Gonadotropin-Releasing Hormone Induction of Extracellular-Signal Regulated Kinase Is Blocked by Inhibition of Calmodulin

Abstract Our previous studies demonstrate that GnRH-induced ERK activation required influx of extracellular Ca2+ in αT3-1 and rat pituitary cells. In the present studies, we examined the hypothesis that calmodulin (Cam) plays a fundamental role in mediating the effects of Ca2+ on ERK activation. Cam inhibition using W7 was sufficient to block GnRH-induced reporter gene activity for the c-Fos, murine glycoprotein hormone α-subunit, and MAPK phosphatase (MKP)-2 promoters, all shown to require ERK activation. Inhibition of Cam (using a dominant negative) was sufficient to block GnRH-induced ERK but not c-Jun N-terminal kinase activity activation. The Cam-dependent protein kinase (CamK) II inhibitor KN62 did not recapitulate these findings. GnRH-induced phosphorylation of MAPK/ERK kinase 1 and c-Raf kinase was blocked by Cam inhibition, whereas activity of phospholipase C was unaffected, suggesting that Ca2+/Cam modulation of the ERK cascade potentially at the level of c-Raf kinase. Enrichment of Cam-interacting proteins using a Cam agarose column revealed that c-Raf kinase forms a complex with Cam. Reconstitution studies reveal that recombinant c-Raf kinase can associate directly with Cam in a Ca2+-dependent manner and this interaction is reduced in vitro by addition of W7. Cam was localized in lipid rafts consistent with the formation of a Ca2+-sensitive signaling platform including the GnRH receptor and c-Raf kinase. These data support the conclusion that Cam may have a critical role as a Ca2+ sensor in specifically linking Ca2+ flux with ERK activation within the GnRH signaling pathway.

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Glucocorticoids Induce Human Glycoprotein Hormone α-Subunit Gene Expression in the Gonadotrope

Endocrinology ◽

10.1210/en.2007-1100 ◽

2008 ◽

Vol 149 (7) ◽

pp. 3643-3655 ◽

Cited By ~ 8

Author(s):

Ravid Sasson ◽

Sang H. Luu ◽

Varykina G. Thackray ◽

Pamela L. Mellon

Keyword(s):

Gene Expression ◽

Dominant Negative ◽

Dependent Manner ◽

Glycoprotein Hormone ◽

Specific Expression ◽

Response Element ◽

Α Subunit ◽

Placental Cells

The human glycoprotein hormone α-subunit (αGSU) gene is transcriptionally regulated by glucocorticoids in a cell type-specific fashion. In direct contrast to repression of αGSU by glucocorticoids in placenta, glucocorticoid receptor (GR) modulation in the pituitary is little understood. We show that glucocorticoids stimulate the αGSU promoter in immortalized pituitary gonadotrope-derived LβT2 cells, whereas estrogens, androgens, and progestins have no significant effect. Moreover, GR acts in a dose-dependent manner at physiological concentrations of glucocorticoids. Transient transfection of GR with dexamethasone (Dex) treatment further stimulates the αGSU promoter, but this induction is severely diminished using a receptor mutated in the DNA-binding domain. Truncation and cis mutations demonstrate that glucocorticoid response element 2 (GRE2) and cAMP-response element 2 (CRE2) within −168 bp of the human αGSU promoter are critical for induction. Moreover, dominant-negative CRE-binding protein markedly inhibits basal but also Dex induction of αGSU promoter activity. Additionally, GR specifically binds to GRE2 in the human αGSU promoter in vitro and to the 5′ region of the endogenous mouse αGSU gene in vivo. Furthermore, overexpression of the homeobox factor, Distal-less 3 that regulates this gene in placental cells through a site partially overlapping GRE2, blocks Dex induction of αGSU in gonadotrope cells, indicating that placenta-specific expression of Dlx3 may interfere with GR, resulting in repression in placental cells vs. induction in gonadotrope cells. These results demonstrate the stimulatory role played by glucocorticoids in αGSU gene expression in the pituitary gonadotrope, in contrast to repression in placental cells, and highlight the tissue-specific nature of steroid hormone action.

