GH3 Cell Line Research Articles

Wnt signaling in estrogen-induced lactotroph proliferation

Prolactinomas are the most common type of functioning pituitary adenoma in humans, but the control of lactotroph proliferation remains unclear. Here, using microarray analysis, we show that estrogen treatment increased expression of Wnt4 mRNA in adult Fischer rat pituitary tissue. Dual immunofluorescence analysis revealed that Wnt4 expression was not confined to lactotrophs, but that it was expressed in all anterior pituitary cell types. Estradiol induced proliferation in the somatolactotroph GH3 cell line, in parallel with Wnt4 mRNA and protein induction. A reporter gene assay for TCF- and LEF-dependent transcription revealed that there was no activation of the canonical Wnt pathway in GH3 cells upon stimulation with Wnt-conditioned culture medium or coexpression of constitutively active mutant β-catenin. Expression of β-catenin in both GH3 cells and normal rat anterior pituitary cells was restricted to the cell membrane and was unaltered by treatment with estradiol, with no nuclear β-catenin being detected under any of the conditions tested. We show for the first time that Wnt4 affects non-canonical signaling in the pituitary by inhibiting Ca(2+) oscillations in GH3 cells, although the downstream effects are as yet unknown. In summary, Wnt4 is expressed in the adult pituitary gland, and its expression is increased by estrogen exposure, suggesting that its involvement in adult tissue plasticity is likely to involve β-catenin-independent signaling pathways.

Detection of thyroid hormone receptor disruptors by a novel stable in vitro reporter gene assay

A stable luciferase reporter gene assay was developed based on the thyroid hormone responsive rat pituitary tumor GH3 cell line that constitutively expresses both thyroid hormone receptor isoforms. Stable transfection of the pGL4CP-SV40-2xtaDR4 construct into the GH3 cells resulted in a highly sensitive cell line (GH3.TRE-Luc), which was further optimized into an assay that allowed the detection of Triiodothyronine (T 3) and Thyroxine (T 4) concentrations in the picomolar range after only 24 h of exposure. The greater than 20-fold induction of T 3 relative to the solvent control is illustrative of the high responsiveness of the system. The assay was validated by the quantification of the agonistic effect of the natural hormones (T 3 and T 4), the acetic acid derivatives of T 3 (triiodothyroaceticacid, or Triac) and T 4 (tetraiodothyroacetic acid, or Tetrac), hydroxy polybrominated diphenylethers (OH-PBDEs), hydroxy polychlorinated biphenyls (OH-PCBs) and the antagonistic action of sodium arsenite (NaAsO 2). The putative antagonist Amiodarone, Bisphenol A (BPA) and its halogenated derivatives (TCBPA and TBBPA) for which effects reported in the literature are not consistent, showed comparable dose–response curves with a slight agonistic effect (5% of T 3-max) followed by a slight antagonistic effect. The magnitude and reproducibility of the responses to various compounds confirms this assay as a promising tool for the identification and quantification of specific thyroid hormone receptor disrupting potency of compounds.

Temozolomide-induced inhibition of pituitary adenoma cells

Aggressive pituitary adenomas frequently require multimodality treatment including pituitary-suppressive medications, microsurgery, and radiation therapy or radiosurgery. The effectiveness of temozolomide in terms of growth suppression and decreased hormonal production is evaluated. Three pituitary adenoma cell lines--MMQ, GH3, and AtT20--were used. A dose escalation of temozolomide was performed for each cell line, and inhibition of cell proliferation was assessed using an MTT assay. Concentrations of temozolomide that produced statistically significant inhibition of cell proliferation for each cell type were identified. Extent of apoptosis for each selected temozolomide concentration was studied using TUNEL staining. The effect of temozolomide on prolactin secretion in MMQ and GH3 cells was also measured via ELISA. Significant inhibition of cell proliferation was noted for MMQ and GH3 cells at a concentration of 250 μM temozolomide. The AtT20 cells demonstrated statistically significant cell inhibition at a concentration of only 50 μM temozolomide (p < 0.05). Apoptosis significantly increased in all cell lines in as little as 24 hours of incubation at the respective temozolomide concentrations (p < 0.05). Prolactin secretion in the prolactin secreting MMQ and GH3 cell lines was inhibited by 250 μM temozolomide. Temozolomide inhibits cell proliferation and induces apoptotic cell death in aggressive pituitary adenoma cells. A reduction in hormonal secretion in prolactinoma cells was also afforded by temozolomide. Temozolomide may prove useful in the multimodality management of aggressive pituitary adenomas.

