Abstract
Here we investigated roles of the pituitary bone morphogenetic protein (BMP) system in modulating GH production regulated by a somatostatin analog, octreotide (OCT) and a dopamine agonist, bromocriptine (BRC) in rat pituitary somatolactotrope tumor GH3 cells. The GH3 cells were found to express BMP ligands, including BMP-4 and BMP-6; BMP type-1 and type-2 receptors (except the type-1 receptor, activin receptor-like kinase (ALK)-6); and Smad signaling molecules. Forskolin stimulated GH production in accordance with cAMP synthesis. BRC, but not OCT, suppressed forskolin-induced cAMP synthesis by GH3 cells. Individual treatment with OCT and BRC reduced forskolin-induced GH secretion. A low concentration (0.1 μM) of OCT in combination with BRC (1–100 μM) exhibited additive effects on reducing GH and cAMP production induced by forskolin. However, a high concentration (10 μM) of OCT in combination with BRC failed to suppress GH and cAMP production. BMP-4 specifically enhanced GH secretion and cAMP production induced by forskolin in GH3 cells. BRC, but not OCT, inhibited BMP-4-induced activation of Smad1,5,8 phosphorylation and Id-1 transcription and decreased ALK-3 expression. Of note, in the presence of a high concentration of OCT, the BRC effects suppressing BMP-4-Smad1,5,8 signaling were significantly impaired. In the presence of BMP-4, a high concentration of OCT also attenuated the BRC effects suppressing forskolin-induced GH and cAMP production. Collectively, a high concentration of OCT interferes with BRC effects by reducing cAMP production and suppressing BMP-4 signaling in GH3 cells. These findings may explain the mechanism of resistance of GH reduction to a combination therapy with OCT and BRC for GH-producing pituitary adenomas.
Introduction
Acromegaly is caused by excessive growth hormone (GH) secretion from pituitary GH-secreting adenomas. Treatment of acromegaly patients often involves combining surgical, pharmacological, and radiotherapeutic approaches to control endocrine profiles and reduce mortality. Somatostatin analogs and dopamine agonists are utilized as principal medications for acromegaly patients before and/or after pituitary surgery.
It has been generally recognized that a combination therapy with somatostatin analogs and dopamine agonists is potentially effective to reduce GH overproduction in acromegaly patients who are resistant to single medication regimens (Lamberts et al. 1986, Chiodini et al. 1987, Wagenaar et al. 1991, Marzullo et al. 1999). The combination treatment is also effective in in vitro studies using cultured pituitary tumor cells of acromegaly patients (Lamberts et al. 1987). For instance, a regimen using low-dose octreotide (OCT) and bromocriptine (BRC) is reported to be as efficacious as high-dose OCT, indicating usefulness and tolerability of combination therapy in acromegaly patients (Li et al. 2000). Increased bioavailability of BRC during the combination with OCT has been suggested as a possible mechanism to explain the efficacy of the combination treatments (Flogstad et al. 1994).
Since dopamine agonists inhibit GH hypersecretion in 20% of acromegalic patients (Jaffe & Barkan 1992), there is a basis to expect that a combination regimen with dopamine agonists and somatostatin analogs would be an effective treatment. However, the efficacy of such combined treatment has yet to be firmly established due to some conflicting data. It was reported that when OCT is administered in three daily injections the additive effect of BRC on GH and insulin-like growth factor-I suppression is only negligible (Fredstorp et al. 1994). The presence of some resistant cases to a combination therapy in patients who were initially treated with OCT subsequently treated with dopamine agonists has also been observed (Cozzi et al. 2004). Thus, the detailed cellular mechanism by which combination therapy suppresses GH production needs to be elucidated in order to distinguish medication-sensitive patients from non-responsive patients to the combination therapy. To approach the cellular mechanism of GH reduction and the resistance to combination treatment with OCT and BRC, we focused on the bone morphogenetic protein (BMP) system in the pituitary, which has recently received attention as a critical factor in the pathogenesis of certain functioning pituitary adenomas.
BMPs, which belong to the transforming growth factor-β superfamily, were originally identified as the active components in bone extracts capable of inducing bone formation at ectopic sites. A variety of physiological BMP actions in many endocrine tissues including the ovary (Otsuka et al. 2000, 2001, Miyoshi et al. 2007), pituitary (Otsuka & Shimasaki 2002, Takeda et al. 2003, 2007), thyroid (Suzuki et al. 2005), and adrenal (Suzuki et al. 2004, Kano et al. 2005, Inagaki et al. 2006) have been discovered. There is increasing evidence that locally produced BMPs play key roles in differentiation of the pituitary. For instance, BMP-4 promotes pituitary prolactinoma pathogenesis through crosstalk between the BMP-4-mediated Smads and the estrogen receptor (Paez-Pereda et al. 2003). BMP-4 also inhibits corticotropic pathogenesis and Cushing's disease in adult pituitary tumor cells (Giacomini et al. 2006). Thus, the pituitary BMP system likely acts as a regulator not only for organogenesis and differentiation process of pituitary cells, but also for transformation and tumorigenesis of the differentiated pituitary cells. Here, we uncovered effects of the pituitary BMP system on GH reduction elicited by OCT and BRC treatments.
Materials and Methods
Reagents and supplies
A 1:1 mixture of Dulbecco's Modified Eagle Medium/Ham F-12 medium (DMEM/F12), penicillin–streptomycin solution, forskolin (FSK), and 3-isobutyl-1-methylxanthine (IBMX) were purchased from Sigma–Aldrich Corp. Recombinant human BMP-4 and BMP-6 were purchased from R&D Systems (Minneapolis, MN, USA) and activin A was from Sigma–Aldrich Corp. Control rat pituitary and ovary tissues were collected from 8-week-old female Wister rats (CLEA Japan Inc., Tokyo, Japan). BRC mesylate and OCT acetate were provided by Novartis International Pharmaceutical Ltd (Basel, Switzerland). Plasmids of Id-1-Luc were gifts from Dr Tetsuro Watabe and Dr Kohei Miyazono, Tokyo University, Japan.
