GnRH Induces Citrullination of the Cytoskeleton in Murine Gonadotrope Cells.

Peptidylarginine deiminases (PADs or PADIs) catalyze the conversion of positively charged arginine to neutral citrulline, which alters target protein structure and function. Our previous work established that gonadotropin-releasing hormone agonist (GnRHa) stimulates PAD2-catalyzed histone citrullination to epigenetically regulate gonadotropin gene expression in the gonadotrope-derived LßT2 cell line. However, PADs are also found in the cytoplasm. Given this, we used mass spectrometry (MS) to identify additional non-histone proteins that are citrullinated following GnRHa stimulation and characterized the temporal dynamics of this modification. Our results show that actin and tubulin are citrullinated, which led us to hypothesize that GnRHa might induce their citrullination to modulate cytoskeletal dynamics and architecture. The data show that 10 nM GnRHa induces the citrullination of ß-actin, with elevated levels occurring at 10 min. The level of ß-actin citrullination is reduced in the presence of the pan-PAD inhibitor biphenyl-benzimidazole-Cl-amidine (BB-ClA), which also prevents GnRHa-induced actin reorganization in dispersed murine gonadotrope cells. GnRHa induces the citrullination of ß-tubulin, with elevated levels occurring at 30 min, and this response is attenuated in the presence of PAD inhibition. To examine the functional consequence of ß-tubulin citrullination, we utilized fluorescently tagged end binding protein 1 (EB1-GFP) to track the growing plus end of microtubules (MT) in real time in transfected LßT2 cells. Time-lapse confocal microscopy of EB1-GFP reveals that the MT average lifetime increases following 30 min of GnRHa treatment, but this increase is attenuated by PAD inhibition. Taken together, our data suggest that GnRHa-induced citrullination alters actin reorganization and MT lifetime in gonadotrope cells.

Peptidylarginine deiminase 2 regulates expression of DGCR8 affecting miRNA biogenesis in gonadotrope cells.

In brief: DGCR8 microprocessor complex, which is important for miRNA biogenesis, is regulated by peptidylarginine deiminase 2 and expression fluctuates in gonadotrope cells across the mouse estrous cycle. Abstract: Canonical miRNA biogenesis requires DGCR8 microprocessor complex subunit, which helps cleave pri-miRNAs into pre-miRNAs. Previous studies found that inhibiting peptidylarginine deiminase (PAD) enzyme activity results in increased DGCR8 expression. PADs are expressed in mouse gonadotrope cells, which play a central role in reproduction by synthesizing and secreting the luteinizing and follicle stimulating hormones. Given this, we tested whether inhibiting PADs alters expression of DGCR8, DROSHA, and DICER in the gonadotrope-derived LßT2 cell line. To test this, LßT2 cells were treated with vehicle or 1 µM pan-PAD inhibitor for 12 h. Our results show that PAD inhibition leads to an increase in DGCR8 mRNA and protein. To corroborate our results, dispersed mouse pituitaries were also treated with 1 µM pan-PAD inhibitor for 12 h which increases DGCR8 expression in gonadotropes. Since PADs epigenetically regulate gene expression, we hypothesized that histone citrullination alters Dgcr8 expression thereby affecting miRNA biogenesis. LßT2 samples were subjected to ChIP using an antibody to citrullinated histone H3, which shows that citrullinated histones are directly associated with Dgcr8. Next, we found that when DGCR8 expression is elevated in LßT2 cells, pri-miR-132 and -212 are reduced, while mature miR-132 and -212 are increased suggesting heightened miRNA biogenesis. In mouse gonadotropes, DGCR8 expression is higher in diestrus as compared to estrus, which is the inverse of PAD2 expression. Supporting this idea, treatment of ovariectomized mice with 17ß-estradiol results in an increase in PAD2 expression in gonadotropes with a corresponding decrease in DGCR8. Collectively, our work suggests that PADs regulate DGCR8 expression leading to changes in miRNA biogenesis in gonadotropes.

Peptidylarginine deiminase enzymes and citrullinated proteins in female reproductive physiology and associated diseases†.