The Expression of AIP-Related Molecules in Elucidation of Cellular Pathways in Pituitary Adenomas

Germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene predispose to the development of pituitary adenomas. Here, we characterized AIP mutation positive (AIPmut+) and AIP mutation negative (AIPmut-) pituitary adenomas by immunohistochemistry. The expressions of the AIP-related proteins aryl hydrocarbon receptor (AHR), AHR nuclear translocator (ARNT), cyclin-dependent kinase inhibitor 1B encoding p27(Kip1), and hypoxia-inducible factor 1-alpha were examined in 14 AIPmut+ and 53 AIPmut- pituitary adenomas to detect possible expression differences. In addition, the expression of CD34, an endothelial and hematopoietic stem cell marker, was analyzed. We found ARNT to be less frequently expressed in AIPmut+ pituitary adenomas (P = 0.001), suggesting that AIP regulates the ARNT levels. AIP small interfering RNA-treated HeLa, HEK293, or Aip-null mouse embryonic fibroblast cells did not show lowered expression of ARNT. Instead, in the pituitary adenoma cell line GH3, Aip silencing caused a partial reduction of Arnt and a clear increase in cell proliferation. We also observed a trend for increased expression of nuclear AHR in AIPmut+ samples, although the difference was not statistically significant (P = 0.06). The expressions of p27(Kip1), hypoxia-inducible factor 1-alpha, or CD34 did not differ between tumor types. The present study shows that the expression of ARNT protein is significantly reduced in AIPmut+ tumors. We suggest that the down-regulation of ARNT may be connected to an imbalance in AHR/ARNT complex formation arising from aberrant cAMP signaling.

The proliferative effect of Ghrelin/GHS-R on rat GH3 cell line and its molecular mechanism

Objective To explore the proliferative effect of Ghrelin/GHS-R1α on rat GH3 cell line and its molecular mechanism.Methods The effect of ghrelin(at 1×10-10-1×10-6 mol/L concentrations)on proliferation of GH3 cells was studied by using MTT methods.The protein of total ERK and phosphorylated ERK was detected by Western blot.The activation of the MAPK pathway was studied by using Western blot and inhibitors of ERK,PKC and tyrosine phosphatase pathways.Results Ghrelin significantly increased proliferation at 1×10-10 mol/L to 1×10-6 mol/L concentrations(1×10-10 mol/L for 107±5.1×10-9 mol/L for 157±3.1×10-8 mol/L for 135±18.1×10-7 mol/L for 197±8,and 1×10-6 mol/L for 171±10,respectively)as compared with untreated controls(P ＜0.01).And ghrelin also caused a significant increase in phosphorylated ERK in Western-blot at 1×10-10 mol/L to 1×10-6 mol/L concentrations(1×10-10 mol/L for 0.412±0.064,1×10-9 mol/L for 0.612± 0.045,1×10-8 mol/L for 0.573±0.038,1×10-7 mol/L for 0.675±0.034,and 1×10-6 mol/L for 0.631±0.053,respectively)as compared with untreated controls(P ＜ 0.05).Ghrelin(at 1×10-7 mol/L concentration)caused a significant increase in phosphorylated ERK1/2.The positive effect of ghrelin on MTT was abolished by the MAPK kinase inhibitor UO126,the PKC inhibitor GF109203X and the tyrosine kinase inhibitor Genistein.This could also be clearly demonstrated by Western blot analysis,where a transient increase in ERK1/2 phosphorylation by ghrelin was attenuated by all three inhibitors.Conclusion This study has shown a novel role for ghrelin in stimulating the proliferation of rat GH3 cell line,suggesting that ERK activation is involved in mediating the effects of ghrelin on cell proliferation. Key words: Ghrelin; Growth hormone secretagogue receptor; Proliferation

