alphaT3-1 Cells Research Articles

Nonclassical actions of estradiol-17beta are not detectable in the alphaT3-1 and LbetaT2 immortalized gonadotrope cell lines†.

The immortalized mouse gonadotrope cell lines alphaT3-1 and LbetaT2 cells have been a substitute model for primary gonadotropes. These cell lines have provided a homogeneous cell population, as compared to the dissociated anterior pituitaries, which contain a heterogeneous population of cells potentially responsive to estradiol-17beta (E2). Nonclassical actions of E2 assumed to occur through the plasma membrane estrogen receptor 1 (ESR1, also known as ERalpha). These actions have included inhibition of gonadotropin-releasing hormone (GnRH)-induced increases in intracellular calcium concentrations and phosphorylation of p44/42 mitogen-activated protein kinase (ERK-1/2) in ovine pituitaries including primary gonadotropes in vitro. The objective of the present experiment was to determine if alphaT3-1 and LbetaT2 are cell models with limitations to examine the nonclassical actions of E2 occurring in gonadotropes. Experiments were conducted to determine if the cells have ESR1 at the plasma membrane using biotinylation cell and isolation of surface protein and staining with a fluorescently labeled E2 conjugate. The alphaT3-1 cells contain ESR1 associated with but not enriched within lipid rafts of the plasma membrane and do not translocate to lipid rafts upon binding of E2. In contrast, LbetaT2 cells lack ESR1 associated with the plasma membrane. Pretreatment with E2 did not cause inhibition of GnRH-stimulated increases in intracellular concentrations of calcium for either cell type. Phosphorylation of ERK-1/2 was not stimulated by E2 in either cell type. Although these cells lines have been used extensively to study GnRH signaling, in vitro or in vivo effects of nonclassical actions of E2 cannot be replicated in either cell line.

Activity of the porcine gonadotropin-releasing hormone receptor gene promoter is partially conferred by a distal gonadotrope specific element (GSE) within an upstream enhancing region, two proximal GSEs and a retinoid X receptor binding site.

BackgroundRegulation of gonadotropin-releasing hormone (GnRH) receptor (GnRHR) numbers on gonadotropes within the anterior pituitary gland represents a critical point for control of reproductive function. Binding of GnRH to its receptor regulates follicle stimulating hormone (FSH) and luteinizing hormone (LH) release and levels of this G-protein coupled receptor on the surface of gonadotropes determines their sensitivity to GnRH pulses. While transcriptional regulation of this gene has been studied in mice, rats, humans and sheep, little is known about its regulation in the pig, an important agricultural species and human research model.MethodsWe isolated 5118 bp of 5′ flanking sequence for the porcine GnRHR gene and generated luciferase reporter vectors. Deletion and mutation constructs were evaluated in gonadotrope-derived alphaT3-1 cells to determine regions important for gene transcription. Additionally, electrophoretic mobility shift assays (EMSAs) were performed to identify transcription factors binding to the GnRHR promoter.ResultsTransient transfections revealed that the GnRHR promoter was functional in alphaT3-1 cells but not in cells of non-gonadotrope origin. Mutation of the highly conserved gonadotrope specific element (GSE) located at -179/-171 of proximal promoter completely ablated luciferase activity, whereas mutation of another GSE at -315/-310 reduced activity by 34%. Consistent with this, EMSAs using alphaT3-1 nuclear extracts and a steroidogenic factor (SF)1 antibody confirmed SF1 binding to both GSEs. EMSAs also demonstrated that a retinoid X receptor (RXR) binding site at -279/-274 binds RXRalpha and RXRbeta and mutation of this site eliminated promoter activity. Transient transfection of alphaT3-1 cells with reporter vectors containing selective removal of 5′ flanking region for the porcine GnRHR gene indicated that the -1915/-1431 segment was important for promoter activity. Definition of this region via transfection assays and EMSAs revealed an upstream enhancing region located at -1779/-1667 that increases porcine GnRHR gene expression in alphaT3-1 cells and includes a SF1 binding site at -1760/-1753.ConclusionsPorcine GnRHR promoter activity in alphaT3-1 cells is partially conferred by a distal GSE, two proximal GSEs and a RXR binding site. Basal gonadotrope expression of the porcine GnRHR gene uniquely involves three GSEs and RXR is newly identified as a regulator of GnRHR promoter activity.