Citrullination, the post-translational modification of arginine residues, is catalyzed by the four catalytically active peptidylarginine deiminase (PAD or PADI) isozymes and alters charge to affect target protein structure and function. PADs were initially characterized in rodent uteri and, since then, have been described in other female tissues including ovaries, breast, and the lactotrope and gonadotrope cells of the anterior pituitary gland. In these tissues and cells, estrogen robustly stimulates PAD expression resulting in changes in levels over the course of the female reproductive cycle. The best-characterized targets for PADs are arginine residues in histone tails, which, when citrullinated, alter chromatin structure and gene expression. Methodological advances have allowed for the identification of tissue-specific citrullinomes, which reveal that PADs citrullinate a wide range of enzymes and structural proteins to alter cell function. In contrast to their important physiological roles, PADs and citrullinated proteins are also involved in several female-specific diseases including autoimmune disorders and reproductive cancers. Herein, we review current knowledge regarding PAD expression and function and highlight the role of protein citrullination in both normal female reproductive tissues and associated diseases.

Progesterone stimulates histone citrullination to increase IGFBP1 expression in uterine cells.

Peptidylarginine deiminases (PAD) enzymes were initially characterized in uteri, but since then little research has examined their function in this tissue. PADs post-translationally convert arginine residues in target proteins to citrulline and are highly expressed in ovine caruncle epithelia and ovine uterine luminal epithelial (OLE)-derived cell line. Progesterone (P4) not only maintains the uterine epithelia but also regulates the expression of endometrial genes that code for proteins that comprise the histotroph and are critical during early pregnancy. Given this, we tested whether P4 stimulates PAD-catalyzed histone citrullination to epigenetically regulate expression of the histotroph gene insulin-like growth factor binding protein 1 (IGFBP1) in OLE cells. 100 nM P4 significantly increases IGFBP1 mRNA expression; however, this increase is attenuated by pre-treating OLE cells with 100 nM progesterone receptor antagonist RU486 or 2 µM of a pan-PAD inhibitor. P4 treatment of OLE cells also stimulates citrullination of histone H3 arginine residues 2, 8, and 17 leading to enrichment of the ovine IGFBP1 gene promoter. Since PAD2 nuclear translocation and catalytic activity require calcium, we next investigated whether P4 triggers calcium influx in OLE cells. OLE cells were pre-treated with 10 nM nicardipine, an L-type calcium channel blocker, followed by stimulation with P4. Using fura2-AM imaging, we found that P4 initiates a rapid calcium influx through L-type calcium channels in OLE cells. Furthermore, this influx is necessary for PAD2 nuclear translocation and resulting citrullination of histone H3 arginine residues 2, 8, and 17. Our work suggests that P4 stimulates rapid calcium influx through L-type calcium channels initiating PAD-catalyzed histone citrullination and an increase in IGFBP1 expression.

GnRH signaling, the gonadotrope and endocrine control of fertility.

Mammalian reproductive cycles are controlled by an intricate interplay between the hypothalamus, pituitary and gonads. Central to the function of this axis is the ability of the pituitary gonadotrope to appropriately respond to stimulation by gonadotropin-releasing hormone (GnRH). This review focuses on the role of cell signaling and in particular, mitogen-activated protein kinase (MAPK) activities regulated by GnRH that are necessary for normal fertility. Recently, new mouse models making use of conditional gene deletion have shed new light on the relationships between GnRH signaling and fertility in both male and female mice. Within the reproductive axis, GnRH signaling is initiated through discrete membrane compartments in which the receptor resides leading to the activation of the extracellular signal-regulated kinases (ERKs 1/2). As defined by gonadotrope-derived cellular models, the ERKs appear to play a central role in the regulation of a cohort of immediate early genes that regulate the expression of late genes that, in part, define the differentiated character of the gonadotrope. Recent data would suggest that in vivo, conditional, pituitary-specific disruption of ERK signaling by GnRH leads to a gender-specific perturbation of fertility. Double ERK knockout in the anterior pituitary leads to female infertility due to LH biosynthesis deficiency and a failure in ovulation. In contrast, male mice are modestly LH deficient; however, this does not have an appreciable impact on fertility.