Cell culture and measurement of cAMP production
Rat pituitary somatolactotrope tumor GH3 cells were provided from Prof. Joseph Majzoub, Children's Hospital, Harvard Medical School. Cells were cultured in DMEM/F12 medium supplemented with 10% fetal calf serum (FCS) and antibiotics in a 5% CO2 atmosphere at 37 °C. GH3 cells (1×105 viable cells) were seeded in 24-well plates with DMEM/F12 containing 10% FCS and penicillin–streptomycin. After preculture, the medium was changed into serum-free DMEM/F12 containing penicillin–streptomycin and 0.1 mM IBMX (a specific inhibitor of phosphodiesterase activity), and then cells were treated with indicated concentrations and combinations of FSK, BRC, OCT, BMP-4, BMP-6, and activin A. After indicated culture periods, the supernatant of the culture media was collected and stored at −80 °C until assay. The extracellular contents of cAMP were determined by enzyme immunoassay (Assay Designs Inc., Ann Arbor, MI, USA) after the acetylation of each sample with assay sensitivity of 0.039 nM.
Determination of GH and prolactin levels
The GH3 cells (1×105 viable cells) were cultured in 24-well plates with DMEM/F12 containing 10% FCS and penicillin–streptomycin. After preculture, the medium was changed into serum-free DMEM/F12, and then cells were treated with indicated concentrations and combinations of FSK, BRC, OCT, BMP-4, BMP-6, and activin A. After 24-h culture, the supernatant of the culture media was collected and stored at −80 °C until assay. The levels of GH and prolactin in the cultured media were determined by rat-specific enzyme immunoassay (Duhau et al. 1991, Ezan et al. 1997; SPI-BIO, Montigny-le-Bretonneux, France).
RNA extraction, RT-PCR, and quantitative real-time PCR analysis
After preculture, cells (3×105 viable cells) were treated with indicated concentrations of FSK, BRC, and OCT in serum-free DMEM/F12. After 24-h culture, the medium was removed and total cellular RNA was extracted using TRIzol (Invitrogen Corp). The whole rat pituitary and ovary tissues were homogenized and total tissue RNA was extracted using TRIzol. Total RNA was quantified by measuring the absorbance of the sample at 260 nm, and stored at −80 °C until assay. The expression of dopamine D2 receptor (D2R), somatostatin receptors (SSTRs), BMP/activin ligands, BMP/activin receptors, Smads, a BMP-binding protein follistatin (FST), and housekeeping gene ribosomal L19 (RPL19) mRNAs were detected by reverse transcription-PCR (RT-PCR) analysis. The extracted RNA (1 μg) was subjected to a RT reaction using First-Strand cDNA Synthesis System (Invitrogen Corp) with random hexamer (2 ng/μl), reverse transcriptase (200 U), and deoxyNTP (dNTP; 0.5 mM) at 42 °C for 50 min, 70 °C for 10 min. Subsequently, hot start PCR was performed using MgCl2 (1.5 mM), dNTP (0.2 mM), and 2.5 U of Taq DNA polymerase (Invitrogen Corp). Oligonucleotides used for RT-PCR were custom ordered from Invitrogen Corp. PCR primer pairs were selected from different exons of the corresponding genes as follows: D2R: 542–562 and 851–871 (from GenBank accession No.
Immunofluorescence microscopy
GH3 cells were precultured in serum-free DMEM/F12 using chamber slides (Nalge Nunc Int., Naperville, IL, USA) and cells at ∼50% confluency were treated with BMP-4 (100 ng/ml). After 1-h stimulation, the cells were fixed with 4% formaldehyde in PBS, permeabilized with 0.5% Triton X-100 in PBS at room temperature, and washed three times with PBS. The cells were then incubated with anti-phospho-Smad1,5,8 antibody (Cell signaling Technology Inc., Beverly, MA, USA) for 1 h and washed three times with PBS. The cells were then incubated with Alexa Fluor 488 anti-rabbit IgG (Invitrogen Corp) in humidified chamber for 1 h and washed with PBS, and then stained cells were visualized under a fluorescent microscope.
Western immunoblot analysis
The cells (3×105 viable cells) were cultured in 24-well plates in DMEM/F12 containing penicillin–streptomycin. After preculture, the medium was changed into serum-free DMEM/F12 and cultured for 24 h, and then the cells were treated with indicated concentrations and combinations of OCT, BRC, and BMP-4. After 1- and 3-h culture, the cells were solubilized in 100 μl RIPA lysis buffer (Upstate Biotechnology Inc., Lake Placid, NY, USA) containing 1 mM Na3VO4, 1 mM NaF, 2% SDS, and 4% β-mercaptoethanol. The cell lysates were then subjected to SDS-PAGE immunoblotting analysis using anti-phospho-Smad1,5,8 antibody (Cell signaling Technology Inc).
Transient transfection and luciferase assay
The cells (1×105 viable cells) were cultured in 24-well plates in DMEM/F12 with 10% FCS containing penicillin–streptomycin. The cells were then transiently transfected with 250 ng each luciferase reporter plasmid (Id-1-Luc) and 25 ng cytomegalovirus-β-galactosidase plasmid (pCMV-β-gal) using FuGENE6 (Roche Molecular Biochemicals). After 16-h incubation, the culture medium was changed into serum-free DMEM/F12 and then the cells were treated with indicated concentrations of BRC and OCT in the presence or absence of BMP-4 for 24 h. The cells were washed with PBS and lysed with Cell Culture Lysis Reagent (TOYOBO, Osaka, Japan). Luciferase activity and β-galactosidase (β-gal) activity of the cell lysate were measured by luminometer. The data were shown as the ratio of luciferase and β-gal activities.