Cold-Shock-Domain Protein A (CSDA) Contributes Posttranscriptionally to Gonadotropin-Releasing Hormone-Regulated Expression of Egr1 and Indirectly to Lhb1

Gonadotropin-releasing hormone (GnRH), a hypothalamic neurohormone, regulates transcription of Lhb in gonadotrophs indirectly through transient induction and accumulation of EGR1, a zinc finger transcription factor. AlphaT3 and LbetaT2 cell lines model gonadotrophs at two distinct stages of development, prenatal and postnatal expression of Lhb. Although GnRH induces EGR1 in both cell lines, the levels of the DNA-binding protein are lower and disappear more quickly in alphaT3 than in LbetaT2 cells. Herein we show that overexpression of Egr1 in alphaT3 cells rescues activity of a transfected LHB promoter-reporter, suggesting that its transcription is dependent on EGR1 crossing a critical concentration threshold. We also show that Csda, a gene that encodes an RNA-binding protein and is a member of the cold-shock-domain (CSD) family, is expressed at higher levels in LbetaT2 compared to alphaT3 cells. Transient expression studies indicate that at least one Csd element, residing in the 3' untranslated region of Egr1 mRNA, increases activity of a chimeric pGL3 luciferase reporter vector in LbetaT2 cells. Additional experiments indicate that CSDA physically interacts with Egr1 mRNA. Furthermore, siRNA-mediated reduction of endogenous Csda mRNA attenuates GnRH regulation of a transiently transfected LHB reporter vector. Taken together, these studies suggest that CSDA contributes posttranscriptionally to GnRH-regulated expression of Egr1, thereby enabling the transcription factor to cross a critical concentration threshold necessary for maximal accumulation of Lhb mRNA in response to the neurohormone.

Poly [ADP-Ribose] Polymerase-1 (PARP-1) Confers Glucocorticoid Responsiveness of the Porcine GnRH Receptor (GnRHR) Gene.

The binding of GnRH to its receptor results in the synthesis and secretion of the gonadotropins, as well as stimulation of the gene encoding its own receptor. Thus, the interaction between GnRH and GnRHR represents a central point for regulation of reproductive function. Glucocorticoids can alter reproduction by reducing GnRH responsiveness of gonadotropes within the anterior pituitary gland, potentially via transcriptional regulation of the GnRHR gene. Investigation of this possible mechanism, however, revealed that transcription of the murine GnRHR gene is stimulated by glucocorticoids. To determine the effect of glucocorticoids on porcine GnRHR gene expression, gonadotrope-derived alphaT3-1 cells were transiently transfected with a vector containing 5118 bp of 5' flanking sequence for the porcine GnRHR gene fused to luciferase for 12 h and treated with increasing concentrations of the glucocorticoid agonist, dexamethasone (0, 1, 10, 100 and 1,000 nM) for an additional 12 h prior to harvest. Maximal induction of luciferase activity was detected at 100 nM of dexamethasone (2-fold over vehicle; P < 0.05), and this response was blocked by the glucocorticoid antagonist, mifepristone (100 pM). Deletion from 274 to 323 bp upstream of the translational start site eliminated glucocorticoid responsiveness, suggesting the presence of a GRE(s) within this region. Electrophoretic mobility shift assays (EMSA) using a 32P-labeled oligonucleotide spanning -290/-270 bp of proximal promoter revealed increased binding of nuclear extracts from alphaT3-1 cells treated with 100 nM dexamethasone compared to vehicle. Sequence analysis of this region indicated putative binding sites for PR, ER, GR, COUP-TF and GATA, as well as RXR alpha, beta, and gamma. However, competitive oligonucleotides for each of these transcription factors, including GR, were unable to compete for binding to the radiolabeled -290/-270 bp probe in EMSAs. Mass spectrometry analysis of isolated proteins from a pulldown using a biotinylated oligonucleotide (-290/-270 bp) indentified PARP-1 as the key binding partner. To confirm the mass spectrometry result, we performed EMSAs with antibodies specific for either GR (sc-1004X; Santa Cruz Biotechnology, Santa Cruz, CA) or PARP-1 (614301; BioLegend, San Diego, CA). Both the GR and PARP-1 antibodies generated a supershift of the specific binding complex. Interestingly, addition of the PARP-1 and GR antibodies together abolished the supershift. Since the competitive oligonucleotide for GR was unable to abrogate the DNA/protein complex whereas inclusion of antibodies confirmed that GR was a member of the specific binding complex, this suggests that GR must recruit PARP-1 in order to bind with the GRE. Inhibition of p38 and ERK1/2 mitogen-activated protein kinase (MAPK) pathways significantly decreased dexamethasone-induced promoter activity (P < 0.05), indicating the involvement of these signaling pathways in glucocorticoid stimulation of the promoter. Thus, our working model for glucocorticoid responsiveness of the porcine GnRHR gene suggests that, upon binding to its receptor, glucocorticoid triggers phosophorylation of GR by p38 and ERK1/2 MAPK pathways, resulting in the recruitment of PARP-1 by phosphorylated, ligand-bound GR to a GRE located within -290/-270 bp of the porcine GnRHR promoter. (platform)

The Non-Classical Actions of Estradiol in Ovine Pituitary Cells Cannot be Mimicked in the Immortalized Mouse Gonadotroph Cell Line, AlphaT3-1 Cells.

