• GnRH;
  • gonadotrophin;
  • gonadotroph;
  • calmodulin;
  • calcineurin;
  • CaMK


  1. Top of page
  2. Abstract
  3. Gonadotrophin-releasing hormone (GnRH), the gonadotroph and calmodulin
  4. CaMKs and gonadotrophin gene expression
  5. Diverse roles for calcineurin in the gonadotroph
  6. Effects of GnRH via Ca2+/CaM signalling on the cell cycle and gonadotroph proliferation
  7. A role for calmodulin in moderating GnRH-induced signalling through negative-feedback?
  8. Conclusions and future directions
  9. Acknowledgements
  10. References

Gonadotrophin-releasing hormone (GnRH) regulates reproduction via binding a G-protein coupled receptor on the surface of the gonadotroph, through which it transmits signals, mostly via the mitogen-activated protein (MAPK) cascade, to increase synthesis of the gonadotrophin hormones: luteinising hormone (LH) and follicle-stimulating hormone (FSH). Activation of the MAPK cascade requires an elevation in cytosolic Ca2+ levels, which is a result of both calcium influx and mobilisation from intracellular stores. However, Ca2+ also transmits signals via an MAPK-independent pathway, through binding calmodulin (CaM), which is then able to bind a number of proteins to impart diverse downstream effects. Although the ability of GnRH to activate CaM was recognised over 20 years ago, only recently have some of the downstream effects been elucidated. GnRH was shown to activate the CaM-dependent phosphatase, calcineurin, which targets gonadotrophin gene expression both directly and indirectly via transcription factors such as nuclear factor of activated T-cells and Nur77, the Transducer of Regulated CREB (TORC) co-activators and also the prolyl isomerase, Pin1. Gonadotrophin gene expression is also regulated by GnRH-induced CaM-dependent kinases (CaMKs); CaMKI is able to derepress the histone deacetylase-inhibition of β-subunit gene expression, whereas CaMKII appears to be essential for the GnRH-activation of all three subunit genes. Asides from activating gonadotrophin gene expression, GnRH also exerts additional effects on gonadotroph function, some of which clearly occur via CaM, including the proliferation of immature gonadotrophs, which is dependent on calcineurin. In this review, we summarise these pathways, and discuss the additional functions that have been proposed for CaM with respect to modifying GnRH-induced signalling pathways via the regulation of the small GTP-binding protein, Gem, and/or the regulator of G-protein signalling protein 2.

Gonadotrophin-releasing hormone (GnRH), the gonadotroph and calmodulin

  1. Top of page
  2. Abstract
  3. Gonadotrophin-releasing hormone (GnRH), the gonadotroph and calmodulin
  4. CaMKs and gonadotrophin gene expression
  5. Diverse roles for calcineurin in the gonadotroph
  6. Effects of GnRH via Ca2+/CaM signalling on the cell cycle and gonadotroph proliferation
  7. A role for calmodulin in moderating GnRH-induced signalling through negative-feedback?
  8. Conclusions and future directions
  9. Acknowledgements
  10. References

Reproduction is regulated through a complex interplay of hormones and feedback operating along the hypothalamic-pituitary-gonadal axis, of which the hypothalamic hormone, gonadotrophin-releasing hormone (GnRH), is the predominant activator. GnRH binds a membrane-bound receptor (GnRHR) on the pituitary gonadotrophs to stimulate transcription of the three gonadotrophin genes: the common α subunit (αGSU) and the hormone specific β-subunits [luteinising hormone (LH)β and follicle-stimulating hormone (FSH)β] (Fig. 1), as well as stimulating the secretion of LH. As part of its regulation of gonadotroph activity and function, GnRH also affects these cells in numerous other ways, including cell proliferation and apoptosis, cell shape and mobility, as well as their responsiveness to other hormones [1-6].


Figure 1. Model of gonadotrophin-releasing hormone (GnRH)-induced pathways downstream of calmodulin towards gonadotrophin gene expression. Upon binding its G-protein coupled receptor on the cell membrane, GnRH induces calcium influx (as well as mobilisation: not shown), which activates calmodulin (CaM). Some of the effects downstream of CaM via the CaM kinases and the phosphatase, calcineurin, which lead to gonadotrophin gene expression, are shown. Solid lines represent effects that are likely direct, and dashed lines represent pathways that may or may not have intermediate elements that are not shown. Blue lines represent transcriptional, and black lines post-translational effects on the target. The pink double-headed arrows indicate proteins that shuttle between cellular compartments. CaMK, calmodulin-dependent kinase; CoR, receptor corepressor; ERK, extracellular-signal regulated kinase; HDAC, histone deacetylase; NFAT, nuclear factor of activated T-cells; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; TORC, transducer of regulated CREB.

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The GnRH arrives at the pituitary in pulses whose amplitude and frequency vary with ensuing sexual maturity and during the oestrous cycle; these distinct pulse frequencies direct the preferential expression of either LHβ or FSHβ [7-10]. Subsequent to their synthesis, the gonadotrophic hormones, LH and FSH, are secreted into the circulation from where they regulate gonadal activity. The pivotal role of GnRH in functioning of the gonadotrophs and the regulation of reproduction is seen at puberty, when GnRH delivery to the gonadotroph is the only endogenous block to the reawakening of the pituitary-gonadal axis and reproductive potential [11].

Upon binding to its G-protein coupled receptor on the gonadotroph, GnRH induces the production of inositol triphosphate (IP3) and diacylglycerol for downstream signalling, much of which is via protein kinase C (PKC) [12]. The elevation of IP3 results in calcium mobilisation from the endoplasmic reticulum (ER), with external calcium also entering through voltage-gated calcium channels on the cell membrane. These GnRH-induced events trigger the major mitogen-activated protein kinase (MAPK) cascades, culminating in activation of extracellular-signal regulated kinase (ERK) 1/2, c-Jun NH2-terminal kinase (JNK), p38 MAPK and ERK5 [10, 12, 13]. The elevation in available intracellular Ca2+ is temporary because the Ca2+ is quickly sequestered by binding proteins and is also pumped out of the cytosol after inhibition of the ER IP3-sensitive Ca2+ channels by the high levels of cytosolic Ca2+ [14]. The resulting short pulses of free Ca2+ play crucial roles in cell signalling in many cell types, and their roles in activating regulated LH secretion have been documented extensively [14, 15], while also playing additional roles in the regulation of other aspects of cell function and gene expression [16, 17]. Diverse studies have indicated the varying involvement of this Ca2+ in expression of the gonadotrophin genes [12] and it was suggested that Ca2+ sensing might play a role in the decoding of GnRH pulse-frequency [18].