Statistical analysis
All results are shown as mean±s.e.m. of data from at least three separate experiments, each performed with triplicate samples. Differences between groups were analyzed for statistical significance using ANOVA with Fisher's protected least significant difference (PLSD) test (StatView 5.0 software, Abacus Concepts Inc., Berkeley, CA, USA). Values of P<0.05 were accepted as statistically significant.
Results
We first examined expression profile of the BMP system in rat pituitary tumor GH3 cells and rat normal pituitary tissues by RT-PCR (Fig. 1). Rat ovarian tissue RNA was utilized as a positive control. The GH3 cells expressed SSTRs including all of SSTR1, 2, 3, 4, and 5 and dopamine receptor D2R as reported previously (Johnston et al. 1991, Yang et al. 2005). The expression level of SSTR2 in GH3 cells was relatively high when compared with that in rat pituitary tissues, while D2R transcript was comparably low in GH3 cells. In addition, both GH3 cells and rat whole pituitaries expressed BMP/activin type-1 (ALK-2, ALK-3, and ALK-4), type-2 (ActRII, ActRIIB, and BMPRII) receptors and Smads (Smad1, Smad2, Smad3, Smad4, Smad5, Smad6, Smad7, and Smad8). Neither GH3 cells nor rat whole pituitaries expressed the BMP type-1B receptor, ALK-6. FST expression was not detected in GH3 cells although it was expressed in the whole pituitary. The whole pituitary expressed all BMP/activin ligands examined, including BMP-2, BMP-4, BMP-6, BMP-7, activinβA/βB, and inhibinα subunits. The predominant BMP ligands expressed in GH3 cells were BMP-4 and BMP-6 (Fig. 1).
Expression of BMP system and somatostatin receptors in GH3 cells. Total cellular RNA was extracted from GH3 cells, rat whole pituitary, and rat whole ovary tissues. Total cellular RNA was quantified by measuring the absorbance of the sample at 260 nm. The expression of BMP/activin (BMP-2, BMP-4, BMP-6, BMP-7/activinβA, activinβB, and inhibinα) ligands, type-1 (ALK-2, ALK-3, ALK-4, and ALK-6) and type-2 (ActRII, ActRIIB, and BMPRII) receptors, a binding protein follistatin (FST), Smad1–8, somatostatin receptor (SSTR) 1–5, dopamine D2 receptor (D2R), and housekeeping gene RPL19 mRNAs was detected by RT-PCR analysis. MM, molecular weight marker.
Citation: Journal of Endocrinology 197, 1; 10.1677/JOE-07-0549
To evaluate cellular responses with respect to GH secretion, we stimulated GH3 cells with FSK and determined the accumulated levels of cAMP and GH in the culture medium using enzyme immunoassays (Fig. 2). As shown in Fig. 2A, FSK (1–10 μM) induced cAMP production in GH3 cells for 24 h in a time- and concentration-dependent manner. In accordance with cAMP synthesis, FSK (1–10 μM) stimulated GH production concentration dependently for 24-h culture (Fig. 2B). We next examined the effect of OCT (0.03–10 μM) and BRC (0.3–100 μM) on FSK-induced cAMP production (Fig. 3). OCT showed a moderate but insignificant reduction of cAMP levels induced by FSK (1 μM), whereas BRC exhibited potent suppression of FSK-induced cAMP production by GH3 cells (Fig. 3A). When cells were treated with BRC in combination with OCT (Fig. 3B), lower concentrations of OCT (0.1–1 μM) exhibited additive effects on BRC-induced cAMP reduction. Interestingly, the effects of BRC were impaired when added in combination with a high concentration (10 μM) of OCT (Fig. 3B).
Forskolin induction of cAMP and GH in GH3 cells. GH3 cells (1×105 viable cells) were precultured in serum-free DMEM/F12 and then treated with forskolin (FSK, 1–10 μM). (A) The extracellular contents of cAMP in the medium were determined after 3–24-h of culture. (B) cAMP levels and GH concentrations in the medium were determined by specific enzyme immunoassays after 24-h culture. For measurement of cAMP levels, the cells were cultured with serum-free medium containing 0.1 mM IBMX. Results are shown as mean±s.e.m. of data from at least three separate experiments, each performed with triplicate samples. *P<0.05 and **P<0.01 versus control in each group or between the indicated groups.
Citation: Journal of Endocrinology 197, 1; 10.1677/JOE-07-0549
Effects of octreotide and bromocriptine on forskolin-induced cAMP levels. GH3 cells (1×105 viable cells) were precultured in serum-free DMEM/F12. (A) Cells were treated with either octreotide (OCT, 0.03–10 μM) or bromocriptine (BRC, 0.3–100 μM) in the presence of forskolin (FSK, 1 μM). (B) The cells were treated with OCT (0.1–10 μM) in combination with BRC (1–100 μM) in the presence of FSK (1 μM). After 24-h treatment, extracellular cAMP levels in the culture medium containing 0.1 mM IBMX were determined by enzyme immunoassay. Results are shown as mean±s.e.m. of data from at least three separate experiments, each performed with triplicate samples. *P<0.05 versus control in each group or between the indicated groups.
Citation: Journal of Endocrinology 197, 1; 10.1677/JOE-07-0549
As shown in Fig. 4A, OCT (0.03–10 μM) and BRC (0.3–100 μM) independently reduced FSK-induced GH production. A significant reduction of prolactin secretion was also observed in BRC-treated GH3 cells (data not shown). When cells were treated with the combination of BRC and OCT, a low concentration (0.1 μM) of OCT exerted additive effects on the BRC effects suppressing FSK (1 μM)-induced GH levels (Fig. 4B). In contrast, a high concentration (10 μM) of OCT impaired the BRC effects reducing FSK-induced GH production (Fig. 4B). The changes of GH reduction were in parallel with the changes of cAMP reduction induced by BRC and OCT (see Figs 3B and 4B).