Plasticity of Anterior Pituitary Gonadotrope Cells Facilitates the Pre-Ovulatory LH Surge.

Gonadotropes cells located in the anterior pituitary gland are critical for reproductive fitness. A rapid surge in the serum concentration of luteinizing hormone (LH) secreted by anterior pituitary gonadotropes is essential for stimulating ovulation and is thus required for a successful pregnancy. To meet the requirements to mount the LH surge, gonadotrope cells display plasticity at the cellular, molecular and morphological level. First, gonadotrope cells heighten their sensitivity to an increasing frequency of hypothalamic GnRH pulses by dynamically elevating the expression of the GnRH receptor (GnRHR). Following ligand binding, GnRH initiates highly organized intracellular signaling cascades that ultimately promote the synthesis of LH and the trafficking of LH vesicles to the cell periphery. Lastly, gonadotrope cells display morphological plasticity, where there is directed mobilization of cytoskeletal processes towards vascular elements to facilitate rapid LH secretion into peripheral circulation. This mini review discusses the functional and organizational plasticity in gonadotrope cells including changes in sensitivity to GnRH, composition of the GnRHR signaling platform within the plasma membrane, and changes in cellular morphology. Ultimately, multimodal plasticity changes elicited by gonadotropes are critical for the generation of the LH surge, which is required for ovulation.

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.

Signaling complexes associated with the type I gonadotropin-releasing hormone (GnRH) receptor: colocalization of extracellularly regulated kinase 2 and GnRH receptor within membrane rafts.

Our previous work demonstrated that the type I GnRH receptor (GnRHR) resides exclusively and constitutively within membrane rafts in alphaT3-1 gonadotropes and that this association was necessary for the ability of the receptor to couple to the ERK signaling pathway. G(alphaq), c-raf, and calmodulin have also been shown to reside in this compartment, implicating a raft-associated multiprotein signaling complex as a functional link between the GnRHR and ERK signaling. In the studies reported here, we used subcellular fractionation and coimmunoprecipitation to analyze the behavior of ERKs with respect to this putative signaling platform. ERK 2 associated partially and constitutively with low-density membranes both in alphaT3-1 cells and in whole mouse pituitary. Cholesterol depletion of alphaT3-1 cells reversibly blocked the association of both the GnRHR and ERKs with low-density membranes and uncoupled the ability of GnRH to activate ERK. Analysis of the kinetics of recovery of ERK inducibility after cholesterol normalization supported the conclusion that reestablishment of the association of the GnRHR and ERKs with the membrane raft compartment was not sufficient for reconstitution of signaling activity. In alphaT3-1 cells, the GnRHR and ERK2 coimmunoprecipitated from low-density membrane fractions prepared either in the presence or absence of detergent. The GnRHR also partitioned into low-density, detergent-resistant membrane fractions in mouse pituitary and coimmunoprecipitated with ERK2 from these fractions. Collectively, these data support a model in which coupling of the GnRHR to the ERK pathway in gonadotropes involves the assembly of a multiprotein signaling complex in association with specialized microdomains of the plasma membrane.

Neuroendocrine plasticity in the anterior pituitary: gonadotropin-releasing hormone-mediated movement in vitro and in vivo.

The secretion of LH is cued by the hypothalamic neuropeptide, GnRH. After delivery to the anterior pituitary gland via the hypothalamic-pituitary portal vasculature, GnRH binds to specific high-affinity receptors on the surface of gonadotrope cells and stimulates synthesis and secretion of the gonadotropins, FSH, and LH. In the current study, GnRH caused acute and dramatic changes in cellular morphology in the gonadotrope-derived alphaT3-1 cell line, which appeared to be mediated by engagement of the actin cytoskeleton; disruption of actin with jasplakinolide abrogated cell movement and GnRH-induced activation of ERK. In live murine pituitary slices infected with an adenovirus-containing Rous sarcoma virus-green fluorescent protein, selected cells responded to GnRH by altering their cellular movements characterized by both formation and extension of cell processes and, surprisingly, spatial repositioning. Consistent with the latter observation, GnRH stimulation increased the migration of dissociated pituitary cells in transwell chambers. Our data using live pituitary slices are a striking example of neuropeptide-evoked movements of cells outside the central nervous system and in a mature peripheral endocrine organ. These findings call for a fundamental change in the current dogma of simple passive diffusion of LH from gonadotropes to capillaries in the pituitary gland.