Apart from its role in the activation of PKC and the downstream MAPK pathways, Ca2+ is also able to induce distinct cellular outcomes via additional binding protein effectors, most notably calmodulin (CaM), which is expressed ubiquitously. CaM does not contain its own enzymatic activity but, in different cell contexts, interacts with a variety of proteins, including kinases, phosphatases and ion channels to transduce the signalling effects to diverse pathways [19]. This is possible because the CaM N-terminal and C-terminal domains are connected by a linker region which allows it to assume distinct conformations when bound to different targets. In the absence of Ca2+, it adopts a closed conformation but, after Ca2+ binding, the conformational change exposes more hydrophobic groups, thus enhancing the ability of CaM to interact with the various effector proteins [19-21].

Early studies in gonadotrophs demonstrated that GnRH induces a change in localisation of CaM from the cytoplasm to the cell membrane and also indicated that it has a role in GnRH-induced LH release, as well as in the expression of primary response genes, c-fos, c-jun and junB, [22-24]. More recently, two of the better characterised signalling pathways downstream of CaM, the CaM-dependent kinases (CaMKs) and the phosphatase, calcineurin, have been shown to play roles in transducing GnRH-activated signals to gonadotrophin gene transcription and other aspects of gonadotroph function. In this review, we summarise these pathways, and examine other evidence for a role of CaM in modulating GnRH-induced ERK signalling, possibly involving the regulator of G-protein signalling (RGS) protein, RGS2, or the small GTP-binding protein, Gem.

CaMKs and gonadotrophin gene expression

  1. Top of page
  2. Abstract
  3. Gonadotrophin-releasing hormone (GnRH), the gonadotroph and calmodulin
  4. CaMKs and gonadotrophin gene expression
  5. Diverse roles for calcineurin in the gonadotroph
  6. Effects of GnRH via Ca2+/CaM signalling on the cell cycle and gonadotroph proliferation
  7. A role for calmodulin in moderating GnRH-induced signalling through negative-feedback?
  8. Conclusions and future directions
  9. Acknowledgements
  10. References

The CaMKs are serine/threonine kinases, which include CaMKI, CaMKII and CaMKIV, as well as the upstream CaMK kinase (CaMKK). These kinases have comprehensive functions and are involved in gene expression, cell proliferation, apoptosis and cytoskeletal reorganisation, as well as synaptic development and plasticity and behaviour [21, 25]. CaMKI, CaMKII and CaMKK are widely expressed, whereas CaMKIV is more tissue-restricted and is not found in the gonadotrophs [26].

All of the CaMKs contain a Ca2+/CaM -binding region that overlaps with an autoinhibitory domain. Binding of Ca2+/CaM disrupts the interaction between the autoinhibitory domain and the catalytic domain, thus activating the kinase [21]. CaMKK is an upstream activator of CaMKI and CaMKIV that shares similar structure with the other CaMKs; however, after its initial activation, the CaMKKβ isoform can also function independently of Ca2+/CaM because of the release of its autoinhibitory domain; moreover, it may also have some autonomous activity through its autophosphorylation [21, 27]. This isoform also acts as a scaffold for a signalling complex including CaM and the CaMKK substrate, which was recently shown to include AMP-dependent kinase (AMPK) [28]. Interestingly, GnRH was shown to induce a prolonged increase in AMPK activity in the gonadotrophs [29], although a role for CaMKK in this activation has yet to be reported.


The activity of CaMKI is entirely dependent on activation by Ca2+/CaM, and CaMKI activity drops as soon as the levels of Ca2+ return to basal levels. This dependency is a result of fact that the Ca2+/CaM-induced displacement of the autoinhibitory domain is required to expose the threonine that is targeted by CaMKK. Phosphorylation of this site by CaMKK increases CaMKI activity, probably by enhancing the stability of the activation loop that increases the competency of the catalytic site [21].

CaMKI is expressed in the αT3-1 partially-differentiated immortalised gonadotroph cells and is notably phosphorylated within 5 min of GnRH treatment; this elevated state is still seen after 60 min of continuous GnRH exposure [26]. CaMKI has a diverse set of protein targets, including transcription factors and the class 2a histone deacetylases (HDACs), which shuttle between the nucleus and cytoplasm. The CaMK-mediated phosphorylation of these class 2a HDACs was shown to signal binding of the HDAC by 14-3-3 chaperone proteins that mask the nuclear localisation signal on the HDAC and may expose a nuclear export signal before exporting the HDAC out of the nucleus [30-32]. HDAC4 and HDAC5 are phosphorylated after GnRH exposure and this is almost completely abolished by a dominant negative (dn) CaMKI. Moreover, over-expression of the dnCaMKI also abolished the ability of GnRH to de-repress expression of the FSHβ gene, although not the LHβ gene [26, 33]. GnRH causes dissociation of these HDACs from the β-subunit gene promoters, and their nuclear export is dependent on the serines targeted by CaMKI (Fig. 1). Given that knockdown of HDAC4 alone was sufficient to allow FSHβ gene expression, it appears that GnRH-activated CaMKI mediates the de-repression of this gene in immature gonadotrophs via targeting these HDACs to cause their removal from the gene promoter. Interestingly, however, this effect appears to be unique to the FSHβ gene because CaMK inhibition failed to block the effect of GnRH on the LHβ gene, indicating that GnRH likely directs de-repression of the LHβ gene downstream of CaMK activation [26, 33].