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In vitro evidence of a role for inhibin in female rabbits

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1984.246.3.e243 ◽

1984 ◽

Vol 246 (3) ◽

pp. E243-E248

Author(s):

A. L. Goodman

Keyword(s):

Granulosa Cells ◽

Pituitary Cells ◽

Dependent Manner ◽

C Cells ◽

Rat Pituitary ◽

Reference Preparation ◽

Lh Release ◽

Rat Pituitary Cells ◽

I Cells

To examine a regulatory role for inhibin in female rabbits, an in vitro bioassay for inhibin activity was modified to use cultured rabbit pituitary cells and charcoal-extracted porcine follicular fluid (pFFx) as a reference preparation. pFFx inhibited follicle-stimulating hormone (FSH) release in a dose-dependent manner in cultures from both intact (I) and castrate (C) does at doses that also inhibited FSH release by cultured rat pituitary cells. Basal FSH release by I cells was inhibited greater than 10% by 0.02% (vol/vol) and greater than 90% by greater than or equal to 0.2% pFFx, whereas in C cells maximal inhibition of FSH release plateaued at only approximately 75%. FSH secretion was restored after removal of pFFx in day 2 media. Luteinizing hormone (LH) release by C cells was not inhibited at any dose of pFFx, but in I cells LH was progressively inhibited to approximately 60% of control levels during day 2 (but not day 1). Charcoal-extracted media (0.25-1%) in which 5 X 10(5) rabbit granulosa cells had been earlier cultured for 72 h produced a parallel inhibition of FSH release. The present findings demonstrate that 1) rabbit pituitary cells are responsive to inhibin, i.e., pFFx preferentially inhibited FSH secretion in a direct, graded, and reversible manner and 2) rabbit follicular granulosa cells secrete an inhibin-like substance.

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Direct Stimulatory Effects of Oxytocin in Female Rat Gonadotrophs and Somatotrophs In Vitro: Comparison With Lactotrophs

Endocrinology ◽

10.1210/en.2014-1543 ◽

2014 ◽

Vol 156 (2) ◽

pp. 600-612 ◽

Cited By ~ 17

Author(s):

Arturo E. Gonzalez-Iglesias ◽

Patrick A. Fletcher ◽

José A. Arias-Cristancho ◽

Ruth Cristancho-Gordo ◽

Cleyde V. Helena ◽

...

Keyword(s):

Receptor Antagonist ◽

Cell Types ◽

Pituitary Hormones ◽

Prolactin Secretion ◽

Female Rats ◽

Pituitary Cells ◽

Female Rat ◽

Dependent Manner ◽

Rat Pituitary Cells

The peptide oxytocin (OT) is secreted by hypothalamic neurons and exerts numerous actions related to reproduction. OT stimulation of prolactin secretion in female rats is important during the estrous cycle, pregnancy, and lactation. Here we report that OT also stimulates transients of intracellular Ca2+ concentration in somatotrophs and gonadotrophs as well as the release of GH and LH in a dose-dependent manner with EC50 values that closely correspond to the ligand affinity of the OT receptor (OTR). Remarkably, the hormone-releasing effect of OT in these two cell types is 2 orders of magnitude more sensitive than that in lactotrophs. The specific OTR agonist [Thr4,Gly7]-oxytocin acutely stimulated the release of LH, GH, and prolactin from female rat pituitary cells in primary culture and increased intracellular Ca2+ concentration in gonadotrophs, somatotrophs, and lactotrophs. In these three cell types, the effects on hormone release and intracellular Ca2+ of both OT and [Thr4,Gly7]oxytocin were abolished by the specific OT receptor antagonist desGly-NH2-d(CH2)5[D-Tyr2,Thr4]OVT but not by the highly selective vasopressin V1a receptor antagonist, d(CH2)5[Tyr(Me)2,Dab5]AVP. Furthermore, 10 nM arginine vasopressin stimulated LH and GH release comparably with a dose of OT that was at least 10 times lower. Finally, the presence of the OTR-like immunoreactivity could be observed in all three cell types. Taken together, these results show that OT directly stimulates gonadotrophs, somatotrophs, and lactotrophs through OT receptors and suggest that OT signaling may serve to coordinate the release of different pituitary hormones during specific physiological conditions.