Adiponectin Regulate Growth Hormone Secretion Via Adiponectin Receptor Mediated Ca2+ Signalling in Rat Somatotrophs In Vitro

Obesity is associated with reduced levels of growth hormone (GH) and the disruption of pulsatile GH secretion. This results in relative GH deficiency. It is likely that a regulatory relationship between GH secretion and adipose tissue exists as the secretion of GH recovers to normal levels after a reduction in body weight. This report characterise the expression and interaction of adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2) and adiponectin, respectively, in regulating the activity of GH secreting cells. Polymerase chain reaction analysis of the GH3 cell line, rat anterior pituitary gland and isolated somatotroph cells from transgenic GFP expressing mice confirmed the expression of both AdipoR1 and AdipoR2 in GH secretory cells. Because GH cells expressed both receptors, it is likely that the measured increase in GH secretion, observed in primary cultured rat pituitary cells after 30 min of incubation with full-length murine adiponectin, was mediated by a direct receptor regulated process. Adiponectin induced an increase in intracellular Ca(2+) through both the influx of extracellular Ca(2+) and the release of intracellular Ca(2+) stores resulting in the secretion of GH. Furthermore, results confirm that this increase in GH secretion depended mainly on an increase in Ca(2+) influx through L-type Ca(2+) channels. It is concluded that adiponectin directly regulates GH secretion from somatotrophs by binding to either adiponectin receptor, and that this is mediated via a similar process observed after the stimulation of GH secretion by GH-releasing hormone.

Characterization of Intracellular Signaling Mediated by Human Somatostatin Receptor 5: Role of the DRY Motif and the Third Intracellular Loop

Somatostatin (SST) exerts inhibitory effects on hormone secretion and cell proliferation by interacting with five different receptors (SST1-SST5) linked to multiple cellular effectors. The receptor structural domains involved in these effects have been only partially elucidated. The aim of the study was to investigate the molecular determinants mediating the interaction of the human SST5 with intracellular signaling in the pituitary cell line GH3, focusing on the BBXXB domain in the third intracellular loop and the DRY motif in the second intracellular loop. We analyzed the effects of the SST5 agonist BIM23206 on cAMP accumulation, intracellular calcium, GH secretion, cell proliferation, and ERK1/2 phosphorylation in cells expressing either wild-type SST5 or mutant receptors, in particular the naturally occurring mutant R240W in the BBXXB domain and the D136A and R137A mutants in the DRY motif. We found that residues D136 and R137 were critical for SST5 signaling because their substitutions abolished all the intracellular responses. Conversely, third intracellular loop mutations resulted in receptor that inhibited intracellular cAMP levels similar to the wild-type (50 +/- 9 vs. 53 +/- 12% inhibition) but failed to mediate the other responses elicited by wild-type SST5, i.e. reduction of intracellular calcium levels as well as inhibition of ERK1/2. These events resulted in an absent inhibition of GH release and an impaired reduction of cell proliferation (38 +/- 7 vs. 76 +/- 6% inhibition in wild type, P < 0.05). These data indicate that different regions of SST5 are required for the activation of different signaling pathways.

Effects of DTPA on zinc flux in leukemia cell lines

Previous studies from our laboratory with Zn‐65 have shown that H4IIE (rat hepatoma) and GH3 (rat pituitary tumor) cell lines reduce zinc efflux in the presence of the extracellular metal chelator DTPA (diethylenetriaminepentaacetic acid). Primary rat hepatocytes and anterior pituitary cells give out more zinc under the same conditions. This may reflect the greater zinc requirements of the rapidly dividing transformed cells. Here, we examined the effects of DTPA on radioactive Zn‐65 flux in C58(NT) (rat thymic lymphoma) and HL‐60 (human promyelocytic leukemia) cell lines. When these cells were co‐incubated with DTPA (50 µM) and Zn‐65, uptake of label was reduced. But when cells were pre‐labeled with Zn‐65 and then incubated with DTPA, the chelator increased efflux of Zn‐65. Despite being transformed, these cell lines behaved similarly to primary cells. Since the leukemia cells grow in suspension unlike the monolayer cell lines previously examined, we determined whether non‐adherence contributed to the difference in response to zinc deprivation. Experiments were repeated with H4IIE cells kept in suspension by growth in roller bottles. However, even under these conditions, DTPA enhanced retention of cellular zinc in the hepatoma cells. Thus, leukemia cells differ from other transformed cell lines in their homeostatic response to zinc deprivation and this is not due to their inability to form a monolayer.