The Non-Classical Actions of Estradiol in Ovine Pituitary Cells Cannot be Mimicked in the Immortalized Mouse Gonadotroph Cell Line, AlphaT3-1 Cells.

Multiple core homeodomain binding motifs differentially contribute to transcriptional activity of the murine gonadotropin-releasing hormone receptor gene promoter

Multiple homeodomain (Hbox) proteins have been shown to organize expression of key markers of gonadotropes. Nine putative Hbox-binding sites, characterized by the homeospecific TAAT motif, are located within the proximal 600 bp of the murine GnRHR promoter. Homeoproteins bind separate Hbox sites within this promoter, supporting basal- and endocrine-directed transcription. The function of the most proximal sites (Hbox1 and Hbox2) in the murine GnRHR is unknown; thus, understanding of the global contribution of homeospecific TAAT sites to promoter function is incomplete. Site-directed mutagenesis revealed that loss of Hbox2 reduced promoter activity in a cell-specific manner, having no effect in alphaT3-1 cells but reducing promoter function in LbetaT2 cells, another gonadotrope-derived cell line representing a later developmental stage. In contrast, eliminating Hbox1 reduced basal activity in both lines. This region displayed specific binding to homeoprotein Oct-1. Mutagenesis of a previously identified Oct-1-binding site in concert with Hbox1 led to further reduction in activity. We suggest that the two most proximal homeodomain-binding sites in the murine GnRHR promoter may regulate the promoter in a developmentally dependent fashion and that Oct-1 acts at multiple but distinct TAAT sites to support basal transcription.

FoxL2 and Smad3 Coordinately Regulate Follistatin Gene Transcription

Follistatin is a transcriptional target and a modulator of activin action. Through an autocrine/paracrine loop, activin controls follistatin levels and thus regulates its own bioavailability. In gonadotropic alphaT3-1 cells, activin induces follistatin transcription primarily through the action of Smad3 at an intronic Smad-binding element (SBE1). Using a proteomics approach, we searched for endogenous alphaT3-1 proteins that participate in SBE1-mediated transcription. We identified FoxL2, a member of the forkhead family, as a candidate modulator of SBE1 function. Mutations of FoxL2 are associated with the blepharophimosis/ptosis/epicanthus inversus syndrome characterized with craniofacial defects and premature ovarian failure. FoxL2 localizes to alpha-glycoprotein subunit- and follicle-stimulating hormone beta-positive cells of the adult mouse pituitary and is present in alphaT3-1 and LbetaT2 cells, but its pituitary actions remain largely unknown. We have determined that FoxL2 binds to a forkhead-binding element (FKHB) located just downstream of the SBE1 site of the follistatin gene and functions as a Smad3 partner to drive SBE1-mediated transcription in alphaT3-1 cells treated with activin. Chromatin immunoprecipitation assays confirm that endogenous FoxL2 and Smad3 are recruited to the intronic enhancer of the follistatin gene where the SBE1 and FKHB sites are located. Exogenous FoxL2 enhances SBE1-mediated transcription, and short hairpin RNA-mediated knockdown of endogenous FoxL2 protein compromises this effect in alphaT3-1 cells. FoxL2 directly associates with Smad3 but not Smad2 or Smad4. This association between Smad3 and FoxL2 is mediated by the MH2 domain of Smad3 and is dependent on an intact forkhead domain in FoxL2. Altogether, these observations highlight a novel role for FoxL2 and suggest that it may function as a transcriptional regulator and a coordinator of Smad3 targets.

Analysis of the Calcium-Dependent Regulation of Proline-Rich Tyrosine Kinase 2 by Gonadotropin-Releasing Hormone