CaMKII differs in structure from the other CaMKs and, unlike other CaMKs, it does not act as a monomer but forms part of a complex of 12 or more subunits with at least six catalytic domains extending away from the core. The ability of this complex to contain diverse proteins likely endows its ability to activate multiple downstream effects [21]. Similar to other CaMKs, CaMKII also contains an autoinhibitory domain that blocks catalytic activity until Ca2+/CaM levels are increased. After the initial activation of one subunit of the holoenzyme by Ca2+/CaM, it can then phosphorylate a neighbouring Ca2+/CaM-bound subunit in the same complex. This phosphorylation disrupts the interaction between the autoinhibitory domain and the catalytic site, such that the subunit subsequently retains activity independent of Ca2+/CaM-binding, at the same time as also increasing the affinity of CaMKII for Ca2+/CaM. Thus, Ca2+/CaM must bind two molecules in the same holoenzyme to attain initial activation of CaMKII, which reflects the amount of available Ca2+; however, subsequent CaMKII activity is maintained even after transiently-elevated Ca2+ levels drop [21, 34]. Because of the unique characteristics of CaMKII, which render it highly sensitive to Ca2+ pulse frequency, it was proposed that CaMKII might provide a mechanism of decoding Ca2+ oscillations that occur in response to diverse stimuli [35, 36].

GnRH activates CaMKII in rat primary pituitary cells and in gonadotroph cell lines, and treatment with the CaMK inhibitor, KN-93, decreased the GnRH induction of promoter activity and/or mRNA levels of all three gonadotrophin subunit genes [9, 26, 37, 38]. Moreover, the differential expression of these genes was mimicked by the administration of Ca2+ pulses at frequencies similar to those seen after GnRH treatment [18]. It was therefore suggested by several groups that Ca2+/CaM- CaMKII might play a role in decoding GnRH pulse frequency towards gonadotrophin gene expression. In one model, it was proposed that CaMKII activation might favour αGSU and LHβ gene expression, which are dominant at higher GnRH pulse frequencies, whereas at lower frequencies that favour FSHβ transcription, the activated CaMKII, which might be less crucial for FSHβ expression, would have time to decay [8]. However, a latter study showed that CaMKII activation by GnRH at 30- or 240 min-pulses, which are optimal for LHβ or FSHβ activation, respectively, is not frequency-dependent [9]. Moreover, a number of subsequent studies, both experimental and computational, have indicated that feedback may be more crucial to the frequency decoding, and have implicated MAPK phosphatases to play a key role [10, 39, 40], whereas feedback inhibition of gene-specific transcription factors has also been suggested [41, 42].

The ability of the CaMKII inhibitor to reduce GnRH-induced transcription of all three gonadotrophin β-subunit genes indicates that, even if it does not decode the GnRH pulse frequency, CaMKII does mediate some of the effects of GnRH, although its targets in this pathway largely remain to be elucidated. Recently, however, GnRH-activation of c-fos was shown to occur via CaMKII phosphorylation of serum response factor, which likely plays a role in GnRH induction of FSHβ and possibly αGSU transcription [43-46].

Diverse roles for calcineurin in the gonadotroph

  1. Top of page
  2. Abstract
  3. Gonadotrophin-releasing hormone (GnRH), the gonadotroph and calmodulin
  4. CaMKs and gonadotrophin gene expression
  5. Diverse roles for calcineurin in the gonadotroph
  6. Effects of GnRH via Ca2+/CaM signalling on the cell cycle and gonadotroph proliferation
  7. A role for calmodulin in moderating GnRH-induced signalling through negative-feedback?
  8. Conclusions and future directions
  9. Acknowledgements
  10. References

Calcineurin is a Ca2+/CaM-dependent serine/threonine protein phosphatase comprising two subunits: the catalytic calcineurin A subunit (CnA), and a Ca2+-binding regulatory subunit, calcineurin B (CnB). The CnA contains three regulatory domains: the CaM-binding domain, the autoinhibitory domain and a domain that interacts with CnB. In the absence of Ca2+/CaM, the autoinhibitory domain binds in the active site and inhibits phosphatase activity. A rise in intracellular Ca2+, which binds CnB, facilitates Ca2+/CaM binding by the CaM-binding domain of CnA, resulting in a displacement of the autoinhibitory domain and activation of the phosphatase, which is subsequently independent of CnB [47, 48].

CnA expression levels increase after GnRH treatment in both αT3-1 cells and the more fully differentiated immortalised gonadotrope LβT2 cells [26, 49, 50]. The activity of calcineurin, as well as other phosphatases, in mediating GnRH signals to gonadotrophin genes are clearly tightly regulated by GnRH and likely crucial for maintaining the integrity of the signal [10, 40, 51-54]. It has been proposed that a continual phosphorylation/dephosphorylation cycle of key substrates is required for GnRH stimulation of gonadotrophin genes to avoid desensitisation, that they act to down-regulate and constrain the magnitude or duration of GnRH signalling, and also that they play a role in decoding the GnRH pulse frequency [10, 40, 51, 55, 56]. However, unlike the MAPK phosphatases, the effects of calcineurin in these cells all appear to be stimulatory to gonadotrophin gene expression and are mediated through multiple targets. Inhibition of calcineurin is repressive to transcription and/or promoter activation of all three gonadotrophin subunits [26, 50, 54, 57]. For the αGSU, calcineurin is both necessary and sufficient to induce gene expression, whereas, for FSHβ, it is necessary but not sufficient, although it clearly augments the effect of GnRH. For the LHβ gene, its exact role remains to be elucidated, although it likely includes indirect effects such as those occurring via Pin1, which also impacts other aspect of gonadotroph cell function (see below) (Fig. 1).

Calcineurin regulates gonadotrophin gene expression via nuclear factor of activated T-cells (NFAT) and Nur77

NFAT is the most extensively studied calcineurin target; first identified in T-cells, it is rapidly induced to activate the IL-2 promoter. However, it is widely expressed and plays extensive roles in regulating gene expression for diverse aspects of cell function. Four NFAT isoforms have been identified, NFAT1 (NFATc2), NFAT2 (NFATc1), NFAT3 (NFATc4) and NFAT4 (NFATc3), all of which are comprised of an N-terminal transactivation domain, a DNA-binding domain and a NFAT regulatory region. This regulatory region is phosphorylated on multiple serines by various kinases, including casein kinase 1 and glycogen synthase kinase 3, which act to repress NFAT activity by keeping it in the cytoplasm. NFAT is activated by calcineurin-mediated dephosphorylation of these same residues, allowing the NFAT to translocate to the nucleus to activate transcription of its target genes. In both the cytoplasm and nucleus, NFAT interacts with a variety of other transcription factors, which include AP-1, GATA4, Foxp and MEF2, and these interactions play a crucial role in determining the specificity of the NFAT-binding to its target genes. Nuclear NFAT is inactivated by phosphorylation, which causes it to return to the cytoplasm [58, 59].