Effects of octreotide and bromocriptine on forskolin-induced GH production by GH3 cells. GH3 cells (1×105 viable cells) were precultured in serum-free DMEM/F12. (A) The cells were treated with either octreotide (OCT, 0.03–10 μM) or bromocriptine (BRC, 0.3–100 μM) in the presence of forskolin (FSK, 1 μM). (B) The cells were treated with OCT (0.1–10 μM) in combination with BRC (1–100 μM) in the presence of FSK (1 μM). And then, GH levels in the culture medium for 24-h treatment were determined by enzyme immunoassay. Results are shown as mean±s.e.m. of data from at least three separate experiments, each performed with triplicate samples. *P<0.05 and **P<0.01 versus control in each group or between the indicated groups.
Citation: Journal of Endocrinology 197, 1; 10.1677/JOE-07-0549
To elucidate functional roles of the BMP system expressed in GH3 cells, we treated cells with BMP-4, BMP-6, and activin A in the presence or absence of FSK and determined GH and cAMP production (Fig. 5). As shown in Fig. 5A, BMP-4, BMP-6, and activin A (10–100 ng/ml) had no significant effects on basal GH production by GH3 cells. It was of note that BMP-4 increased GH production induced by FSK (1 μM) for 24-h culture (Fig. 5B). BMP-4 also increased FSK-induced cAMP synthesis by GH3 cells (Fig. 5C). In contrast, BMP-6 and activin A had no significant effects on GH and cAMP levels induced by FSK in GH3 cells (Fig. 5B and C).
Effects of BMP-4 on GH and cAMP production. GH3 cells (1×105 viable cells) were precultured in serum-free DMEM/F12. The cells were then treated with BMP-4 (10–100 ng/ml), BMP-6 (10–100 ng/ml), and activin A (10–100 ng/ml) in the presence or absence of forskolin (FSK, 1 μM). After 24-h culture, the culture media were collected and (A and B) GH production and (C) cAMP levels were determined by specific enzyme immunoassays. For measurement of cAMP levels, the cells were cultured with serum-free medium containing 0.1 mM IBMX. Results are shown as mean±s.e.m. of data from at least three separate experiments, each performed with triplicate samples. *P<0.05 and **P<0.01 versus control in each group.
Citation: Journal of Endocrinology 197, 1; 10.1677/JOE-07-0549
The effects of OCT and BRC on BMP–Smad signaling were further examined by immunoblot and reporter-gene assays (Fig. 6). As shown in Fig. 6A, Smad1,5,8 phosphorylation was readily activated by BMP-4 (100 ng/ml) in GH3 cells. Immunofluorescence studies clearly demonstrated nuclear localization of the phosphorylated Smad1,5,8 molecules in response to BMP-4 addition. As shown by immunoblot analysis (Fig. 6A), BMP-4-induced Smad1,5,8 phosphorylation was not affected by adding OCT (10 μM); however, BRC (100 μM) inhibited BMP-4-induced Smad1,5,8 phosphorylation. Furthermore, transcription of the BMP-target gene, Id-1, was readily activated by BMP-4 (100 ng/ml; Fig. 6B). The Id-1-Luc activity induced by BMP-4 was not affected by OCT (0.1–100 μM), whereas it was significantly suppressed by BRC treatment (1–100 μM) in a concentration-dependent manner (Fig. 6B). As shown in Fig. 6C, a low concentration of OCT (0.1 μM) had additive effects on the BRC suppression of Id-1-Luc activity induced by BMP-4. However, the BRC suppression of BMP-4-induced Id-1-Luc activity was impaired in the presence of a high concentration (10 μM) of OCT (Fig. 6C).
Effects of octreotide and bromocriptine on BMP-4-Smad signaling. (A) GH3 cells were precultured in serum-free DMEM/F12 using chamber slides and treated with BMP-4 (100 ng/ml) for 1 h. The cells were then fixed, permeabilized, and incubated with anti-phospho-Smad1,5,8 (pSmad1,5,8) antibody for immunofluorescent (IF) studies. For immunoblotting analysis, GH3 cells (3×105 viable cells) were precultured in serum-free DMEM/F12. After pretreatment with octreotide (OCT, 10 μM) or bromocriptine (BRC, 100 μM), the cells were treated with BMP-4 (100 ng/ml) for 60–180 min. The cell lysates were then subjected to SDS-PAGE immunoblotting (IB) analysis using anti-pSmad1,5,8 antibody. (B) GH3 cells (1×105 viable cells/ml) were transiently transfected with Id-1-Luc and pCMV-β-gal. The cells were then treated with either OCT (0.1–100 μM) or BRC (1–100 μM) in the presence of BMP-4 (100 ng/ml) for 24 h. (C) GH3 cells transiently transfected with Id-1-Luc and pCMV-β-gal were treated with OCT (0.1–10 μM) in combination with BRC (1–100 μM) in the presence of BMP-4 (100 ng/ml) for 24 h. Luciferase activity and β-galactosidase (β-gal) activity of the cell lysates were measured. The data were shown as the ratio of luciferase to β-gal activity. Results are shown as mean±s.e.m. of data from at least three separate experiments, each performed with triplicate samples. *P<0.05 and **P<0.01 versus control in each group.