Gonadotropin releasing hormone activation of the mTORC2/Rictor complex regulates actin remodeling and ERK activity in LßT2 cells.

The mammalian target of rapamycin (mTOR) assembles into two different multi-protein complexes, mTORC1 and mTORC2. The mTORC2 complex is distinct due to the unique expression of the specific core regulatory protein Rictor (rapamycin-insensitive companion of mTOR). mTORC2 has been implicated in regulating actin cytoskeletal reorganization but its role in gonadotrope function is unknown. Using the gonadotrope-derived LßT2 cell line, we find that the GnRH agonist buserelin (GnRHa) phosphorylates both mTOR and Rictor. Interestingly, inhibition of mTORC2 blunts GnRHa-induced cyto-architectural rearrangements. Coincident with blunting of actin reorganization, inhibition of mTORC2 also attenuates GnRHa-mediated activation of both protein kinase C (PKC) and extracellular signal regulated kinase (ERK). Collectively, our data suggests that GnRHa-mediated mTORC2 activation is important in facilitating actin reorganization events critical for initiating PKC activity and subsequent ERK phosphorylation in the gonadotrope-derived LßT2 cell line.

Functional Role of Gonadotrope Plasticity and Network Organization.

Gonadotrope cells of the anterior pituitary are characterized by their ability to mount a cyclical pattern of gonadotropin secretion to regulate gonadal function and fertility. Recent in vitro and in vivo evidence suggests that gonadotropes exhibit dramatic remodeling of the actin cytoskeleton following gonadotropin-releasing hormone (GnRH) exposure. GnRH engagement of actin is critical for gonadotrope function on multiple levels. First, GnRH-induced cell movements lead to spatial repositioning of the in vivo gonadotrope network toward vascular endothelium, presumably to access the bloodstream for effective hormone release. Interestingly, these plasticity changes can be modified depending on the physiological status of the organism. Additionally, GnRH-induced actin assembly appears to be fundamental to gonadotrope signaling at the level of extracellular signal-regulated kinase (ERK) activation, which is a well-known regulator of luteinizing hormone (LH) ß-subunit synthesis. Last, GnRH-induced cell membrane projections are capable of concentrating LHß-containing vesicles and disruption of the actin cytoskeleton reduces LH secretion. Taken together, gonadotrope network positioning and LH synthesis and secretion are linked to GnRH engagement of the actin cytoskeleton. In this review, we will cover the dynamics and organization of the in vivo gonadotrope cell network and the mechanisms of GnRH-induced actin-remodeling events important in ERK activation and subsequently hormone secretion.

Gonadotropin-releasing hormone induction of extracellular-signal regulated kinase is blocked by inhibition of calmodulin.

Our previous studies demonstrate that GnRH-induced ERK activation required influx of extracellular Ca2+ in alphaT3-1 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 alpha-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.

GnRH Stimulates Peptidylarginine Deiminase Catalyzed Histone Citrullination in Gonadotrope Cells.