In αT3-1 cells, NFAT 2, 3 and 4 are expressed, with NFAT3 being notably translocated into the nucleus, in a calcineurin-dependent manner, after GnRH treatment [50] (Fig. 1). In these cells, NFAT 2 and 4 appear to be constitutively nuclear, although, in studies in which NFAT2-EFP was transfected into LβT2 cells, the over-expressed protein did oscillate between cellular compartments in response to GnRH [57]. NFAT3 is required for GnRH-induced αGSU expression in the αT3-1 cells, whereas NFAT4 may play a role in its basal expression. In support of a role for NFAT3 in mediating the effect of GnRH on αGSU gene expression, the calcineurin-responsive region of the promoter maps to a sequence (−420 to −346 bp upstream of the tss) already shown to mediate both the GnRH and calcium responses, and this common sequence (tTTCCTGTT) contains a consensus NFAT-binding element [50, 60, 61].

Recently, calcineurin-driven NFAT was also reported to mediate GnRH-induced expression of the early response genes, Jun and Aft3. It was shown that NFAT binds the Jun promoter and interacts with Jun through AP-1 sites [62]. Given that AP-1 sites also regulate αGSU expression, this suggests an additional mechanism of the role of NFAT with respect to transducing the effect of GnRH to this gene.

NFAT also regulates FSHβ gene transcription, although this effect is indirect, mediating GnRH stimulation of the Nur77 transcription factor. The expression of the Nur77 is elevated by GnRH in a potent and rapid manner, and this is calcineurin-dependent [26, 50, 52, 63] (Fig. 1). In T-cells, the transcription of Nur77 occurs via calcineurin-activated NFAT, which interacts with MEF2D at the Nur77 gene promoter [64]. Also in the gonadotroph, NFAT3 is recruited to the Nur77 promoter after GnRH treatment concomitant with the association of RNAPII, and NFAT appears to play a central role because its knockdown reduced the GnRH stimulatory effect on Nur77 expression. In the αT3-1 cells, Nur77 over-expression is sufficient to de-repress expression of the FSHβ gene, whereas FSHβ promoter activity and transcript levels are reduced after NFAT3 knockdown in LβT2 cells, suggesting that NFAT3-activated Nur77 is required for GnRH-induction of the FSHβ gene [26, 50].

Apart from the involvement of calcineurin in the GnRH stimulation of Nur77 expression, it is likely also involved in the GnRH-activated dephosphorylation of Nur77 at S354 (Fig. 1). Mutation of this residue indicates that the dephosphorylation is a means of activating Nur77 transcriptional activity [26]. This is also the mechanism of Nur77 activation by adrenocorticotrophic hormone, involving dephosphorylation at the same residue, although, unlike at the FSHβ gene, the phosphorylation prevented DNA binding in the contexts tested [65]. Although we have yet to determine the function of this dephosphorylation in FSHβ gene activation, Nur77 is known to interact with SMRT, which represses expression of the FSHβ gene in these cells [26, 66, 67]. It is therefore quite possible that the dephosphorylation causes a switch in Nur77 interacting proteins, allowing it to dissociate from SMRT and recruit coactivators.

A role for calcineurin-regulated Transducer of Regulated CREB (TORC)

The TORCs are transcriptional coactivators whose activity is regulated by calcineurin-mediated dephosphorylation. Also known as CREB-Regulated Transcription Coactivators, this group of proteins was originally identified as interacting with, and potently increasing the activity of CREB, however, they are now known to coactivate additional transcription factors, including c-jun, c-fos, ATF1 and ATF4 [68, 69]. Unlike the CREB coactivation by CBP, the actions of TORC are independent of CREB phosphorylation at Ser 133, although TORC appears to coactivate at only a subset of CREB targets [70, 71]. Dephosphorylation of the TORC proteins by calcineurin allows their nuclear import and thus their transcriptional activity. Conversely, they are negatively regulated by phosphorylation, for example by protein kinase A-induced salt-inducible kinase, which prevents their nuclear localisation [72, 73].

Although enriched in the brain and lymphocytes, TORC proteins are expressed quite ubiquitously at low levels, and we were able to detect TORC1 and TORC3 proteins (but not TORC2) in the αT3-1 cells (Melamed, unpublished data). TORC1 over-expression was sufficient to increase αGSU promoter activity, whereas its knockdown indicated that TORC1 plays an essential role in GnRH-activated αGSU transcription. However, this gene is not regulated by CREB, and TORC does not appear to be recruited to the αGSU promoter by CREB. Because we were unable to discern synergistic effects of TORC with any transcription factor in the activation of this gene, the factor responsible for recruiting TORC remains to be identified, although it may already be found at the promoter in untreated cells [50].

CREB clearly does play a role in GnRH-mediated activation of the FSHβ gene [74], and it acts synergistically with TORC1 [50]. The CREB-TORC1 synergy is further enhanced by Nur77, although Nur77 and TORC also exert a powerful synergistic effect without the addition of CREB. TORC1 over-expression also increased the effect of GnRH, although its knockdown revealed more of an effect on basal promoter activity than on that induced by GnRH. Notably, the GnRH-induced phosphorylation of CREB at S133 prevents its interaction with TORC1, presumably favouring the recruitment of CBP [74]. Given that this serine is required for the full effect of GnRH but not for that of TORC1, TORC1 may not be required for the GnRH-stimulated FSHβ promoter activity. However, TORC1 does appear to play a role in regulating FSHβ gene expression because TORC1 knockout male mice have significantly lower circulating FSH levels than their wild-type counterparts; although this was not seen in female mice, which were tested only at pro-oestrous [75]. The role of TORC in FSHβ gene expression has thus to be fully elucidated, although it might provide a different mechanism of activating transcription as part of an alternatively regulated GnRH-independent pathway, at the same time as still allowing for cross-talk with the GnRH-activated cascade.