Citation: Journal of Endocrinology 197, 1; 10.1677/JOE-07-0549
We further examined the combination effects of OCT and BRC on cAMP and GH production in the presence of BMP-4 (Fig. 7). As shown in Fig. 7A, BMP-4 (100 ng/ml) enhanced FSK (1 μM)-induced cAMP levels, which was dose dependently suppressed by BRC treatment. In GH3 cells activated by FSK and BMP-4, a low concentration of OCT (0.1 μM) had additive effects on the cAMP reduction evoked by BRC, whereas a high concentration (10 μM) of OCT rather impaired the BRC effects reducing cAMP synthesis (Fig. 7A). Likewise, BMP-4 (100 ng/ml) enhanced FSK (1 μM)-induced GH levels and BRC treatment reduced the GH production stimulated by FSK and BMP-4 (Fig. 7B). Furthermore, a low concentration of OCT (0.1 μM) facilitated BRC-induced GH reduction in GH3 cells activated by FSK and BMP-4, whereas a high concentration (10 μM) attenuated the BRC effects reducing GH production (Fig. 7B).
Effects of octreotide and bromocriptine on BMP-4 enhancement of cAMP and GH production induced by forskolin. GH3 cells (1×105 viable cells) were precultured in serum-free DMEM/F12. The cells were treated with octreotide (OCT, 0.1–10 μM) in combination with bromocriptine (BRC, 1–100 μM) under the presence of BMP-4 (100 ng/ml) and forskolin (FSK, 1 μM). After 24-h treatment, (A) extracellular cAMP levels and (B) GH production in culture medium were determined by enzyme immunoassays. For measurement of cAMP levels, the cells were cultured with serum-free medium containing 0.1 mM IBMX. Results are shown as mean±s.e.m. of data from at least three separate experiments, each performed with triplicate samples. *P<0.05 and **P<0.01 versus control in each group or between the indicated groups.
Citation: Journal of Endocrinology 197, 1; 10.1677/JOE-07-0549
To assess the possible mechanism by which OCT and BRC affect BMP-4 signaling in GH3 cells, changes of mRNA levels of endogenous BMP ligands including BMP-4, BMP-6; ALK-2, ALK-3; ActRII; BMPRII, and inhibitory Smad6 and Smad7 were examined by quantitative PCR (Fig. 8). FSK (1 μM) treatment had no significant effects on the expression of BMP ligands or BMP signaling molecules in GH3 cells. In the presence of FSK, BRC (100 μM) caused a decrease in ALK-3 expression levels, while OCT had no significant effects on the expression levels of these BMP molecules examined.
Effects of octreotide and bromocriptine on BMP system expression in GH3 cells. GH3 cells (3×105 viable cells) were cultured in serum-free DMEM/F12 with forskolin (FSK, 1 μM), octreotide (OCT, 0.1–10 μM), and bromocriptine (BRC, 1–100 μM). After 24-h culture, total cellular RNA was extracted and subjected to RT-PCR. The mRNA expression levels of BMP-4, BMP-6, ALK-2, ALK-3, ActRII, BMPRII, Smad6, and Smad7 were quantified by real-time PCR analysis. The expression levels of target genes were standardized by RPL19 levels in each sample. Results are shown as mean±s.e.m. of data from at least three separate experiments, each performed with triplicate samples. *P<0.05 versus control in each group.
Citation: Journal of Endocrinology 197, 1; 10.1677/JOE-07-0549
Discussion
In the present study, we found that BMP-4 facilitates FSK-induced cAMP and GH production through BMP receptor and Smad1,5,8 pathway of GH3 cells (Fig. 9). BRC suppressed FSK-induced cAMP synthesis, leading to the reduction of GH secretion, whereas OCT reduced GH production possibly by a cAMP-independent mechanism.
A possible involvement of BMP-4 in regulating GH secretion by octreotide and bromocriptine. BMP-4 facilitates forskolin (FSK)-induced cAMP and GH production through BMP receptors, ALK-3/BMPRII, and Smad1,5,8 pathway of GH3 cells. Bromocriptine (BRC) suppresses FSK-induced cAMP synthesis, leading to the reduction of GH secretion. Octreotide (OCT) reduces GH production possibly by cAMP-independent mechanism. Importantly, exposure to a high concentration of OCT impairs the BRC effects suppressing FSK-induced cAMP production and BMP-4-Smad1,5,8 signaling in GH3 cells. AC, adenylyl cyclase; PKA, protein kinase-A.
Citation: Journal of Endocrinology 197, 1; 10.1677/JOE-07-0549
The efficacy of somatostatin analogs is linked to the SSTR selectivity profile, in which binding capability to SSTR2 and SSTR5 appears critical (Shimon et al. 1997). SSTR2 and SSTR5 are negatively coupled to adenylyl cyclase, activation of which results in a reduction of intracellular cAMP concentrations (Reisine & Bell 1995). The SSTR effects are further mediated by Ca++ influx through a direct action on Ca++ channels (Chen et al. 1997) and/or indirectly through activating K+ channels (Takano et al. 1997; Fig. 9). On the other hand, D1 and D2 dopamine receptors are widely distributed in the central nervous system and the gastrointestinal tract. D2 dopamine receptors are the predominant mediators of GH regulation in the pituitary. Since GH-producing adenomas having a high activity of adenylyl cyclase exhibit more sensitivity to dopamine agonists (Spada et al. 1990), the capability of cAMP suppression is likely to be a key factor for GH reduction by dopamine agonists.
Combination treatment with somatostatin analogs and dopamine agonists is reserved for acromegaly patients who are resistant to single medical treatment. However, the mechanism of action underlying the efficaciousness of the combined treatment has been uncertain. Balsa and colleagues reported interesting findings regarding the effects of combination therapy on GH release in cultures of surgically removed GH-producing adenomas (Balsa et al. 2002). In their study, the combination treatment with somatostatin analogs and BRC was found to be effective in 18–30% tumor cell cultures. However, the significant correlation between cAMP levels and GH reduction in the cultures were not clearly displayed, implying the involvement of other pathways than adenylyl cyclase–cAMP signaling in the mechanism of GH reduction by the combination treatment.
