTTGCTCACAAGTGGAG-3'). For analysis of CRLR and RAMP transcripts in pituitary tissues, rat transcript-specific TaqMan primers were designed based on rat cDNA sequences (GenBank Accession nos.: RAMP1, NP_113833; RAMP3, NP_064485; CRLR, NP_036849). Similar to studies of IMD transcript, rat CRLR and RAMP cDNA fragments also were PCR amplified and subcloned in pUC18 vector as the copy number standard. The probes were labeled with the reporter fluorochrome 6-carboxyfluorescein at the 5'-end and the quencher fluorochrome 6-carboxy-tetramethyl-rhodamine at the 3'-end. The probes for IMD, RAMP1, RAMP3, CRLR, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are 5'-TGTGCCTAAAGAAAATCGGCAAAAG-3', 5'-TGAAAGCAGATCTGCATTACACTAA-3', 5'-AGCACATTCTCTGTTGGACATGAAG-3', 5'-ACGGTTTGTCCAGAAACACTTTAAC-3', and 5'-ACAACTTTGTGAAGCTCATTTCCTG-3', respectively. Forward TaqMan primers used for amplification of the rat RAMP1, RAMP3, CRLR, and GAPDH cDNAs are rRAMP1FW (5'-CATCCTGCTCTAGCCTAGTTAG-3'), rRAMP3FW (5'-GCAGGCAAGGTCATCTGGAAG-3'), rCRLRFW (5'-GCTCAGTCTTACAGACATGAAA-3'), and rGAPDHfw (5'-CCCATTCTTCCACCTTTGATGCTGG-3'), respectively. Reverse TaqMan (TM) primers are rRAMP1RW (5'-AGACCTGGCGCTTAGCGCTCTTT-3'), rRAMP3RW (5'-TCATGGCAGATCGCCTAGAAGA-3'), rCRLRRW (5'-TTCTTAAAGAAAGGAACACGAGG-3'), and rGAPDHRW (5'-CATGTAGGCCATGAGGTCCACCACC-3').

Peptide Synthesis
IMD related peptides (IMD17–47 and IMD28–47) were synthesized based on the solid-phase fluorenylmethoxycarbonyl protocol and analyzed by reverse phase HPLC with Vydac C18 analytical column and mass spectrometry using a MALDI-TOF Voyager-DE RP Workstation (Stanford University Protein and Nucleic Acid Biotechnology Facility). Synthetic adrenomedullin, ßCGRP, and related peptides were obtained from AnaSpec, Inc. (San Jose, CA), Sigma-Aldrich (St. Louis, MO), and Bachem California, Inc. (Torrance, CA). Radiolabeled [125I]CGRP (2000 Ci/mmol) was purchased from Amersham Pharmacia (Little Chalfont, Buckinghamshire, UK). Stocks of different hormones were prepared in distilled water and diluted in culture medium.

Immunoanalysis
Rabbit anti-IMD antibodies were generated using synthetic peptides corresponding to residues 28–47, MGPAGRQDSAPVDPSSPHSY, of human IMD (Strategic Biosolutions, Ramona, CA). In immunoblot analysis, the antihuman IMD antibody showed negligible cross-reaction with paralogous peptides including calcitonin, CGRP, adrenomedullin, and amylin (1). The mouse anti-ACTH monoclonal antibody was obtained from Research Diagnostic, Inc. (Flanders, NJ). For immunohistochemical analysis, tissues were obtained from rats at different ages and analyzed as described (56). To determine the cell types expressing IMD and/or ACTH in the anterior pituitary, paraffin-embedded pituitary sections were incubated with primary polyclonal anti-IMD and monoclonal anti-ACTH antibodies, followed by incubation with rhodamine-conjugated goat antirabbit secondary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and/or fluorescein isothiocyanate-conjugated goat antimouse secondary antibody (Sigma-Aldrich). For comparison of {alpha}-MSH and IMD expression patterns, adjacent sections were stained with the anti-IMD antibody or with an anti-{alpha}-MSH antibody followed by a rhodamine-conjugated donkey antirabbit secondary antibody (Sigma-Aldrich). For analysis of prolactin expression, a rabbit antiprolactin polyclonal antibody (National Hormone and Peptide Program) was used in conjunction with a rhodamine-conjugated donkey antirabbit secondary antibody. The signal was observed using a Leica MZ-FLIII fluorescent microscope (Leica Microsystems, Inc., Bannockburn, IL). Images were captured using a Leica DFC300 FX digital camera and Image-Pro PLUS 5.0 software (Media Cybernetics, Inc. Silver Spring, MD). To avoid erroneous interpretation of the results, various negative controls including staining without the first antibody or with mismatched secondary antibodies were performed routinely. In addition, signals derived from two separate antibodies were verified based on staining with individual antibodies using adjacent sections to ensure that there was no secondary antibody cross-talk.