Peptidylarginine deiminase (PAD) enzymes convert histone tail arginine residues to citrulline resulting in chromatin decondensation. Our previous work found that PAD isoforms are expressed in female reproductive tissues in an estrous cycle-dependent fashion, but their role in the anterior pituitary gland is unknown. Thus, we investigated PAD expression and function in gonadotrope cells. The gonadotrope-derived LßT2 cell line strongly expresses PAD2 at the protein level compared with other PAD isoforms. Consistent with this, PAD2 protein expression is highest during the estrous phase of the estrous cycle and colocalizes with the LH ß-subunit in the mouse pituitary. Using the GnRH agonist buserelin (GnRHa), studies in LßT2 and mouse primary gonadotrope cells revealed that 30 minutes of stimulation caused distinct puncta of PAD2 to localize in the nucleus. Once in the nucleus, GnRHa stimulated PAD2 citrullinates histone H3 tail arginine residues at sites 2, 8, and 17 within 30 minutes; however, this effect and PAD2 nuclear localization was blunted by incubation of the cells with the pan-PAD inhibitor, biphenyl-benzimidazole-Cl-amidine. Given that PAD2 citrullinates histones in gonadotropes, we next analyzed the functional consequence of PAD2 inhibition on gene expression. Our results show that GnRHa stimulates an increase in LHß and FSHß mRNA and that this response is significantly reduced in the presence of the PAD inhibitor biphenyl-benzimidazole-Cl-amidine. Overall, our data suggest that GnRHa stimulates PAD2-catalyzed histone citrullination in gonadotropes to epigenetically regulate gonadotropin gene expression.

The F0F1 ATP Synthase Complex Localizes to Membrane Rafts in Gonadotrope Cells.

Fertility in mammals requires appropriate communication within the hypothalamic-pituitary-gonadal axis and the GnRH receptor (GnRHR) is a central conduit for this communication. The GnRHR resides in discrete membrane rafts and raft occupancy is required for signaling by GnRH. The present studies use immunoprecipitation and mass spectrometry to define peptides present within the raft associated with the GnRHR and flotillin-1, a key raft marker. These studies revealed peptides from the F0F1 ATP synthase complex. The catalytic subunits of the F1 domain were validated by immunoprecipitation, flow cytometry, and cell surface biotinylation studies demonstrating that this complex was present at the plasma membrane associated with the GnRHR. The F1 catalytic domain faces the extracellular space and catalyzes ATP synthesis when presented with ADP in normal mouse pituitary explants and a gonadotrope cell line. Steady-state extracellular ATP accumulation was blunted by coadministration of inhibitory factor 1, limiting inorganic phosphate in the media, and by chronic stimulation of the GnRHR. Steady-state extracellular ATP accumulation was enhanced by pharmacological inhibition of ecto-nucleoside triphosphate diphosphohydrolases. Kisspeptin administration induced coincident GnRH and ATP release from the median eminence into the hypophyseal-portal vasculature in ovariectomized sheep. Elevated levels of extracellular ATP augmented GnRH-induced secretion of LH from pituitary cells in primary culture, which was blocked in media containing low inorganic phosphate supporting the importance of extracellular ATP levels to gonadotrope cell function. These studies indicate that gonadotropes have intrinsic ability to metabolize ATP in the extracellular space and extracellular ATP may serve as a modulator of GnRH-induced LH secretion.

Peptidylarginine Deiminase 3 (PAD3) Is Upregulated by Prolactin Stimulation of CID-9 Cells and Expressed in the Lactating Mouse Mammary Gland.

Peptidylarginine deiminases (PADs) post-translationally convert arginine into neutral citrulline residues. Our past work shows that PADs are expressed in the canine and murine mammary glands; however, the mechanisms regulating PAD expression and the function of citrullination in the normal mammary gland are unclear. Therefore, the first objective herein was to investigate regulation of PAD expression in mammary epithelial cells. We first examined PAD levels in CID-9 cells, which were derived from the mammary gland of mid-pregnant mice. PAD3 expression is significantly higher than all other PAD isoforms and mediates protein citrullination in CID-9 cells. We next hypothesized that prolactin regulates PAD3 expression. To test this, CID-9 cells were stimulated with 5 µg/mL of prolactin for 48 hours which significantly increases PAD3 mRNA and protein expression. Use of a JAK2 inhibitor and a dominant negative (DN)-STAT5 adenovirus indicate that prolactin stimulation of PAD3 expression is mediated by the JAK2/STAT5 signaling pathway in CID-9 cells. In addition, the human PAD3 gene promoter is prolactin responsive in CID-9 cells. Our second objective was to investigate the expression and activity of PAD3 in the lactating mouse mammary gland. PAD3 expression in the mammary gland is highest on lactation day 9 and coincident with citrullinated proteins such as histones. Use of the PAD3 specific inhibitor, Cl4-amidine, indicates that PAD3, in part, can citrullinate proteins in L9 mammary glands. Collectively, our results show that upregulation of PAD3 is mediated by prolactin induction of the JAK2/STAT5 signaling pathway, and that PAD3 appears to citrullinate proteins during lactation.