Calcium influx through L-type voltage-gated calcium channels (VGCC) is required for ERK activation induced by GnRH in pituitary gonadotropes. The current studies investigate VGCC-sensitive catalytic activities that may lie upstream of ERKs within the GnRH signaling network. Ion exchange fractionation of alphaT3-1 cell lysates subjected to anti-phosphotyrosine Western blot analysis revealed a nifedipine-sensitive activity that colocalized with proline-rich tyrosine kinase (Pyk) 2 immunoreactivity. Phosphorylated Pyk2 was present in alphaT3-1 cells after GnRH agonist administration for a time course that lasted up to 4 h. Pyk2 phosphorylation was also evident in gonadotropes in vivo after administration of a bolus of GnRH. Knockdown of Pyk2 using specific small interfering RNAs revealed that Pyk2 contributed to modulation of GnRH-induced ERK but not c-Jun N-terminal kinase activation. Using pharmacological approaches, calmodulin (Cam) was also demonstrated to be required for the phosphorylation of Pyk2. Pyk2 was shown to bind specifically to a Cam agarose affinity column in a calcium-dependent manner, suggesting Cam and Pyk2 are capable of forming a complex. Specific mutation of a putative Cam binding motif within the catalytic domain of Pyk2 blocked association with Cam and uncoupled Pyk2's ability to activate ERK-dependent gene transcription. Thus, GnRH induces Pyk2 tyrosine phosphorylation dependent upon calcium flux within gonadotropes. Furthermore, association of Pyk2 and Cam may be required to mediate the effects of calcium on Pyk2 phosphorylation and subsequent activation of ERKs by GnRH.

Molecular, pharmacological and functional properties of GABAA receptors in anterior pituitary cells

Anterior pituitary cells express gamma-aminobutyric acid (GABA)-A receptor-channels, but their structure, distribution within the secretory cell types, and nature of action have not been clarified. Here we addressed these questions using cultured anterior pituitary cells from postpubertal female rats and immortalized alphaT3-1 and GH(3) cells. Our results show that mRNAs for all GABA(A) receptor subunits are expressed in pituitary cells and that alpha1/beta1 subunit proteins are present in all secretory cells. In voltage-clamped gramicidin-perforated cells, GABA induced dose-dependent increases in current amplitude that were inhibited by bicuculline and picrotoxin and facilitated by diazepam and zolpidem in a concentration-dependent manner. In intact cells, GABA and the GABA(A) receptor agonist muscimol caused a rapid and transient increase in intracellular calcium, whereas the GABA(B) receptor agonist baclofen was ineffective, suggesting that chloride-mediated depolarization activates voltage-gated calcium channels. Consistent with this finding, RT-PCR analysis indicated high expression of NKCC1, but not KCC2 cation/chloride transporter mRNAs in pituitary cells. Furthermore, the GABA(A) channel reversal potential for chloride ions was positive to the baseline membrane potential in most cells and the activation of ion channels by GABA resulted in depolarization of cells and modulation of spontaneous electrical activity. These results indicate that secretory pituitary cells express functional GABA(A) receptor-channels that are depolarizing.

A Proteomic Comparison of Immature and Mature Mouse Gonadotrophs Reveals Novel Differentially Expressed Nuclear Proteins that Regulate Gonadotropin Gene Transcription and RNA Splicing1

The alphaT3-1 and LbetaT2 gonadotroph cell lines contain all the known factors required for expression of gonadotropin genes, yet only the LbetaT2 cells express the beta subunits. We hypothesized that comparison of their nuclear proteomes would reveal novel proteins and/or modifications that regulate expression of these genes. We identified nine proteins with different expression profiles in the two cell lines, of which several were chosen for further functional studies. Of those found at higher levels in alphaT3-1 nuclei, 1110005A23RIK was found associated with the Fshb gene promoter and repressed its expression. Transgelin 3 overexpression reduced transcript levels of Fshb, and its knockdown elevated Lhb and Cga transcript levels, indicating an ongoing repressive effect on these more highly expressed genes, possibly through altering levels of phosphorylated mitogen-activated protein kinase. Heterogeneous nuclear ribonucleoprotein A2/B1 repressed splicing of the Fshb primary transcript, which it binds in the first intron. Proteins at higher levels in LbetaT2 nuclei included prohibitin, the overexpression of which reduced promoter activity of all three gonadotropin subunits, and appeared to mediate the differential effect of GnRH on proliferation of the two cell lines; its knockdown also altered cell morphology. Two other splicing factors were also found at higher levels in LbetaT2 nuclei: the knockdown of PRPF19 or EIF4A3 decreased splicing of Lhb, or of both beta subunit transcripts, respectively. The levels of Eif4a3 mRNA were increased by activin, and both factors increased Fshb splicing. This study has revealed a number of novel factors that alter gonadotropin expression and gonadotroph function, and likely mediate or moderate effects of the regulatory hormones.

SF-1 Expression Is Mostly Directed by Distal Regulatory Elements.