Modulation of Growth Hormone and Prolactin Secretion in Trypanosoma cruzi-Infected Mammosomatotrophic Cells

Objective: In a previous study, we reported an imbalance in the hypothalamus-pituitary-adrenal axis of mice acutely infected with the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease. Methods: Possible effects of this parasitic infection on the endocrine function of other pituitary cell types were studied, in particular regarding the production of prolactin (PRL) and growth hormone (GH). Results: In the mammosomatotrophic cell line GH3, both GH and PRL secretion were decreased, reflecting the diminished PRL concentrations in the pituitary glands of infected mice. Additionally, expression of extracellular matrix proteins, e.g. laminin, was increased in T. cruzi-infected GH3 cells, which may be related to the diminished secretory function of these cells. Lastly, the expression of Pit-1, a major transcription factor for the PRL and GH genes, is also decreased in T. cruzi-infected cultures. Conclusion: T. cruzi infection downregulates PRL and GH production. Combined with our previous data showing increased glucocorticoid levels following T. cruzi infection, the immunosuppression induced by T. cruzi infection may be partially related to multiple endocrine changes involving the hypothalamus-pituitary axis and corresponding target endocrine glands.

Effect of 9-cis Retinoic Acid on Dopamine D2 Receptor Expression in Pituitary Adenoma Cells

The dopamine receptor subtype 2 (D2R) promoter contains a functional retinoic acid response element involved in the control of D2R expression. The aim of the study was to evaluate the effect of 9-cis retinoic acid (9-cis RA) on D2R protein expression in human pituitary adenomas and GH3 cell line. Treatment with 9-cis RA (100 nM for 48 hrs) caused a 109 +/- 32% increase of basal D2R levels in five of eight growth hormone (GH)-secreting adenomas (GH-omas), a 129 +/- 28% increase in 7 of 11 nonfunctioning adenomas, and no effect in two resistant prolactinomas by Western blotting. The lack of D2R induction in some tumors was not associated with a different pattern of retinoid x receptor (RXR) and retinoic acid receptor (RAR) isoform expression that was similar in all tumors by immunohistochemistry. While the induction of D2R did not affect the slight but significant inhibitory effect exerted by dopamine (10 nM) on in vitro GH release by GH-oma cultured cells, in pituitary GH3 cell lines cis-9 RA enhanced the dopamine-induced inhibition of in vitro GH release (% inhibition: 16 +/- 2 versus 26 +/- 5, P < 0.05), cell proliferation (25 +/- 2% versus 44 +/- 5%, P < 0.05) and cell viability (16 +/- 0.8% versus 29 +/- 1%, P < 0.05), likely by activating caspase-3 (28 +/- 3% versus basal, P < 0.05). In conclusion, this study provides novel evidence for a permissive role of retinoids on the expression of D2R in a good proportion of pituitary tumors and on the generation of pro-apoptotic signals in GH3 cell line.

The Third Intracellular Loop of the Human Somatostatin Receptor 5 Is Crucial for Arrestin Binding and Receptor Internalization after Somatostatin Stimulation