Receptor-Binding Assay
Ligand-binding assays were done in siliconized microcentrifuge tubes at 22 C for 2 h. Dissociated pituitary cells cultured for 2 d in DMEM/F12 medium with 10% fetal bovine serum (FBS) were scraped and resuspended in binding buffer [20 mM Tris-HCl (pH 7.4), 2 mM MgCl2, 0.1% BSA, and an aliquot of a protease inhibitor cocktail (Sigma-Aldrich)] with 0.06 µg of [125I]CGRP and various concentrations of nonradioactive peptides. After repeated washing, the cell membrane-associated radioligand was estimated. Radioactivity was determined using a {gamma}-counter (EG&G Wallace, Gaithersburg, MD).

In Situ Hybridization Study
Rat pituitaries from 8-wk-old rats were fixed at 4 C for 6 h in 4% paraformaldehyde in PBS, followed by immersion in 0.5 M sucrose in PBS overnight. Cryostat sections of fixed pituitaries were mounted on microscope slides (Sigma Chemical Co., St. Louis, MO), fixed in 4% paraformaldehyde in PBS, and stored at –80 C before the hybridization procedure. Sections were pretreated serially with 0.2 M HCl, 2x saline sodium citrate, pronase E (0.125 mg/ml), 4% paraformaldehyde, and acetic anhydride in triethanolamine before dehydration in ascending grades of ethanol. The antisense and sense probes were generated and labeled with [35S]uridine 5-triphosphate (1000 Ci/mmol, Amersham Pharmacia Biotech, Piscataway, NJ) by in vitro transcription using the Riboprobe System (Promega Corp., Madison, WI). The sections were hybridized overnight at 52–55 C in 50% formamide, 0.3 M NaCl, 10 mM Tris-HCl, 5 mM EDTA, 1x Denhardt’s solution, 10% dextran sulfate, 1 µg/ml carrier transfer RNA, and 10 mM dithiothreitol. After ribonuclease A (25 µg/ml) treatment at 37 C for 30 min, posthybridization washing was performed to a final stringency of 0.1x saline sodium citrate. Slides were dipped into NTB-2 emulsion (Eastman Kodak Co., Rochester, NY) and exposed at 4 C for 1–4 wk before development. The slides were subsequently stained with hematoxylin and eosin and mounted with Permount (Fisher Scientific, Fair Lawn, NJ). Photographs of the slides were taken using a 35-mm camera and microscope (Zeiss, Oberkochen, Germany) with bright- and dark-field illumination. To allow for direct comparison among the pituitary sections hybridized with different probes, all slides were processed simultaneously and under identical conditions.

Effects of IMD on Hormone Secretions in Vitro
Eight-week-old male and female Sprague Dawley rats were killed by CO2 and decapitation. Pituitary glands were collected into DMEM/F12 medium (Life Technologies, Inc., Gaithersburg, MD) containing HEPES, 1% penicillin-streptomycin (Life Technologies, Inc.), and 10% FBS, and the anterior lobe was mechanically dispersed and digested with collagenase (Sigma-Aldrich) until a single-cell suspension was obtained. Cell suspensions were aliquoted into 24-well culture dishes (200,000 cells per well) and incubated for 4–5 d at 37 C in DMEM/F12 medium containing 10% FBS. On the day of experimentation, cells were washed, and replaced with serum-free DMEM/F12 medium containing testing hormones. After a 30-min incubation, the supernatant was collected after centrifugation and assayed for hormone content using specific RIAs including a rat prolactin-specific Biotrak RIA (Amersham Pharmacia), a rat GH-specific Biotrak RIA (Amersham Pharmacia), an ACTH RIA (DSL, Inc., Webster, TX), and a FSH RIA (DSL, Inc.).

The treatment of IMD leads to the activation of adenylyl cyclase and the PKA-dependent pathway in target cells (1). To study the specificity of IMD action on lactotrophs, we analyzed the effect of an IMD receptor-binding domain-based antagonist, IMD17–47, on IMD-induced prolactin release (1). Cultured pituitary cells were pretreated with IMD17–47 or a nonfunctional truncated IMD peptide IMD28–47 for 1 h in serum-free DMEM/F12 before IMD treatment. Conditioned media were harvested for hormone assays 30 min after IMD treatment. To verify that IMD stimulates prolactin release via the PKA-dependent pathway, cultures of pituitary cells in 24-well plates were preincubated for 1 h with the PKA-selective inhibitor, H89 (10 µM) (23) or a MAPK kinase kinase inhibitor, PD98059 (10 µM) (24) (Calbiochem, San Diego, CA), before IMD treatment.

Effects of IMD on Hormone Secretions in Vivo
To study the effect of IMD on prolactin release in vivo, adult 12-wk-old male Sprague Dawley rats (450–500 g; Charles River Laboratory, Wilmington, MA) were employed. In vivo experiments were begun 4–5 h after lights on. Under anesthesia (0.2 ml ketamine in isotonic saline/100 g body weight by ip injection), the testing agents were administered through the femoral vein. Blood samples were obtained 30 min after iv injections. All blood samples (5 ml, into heparinized tubes) were kept at 4 C before plasma was separated (3000 x g, 3 min). Plasma was stored at –20 C before being assayed for prolactin and GH content using specific RIAs.