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Effects of crude and highly purified bovine inhibin (Mr 32 000 form) on gonadotrophin production by ovine pituitary cells in vitro: inhibin enhances gonadotrophin-releasing hormone-induced release of LH

Journal of Endocrinology ◽

10.1677/joe.0.1270149 ◽

1990 ◽

Vol 127 (1) ◽

pp. 149-159 ◽

Cited By ~ 11

Author(s):

S. Muttukrishna ◽

P. G. Knight

Keyword(s):

Primary Cultures ◽

Suppressive Effect ◽

Pituitary Cells ◽

Dependent Manner ◽

Α Subunit ◽

Releasing Hormone ◽

Lh Release ◽

Gonadotrophin Releasing Hormone

ABSTRACT Primary cultures of ovine pituitary cells (from adult ewes) were used to investigate the actions of steroid-free bovine follicular fluid (bFF) and highly-purified Mr 32 000 bovine inhibin on basal and gonadotrophin-releasing hormone (GnRH)-induced release of FSH and LH. Residual cellular contents of each hormone were also determined allowing total gonadotrophin content/well to be calculated. As in rats, both crude and highly purified inhibin preparations promoted a dose (P < 0·001)- and time (P < 0·001)-dependent suppression of basal and GnRH-induced release of FSH as well as an inhibition of FSH synthesis, reflected by a fall in total FSH content/well. However, while neither inhibin preparation affected basal release of LH or total LH content/well, GnRH-induced LH release was significantly (P< 0·001) increased by the presence of either bFF (+ 75%) or highly-purified inhibin (+ 64%) in a dose- and time-dependent manner. This unexpected action of bFF on GnRH-induced LH release was abolished in the presence of 5 μl specific anti-inhibin serum, confirming that the response was indeed mediated by inhibin. Furthermore, neither oestradiol-17β (1 pmol/l–10 nmol/l) nor monomeric α-subunit of bovine inhibin (2·5–40 ng/ml) significantly affected basal or GnRH-induced release of LH. These in-vitro findings for the ewe lend support to a number of recent in-vivo observations and indicate that, in addition to its well-documented suppressive effect on the synthesis and secretion of FSH, inhibin may actually facilitate LH release in this species, in marked contrast to its action in the rat. Journal of Endocrinology (1990) 127, 149–159

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The effects of GH-releasing peptide-6 (GHRP-6) and GHRP-2 on intracellular adenosine 3′,5′-monophosphate (cAMP) levels and GH secretion in ovine and rat somatotrophs

Journal of Endocrinology ◽

10.1677/joe.0.1480197 ◽

1996 ◽

Vol 148 (2) ◽

pp. 197-205 ◽

Cited By ~ 33

Author(s):

D Wu ◽

C Chen ◽

J Zhang ◽

C Y Bowers ◽

I J Clarke

Keyword(s):