AbstractSomatostatin (SS) is a widely distributed polypeptide that exerts inhibitory effects on hormone secretion and cell proliferation by interacting with five different receptors (SST1-SST5). β-Arrestins have been implicated in regulating SST internalization, but the structural domains mediating this effect are largely unknown. The aim of this study was to characterize the intracellular mechanisms responsible for internalization of human SST5 in the rat pituitary cell line GH3 and to identify the SST5 structural domains involved in this process. To this purpose we evaluated, by fluorescence microscopy and biochemical assay, the ability of wild-type, progressive C-terminal truncated and third cytoplasmatic loop mutants SST5-DsRed to associate with β-arrestin-enhanced green fluorescent protein and to internalize under SS28 stimulation. The truncated mutants were comparable to the wild-type receptor with respect to recruitment of β-arrestin-2 and internalization, whereas the third loop mutants R240W, S242A, and T247A showed the abolishment or reduction of arrestin association and a significant reduction of receptor internalization (14.4%, 29%, and 30.9% vs. 52.4% of wild type) and serine phosphorylation upon SS28 stimulation. Moreover, we evaluated the ability of simultaneous mutation of these three residues (R240, S242, and T247) and C-terminal truncated receptors to internalize. The progressive truncation of the C-terminal tail resulted in a progressive increased internalization (21.6%, 36.7%, and 41%, respectively) with respect to the full-length total third-loop mutant (15%). In conclusion, our results indicate the SST5 third intracellular loop as an important mediator of β-arrestin/receptor interaction and receptor internalization, whereas they suggest that residues 328–347 within the C terminus may play an inhibitory role in receptor internalization.

RGD-FasL Induces Apoptosis of Pituitary Adenoma Cells

This study was to investigate the cytotoxic effects on pituitary adenoma cell lines GH3/MMQ/AtT20 induced by RGD-FasL and the underlying mechanism. Fas/DcR3 mRNAs were detected by RT-PCR and their surface expressions were measured by flow cytometry. Cytotoxicity exerted by RGD-FasL on tumor cells was measured with MTT assay and the induced apoptosis was determined by agarose gel electrophoresis. The cell cycle and apoptosis was assessed by flow cytometry with PI staining. The expressions of caspase8/9/3, Bcl-2, RANKL and JNK2 were detected by Western blotting. Approximately 13.7% of GH3 cells, 25.5% of MMQ cells, 22.2% of AtT20 cells express Fas, while 23.9% of GH3 cells, 24.1% of MMQ cells, 4.6% of AtT20 cells express DcR3. The cytotoxic effects of FasL/RGD-FasL on tumor cells were all taken in a dose-dependent manner. Cell lines MMQ/AtT20 showed the same sensitivity to RGD-FasL as to FasL, while cell line GH3 was less sensitive to RGD-FasL. The cell cycle analysis indicated that RGD-FasL could inhibit cells in G0/G1 phase and G2/M phase. In MMQ and AtT20 cells treated with RGD-FasL, the AI was not significantly different from that treated with FasL, while in GH3 cells treated with RGD-FasL, the AI was lower than that treated with FasL. The expressions of caspase-8/9/3, RANKL and JNK2 were increased while that of Bcl-2 was decreased after treatment with RGD-FasL, suggesting that RGD-FasL induces apoptosis through caspase activation. We concluded that RGD-FasL could possibly be considered as a novel therapeutical candidate for the treatment of pituitary adenomas.

RSUME, a Small RWD-Containing Protein, Enhances SUMO Conjugation and Stabilizes HIF-1α during Hypoxia

SUMO conjugation to proteins is involved in the regulation of diverse cellular functions. We have identified a protein, RWD-containing sumoylation enhancer (RSUME), that enhances overall SUMO-1, -2, and -3 conjugation by interacting with the SUMO conjugase Ubc9. RSUME increases noncovalent binding of SUMO-1 to Ubc9 and enhances Ubc9 thioester formation and SUMO polymerization. RSUME enhances the sumoylation of IkB in vitro and in cultured cells, leading to an inhibition of NF-kB transcriptional activity. RSUME is induced by hypoxia and enhances the sumoylation of HIF-1alpha, promoting its stabilization and transcriptional activity during hypoxia. Disruption of the RWD domain structure of RSUME demonstrates that this domain is critical for RSUME action. Together, these findings point to a central role of RSUME in the regulation of sumoylation and, hence, several critical regulatory pathways in mammalian cells.