Although calcineurin is known to promote TORC nuclear localisation, and GnRH treatment causes dephosphorylation of TORC1 in a calcineurin-dependent manner, the levels of nuclear TORC1 were not increased by GnRH treatment. TORC1 appears to be inherently unstable in these cells and, after GnRH is added, the existing TORC1 is rapidly degraded. Subsequently, the newly synthesised protein appears to have a protected N-terminus which is associated with its enhanced activity [50]. Instability of the TORC1 before GnRH treatment is a result of the actions of calpain and the proteasome, and we have proposed that the degradation of the newly synthesised TORC is regulated by GnRH actions on the calpain-calpastatin system, possibly through ERK-activated phosphorylation. This is similar to the regulation of PKCε degradation in rat pituitary GH4C1 cells by thyrotrophin-releasing hormone [76]. However, other explanations are also quite possible.

Wide-ranging effects of calcineurin via dephosphorylation of Pin1

We recently reported a novel target of calcineurin in the gonadotrophs: the ubiquitously expressed peptidyl-prolyl isomerase, Pin1 [49]. Pin1 is the only known enzyme that binds a phosphorylated serine or threonine preceding a proline (pSer/Thr-Pro) and isomerises the cis-trans conformation of the prolyl-peptidyl bond. It consists of a short N-terminal WW domain that binds only to pSer/Thr-Pro motifs, providing target specificity, and a C-terminal catalytic domain that isomerises the prolyl-peptidyl bond. Pin1 thus mediates a phosphorylation-induced conformational change in the target protein, which leads to altered activity, providing additional insight into the role of this post-translational modification [77, 78]. Notably, Ser/Thr-Pro is a common motif that is phosphorylated by various kinases, particularly by the MAPKs which form an integral part of GnRH-induced signal transduction, and Pin1-catalysed prolyl isomerisation has been shown to be involved in a wide variety of contexts including the regulation of transcription, protein–protein interaction and/or protein subcellular localisation [77, 78].

Pin1 activity is tightly regulated via several mechanisms that determine its ability to bind the substrate, its catalytic activity and/or its cellular localisation (Fig. 1). Phosphorylation of Pin1 at S16, in the pSer/Thr-Pro binding pocket, is a key mechanism of control because it prevents the WW domain from interacting with pSer/Thr-Pro motifs on its substrates and may facilitate Pin1 nuclear export [79]. In the gonadotrophs, intriguingly, GnRH increases levels of phosphoPin1, as does forskolin or, to a lesser extent, phorbol 12-myristate 13-acetate (PMA) treatment. This suggests a role for GnRH-activated PKC, or protein kinase A which has been implicated in Pin1 phosphorylation in the past [79]. Conversely however, Pin1 and calcineurin co-precipitate and this interaction increases after GnRH treatment. The GnRH-activated calcineurin acts to dephosphorylate Pin1, thus keeping it in its active state; this may be crucial to counteract the apparently paradoxical increase in Pin1 phosphorylation after GnRH treatment [49].

One of the targets of Pin1 in the gonadotrophs is the transcription factor Sf-1. Pin1 facilitates the phosphorylation-to-ubiquitination transition of Sf-1 (Fig. 1), which is required for Sf-1 interaction with Pitx-1; this interaction is essential for the synergistic transcriptional activation of the LHβ gene [49, 80]. However, Sf-1 is required for the full GnRH response of all three gonadotrophin subunit genes, while many of the other GnRH-activated transcription factors that regulate gene expression in the gonadotrophs also contain pSer/Thr-Pro motifs and are activated through phosphorylation by MAPKs, making them likely Pin1 targets.

In other contexts, Pin1 has been shown to bind and regulate the activity of additional modulators of gonadotrophin gene expression, including ERα, c-Jun, β-catenin, SMADs 2 and 3, SMRT, NFAT and TORCs [2, 3, 43, 44, 50, 78, 81-84]. For NFAT and the TORCs, the effect of the Pin1 is to repress transcriptional activity of the factor by keeping it in the cytoplasm; moreover, the interaction of NFAT with Pin1 specifically prevents NFAT activation by calcineurin [81, 83]. Although the role of Pin1 in regulating NFAT and/or TORC activity in the gonadotrophs has yet to be verified, these studies indicate that cytoplasmic, presumably phosphorylated, Pin1 also plays a role in determining the signalling pathways to differential gene expression, and may help contribute an explanation for the GnRH-induction of Pin1 phosphorylation. This paradoxical regulation further highlights the important role of calcineurin in the regulation of Pin1 function by phosphorylation/dephosphorylation and indicates that Pin1 plays a likely widespread role not only in the gonadotroph, but also in MAPK signalling cascades in more diverse contexts.

Effects of GnRH via Ca2+/CaM signalling on the cell cycle and gonadotroph proliferation

  1. Top of page
  2. Abstract
  3. Gonadotrophin-releasing hormone (GnRH), the gonadotroph and calmodulin
  4. CaMKs and gonadotrophin gene expression
  5. Diverse roles for calcineurin in the gonadotroph
  6. Effects of GnRH via Ca2+/CaM signalling on the cell cycle and gonadotroph proliferation
  7. A role for calmodulin in moderating GnRH-induced signalling through negative-feedback?
  8. Conclusions and future directions
  9. Acknowledgements
  10. References

Ca2+/CaM signalling plays a variety of roles in the regulation of the cell cycle, and CaM levels vary through the cell cycle, increasing just before the S-phase. CaM over-expression accelerates proliferation, apparently due to a shortening of progression through G1, whereas, conversely, a reduction of CaM levels inhibits cell proliferation [25]. We have already shown that GnRH induces an increase in the number of αT3-1 cells [6] and we now report preliminary experiments showing that this effect is abolished by treatment with cyclosporin A (Fig. 2a), indicating that it is likely mediated primarily by the Ca2+/CaM activation of calcineurin.