Additive Effect ◽

Intracellular Camp ◽

Pituitary Cells ◽

Dependent Manner ◽

Camp Accumulation ◽

Gh Secretion ◽

Rat Pituitary ◽

Rat Pituitary Cells ◽

Camp Levels

Abstract The mechanism of action of GH-releasing peptide-6 (GHRP-6) and GHRP-2 on GH release was investigated in ovine and rat pituitary cells in vitro. In partially purified sheep somatotrophs, GHRP-2 and GH-releasing factor (GRF) increased intracellular cyclic AMP (cAMP) concentrations and caused GH release in a dose-dependent manner; GHRP-6 did not increase cAMP levels. An additive effect of maximal doses of GRF and GHRP-2 was observed in both cAMP and GH levels whereas combined GHRP-6 and GHRP-2 at maximal doses produced an additive effect on GH release only. Pretreatment of the cells with MDL 12,330A, an adenylyl cyclase inhibitor, prevented cAMP accumulation and the subsequent release of GH that was caused by either GHRP-2 or GRF. The cAMP antagonist, Rp-cAMP also blocked GH release in response to GHRP-2 and GRF. The cAMP antagonist did not prevent the effect of GHRP-6 on GH secretion whereas MDL 12,330A partially reduced the effect. An antagonist for the GRF receptor, [Ac-Tyr1,d-Arg2]-GRF 1–29, significantly diminished the effect of GHRP-2 and GRF on cAMP accumulation and GH release, but did not affect GH release induced by GHRP-6. Somatostatin prevented cAMP accumulation and GH release responses to GHRP-2, GRF and GHRP-6. Ca2+ channel blockade did not affect the cAMP increase in response to GHRP-2 or GRF but totally prevented GH release in response to GHRP-2, GRF and GHRP-6. These results indicated that GHRP-2 acts on ovine pituitary somatotrophs to increase cAMP concentration in a manner similar to that of GRF; this occurs even during the blockade of Ca2+ influx. GHRP-6 caused GH release without an increase in intracellular cAMP levels. GH release in response to all three secretagogues was reduced by somatostatin and was dependent upon the influx of extracellular Ca2+. The additive effect of GHRP-2 and GRF or GHRP-6 suggested that the three peptides may act on different receptors. In rat pituitary cell cultures, GHRP-6 had no effect on cAMP levels, but potentiated the effect of GRF on cAMP accumulation. The synergistic effect of GRF and GHRP-6 on cAMP accumulation did not occur in sheep somatotrophs. Whereas GHRP-2 caused cAMP accumulation in sheep somatotrophs, it did not do so in rat pituitary cells. These data indicate species differences in the response of pituitary somatotrophs to the GHRPs and this is probably due to different subtypes of GHRP receptor in rat or sheep. Journal of Endocrinology (1996) 148, 197–205

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The Biological and Immunological Characterization of Inhibin A and B Forms in Human Follicular Fluid and Plasma1

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jcem.82.3.3801 ◽

1997 ◽

Vol 82 (3) ◽

pp. 889-896 ◽

Cited By ~ 2

Author(s):

D. M. Robertson ◽

N. Cahir ◽

J. K. Findlay ◽

H. G. Burger ◽

N. Groome

Keyword(s):

Molecular Weight ◽

Inhibin B ◽

Pituitary Cells ◽

In Vitro Bioactivity ◽

Serum Samples ◽

Human Follicular Fluid ◽

Inhibin A ◽

Α Subunit ◽

Rat Pituitary Cells

Abstract In a previous study (see Ref. 7), the molecular weight distribution of inhibin activity in fractionated human follicular fluid (hFF) and human male and female plasma/serum was determined by in vitro bioassay using ovine pituitary cells in culture and various specific inhibin A and inhibin α-subunit-directed immunoassays. It was shown, however, that the ovine in vitro bioassay detected inhibin B poorly. These findings are extended in the present study by the determination of the molecular weight profile of in vitro bioactivity using rat pituitary cells, which detects both inhibin A and B, a specific inhibin B enzyme-linked immunosorbent assay (ELISA), an RIA detecting the αN region of the α-subunit, anα -subunit ELISA (Pro-αC) directed to the inhibin forms containing the Pro sequence, and an αC subunit immunofluorometric assay that detects all inhibin forms. The profile in hFF of inhibin in vitro bioactivity, using rat pituitary cells in culture, significantly (P < 0.001) correlated with in vitro bioactivity using ovine pituitary cells (r= 0.85), inhibin A immunoactivity (r = 0.70), inhibin B immunoactivity (r = 0.89), and the combination of inhibin A+B immunoactivities (r = 0.93), with peaks of activity identified at 66K, 55K, 36K and 33K, consistent with presumed known mol wt forms of inhibin. Inhibin B profiles in fractionated serum from women stimulated with gonadotropins and male plasma consisted of two forms (66K and 36K), whereas inhibin A in female serum included, in addition, the 55K form. These findings indicated that higher molecular weight forms of inhibin B are present in biological samples, and their distribution differs from that of inhibin A, suggesting a differential processing of the precursor forms in the circulation. Pro-αC immunoactivity was identified in serum samples with prominent peaks at 36K and 29K (known Pro-αC subunit forms) and not with any high mol wt dimeric forms of inhibin. If this observation applies to a wider range of serum samples, the Pro-αC ELISA may provide an appropriate and specific assay for the measurement of free α-subunit. To compare immunoactivity levels between assays, the inhibins A, B, and Pro-αC standards were calibrated in terms of their αC subunit content, as determined by anα C subunit immunoassay, with the inhibin B standard containing 60% of the αC subunit content compared with either the inhibin A or Pro-αC standard. After adjustments of the various standards for this difference in αC subunit content, a comparison was undertaken of the combined levels of inhibins A, B, and Pro-αC immunoactivity across the hFF and serum chromatograms and compared with levels determined by the α-subunit-directed immunoassays. A high correlation (r = 0.59–0.96) was observed, indicating that the α-subunit immunoactivity in serum consists largely of a composite of presumed known molecular weight forms of inhibins A, B, and Pro-αC. It is concluded that: 1) inhibin in vitro bioactivity in hFF is largely attributed to the presence of 33–36K and 50–66K forms of inhibins A and B; and 2) inhibin α-subunit immunoactivity in hFF and serum is a composite of presumed known forms of inhibin A, inhibin B, and the α-subunit.