Dynamin Is Required for GnRH Signaling to L-Type Calcium Channels and Activation of ERK.

We have shown that GnRH-mediated engagement of the cytoskeleton induces cell movement and is necessary for ERK activation. It also has previously been established that a dominant negative form of the mechano-GTPase dynamin (K44A) attenuates GnRH activation of ERK. At present, it is not clear at what level these cellular events might be linked. To explore this, we used live cell imaging in the gonadotrope-derived αT3-1 cell line to determine that dynamin-green fluorescent protein accumulated in GnRH-induced lamellipodia and plasma membrane protrusions. Coincident with translocation of dynamin-green fluorescent protein to the plasma membrane, we demonstrated that dynamin colocalizes with the actin cytoskeleton and the actin binding protein, cortactin at the leading edge of the plasma membrane. We next wanted to assess the physiological significance of these findings by inhibiting dynamin GTPase activity using dynasore. We find that dynasore suppresses activation of ERK, but not c-Jun N-terminal kinase, after exposure to GnRH agonist. Furthermore, exposure of αT3-1 cells to dynasore inhibited GnRH-induced cyto-architectural rearrangements. Recently it has been discovered that GnRH induced Ca(2+) influx via the L-type Ca(2+) channels requires an intact cytoskeleton to mediate ERK phosphorylation. Interestingly, not only does dynasore attenuate GnRH-mediated actin reorganization, it also suppresses Ca(2+) influx through L-type Ca(2+) channels visualized in living cells using total internal reflection fluorescence microscopy. Collectively, our data suggest that GnRH-induced membrane remodeling events are mediated in part by the association of dynamin and cortactin engaging the actin cytoskeleton, which then regulates Ca(2+) influx via L-type channels to facilitate ERK phosphorylation.

Foxo1 Is Required for Normal Somatotrope Differentiation.

The etiology for half of congenital hypopituitarism cases is unknown. Our long-term goal is to expand the molecular diagnoses for congenital hypopituitarism by identifying genes that contribute to this condition. We have previously shown that the forkhead box transcription factor, FOXO1, is present in approximately half of somatotropes at embryonic day (e) 18.5, suggesting it may have a role in somatotrope differentiation or function. To elucidate the role of FOXO1 in somatotrope differentiation and function, Foxo1 was conditionally deleted from the anterior pituitary (Foxo1Δpit). Uncommitted progenitor cells are maintained and able to commit to the somatotrope lineage normally based on the expression patterns of Sox2, a marker of uncommitted pituitary progenitors, and Pou1f1 (also known as Pit1), which marks committed progenitors. Interestingly, Foxo1Δpit embryonic mice exhibit delayed somatotrope differentiation as evidenced by an almost complete absence of GH immunoreactivity at e16.5 and reduced expression of Gh at e18.5 and postnatal day (P) 3. Consistent with this conclusion, expression of GHRH receptor, a marker of terminally differentiated somatotropes, is significantly reduced at e18.5 and P3 in the absence of FOXO1. The mechanism of FOXO1 regulation of somatotrope differentiation may involve the basic helix-loop-helix transcription factor, Neurod4, which has been implicated in somatotrope differentiation and is significantly reduced in Foxo1Δpit mice. Foxo1Δpit mice do not exhibit growth defects, and at P21 their pituitary glands exhibit a normal distribution of somatotropes. These studies demonstrate that FOXO1 is important for initial somatotrope specification embryonically but is dispensable for postnatal somatotrope expansion and growth.

Loss of Foxm1 Results in Reduced Somatotrope Cell Number during Mouse Embryogenesis.