Expression of IMD and IMD Receptor Genes in the Pituitary of Lactating and Ovariectomized Rats
To analyze the effect of lactation on pituitary IMD expression, 10-wk-old lactating female Sprague Dawley rats at 15 d postpartum and age-matched nonlactating females were killed for the collection of pituitaries. Aliquots of pituitary samples from individual animals were extracted for the analysis of IMD transcript expression using real-time PCR or fixed for fluorescent immunohistochemical analysis of IMD peptide.

To study whether estrogen or other ovarian factors regulate pituitary IMD expression, ovaries of 9-wk-old female Sprague Dawley rats were removed after anesthesia with ketamine. One week after ovariectomy, subgroups of ovariectomized rats were injected daily with 17ß-estradiol (10 µg/kg body weight) or implanted with a diethylstilbestrol capsule (2 mm x 1 cm) for 4 d (57). At 11 d after ovariectomy, pituitaries were removed and analyzed as described for lactating animals.

Subcloning of the Proximate Region of Mouse IMD Gene Promoter
To identify putative ERE and binding sites for different transcription factors in the IMD gene promoter, the IMD gene sequences from human, mouse, and rat were retrieved from the GenBank and analyzed using TRANSFAC database (http://www.cbil.upenn.edu/cgi-bin/tess/tess33?RQ=SEA-FR-Query) and Perl-based scripts. To investigate the effect of 17ß-estradiol on IMD gene promoter activity, a 2.7 kb (–1 ~ –2650) and a 1.1 kb (–1 ~ –1131) proximate region of mouse IMD gene promoter were amplified by PCR with sequence-specific primer sets and Advantage DNA polymerase (BD Biosciences, Bedford, MA) using mouse genomic DNA as the template (Fig. 10AGo). The PCR product was electrophoresed, eluted, and purified using QIAquick gel extraction kit (QIAGEN). Both DNA fragments were directionally subcloned into the XhoI and EcoRI sites of the SK pBluescript vector (Invitrogen, Carlsbad, CA) and confirmed by sequencing. For the analysis of promoter activity, the two putative promoter DNA fragments (2650 bp and 1131 bp) were subsequently subcloned into a pGL2-Enhancer luciferase vector (Promega Corp.).

Analysis of IMD Gene Promoter Reporter Activity
Rat pituitary cells from 8-wk-old female rats were cultured in DMEM/F12 supplemented with 10% FBS, together with 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine in 12-well plates. At 2 d after culture, cells in each well were transfected with 1 µg of pGL2-luc reporter plasmid in serum-free DMEM/F12 using lipofectamine 2000 (Invitrogen). pCMV-ß-galactosidase vector (50 ng) was cotransfected to monitor transfection efficiency. After transfection, cells were treated with the appropriate hormone for 12 h in DMEM/F12 containing 0.1% BSA. To analyze luciferase activity in transfected cells, culture media were aspirated and 200 µl of lysis buffer (Promega) was added to each well, and 40 µl of the supernatant was used for luciferase determination using a luminometer (Luminark microplate reader; Bio-Rad Laboratories, Inc., Hercules, CA). Cell lysate (50 µl) from each sample was also used to measure the ß-galactosidase activity. Cell lysate was added to a well in a 96-well plate and mixed with 50 µl of 1x ß-galactosidase assay buffer (2x assay buffer contains 200 mM sodium phosphate buffer at pH 7.4, 2 mM MgCl2, 100 mM ß-mercaptoethanol, and 1.33 mg/ml ONPG dissolved in H2O). After incubation at room temperature for 30–60 min until solutions began to turn yellow, optical density was read using a Luminark microplate reader. The reporter activity is expressed as the ratio of relative luciferase unit/ß-galactosidase activity.

Statistical Analysis
Differences between treatment groups were analyzed by one-way ANOVA and Scheffe’s multiple comparison testing or Student’s t test. Significance was assigned to results that occurred with less than 5% probability.


    ACKNOWLEDGMENTS
 
We thank Augustin Sanchez and Caren Spencer for technical and editorial assistance. We also thank Drs. Anita Payne and Aaron Hsueh for comments on the manuscript.


    FOOTNOTES
 
C.L.C. is supported by a Dean’s Fellowship from the Chang Gung Memorial Hospital, Department of Obstetrics and Gynecology, Tao-Yuan, Taiwan.

Present address for R.V.H.: Institute for Anatomy and Cell Biology, Justus-Liebig-University Giessen, Aulweg 123, 35385 Giessen, Germany.

First Published Online July 7, 2005

Abbreviations: CGRP, Calcitonin gene-related peptide; CRLR, calcitonin receptor-like receptor; ERE, estrogen response element; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IMD, intermedin; IMDL, long intermedin isoform; IMDS, short intermedin isoform; PKA, protein kinase A; RAMP, receptor activity-modifying protein.

Received for publication May 6, 2004. Accepted for publication June 30, 2005.


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