Down-regulation of goldfish ( Carassius auratus) prolactin gene expression by dopamine and thyrotropin releasing hormone

Prolactin (PRL) is the most versatile hormone identified with multiple functions from osmoregulation in euryhaline fish to lactation in mammals. However, little is known about the basic physiological control of PRL in stenohaline freshwater fish. In this study, goldfish is used as a model for the study of PRL gene expression in stenohaline fish. We report herein the identification of the goldfish PRL (gfPRL) genomic sequence which possesses five exons with its 5′-flanking gene promoter region characterized in vitro using GH3 and GH4 cell-lines. Dopamine and thyrotropin releasing hormone were found to down-regulate the transcription of this gfPRL gene promoter in vitro. This was further confirmed by the decrease of PRL mRNA levels in vitro (in goldfish pituitary primary cells) and in vivo (intra-peritoneal injection) following the administrations of dopamine and thyrotropin releasing hormone. The Pit-1 binding sites of gfPRL are highly conserved with a consensus DNA sequence of 5′TATNCAT-3′, as confirmed with the electrophoretic mobility shift assay using nuclear extract from the GH3 cell-line, may be responsible for the control of gfPRL gene promoter.

Different kinases regulate activation of voltage-dependent calcium channels by depolarization in GH3 cells

The L-type Ca(2+) channel is the primary voltage-dependent Ca(2+)-influx pathway in many excitable and secretory cells, and direct phosphorylation by different kinases is one of the mechanisms involved in the regulation of its activity. The aim of this study was to evaluate the participation of Ser/Thr kinases and tyrosine kinases (TKs) in depolarization-induced Ca(2+) influx in the endocrine somatomammotrope cell line GH3. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured using a spectrofluorometric method with fura 2-AM, and 12.5 mM KCl (K(+)) was used as a depolarization stimulus. K(+) induced an abrupt spike (peak) in [Ca(2+)](i) that was abolished in the presence of nifedipine, showing that K(+) enhances [Ca(2+)](i), preferably activating L-type Ca(2+) channels. H89, a selective PKA inhibitor, significantly reduced depolarization-induced Ca(2+) mobilization in a concentration-related manner when it was applied before or after K(+), and okadaic acid, an inhibitor of Ser/Thr phosphatases, which has been shown to regulate PKA-stimulated L-type Ca(2+) channels, increased K(+)-induced Ca(2+) entry. When PKC was activated by PMA, the K(+)-evoked peak in [Ca(2+)](i), as well as the plateau phase, was significantly reduced, and chelerythrine (a PKC inhibitor) potentiated the K(+)-induced increase in [Ca(2+)](i), indicating an inhibitory role of PKC in voltage-dependent Ca(2+) channel (VDCC) activity. Genistein, a TK inhibitor, reduced the K(+)-evoked increase in [Ca(2+)](i), but, unexpectedly, the tyrosine phosphatase inhibitor orthovanadate reduced not only basal Ca(2+) levels but, also, Ca(2+) influx during the plateau phase. Both results suggest that different TKs may act differentially on VDCC activation. Activation of receptor TKs with epidermal growth factor (EGF) or vascular endothelial growth factor potentiated K(+)-induced Ca(2+) influx, and AG-1478 (an EGF receptor inhibitor) decreased it. However, inhibition of the non-receptor TK pp60 c-Src enhanced K(+)-induced Ca(2+) influx. The present study strongly demonstrates that a complex equilibrium among different kinases and phosphatases regulates VDCC activity in the pituitary cell line GH3: PKA and receptor TKs, such as vascular endothelial growth factor receptor and EGF receptor, enhance depolarization-induced Ca(2+) influx, whereas PKC and c-Src have an inhibitory effect. These kinases modulate membrane depolarization and may therefore participate in the regulation of a plethora of intracellular processes, such as hormone secretion, gene expression, protein synthesis, and cell proliferation, in pituitary cells.

A Calcium-Binding Protein, Calbindin-D9k, Is Regulated through an Estrogen-Receptor Mediated Mechanism following Xenoestrogen Exposure in the GH3 Cell Line