Steroidogenic factor 1 (SF-1/Ad4BP/NR5A1) is a nuclear hormone receptor that has a pivotal role in endocrine regulation and development of the adrenal glands and gonads. Considering the critical nature of SF-1, many studies have focused on uncovering transcriptional mechanisms that drive SF-1 expression. Despite these efforts, little has been revealed regarding the mechanism that direct SF-1 to its appointed cell types, a result largely due to its requirement for distal regulatory sequences in directing tissue-specific expression. Previous transgenic studies identified a 153kb region, containing Nr5a1, that was sufficient for proper SF-1 expression, which provided an important initiation point for the identification of essential regulatory elements. Comparative genomics through the web-based tool ECR browser (http://ecrbrowser.dcode.org/) was performed and identified within the Nr5a1 locus several evolutionarily conserved non-coding regions called "ECR"s. Four of the most conserved ECRs were examined for transcriptional regulatory activity. The ECRs were cloned into a vector that contained SF-1 promoter sequence from -734 to +60 upstream of a luciferase reporter. Transcriptional activity of each ECR was tested individually using transient transfection analysis in different SF-1-expressing cell types. Our results identified one ECR (ECR3) with activity that increased transcription in alpha T3(gonadotrope) and Y-1(adrenal) cells and decreased it in MA-10(Leydig), Myoid, MSC-1(Sertoli), and primary Sertoli cells. The data indicate that ECR3 contains control elements necessary for SF-1 transcriptional induction in the pituitary and adrenal cells and repression in the testis cells. Mutagenesis and DNA/protein interaction studies identified sequences within ECR3 that were important for activity in alpha T3 cells. One such sequence contains an E-box that bound the class A basic-helix-loop-helix (bHLH) protein E2A, suggesting studies will reveal a class B bHLH, which exhibit tissue-specific expression, as its dimeric partner. This Research was supported by a Center Grant in Reproductive Sciences (U54 HD33994 from NICHD to LLH) and a KUMC Biomedical Research Training Grant to R.K.

β1-Adrenoceptor Expression in Rat Anterior Pituitary Gonadotrophs and in Mouse αT3-1 and LβT2 Gonadotrophic Cell Lines

The rat anterior pituitary expresses beta(2)-adrenoceptors (ARs) on somatotrophs, lactotrophs, and corticotrophs. The present study investigates whether beta(1)-ARs exist in the anterior pituitary, in which cell type(s) they are found, and whether they are regulated by glucocorticoids. As determined by quantitative RT-PCR and Western immunoblotting, the rat anterior pituitary expressed beta(1)-AR mRNA and protein. Unlike the beta(2)-AR, expression decreased to very low levels after 5-d aggregate cell culture but was strongly up-regulated in a dose- and time-dependent manner by dexamethasone (DEX). Glucocorticoids attenuated isoproterenol-induced down-regulation of beta(1)-AR mRNA levels. As examined by immunofluorescence confocal microscopy, beta(1)-AR immunoreactivity was detected in a subpopulation of gonadotrophs, but not in somatotrophs, lactotrophs, corticotrophs, thyrotrophs, or folliculo-stellate cells. beta(1)-AR-immunoreactivity cells were often surrounded by cup-shaped lactotrophs. Consistent with these findings, beta(1)-AR mRNA was considerably more abundant in the gonadotrophic alphaT3-1 and LbetaT2 cell lines than in the GHFT, GH3, and TtT/GF cell lines. DEX did not affect expression level in the cell lines. DEX also failed to up-regulate beta(1)-AR mRNA levels in aggregates from a subpopulation enriched in large gonadotrophs obtained by gradient sedimentation. In contrast, excessive DEX-dependent up-regulation of beta(1)-AR mRNA was found in a subpopulation enriched in small nonhormonal cells. The present data indicate that beta(1)-AR is expressed in a subpopulation of gonadotrophs with a topographical relationship to lactotrophs. However, the glucocorticoid-induced up-regulation does not seem to occur directly in the gonadotrophs but within (an)other unidentified cell type(s), or is transduced by that cell type on gonadotrophs.

Single-Stranded DNA-Binding Proteins (SSBPs) Regulate the Abundance of the LIM-Homeodomain Protein LHX2 and Augment Its Transcriptional Activity.