In the present study, a novel role of endogenous BMP-4 mediating the effects of OCT and BRC combination treatment with respect to GH production was elucidated in the GH3 cell model (Fig. 9). Specifically, BMP-4 stimulated GH and cAMP synthesis induced by FSK. Since GH-releasing hormone elicits GH synthesis and secretion by activating cAMP in the pituitary somatotrope, this interrelationship between cAMP and BMP-4 would be physiologically critical in regulating GH secretion by the normal pituitary and GH-producing adenomas. Importantly, BRC but not OCT inhibited BMP-4-induced activation of Smad1,5,8 phosphorylation and Id-1 transcription. The reduction of ALK-3 expression may be involved in this mechanism. Furthermore, in cases of combination treatment with OCT and BRC, addition of a high concentration of OCT resulted in decreasing the major BRC effects including the inhibition of BMP-4 signaling and the reduction of FSK-induced cAMP production. These findings suggested the involvement of endogenous BMP-4 actions in controlling FSK-induced GH levels by OCT and BRC.
BMP-2 and BMP-4 have been known to play a key role in the initial development of the anterior pituitary (Scully & Rosenfeld 2002). BMP-4 is required during the first stage of pituitary organogenesis for the proliferation of the Rathke's pouch that gives rise to Pit-1 lineage cells including lactotrope cells. Overexpression of noggin or a dominant-negative ALK-3 in the anterior pituitary leads to the arrest of the development of Pit-1-expressing lineage (Scully & Rosenfeld 2002). During the following stages of pituitary organogenesis, an inhibition of BMP-2 by fibroblast growth factor-8 leads to differentiation of corticotrope cells (Kioussi et al. 1999, Dasen & Rosenfeld 2001). BMP-4 not only governs this pituitary organogenesis but also plays a key role in the pathogenesis of differentiated pituitary lineages. For instance, BMP-4 is overexpressed in lactotrope adenomas derived from D2R-null mice as well as human prolactinomas (Paez-Pereda et al. 2003). A binding protein for BMPs, noggin expression is conversely downregulated in the prolactinoma from the D2R-null mouse (Paez-Pereda et al. 2003), suggesting that BMP-4 promotes cell proliferation in lactotropes in conjunction with Smad–estrogen receptor interaction under the influence of its binding protein. BMP-4 also inhibits adrenocorticotropin secretion and cell proliferation of corticotropinoma cells (Giacomini et al. 2006). This BMP-4 action seems to be involved in the anti-proliferative effect induced by retinoic acid on corticotropinomas. Thus, BMP-4 promotes pituitary prolactinoma through Smad–estrogen receptor crosstalk (Paez-Pereda et al. 2003) while BMP-4 inhibits corticotrope pathogenesis of Cushing's disease (Giacomini et al. 2006).
Several preferential combinations of BMP ligands and receptors have been recognized to date, for example, BMP-2 and BMP-4 preferentially bind to ALK-3 and/or ALK-6, BMP-6, and BMP-7 most readily bind to ALK-2 and/or ALK-6 (ten Dijke et al. 1994, Yamashita et al. 1995, Ebisawa et al. 1999, Aoki et al. 2001), and BMP-15 efficiently binds to ALK-6 with much lower affinity for ALK-3 (Moore et al. 2003). Regarding type II receptors, ActRII, which was originally identified as activin receptors, also acts as receptors for BMP-6 and BMP-7 (Yamashita et al. 1995, Ebisawa et al. 1999), while BMPRII binds exclusively to BMP ligands including BMP-2, BMP-4, BMP-6, BMP-7, and BMP-15 (Liu et al. 1995, Nohno et al. 1995, Rosenzweig et al. 1995, Moore et al. 2003). Since ALK-6 is not expressed in GH3 cells, the receptor pairs of ALK-3 and BMPRII are likely to be the major functional complex for BMP-4 for activating cAMP and GH production by GH3 cells. The reduction of ALK-3 expression may be involved, at least in part, in the mechanism by which BRC suppresses BMP-4 signaling in GH3 cells. Notably, the exposure to a high concentration of OCT impairs the BRC effects suppressing cAMP-to-GH production induced by FSK and BMP-4 as well as BMP-4–Smad1,5,8 signaling in GH3 cells. However, the molecular mechanism by which a high concentration of OCT inactivates BRC actions has yet to be elucidated. In this regard, recent studies have shown the presence of a heterodimeric association between SSTR5 and D2R, leading to the enhanced biological activity (Rocheville et al. 2000). Further investigation is necessary to approach the impact and possibility of the receptor heteromerization of OCT and BRC on GH regulation and its interaction with pituitary BMP system.
Collectively, a new interaction between cAMP and BMP-4 signaling for GH production was uncovered. Treatment with high concentrations of OCT interferes with key BRC effects that inhibit cAMP production as well as the BMP-4 pathway in vitro. This interaction may be involved in the mechanism of ineffectiveness of GH reduction in combination therapy with OCT and BRC for acromegaly patients when treated with high concentrations of OCT.
Acknowledgements
We thank Dr R Kelly Moore for helpful discussion and critical reading of the manuscript. We also thank Drs Tetsuro Watabe and Kohei Miyazono for providing Id-1-Luc plasmid. This work was supported in part by Grants-in-Aid for Scientific Research, The Kawasaki Foundation for Medical Science & Medical Welfare, The Ichiro Kanahara Foundation, Kato Memorial Bioscience Foundation, Terumo Lifescience Foundation, and The Naito Foundation. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
References
Aoki H, Fujii M, Imamura T, Yagi K, Takehara K, Kato M & Miyazono K 2001 Synergistic effects of different bone morphogenetic protein type I receptors on alkaline phosphatase induction. Journal of Cell Science 114 1483–1489.