Figure 2. Calcineurin plays a role in gonadotrophin-releasing hormone (GnRH)-activated αT3-1 cell proliferation. (a) An XTT assay to assess cell numbers was carried out in αT3-1 cells after some were exposed to GnRH for 24 h, with or without 30 min cyclosporine A (CsA) pretreatment. Absorbance is expressed relative to that in nontreated (NT) cells without GnRH; mean ± SEM (n = 7–9). anova followed by a Bonferroni t-test compared all means and those that are similar (P > 0.05) are designated the same letter. (b) Cyclin D1 protein levels (with β-actin as internal control) were assessed by western analysis in αT3-1 cells treated with GnRH (10 nm) for 0–8 h. (c) Real-time quantitative polymerase chain reaction (PCR) analysis was performed to measure the effects of GnRH (8 h) with and without CsA pre-treatment (10 min before GnRH) on mRNA levels of Cdk4. Transcript levels were quantified using a standard curve and are represented relative to levels in untreated controls, after normalisation to levels of large ribosomal protein (RPLPO). Mean ± SEM (n = 3–4); statistical analysis as in Fig. 2(a). (d) Small interfering (si)RNA targeting β-catenin, or green fluorescent protein (GFP) as control (both in pSUPER) was transfected 48 h before GnRH treatment (3 h), after which RNA was extracted, reverse transcribed and PCR amplification carried out for cyclin D1 and β-actin. PCR products were resolved in agarose gel and the intensity of each band quantified relative to untreated siGFP-control, after normalisation with β-actin levels. Western analysis for the β-catenin knockdown is also shown. (e) The siRNA targeting β-catenin was transfected and, after 40 h, some cells were treated with GnRH (20 nm for 16 h) before a bromodeoxyuridine assay was performed to assess cell proliferation. Absorbance is expressed and the data are presented as in Fig 2(a); mean ± SEM (n = 4). (f, g) Real-time quantitative PCR analysis was carried out as in Fig. 2(c) to assess effects of CsA and/or GnRH on mRNA levels of (f) p27 and (g) p21. Experimental details and data presentation are as in Fig. 2(c). All experiments were carried out on at least three independent occasions and representative results are shown.

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It has been suggested that Ca2+ and CaM are required for the activation of cyclin D1/cdk4 and pRb hyperphosphorylation and, indeed, we show here in a pilot study that GnRH increases the expression of cyclin D1, although not cdk4 (Fig. 2b,c). Paradoxically, the cdk4 promoter is reportedly inhibited by calcineurin-regulated NFATc2, while calcineurin is also able to dephosphorylate cdk4 directly, leading to its inactivation [85, 86]. GnRH-induced NFAT activity does not appear to play a similar role in GnRH-activated proliferation of the immature gonadotrophs because these initial studies reveal that cdk4 expression is reduced by cyclosporin A but not affected by GnRH (Fig. 2c). Any role for GnRH-activated post-translational regulation of cdk4 activity via calcineurin remains to be investigated.

GnRH likely also affects cell cycle progression via its calcineurin-mediated dephosphorylation of Pin1, which regulates transcription of cyclin D1, as well as the accumulation of both cyclin D1 and β-catenin in the nucleus [78, 87-89]. Pin1 stimulates cyclin D1 gene expression via enhancing the activity of JNK, as well as stabilising c-jun, both of which are activated downstream of GnRH in a manner that is dependent on β-catenin [4, 52, 82, 90-92]. GnRH and Pin1 have been shown to stimulate translocation of β-catenin to the nucleus [91, 93], and we show here in a novel study that β-catenin plays a role in GnRH-induced cyclin D1 gene expression, as well as its induction of the proliferation of αT3-1 cells (Fig. 2d–e).

Additional anti-proliferative effects of calcineurin-inhibition, as shown in various cell types, are mediated via growth factor-mediated increases in p21 and/or the accumulation of p27, which are both cyclin-dependent kinase inhibitors (CKIs) that prevent activation of the cyclin/cdk complexes [25, 94]. In our preliminary studies, GnRH treatment led to a marginal increase in p27 and p21 levels in αT3-1 cells, and this was inhibited by cyclosporin A treatment (Fig. 2f-g), suggesting an involvement of calcineurin in their transcription, as seen previously in other cell contexts [95, 96]. At low levels, these CKIs may not suffice to inactivate the cyclin/cdk complex and may actually promote their activation through facilitating assembly of the complexes, increasing their nuclear import and by stabilising cyclin D [25].

In LβT2 cells, GnRH no longer induces cell proliferation but reduces cell numbers through inhibiting proliferation and/or inducing apoptosis [1, 6]. GnRH is seen to have anti-proliferative/pro-apoptotic effects in a variety of other cells as well, although, for the most part, the mechanisms have either not been elucidated or appear to vary between the different cell types [97-100]. We are currently investigating the mechanisms through which GnRH reduces cell number of the LβT2 cells, and the reasons for the apparent shift in response with differentiation. However, GnRH was reported to increase p21 mRNA levels quite significantly (three- to six-fold) in LβT2 cells [4, 63, 101], suggesting that this CKI induction might play a role in blocking cell cycle progression. GnRH was also shown to reduce expression of a number of the cyclins, cyclin-associated kinases and several subunits of the anaphase-promoting complex [4]. Although these cells were immortalised using the SV40 large T antigen, which could potentially alter their proliferative response, the vast majority of large T-responsive genes are not affected by GnRH and/or activin treatments [4], indicating that the presence of SV40 large T antigen is unlikely to confound these conclusions. Confirmation of this effect of GnRH in primary partially-differentiated embryonic gonadotroph cells in embryos is technically difficult, however GnRH was seen to increase gonadotrope proliferation in adult mice and rats under certain circumstances, including at specific stages of the oestrous cycle or after castration [102-104], suggesting a latent ability to proliferate in response to GnRH that can be moderated by other factors including steroids.