FOXM1, a member of the forkhead box transcription factor family, plays a key role in cell cycling progression by regulating the expression of critical G1/S and G2/M phase transition genes. In vivo studies reveal that Foxm1 null mice have a 91% lethality rate at e18.5 due to significant cardiovascular and hepatic hypoplasia. Thus, FOXM1 has emerged as a key protein regulating mitotic division and cell proliferation necessary for embryogenesis. In the current study, we assess the requirement for Foxm1 in the developing pituitary gland. We find that Foxm1 is expressed in the pituitary at embryonic days 10.5-e18.5 and localizes with markers for active cell proliferation (BrdU). Interestingly, direct analysis of Foxm1 null mice at various embryonic ages, reveals no difference in gross pituitary morphology or cell proliferation. We do observe a downward trend in overall pituitary cell number and a small reduction in pituitary size in e18.5 embryos suggesting there may be subtle changes in pituitary proliferation not detected with our proliferation makers. Consistent with this, Foxm1 null mice have reductions in both the somatotrope and gonadotrope cell populations.

Role of cortactin in dynamic actin remodeling events in gonadotrope cells.

GnRH induces marked activation of the actin cytoskeleton in gonadotropes; however, the physiological consequences and cellular mechanisms responsible have yet to be fully elucidated. The current studies focus on the actin scaffolding protein cortactin. Using the gonadotrope-derived αT3-1 cell line, we found that cortactin is phosphorylated at Y(421), S(405), and S(418) in a time-dependent manner in response to the GnRH agonist buserelin (GnRHa). GnRHa induced translocation of cortactin to the leading edge of the plasma membrane where it colocalizes with actin and actin-related protein 3 (Arp3). Incubation of αT3-1 cells with the c-src inhibitor phosphoprotein phosphatase 1, blocked tyrosine phosphorylation of cortactin, reduced cortactin association with Arp3, and blunted actin reorganization in response to GnRHa. Additionally, we used RNA silencing strategies to knock down cortactin in αT3-1 cells. Knockdown of cortactin blocked the ability of αT3-1 cells to generate filopodia, lamellipodia, and membrane ruffles in response to GnRHa. We show that lamellipodia and filopodia are capable of LHß mobilization in primary pituitary culture after GnRHa treatment, and disruption of these structures using jasplakinolide reduces LH secretion. Collectively, our findings suggest that after GnRHa activation, src activity leads to tyrosine phosphorylation of cortactin, which facilitates its association with Arp3 to engage the actin cytoskeleton. The reorganization of actin by cortactin potentially underlies GnRHa-induced secretory events within αT3-1 cells.

GnRH evokes localized subplasmalemmal calcium signaling in gonadotropes.

The binding of GnRH to its receptor initiates signaling cascades in gonadotropes, which result in enhanced LH and FSH biosynthesis and secretion. This process is necessary for follicular maturation and ovulation. Calcium influx activates MAPKs, which lead to increased transcription of LH and FSH genes. Previous research suggests that two MAPK signaling pathways, ERK and jun-N-terminal kinase, are activated by either calcium influx through L-type calcium channels or by global calcium signals originating from intracellular stores, respectively. Here we continued this investigation to further elucidate molecular mechanisms transducing GnRH receptor stimulation to ERK activation. Although it is known that GnRH activation of ERK requires calcium influx through L-type calcium channels, direct evidence supporting an underlying local calcium signaling mechanism was lacking. Here we used a combination of electrophysiology and total internal reflection fluorescence microscopy to visualize discrete sites of calcium influx (calcium sparklets) in gonadotrope-derived αT3-1 cells in real time. GnRH increased localized calcium influx and promoted ERK activation. The L-type calcium channel agonist FPL 64176 enhanced calcium sparklets and ERK activation in a manner indistinguishable from GnRH. Conversely, the L-type calcium channel antagonist nicardipine inhibited not only localized calcium sparklets but also ERK activation in response to GnRH. GnRH-dependent stimulation of L-type calcium channels was found to require protein kinase C and a dynamic actin cytoskeleton. Taken together, we provide the first direct evidence for localized L-type calcium channel signaling in αT3-1 cells and demonstrate the utility of our approach for investigating signaling mechanisms and cellular organization in gonadotropes.