A small family of proteins with putative single-stranded DNA-binding activity has been shown to augment the biological actions of LIM-homeodomain (LIM-HD) transcription factors through the mediation of the LIM domain-binding protein LDB1. We recently established that two of these SSBPs, Ssbp2 and Ssbp3, were components of an E-box-GATA DNA-binding complex in murine erythroid progenitors containing transcription factors Tal1, E2A, and Gata-1 and LIM-only protein Lmo2 and showed that Ssbp2 stimulated E box-GATA DNA-binding activity by inhibiting Ldb1 ubiquitination and Ldb1 and Lmo2 turnover (Genes & Dev. 21:942–955, 2007). Since LIM-HD proteins are substrates of different E3 ubiquitin ligases than LIM-only proteins and have the additional property of binding DNA, we sought to determine the effect of SSBPs on LIM-HD expression and function. Using the prototype LIM-HD protein Lhx2 and one of its best-characterized target genes, Cga, for analysis, we found that an Ssbp3-, Ldb1-, and Lhx2-containing complex associated with an Lhx2 binding element in the Cga promoter in vitro and in mouse pituitary cells (alphaT3-1 cell line) in vivo. We then showed that enforced expression of Ssbp2 and Ssbp3 in alphaT3-1 cells increased Lhx2 and Ldb1 protein abundance, Lhx2 DNA-binding activity, and Cga expression and augmented Lhx2 transcriptional activity in an Ldb1-dependent fashion. While Lhx2-Ldb1-Ssbp3 DNA-binding activity increased in Ssbp3- relative to vector-transfected cells, the affinity of this complex for DNA was unaltered. Similar to the effect of Ssbp2 on Lmo2 in murine erythroleukemia (MEL) cells, overexpressed Ssbp3 reduced Lhx2 protein turnover in cycloheximide-treated alphaT3-1 cells without affecting Lhx2 RNA levels. In contrast, knockdown of endogenous Ssbp3, but not Ssbp2 which is expressed at much lower levels in these cells, reduced Lhx2 and Ldb1 abundance, Lhx2 DNA-binding activity, Lhx2, Ldb1, and Ssbp3 loading onto the Cga promoter, Cga promoter activity, and endogenous Cga gene expression. Significantly, neither overexpression nor knockdown of Ssbp2 in MEL cells, which express both the LIM-only protein Lmo2 and LIM-HD protein Lhx2, affected Lhx2 protein abundance, and Lhx2 DNA-binding activity was undetectable in nuclear extracts from these cells despite the presence of immunoreactive Lhx2. These studies indicate that SSBP augmentation of LIM-HD function results from Ldb1-mediated inhibition of LIM-HD protein turnover and increased assembly of a LIM-HD/LDB1/SSBP DNA-binding complex. The much greater affinity for LDB1 of LIM-only compared to LIM-HD proteins is likely a major determinant of the SSBP effect on LIM-HD protein abundance. Finally, these findings are consistent with cell type-specific contributions of different SSBPs, even for similar LDB1-dependent actions.

3′, 5′-Cyclic Adenosine 5′-Monophosphate Response Element-Dependent Transcriptional Regulation of the Secretogranin II Gene Promoter Depends on Gonadotropin-Releasing Hormone-Induced Mitogen-Activated Protein Kinase Activation and the Transactivator Activating Transcription Factor 3

Previous studies demonstrated that GnRH-induced secretogranin II (SgII) promoter regulation required a consensus cAMP response element (CRE) and protein kinase A/CRE binding protein. The present studies examined the role of additional components of the GnRH signaling network on SgII promoter activity with particular attention devoted to CRE-dependent gene regulation. Disruption of the SgII CRE by mutagenesis resulted in inhibition of GnRH agonist (GnRHa) induction of this promoter in alphaT3-1 cells. Pharmacological and dominant-negative inhibition of the ERK and c-Jun N-terminal kinase (JNK) signaling pathways revealed that GnRHa-induced SgII promoter activity required functional JNK and ERK modules. Combined inhibition of both pathways nearly abolished GnRHa-induced SgII promoter activity. Specific induction of the ERK cascade alone using overexpression of Raf-CAAX was not sufficient to activate the SgII gene promoter. In contrast, overexpression of the catalytic domain of the more pleiotropic MAPK activator, MAPK/ERK kinase-1, was sufficient to induce SgII promoter activity. The effect(s) of mitogen-activated protein/ERK kinase-1 on SgII promoter activity was CRE dependent and was reversed by the combined pharmacological inhibition of both JNK and ERK modules. CRE DNA binding studies demonstrated the recruitment of activating transcription factor (ATF)-3 and c-Jun to the CRE after administration of GnRHa to alphaT3-1 cells. Specific small interfering RNA knockdown of ATF3 reduced ATF3 DNA binding and the effect of GnRHa on the SgII promoter. These studies support the conclusion that MAPK signaling and ATF3 action are essential for full SgII promoter activation by GnRHa through a canonical CRE. Moreover, we suggest that within the GnRH signaling network, CRE-dependent gene regulation in general may be mediated primarily through the immediate early response gene ATF3.