Calcineurin is clearly not the only way in which calcium influx stimulates an apoptotic response, which is driven in many cases by CaM-independent mechanisms via caspase activation. The Ca2+-influx into the mitochondria increases cytochrome C in the cytoplasm, allowing activation of the caspases and also of the nucleases required for the apoptotic response [16, 105-107]. However, other forms of cell death triggered by Ca2+-mobilisation are CaM-dependent, including the activation of death-associated protein kinase and AMPK, which are involved in autophagic cell death [108]. AMPK is activated in gonadotrophs in response to GnRH, although neither protein has yet been linked to cell death after GnRH treatment [29]. A specialised form of apoptosis, anoikis, involving detachment of cells from their surrounding matrix, is also regulated by Ca2+-CaM, in which changes in Ca2+ homeostasis lead to the activation of Rho kinase signalling [108]. Although the induction of this form of apoptosis by GnRH has yet to be reported, GnRH does induce major cytoskeletal rearrangement and gonadotroph cell migration, likely involving Rho signalling cascades [109, 110]. GnRH-induced Rho signalling may be regulated in part by a small GTP-binding protein, Gem, which is activated by GnRH and regulated by CaM, and was shown, in other contexts, to bind Rho kinase to alter downstream signalling [111].

A role for calmodulin in moderating GnRH-induced signalling through negative-feedback?

  1. Top of page
  2. Abstract
  3. Gonadotrophin-releasing hormone (GnRH), the gonadotroph and calmodulin
  4. CaMKs and gonadotrophin gene expression
  5. Diverse roles for calcineurin in the gonadotroph
  6. Effects of GnRH via Ca2+/CaM signalling on the cell cycle and gonadotroph proliferation
  7. A role for calmodulin in moderating GnRH-induced signalling through negative-feedback?
  8. Conclusions and future directions
  9. Acknowledgements
  10. References

Gem and its regulation by GnRH

Gem belongs to the RGK (comprising Rad, Gem and Kir, as well as Rem1 and Rem2) subfamily of small Ras-related GTP-binding proteins that moderate various G-protein coupled pathways. These differ structurally from other GTPases in that they do not contain lipid modification for anchorage to the membrane. Gem and other members of this group have several roles, including inhibiting Ca2+ channels, while Gem also affects rearrangement of the cytoskeleton through altering Rho signalling. The activity and localisation of Gem are regulated through CaM and 14-3-3 proteins [111, 112].

Gem is expressed at higher levels in αT3-1 than in LβT2 cells, while in the LβT2 cells, its mRNA levels increase after GnRH treatment [4, 6, 52]. Given that Gem inactivates the L-type VSCC that are utilised by GnRH for Ca2+ influx, it was suggested that Gem might reduce external calcium entry as part of a negative-feedback loop, so controlling pathways downstream of calcium [8, 52, 113]. Consistent with this, we found that Gem, which is initially found throughout αT3-1 cells, moves out of the nucleus after exposure to GnRH for 15–30 min (Fig. 3a). This effect appears to be independent of CaM because a mutant Gem that cannot bind CaM (W269G) [113] translocated to the cytoplasm in a manner similar to the wild-type protein (Fig. 3a). The export is likely therefore driven by GnRH-activated phosphorylation of Gem, which facilitates recognition by 14-3-3 proteins and can be targeted by a number of kinases, as reported in other cell contexts [111, 114, 115]. Both groups of cells also underwent morphological changes after exposure to GnRH (Fig. 3a), which has already been reported in these cells [5], and is in keeping with actions of Gem on cytoskeletal rearrangement [115].


Figure 3. Gonadotrophin-releasing hormone (GnRH)/calmodulin (CaM)-regulated Gem in gonadotrophs. (a) αT3-1 cells plated on glass cover-slips were co-transfected with expression vectors for yellow fluorescent protein-tagged histone deacetylase (HDAC) 7 together with enhanced green fluorescemt protein (EGFP)-tagged wild-type (WT) Gem (first and second columns) or EGFP-tagged W269G Gem mutant (third and fourth columns). After 48 h, GnRH in phenol-red free medium was added. Confocal images of cells were captured initially and after 15 and 30 min. A single representative cell is shown for each treatment/time point; scale bars = 10 μm. (b) αT3-1 cells were transfected with mouse glycoprotein hormone α-subunit promoter-luciferase reporter gene (αGSU), and WT Gem or the W269G mutant were overexpressed, and samples exposed to GnRH treatment (100 nm, 8 h). After 60 h, luciferase assays were performed, and levels normalised to those of Renilla; the results are expressed as the fold induction over the mouse αGSU-luciferase control-transfected and untreated cells. Statistical analysis (anova followed by Bonferroni t-test) compared mean values separately for untreated (upper case) and GnRH-treated (lower case) cells, and those designated by different letters are significantly different (P < 0.05); mean ± SEM (n = 5–6). (c) αT3-1 cells were transfected with mouse αGSU-luciferase (as above) and some with small interfering (si)RNA targeting Gem (in pSUPER). Some of the cells were treated with GnRH, luciferase assays performed, values normalised and presented all as above (n = 6). All experiments were carried out on at least three independent occasions and representative results are shown. Insert: western blot showing levels of Gem in αT3-1 cells 60 h after transfection of siGEM.

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Although GnRH regulates Gem localisation, our preliminary studies did not provide any evidence of Gem exerting a negative effect on gonadotrophin gene expression. GnRH-induced αGSU promoter activity in αT3-1 cells is highly dependent on calcium influx [60], yet, in our preliminary studies using αGSU-promoter-driven luciferase assays, over-expression of wild-type Gem did not reduce GnRH-stimulated promoter activity but, instead, had a minor stimulatory effect, although the W269G mutant reduced promoter activity by approximately 30% (Fig. 3b). Furthermore, small interfering RNA-mediated partial knockdown of Gem also had an inhibitory effect on the promoter activity (Fig. 3c). Taken together, these results indicate that Gem does not exert negative-feedback on the effect of GnRH but may perhaps play a novel positive role, possibly being regulated through its interaction with CaM in the cytoplasm.

RGS proteins

RGS proteins, which act as GTPase-activating proteins of Gα, including Gαq that transduces much of the effect of GnRH, have also been implicated in modulating GnRH-induced signalling and these too are regulated by CaM. RGS2 expression is increased by GnRH and it was proposed that the ability of RGS2 to down-regulate Gαq activity might contribute to the frequency-dependent downstream gene activation [8, 52]. In keeping with this, RGS2 over-expression in αT3-1 cells reduced GnRH signalling via Gαq/11, resulting in a marked reduction in phospholipase C activation, while also decreasing GnRH-induced mRNA levels of Egr-1 and c-fos [116, 117].