A variety of environmental chemicals may possess the potential to interact with various endocrine factors and consequently cause adverse effects on the reproductive, central nervous, and immune systems via the endocrine system(s). In this study, we used the GH3 cell line as an in vitro model to determine the effects of potential endocrine disruptors (EDs) on the induction of calbindin-D9k (CaBP-9k), a useful biomarker for detecting the estrogenic activities of EDs. A rat pituitary cell line, GH3, was treated with octyl-phenol (OP), nonyl-phenol (NP), and bisphenol A (BPA) in a dose-dependent manner (10(-5), 10(-6), and 10(-7)M) for 24 h. To determine the time dependency, the cells were exposed to a high concentration (10(-5)M) of OP, NP, and BPA and harvested at different time points (1, 3, 6, 12, and 24 h). An antiestrogen, ICI 182,780, was used to examine the potential involvement of the estrogen receptor (ER) in the induction of CaBP-9k by EDs via an ER-mediated pathway. Treatment with OP, NP, and BPA induced a significant increase in CaBP-9k expression at both the transcriptional and translational levels in a dose-dependent manner. Interestingly, ED exposure caused a significant increase in CaBP-9k messenger RNA (mRNA) expression at 6 h, whereas induction of CaBP-9k protein was observed as early as 1 h after treatment. However, both CaBP-9k mRNA and protein expression peaked at 24 h following treatment. The differential response of CaBP-9k mRNA and protein to EDs may be explained by translational efficiency. Cotreatment with ICI 182,780 significantly reversed ED-induced CaBP-9k expression in GH3 cells, suggesting that EDs may trigger the induction of CaBP-9k via an ER-mediated pathway in these cells. Taken together, these results demonstrate for the first time that a single in vitro exposure to OP, NP, or BPA results in an increase in CaBP-9k expression in GH3 cells, after 24 h. These results may contribute to our understanding of the relationship between the molecular events evoked by ED exposure and its biological effects on the pituitary gland in humans and animals. This in vitro model, in combination with a single injection in vivo method developed by us, may be important for elucidating additional details of the mode of action of xenoestrogens and may provide reliable tests for screening estrogenic agonists and antagonists.

Caveolin-1 sensitizes rat pituitary adenoma GH3 cells to bromocriptine induced apoptosis

BackgroundProlactinoma is the most frequent pituitary tumor in humans. The dopamine D2 receptor agonist bromocriptine has been widely used clinically to treat human breast tumor and prolactinoma through inhibition of hyperprolactinemia and induction of tumor cell apoptosis, respectively, but the molecular mechanism of bromocriptine induction of pituitary tumor apoptosis remains unclear. Caveolin-1 is a membrane-anchored protein enriched on caveolae, inverted flask-shaped invaginations on plasma membranes where signal transduction molecules are concentrated. Currently, caveolin-1 is thought to be a negative regulator of cellular proliferation and an enhancer of apoptosis by blocking signal transduction between cell surface membrane receptors and intracellular signaling protein cascades. Rat pituitary adenoma GH3 cells, which express endogenous caveolin-1, exhibit increased apoptosis and shrinkage after exposure to bromocriptine. Hence, the GH3 cell line is an ideal model for studying the molecular action of bromocriptine on prolactinoma.ResultsThe expression of endogenous caveolin-1 in GH3 cells was elevated after bromocriptine treatment. Transiently expressed mouse recombinant caveolin-1 induced apoptosis in GH3 cells by enhancing the activity of caspase 8. Significantly, caveolin-1 induction of GH3 cell apoptosis was sensitized by the administration of bromocriptine. Phosphorylation of caveolin-1 at tyrosine 14 was enhanced after bromocriptine treatment, suggesting that bromocriptine-induced phosphorylation of caveolin-1 may contribute to sensitization of apoptosis in GH3 cells exposed to bromocriptine.ConclusionOur results reveal that caveolin-1 increases sensitivity for apoptosis induction in pituitary adenoma GH3 cells and may contribute to tumor shrinkage after clinical bromocriptine treatment.

Alteration of hormone and neurotransmitter production in cultured cells by high and low frequency electrical stimulation