Balsa JA, Varela C, Lucas T, Garcia-Uria J, Barcelo B & Sancho-Rof JM 2002 Varying additive effects of bromocriptine with two somatostatin analogs in cultures of GH-secreting adenomas. Hormone and Metabolic Research 34 435–440.
Chen ZP, Xu S, Lightman SL, Hall L & Levy A 1997 Intracellular calcium ion responses to somatostatin in cells from human somatotroph adenomas. Clinical Endocrinology 46 45–53.
Chiodini PG, Cozzi R, Dallabonzana D, Oppizzi G, Verde G, Petroncini M, Liuzzi A & del Pozo E 1987 Medical treatment of acromegaly with SMS 201–995, a somatostatin analog: a comparison with bromocriptine. Journal of Clinical Endocrinology and Metabolism 64 447–453.
Cozzi R, Attanasio R, Lodrini S & Lasio G 2004 Cabergoline addition to depot somatostatin analogues in resistant acromegalic patients: efficacy and lack of predictive value of prolactin status. Clinical Endocrinology 61 209–215.
Dasen JS & Rosenfeld MG 2001 Signaling and transcriptional mechanisms in pituitary development. Annual Review of Neuroscience 24 327–355.
ten Dijke P, Yamashita H, Sampath TK, Reddi AH, Estevez M, Riddle DL, Ichijo H, Heldin C-H & Miyazono K 1994 Identification of type I receptors for osteogenic protein-1 and bone morphogenetic protein-4. Journal of Biological Chemistry 269 16985–16988.
Duhau L, Grassi J, Grouselle D, Enjalbert A & Grognet JM 1991 An enzyme immunoassay for rat prolactin: application to the determination of plasma levels. Journal of Immunoassay 12 233–250.
Ebisawa T, Tada K, Kitajima I, Tojo K, Sampath TK, Kawabata M, Miyazono K & Imamura T 1999 Characterization of bone morphogenetic protein-6 signaling pathways in osteoblast differentiation. Journal of Cell Science 112 3519–3527.
Ezan E, Laplante E, Bluet-Pajot MT, Mounier F, Mamas S, Grouselle D, Grognet JM & Kordon C 1997 An enzyme immunoassay for rat growth hormone: validation and application to the determination of plasma levels and in vitro release. Journal of Immunoassay 18 335–356.
Flogstad AK, Halse J, Grass P, Abisch E, Djoseland O, Kutz K, Bodd E & Jervell J 1994 A comparison of octreotide, bromocriptine, or a combination of both drugs in acromegaly. Journal of Clinical Endocrinology and Metabolism 79 461–465.
Fredstorp L, Kutz K & Werner S 1994 Treatment with octreotide and bromocriptine in patients with acromegaly: an open pharmacodynamic interaction study. Clinical Endocrinology 41 103–108.
Giacomini D, Paez-Pereda M, Theodoropoulou M, Labeur M, Refojo D, Gerez J, Chervin A, Berner S, Losa M & Buchfelder M et al. 2006 Bone morphogenetic protein-4 inhibits corticotroph tumor cells: involvement in the retinoic acid inhibitory action. Endocrinology 147 247–256.
Inagaki K, Otsuka F, Suzuki J, Kano Y, Takeda M, Miyoshi T, Otani H, Mimura Y, Ogura T & Makino H 2006 Involvement of bone morphogenetic protein-6 in differential regulation of aldosterone production by angiotensin II and potassium in human adrenocortical cells. Endocrinology 147 2681–2689.
Jaffe CA & Barkan AL 1992 Treatment of acromegaly with dopamine agonists. Endocrinology and Metabolism Clinics of North America 21 713–735.
Johnston JM, Wood DF, Bolaji EA & Johnston DG 1991 The dopamine D2 receptor is expressed in GH3 cells. Journal of Molecular Endocrinology 7 131–136.
Kano Y, Otsuka F, Takeda M, Suzuki J, Inagaki K, Miyoshi T, Miyamoto M, Otani H, Ogura T & Makino H 2005 Regulatory roles of bone morphogenetic proteins and glucocorticoids in catecholamine production by rat pheochromocytoma cells. Endocrinology 146 5332–5340.
Kioussi C, Carriere C & Rosenfeld MG 1999 A model for the development of the hypothalamic–pituitary axis: transcribing the hypophysis. Mechanisms of Development 81 23–35.
Lamberts SW, Zweens M, Verschoor L & del Pozo E 1986 A comparison among the growth hormone-lowering effects in acromegaly of the somatostatin analog SMS 201–995, bromocriptine, and the combination of both drugs. Journal of Clinical Endocrinology and Metabolism 63 16–19.
Lamberts SW, Verleun T, Hofland L & Del Pozo E 1987 A comparison between the effects of SMS 201–995, bromocriptine and a combination of both drugs on hormone release by the cultured pituitary tumour cells of acromegalic patients. Clinical Endocrinology 27 11–23.
Li JK, Chow CC, Yeung VT, Mak TW, Ko GT, Swaminathan R, Chan JC & Cockram CS 2000 Treatment of Chinese acromegaly with a combination of bromocriptine and octreotide. Australian and New Zealand Journal of Medicine 30 457–461.
Liu F, Ventura F, Doody J & Massague J 1995 Human type II receptor for bone morphogenic proteins (BMPs): extension of the two-kinase receptor model to the BMPs. Molecular and Cellular Biology 15 3479–3486.
Marzullo P, Ferone D, Di Somma C, Pivonello R, Filippella M, Lombardi G & Colao A 1999 Efficacy of combined treatment with lanreotide and cabergoline in selected therapy-resistant acromegalic patients. Pituitary 1 115–120.