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Inhibin A and B in Vitro Bioactivities Are Modified by Their Degree of Glycosylation and Their Affinities to Betaglycan

Endocrinology ◽

10.1210/en.2006-1612 ◽

2007 ◽

Vol 148 (5) ◽

pp. 2309-2316 ◽

Cited By ~ 36

Author(s):

Yogeshwar Makanji ◽

Craig A. Harrison ◽

Peter G. Stanton ◽

Radha Krishna ◽

David M. Robertson

Keyword(s):

Reversed Phase ◽

Normal Function ◽

Inhibin B ◽

Pituitary Cells ◽

In Vitro Bioactivity ◽

Inhibin A ◽

Α Subunit ◽

Reproductive Axis ◽

Rat Pituitary Cells

Inhibin A and B, important regulators of normal function in tissues of the reproductive axis, are glycosylated at either Asn268 or Asn268 and Asn302 in the α-subunit to produce 31- and 34-kDa isoforms, respectively. In this study, glycosylated isoforms of recombinant human inhibin A and B were purified from conditioned medium using immunoaffinity chromatography and reversed-phase HPLC. The masses of the purified inhibin preparations were determined by several inhibin immunoassays, and their in vitro bioactivities were based on suppression of FSH release by rat pituitary cells in culture. Based on a ratio of in vitro bioactivity to immunoactivity (B:I ratio), the monoglycosylated 31-kDa inhibin A was 5-fold more potent than the diglycosylated 34-kDa inhibin A (B:I ratio, 1.22 ± 0.15 vs. 0.24 ± 0.05; P < 0.001, respectively). The 31-kDa inhibin B was significantly (P < 0.001) more potent (1.75 ± 0.29) than the 34-kDa form (1.08 ± 0.20). Because inhibin biological activity is dependent upon interactions with the coreceptor betaglycan, the effect of inhibin glycosylation on betaglycan binding was assessed. Analogous to the pattern of in vitro bioactivity, 31-kDa inhibin A was 12-fold more active (IC50, 0.68 nm) than the 34-kDa isoform (IC50, 8.2 nm) at displacing [125I]inhibin A from COS7 cells expressing betaglycan. However, the 1.6-fold difference in bioactivity of the inhibin B isoforms was not matched by differences in their affinities for betaglycan. It is concluded that glycosylation of Asn302 of the α-subunit of inhibin A and B results in a decrease in bioactivity, and the effect on inhibin A, at least, is explained by its reduced affinity to betaglycan.

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Lentiviral vectors efficiently transduce human gonadotroph and somatotroph adenomas in vitro. Targeted expression of transgene by pituitary hormone promoters

Journal of Endocrinology ◽

10.1677/joe.1.05759 ◽

2004 ◽

Vol 183 (1) ◽

pp. 217-233 ◽

Cited By ~ 8

Author(s):

Catherine Roche ◽

Alfredo J Zamora ◽

David Taïeb ◽

Esteban Lavaque ◽

Ramahefarizo Rasolonjanahary ◽

...