HFS has become a widely used method in functional neurosurgery. However, its mechanism is not well understood, and its cellular and molecular effects have not yet been investigated. The aim of the study was to understand which cellular events, unrelated to the network organization of cells or neurons, participate in the mechanism of action of HFS. In vitro cellular effects of high (HFS) and low (LFS) frequency electrical stimulation on prolactin secretion in GH3 cell lines (prolactinoma), as well as the catecholaminergic secretion on PC12 cells (pheochromocytoma) were investigated. Cells were cultured in dishes with integrated electrodes to deliver stimulation at the same parameters as those used in clinical conditions to treat advanced forms of Parkinson's disease. Prolactin production was measured in GH3 using a Radio-Immuno-Assay. Dopamine, epinephrine and norepinephrine were measured in PC12 using Enzymo-immuno-assays. HFS for 24 hours reduced prolactin secretion by 40.3%, dopamine by 32.7%, epinephrine by 18.1% (non significant) and norepinephrine by 27.0%. LFS did not induce significant changes. These results suggest that HFS has an inhibitory impact on the cellular machinery responsible for hormone and neurotransmitter production. In this model of isolated cultured cells, network interactions and particularly presynaptic actions are discarded. HFS has inhibitory effects on cellular mechanisms responsible for the production and release of molecules participating in intercellular communication. This HFS-induced inhibition might participate in the lesion-like effect of therapeutic HFS in the basal ganglia during various movement disorders.

Modulation of VEGF/Flk-1 receptor expression in the rat pituitary GH3 cell line by growth factors

Both vascular endothelial growth factor (VEGF) and its receptor Flk-1 are expressed in normal pituitary cells and in the prolactin- and growth hormone-producing GH3 cell line of the rat, thus suggesting autocrine/paracrine function. Regulation of the Flk-1 receptor system in pituitary cells is poorly understood, but evidence suggests that up-regulated growth factors play a role in its expression and activation. To study the role of growth factors in this process, we examined changes in VEGF and Flk-1 expression in GH3 cells following varied exposure to betaFGF, EGF, and TGFbeta1. Immunofluorescence labelling and laser scanning cytometry were used to measure changes in VEGF and Flk-1 expression. Results showed that betaFGF, EGF and TGFbeta up-regulated the VEGF/FLK-1 receptor system. Distinct patterns of activation were detected. At 2 hours, EGF and TGFbeta caused no significant changes in VEGF and Flk-1 expression; however, betaFGF up-regulated VEGF expression in 99% of cells but only induced modest changes in Flk-1 overexpression. A similar percentage of cells overexpressed VEGF after 24-hour incubation with betaFGF, but more prominent Flk-1 overexpression was detected. At 24 hours, EGF and TGFbeta1 induced a significant increase in both VEGF and Flk-1 expression. In summary, our findings show that VEGF/Flk-1 expression in pituitary cells may be altered by different growth factors. This may affect angiogenesis and the progression of pituitary tumors.

Adenoviral vectors encoding tumor necrosis factor-α and FasL induce apoptosis of normal and tumoral anterior pituitary cells

Our previous work showed that tumor necrosis factor (TNF)-alpha and FasL induce apoptosis of anterior pituitary cells. To further analyze the effect of these proapoptotic factors, we infected primary cultures from rat anterior pituitary, GH3 and AtT20 cells with first-generation adenoviral vectors encoding TNF-alpha, FasL or, as a control, beta-galactosidase (beta-Gal), under the control of the human cytomegalovirus promoter. Successful expression of the encoded transgenes was determined by immunocytochemistry. Although we observed basal expression of TNF-alpha and FasL in control cultures of anterior pituitary cells, fluorescence-activated cell sorting (FACS) cell cycle analysis showed that the overexpression of TNF-alpha or FasL increases the percentage of hypodiploid lactotropes and somatotropes. Nuclear morphology and TUNEL staining revealed that the cells undergo an apoptotic death process. We detected strong immunoreactivity for TNFR1 and Fas in the somatolactotrope cell line GH3. TNF-alpha, but not FasL, was expressed in control cultures of GH3 cells. The infection of GH3 cells with adenovirus encoding TNF-alpha or FasL increased the percentages of hypodiploid and TUNEL-positive cells. TNF-alpha or FasL immunoreactivity was not observed in the corticotrope cell line AtT20. However, adenovirus encoding TNF-alpha or FasL efficiently transduced these cells and increased the percentages of hypodiploid and TUNEL-positive cells. The expression of beta-Gal was detected in all these cultures but did not affect cell viability. In conclusion, these results suggest that death signaling cascades triggered by TNF receptor 1 (TNFR1) and Fas are present in both normal and tumoral pituitary cells. Therefore, overexpression of proapoptotic factors could be a useful tool in the therapy of pituitary adenomas.