Estrogen receptor alpha (ERα) and insulin‐like growth factor I receptor (IGF‐IR) cross‐talk in the gonadotropic αT3‐1 cell line

In reproductive tissues such as the breast and the uterus, cell proliferation and differentiation is strongly regulated by complex interactions between estrogen receptor alpha (ERalpha) and growth factor receptors. In the present study, we investigated the potential occurrence of such cross-talk in the murine, gonadotropic alphaT3-1 cell line, which expresses ERalpha and the IGF-I receptor (IGF-IR). Under estrogen-free conditions, basal cell proliferation and ER-mediated gene transcription was strongly inhibited by the selective estrogen receptor modulator (SERM) 4-hydroxy-tamoxifen (4-OH-Tam) and by the pure anti-estrogen ICI 182,780 (ICI). These effects can be reversed by either 17-beta-estradiol (E(2)) or insulin-like growth factor I (IGF-I), both exerting modest mitogenic effects in the alphaT3-1 cell line. Furthermore, IGF-I enhanced both basal and E(2)-induced ER-driven gene transcription. This may be explained, at least in part, by enhanced phosphorylation of ERalpha at serine 118, a prerequisite for the transactivation capacity of the receptor. Finally, the IGF-I-induced response on cell growth and ER-mediated transactivation can be inhibited with either ICI or 4-OH-Tam. In conclusion, our data indicate IGF-IR and ER interactions in the alphaT3-1 cell line, an in vitro model for the pituitary gonadotrophs, hereby suggesting a role of IGF-I in the regulation of gonadotropin synthesis and secretion.

Functional Interaction between Gonadotropin-Releasing Hormone and PACAP in Gonadotropes and αT3-1 Cells

Gonadotropes, like other cells, receive informational input from multiple receptor types, acting through multiple intracellular signaling pathways, and are therefore faced with the task of integrating this input in order to respond appropriately to their environment. In recent years an increasing number of examples of functional interactions occurring between the PIC and adenylyl cyclase signaling pathways in gonadotropes have been described, and the discovery that these cells are targets for PACAP has provided a physiological context for earlier work on gonadotrope regulation by cyclic AMP. The development of the alpha T3-1 cell line has greatly facilitated investigation of the interaction between these signaling systems. In these cells we have obtained no evidence for interaction between the GnRH and PACAP receptor-effector systems at the level of receptor occupancy or expression, but these systems clearly do have reciprocal modulatory effects on second messenger generation and/or mobilization. We are now faced with the challenge of determining the physiological and/or pathophysiological relevance of such interactions.

PACAP/VIP receptor subtypes, signal transducers, and effectors in pituitary cells.

Rat anterior pituitary tissue expresses mRNA for PVR1 and PVR3, as well as a low level of PVR2. The PVR1 appears to be highly expressed in gonadotroph-like cells, while somatotroph-like cells apparently express the PVR3. We have recently demonstrated the expression of mRNA for both PVR2 and PVR3 in corticotroph-like AtT20 cells (FIG.3). If normal corticotrophs express the same mRNA as AtT20 cells, this may partly explain the low levels of PVR2 seen in normal pituitary tissue. Significant levels of at least two PVR1 splice variants mRNAs (PVR1s and PVR1hop) were expressed in clonal gonadotroph-like alpha T3-1 cells and normal rat anterior pituitary tissue. However, these splice variants are reported to have almost identical pharmacological characteristics in terms of binding, and the activation of AC and PLC. Further experiments are necessary to determine the functional consequences of differential splice variant expression in such cells. Interestingly, all three pituitary-cell lines studied expressed mRNA for the PVR3 (FIG.3), whereas earlier binding studies demonstrate a predominance of PACAP-preferring binding sites on normal anterior pituitary-cell membranes. In addition, it is clear that the different PVR subtypes can couple to different intracellular messenger systems. Thus it will be important to determine the expression of the different PVR subtypes in normal anterior pituitary-cell types if we are to begin to understand the regulation of pituitary-cell regulation by PACAP. Such questions form the basis of some of the ongoing studies in our laboratory.