Miyoshi T, Otsuka F, Inagaki K, Otani H, Takeda M, Suzuki J, Goto J, Ogura T & Makino H 2007 Differential regulation of steroidogenesis by bone morphogenetic proteins in granulosa cells: involvement of extracellularly regulated kinase signaling and oocyte actions in follicle-stimulating hormone-induced estrogen production. Endocrinology 148 337–345.
Moore RK, Otsuka F & Shimasaki S 2003 Molecular basis of bone morphogenetic protein-15 signaling in granulosa cells. Journal of Biological Chemistry 278 304–310.
Nohno T, Ishikawa T, Saito T, Hosokawa K, Noji S, Wolsing DH & Rosenbaum JS 1995 Identification of a human type II receptor for bone morphogenetic protein-4 that forms differential heteromeric complexes with bone morphogenetic protein type I receptors. Journal of Biological Chemistry 270 22522–22526.
Otani H, Otsuka F, Inagaki K, Takeda M, Miyoshi T, Suzuki J, Mukai T, Ogura T & Makino H 2007 Antagonistic effects of bone morphogenetic protein-4 and -7 on renal mesangial cell proliferation induced by aldosterone through MAPK activation. American Journal of Physiology. Renal Physiology 292 F1513–F1525.
Otsuka F & Shimasaki S 2002 A novel function of bone morphogenetic protein-15 in the pituitary: selective synthesis and secretion of FSH by gonadotropes. Endocrinology 143 4938–4941.
Otsuka F, Yao Z, Lee TH, Yamamoto S, Erickson GF & Shimasaki S 2000 Bone morphogenetic protein-15: identification of target cells and biological functions. Journal of Biological Chemistry 275 39523–39528.
Otsuka F, Moore RK & Shimasaki S 2001 Biological function and cellular mechanism of bone morphogenetic protein-6 in the ovary. Journal of Biological Chemistry 276 32889–32895.
Paez-Pereda M, Giacomini D, Refojo D, Nagashima AC, Hopfner U, Grubler Y, Chervin A, Goldberg V, Goya R & Hentges ST et al. 2003 Involvement of bone morphogenetic protein 4 (BMP-4) in pituitary prolactinoma pathogenesis through a Smad/estrogen receptor crosstalk. PNAS 100 1034–1039.
Reisine T & Bell GI 1995 Molecular biology of somatostatin receptors. Endocrine Reviews 16 427–442.
Rocheville M, Lange DC, Kumar U, Patel SC, Patel RC & Patel YC 2000 Receptors for dopamine and somatostatin: formation of hetero-oligomers with enhanced functional activity. Science 288 154–157.
Rosenzweig BL, Imamura T, Okadome T, Cox GN, Yamashita H, ten Dijke P, Heldin C-H & Miyazono K 1995 Cloning and characterization of a human type II receptor for bone morphogenetic proteins. PNAS 92 7632–7636.
Scully KM & Rosenfeld MG 2002 Pituitary development: regulatory codes in mammalian organogenesis. Science 295 2231–2235.
Shimon I, Yan X, Taylor JE, Weiss MH, Culler MD & Melmed S 1997 Somatostatin receptor (SSTR) subtype-selective analogues differentially suppress in vitro growth hormone and prolactin in human pituitary adenomas. Novel potential therapy for functional pituitary tumors. Journal of Clinical Investigation 100 2386–2392.
Spada A, Arosio M, Bochicchio D, Bazzoni N, Vallar L, Bassetti M & Faglia G 1990 Clinical, biochemical, and morphological correlates in patients bearing growth hormone-secreting pituitary tumors with or without constitutively active adenylyl cyclase. Journal of Clinical Endocrinology and Metabolism 71 1421–1426.
Suzuki J, Otsuka F, Inagaki K, Takeda M, Ogura T & Makino H 2004 Novel action of activin and bone morphogenetic protein in regulating aldosterone production by human adrenocortical cells. Endocrinology 145 639–649.
Suzuki J, Otsuka F, Takeda M, Inagaki K, Miyoshi T, Mimura Y, Ogura T, Doihara H & Makino H 2005 Functional roles of the bone morphogenetic protein system in thyrotropin signaling in porcine thyroid cells. Biochemical and Biophysical Research Communications 327 1124–1130.
Takano K, Yasufuku-Takano J, Teramoto A & Fujita T 1997 Gi3 mediates somatostatin-induced activation of an inwardly rectifying K+ current in human growth hormone-secreting adenoma cells. Endocrinology 138 2405–2409.
Takeda M, Otsuka F, Suzuki J, Kishida M, Ogura T, Tamiya T & Makino H 2003 Involvement of activin/BMP system in development of human pituitary gonadotropinomas and nonfunctioning adenomas. Biochemical and Biophysical Research Communications 306 812–818.
Takeda M, Otsuka F, Otani H, Inagaki K, Miyoshi T, Suzuki J, Mimura Y, Ogura T & Makino H 2007 Effects of peroxisome proliferator-activated receptor activation on gonadotropin transcription and cell mitosis induced by bone morphogenetic proteins in mouse gonadotrope LβT2 cells. Journal of Endocrinology 194 87–99.
Wagenaar AH, Harris AG, van der Lely AJ & Lamberts SW 1991 Dynamics of the acute effects of octreotide, bromocriptine and both drugs in combination on growth hormone secretion in acromegaly. Acta Endocrinologica 125 637–642.
Yamashita H, ten Dijke P, Huylebroeck D, Sampath TK, Andries M, Smith JC, Heldin C-H & Miyazono K 1995 Osteogenic protein-1 binds to activin type II receptors and induces certain activin-like effects. Journal of Cell Biology 130 217–226.
Yang SK, Parkington HC, Blake AD, Keating DJ & Chen C 2005 Somatostatin increases voltage-gated K+ currents in GH3 cells through activation of multiple somatostatin receptors. Endocrinology 146 4975–4984.