Keyword(s):

Gene Transfer ◽

Promoter Activity ◽

Lentiviral Vectors ◽

Pituitary Cells ◽

Glycoprotein Hormone ◽

Human Pituitary ◽

Α Subunit ◽

Efficient Treatment ◽

Fluorescent Cells

Despite important advances in human therapeutics, no specific treatment for both non-functioning gonadotroph and resistant somatotroph adenomas is available. Gene transfer by viral vectors can be considered as a promising way to achieve a specific and efficient treatment. Here we show the possibility of efficient gene transfer in human pituitary adenoma cells in vitro using a human immunodeficiency virus (HIV)-type 1-derived vector. Using enhanced green fluorescent protein (eGFP) gene as a marker placed under the phosphoglycerate kinase (PGK) promoter, gonadotroph and somatotroph adenomas were transduced even with moderate viral loads. The expression started at day 2, reached a peak at day 5, and it was still present at day 90. For targeting somatotroph and gonadotroph adenomas, human growth hormone (GH) promoter (GH −481, +54 bp) and two fragments of the human glycoprotein hormone α-subunit promoter (α-subunit 1 −520, +33 bp, and α-subunit 2 −907, +33 bp) were tested. In gonadotroph adenomas, the percentage of identified fluorescent cells and the fluorescence intensity analyzed by fluorescence-activated cell sorting indicated that the strength of the α-subunit 1 and α-subunit 2 promoters were comparable to that of the PGK promoter. Primary cultures of rat pituitary cells showed that α-subunit 1 is more selective to thyreotroph and gonadotroph phenotypes than α-subunit 2. GH promoter activity appeared weak in somatotroph adenomas. The human GH enhancer did not increase the GH promoter activity at all but the human prolactin promoter (−250 bp) allowed 4-fold more fluorescent cells to be obtained than the GH promoter. Several cell lines appeared too permissive to test cell-specificity of pituitary promoters. However, on human non-pituitary cell cultures, the tested pituitary promoters seemed clearly selective to target endocrine pituitary phenotypes. This study gives a starting point for a gene-therapy program using lentiviral vectors to transfer therapeutic genes in human pituitary adenomas.

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Stimulation of Combinatorial Expression of Prolactin and Glycoprotein Hormone α-Subunit Genes by Gonadotropin-Releasing Hormone and Estradiol-17β in Single Rat Pituitary Cells during Aggregate Cell Culture

Endocrinology ◽

10.1210/en.2002-220606 ◽

2003 ◽

Vol 144 (1) ◽

pp. 388-399 ◽

Cited By ~ 14

Author(s):

A. Hauspie ◽

E. Seuntjens ◽

H. Vankelecom ◽

C. Denef

Keyword(s):

Cell Culture ◽

Single Cells ◽

Pituitary Hormone ◽

Pituitary Cells ◽

Glycoprotein Hormone ◽

Α Subunit ◽

Regulatory Factors ◽

Fluo Rescence ◽

Rat Pituitary Cells ◽

Combinatorial Expression

Abstract Previously we showed the existence of rat and mouse anterior pituitary cells coexpressing mRNA from two or more hormone genes in which production and/or storage of the corresponding hormones were not detectable. To substantiate a putative function for these cells, we investigated whether these phenotypes were retained during long-term reaggregate cell culture and whether protagonist regulatory factors could expand cell populations expressing particular hormone mRNA combinations. After 4-wk culture and treatments, aggregates were trypsinized and single cells collected by means of a fluo-rescence-activated cell sorter. Hormone mRNAs were detected by single-cell RT-PCR. Combinatorial hormone mRNA expression was retained in culture. Both estradiol (E2) and GnRH (1 nm) markedly augmented the proportion of cells expressing prolactin (PRL) mRNA together with other hormone mRNAs and cells expressing glycoprotein subunit (GSU)-α mRNA together with other hormone mRNAs. GnRH strongly increased the proportion of cells containing αGSU mRNA alone, but E2 did not. GnRH and (E2) affected the expansion of a population (∼20% of all cells) coexpressing PRL and αGSU mRNA without βGSUs. Immunostaining of stored hormone on tissue sections revealed colocalization of PRL and αGSU in the E2- but not in the GnRH-treated cells. The present findings suggest that cells coexpressing different pituitary hormone mRNAs form a distinct population that survives without extrapituitary factors. Their occurrence can be markedly modified by regulatory factors. Certain hormone regimens favor unique coexpressions distinctly at mRNA and protein level. These peculiar characteristics support the notion that combinatorial expression of hormone genes in the pituitary serves a biological role.

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