High Level Expression of Prop-1 Gene in Gonadotropic Cell Lines

Prop-1 acts as an upstream regulator for the Pit-1 gene to induce development of Pit-1 lineage pituitary cell lines, GH-, PRL-, and TSH-producing cells, in the early stage of pituitary organogenesis. Furthermore, Prop-1 is presumed to be involved in the function of FSH/LH-producing cells, gonadotropes, since the defective Prop-1 gene shows hypogonadism. Recently, we reported evidence that Prop-1 directly regulates expression of the porcine FSHbeta gene, thus providing a novel advance in understanding the function of Prop-1 in FSH/LH production and hypogonadism. This study was intended to demonstrate the expressions of Prop-1 gene in pituitary tumor-derived cell lines. RT-PCR analyses were conducted of Pit-1, glycoprotein alpha subunit (alphaGSU), GnRH receptor, and cyclophilin A (a ubiquitously expressing gene). We observed expression of the Pit-1 gene in alphaT1-1, TalphaT1, MtT/S, GH3, and TtT/GF cells, expression of the alphaGSU gene in alphaT1-1, alphaT3-1, LbetaT2, LbetaT4, TalphaT1, and GH3 cells, and expression of GnRH receptor gene in alphaT3-1, LbetaT2, LbetaT4, and GH3 cells, respectively. These expression profiles were in accord with their cell lineages, with only a few exceptions. To accurately measure the expression level of the Prop-1 gene, a quantitative analysis was performed using the real-time PCR method. This analysis demonstrated that the LbetaT2 and LbetaT4 gonadotrope cell lines, which express the FSHbeta gene, express the Prop-1 gene. Taken together with our previous observation that Prop-1 is present in the adult porcine pituitary gonadotropes, Prop-1 might also be involved in development of gonadotropes and hormone production.

Regulation of GnRH I Receptor Gene Expression by the GnRH Agonist Triptorelin, Estradiol, and Progesterone in the Gonadotroph-Derived Cell Line αT3-1

The secretion of luteinizing hormone (LH) and the GnRH receptor (GnRH-R) concentration are modulated by ovarian steroids and GnRH. To elucidate whether this regulation is due to alterations at the transcriptional level, we examined the GnRH I-R mRNA expression in the gonadotroph-derived cell line alphaT3-1 treated with different estradiol and progesterone paradigms and the GnRH I agonist triptorelin. alphaT3-1 cells were treated with different steroid paradigms: 1 nM estradiol or 100 nM progesterone for 48 h alone or in combination. Cells were exposed to 10 nM or 100 pM triptorelin for 30 min, 3 h, 9 h, or, in pulsatile way, with a 5-min pulse per hour. The GnRH I-R mRNA was determined by Northern blot analysis. GnRH I-R mRNA from cells treated with continuous triptorelin decreased in a time- and concentration-dependent manner. Pulsatile triptorelin increased GnRH I-R gene expression. Progesterone alone further enhanced this effect, whereas estradiol and its combination with progesterone diminished it. Continuous combined treatment with estradiol and progesterone lead to a significant decrease of GnRH I-R mRNA by 30% and by 35% for estradiol alone. The addition of 10 nM triptorelin for 30 min or 3 h could not influence that steroid effect. In conclusion, estradiol and progesterone exclusively decreased GnRH I-R mRNA in alphaT3-1 cells no matter whether they are treated additionally with the GnRH I agonist triptorelin. The enhanced sensitivity of gonadotrophs and GnRH I-R upregulation by estradiol is not due to increased GnRH I gene expression because GnRH I-R mRNA is downregulated by estradiol and progesterone. Other pathways of the GnRH I-R signal transduction might be involved.

Gonadotropin-Releasing Hormone (GnRH) Positively Regulates Corticotropin-Releasing Hormone-Binding Protein Expression via Multiple Intracellular Signaling Pathways and a Multipartite GnRH Response Element in αT3-1 Cells

CRH-binding protein (CRH-BP) binds CRH with high affinity and inhibits CRH-mediated ACTH release from anterior pituitary corticotrope-like cells in vitro. In female mouse pituitary, CRH-BP is localized not only in corticotropes, but is also expressed in gonadotropes and lactotropes. To investigate the functional significance of gonadotrope CRH-BP, we examined the molecular mechanisms underlying GnRH-regulated CRH-BP expression in alphaT3-1 gonadotrope-like cells. CRH-BP is endogenously expressed in alphaT3-1 cells, and quantitative real-time RT-PCR and ribonuclease protection assays demonstrate that GnRH induces a 3.7-fold increase in CRH-BP mRNA levels. GnRH also induces intracellular CRH-BP (2.0-fold) and secreted CRH-BP (5.3-fold) levels, as measured by [125I]CRH:CRH-BP chemical cross-linking. Transient transfection assays using CRH-BP promoter-luciferase constructs indicate that GnRH regulation involves protein kinase C-, ERK- and calcium-dependent signaling pathways and is mediated via a multipartite GnRH response element that includes activator protein 1 and cAMP response element (CRE) sites. The CRE site significantly contributes to GnRH responsiveness, independent of protein kinase A, representing a unique form of multipartite GnRH regulation in alphaT3-1 cells. Furthermore, EMSAs indicate that alphaT3-1 nuclear proteins specifically bind at activator protein 1 and CRE sites. These data demonstrate novel regulation of pituitary CRH-BP, highlighting the importance of the pituitary gonadotrope as a potential interface between the stress and reproductive axes.