Although RGS repression of Gαq/11 can be inhibited by various kinases including PKC, as well as by phosphatidylinositol-3,4,5,-trisphosphate (PIP3), it is restored by Ca2+/CaM [118, 119]. It was suggested that, in cardiac myocytes, the cyclical inhibition of Gαq/11 is determined by CaM-stimulated RGS4, which inactivates Gαq/11 leading to a reduction in the formation of PIP3; this results in a drop in Ca2+ levels that leads to CaM dissociation from RGS4, after which PIP3-mediated repression of RGS4 is restored and once more inhibits Gαq/11 [118, 120]. Such a role for RGS2 in negative-feedback during GnRH-induced signalling in the gonadotroph appears feasible, although this has yet to be demonstrated.

Ca2+/CaM-sensitive GnRHR-ERK signalling complexes

RGS proteins are localised in membrane-associated lipid rafts where they interact with various scaffold proteins to concentrate these proteins with the G-protein coupled receptor for regulation of downstream signalling, and disruption of these rafts was seen to inhibit the interaction between RGS4 and CaM [118, 121-123]. In the gonadotrophs, compartmentalisation of a putative Ca2+-sensitive signalling complex into lipid rafts together with the GnRHR was proposed to integrate Ca2+ and ERK signalling and poise them for the GnRH-activated response. Both CaM and 14-3-3b colocalise in these membrane-associated lipid rafts together with the GnRHR, Gq and c-Raf kinase, and it was suggested that CaM serves as a link between the VSCC, Ca2+ and ERK [13, 53]. Indeed, inhibition of CaM blocks GnRH- induced ERK1/2 but not JNK activation in αT3-1 cells, and it also attenuated ERK-dependent gene reporter activity of c-fos, αGSU and MAPK phosphatase-2 promoters, in a manner that is at least partially CaMK-independent [53]. Although RGS2 has yet to examined in the context of these gonadotroph lipid rafts, it appears likely that it is part of this signalling complex, being localised and/or regulated in the rafts by CaM, to provide a mechanism of Ca2+-sensing in GnRH-induced ERK signalling.

Additional signalling complexes associated with ERK-activation by GnRH are expected also to be regulated by CaM-activated pathways [12, 13, 124-127]. GnRH stimulates the rapid assembly of an ERK-activating complex that contains the proline-rich tyrosine kinase, Pyk2, which serves as a scaffold for c-Src, Grb2 and Sos and is essential for ERK-dependent gene transcription. The catalytic domain of Pyk2 interacts directly with Ca2+-CaM, and this is required for its phosphorylation after GnRH exposure, as well as its functional activity. The transmission of the ERK-activating signal to the nucleus by the Pyk complex appears to be via focal adhesion complexes and is dependent on a functional actin cytoskeleton. Its role in ERK activation and translocation to the nucleus led to the suggestion that this complex also may form a link between VSCC Ca2+ signals and the ERK activation pathway [12, 13, 125, 126].

The GnRHR, c-Src, and focal adhesion kinase (FAK) are found localised together in an additional signalling complex, likely also in lipid rafts, that is restructured upon GnRH treatment. It was proposed that this complex acts to restrict the amount of active ERK from moving to the nucleus, and suggested that the ERK might direct phosphorylation of FAK and paxillin to mediate some of the GnRH-induced changes in cell morphology [127, 128]. GnRH has been shown to promote cytoskeletal rearrangement, cell morphology and adhesion, and Rho-kinase pathways were implicated in this effect [110]. It is an interesting speculation that Gem, which is regulated by GnRH and known to alter cell morphology by affecting Rho kinase substrate specificity, might play a role in this complex.

Conclusions and future directions

  1. Top of page
  2. Abstract
  3. Gonadotrophin-releasing hormone (GnRH), the gonadotroph and calmodulin
  4. CaMKs and gonadotrophin gene expression
  5. Diverse roles for calcineurin in the gonadotroph
  6. Effects of GnRH via Ca2+/CaM signalling on the cell cycle and gonadotroph proliferation
  7. A role for calmodulin in moderating GnRH-induced signalling through negative-feedback?
  8. Conclusions and future directions
  9. Acknowledgements
  10. References

GnRH has far-reaching effects on the gonadotrophs beyond merely inducing release and synthesis of the gonadotrophin hormones, and CaM-driven pathways, which are activated in response to increases in Ca2+, clearly play a major role in many of these effects, as highlighted in this review. Although we have focused on recent literature describing the effects of calcineurin and CaMKs, this is likely only a subset of the pathways activated downstream of CaM in response to GnRH. Hundreds of proteins are known to be bound by CaM, including various DEAD/H box proteins and enzymes involved in protein ubiquitination and degradation, and there is particularly strong evidence for its dominant cytoplasmic roles in both signalling and vesicular trafficking [129-131]. However, CaM also translocates to the nucleus in response to various stimuli, and although its nuclear functions have barely been studied, it appears to be involved in signalling to transcription; in neuronal cells, it was suggested to provide a cellular ‘memory’ in facilitating repeated neural activity [132]. Moreover, a novel role for CaM was demonstrated recently, in which it binds the signal peptides of small secretory proteins, and acts as a chaperone for their translocation through the cytosol and into the endoplasmic reticulum [133]. We have likely only just begun to understand the role of CaM in GnRH signalling in the gonadotroph.


  1. Top of page
  2. Abstract
  3. Gonadotrophin-releasing hormone (GnRH), the gonadotroph and calmodulin
  4. CaMKs and gonadotrophin gene expression
  5. Diverse roles for calcineurin in the gonadotroph
  6. Effects of GnRH via Ca2+/CaM signalling on the cell cycle and gonadotroph proliferation
  7. A role for calmodulin in moderating GnRH-induced signalling through negative-feedback?
  8. Conclusions and future directions
  9. Acknowledgements
  10. References
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