Activation of the luteinizing hormone beta promoter by gonadotropin-releasing hormone requires c-Jun NH2-terminal protein kinase

Regulation of the mitogen-activated protein kinase (MAPK) family by gonadotropin-releasing hormone (GnRH) in the gonadotrope cell line LbetaT2 was investigated. Treatment with gonadotropin-releasing hormone agonist (GnRHa) activates extracellular signal-regulated kinase (ERK) and c-Jun NH(2)-terminal kinase (JNK). Activation of ERK by GnRHa occurred within 5 min, and declined thereafter, whereas activation of JNK by GnRHa occurred with a different time frame, i.e. it was detectable at 5 min, reached a plateau at 30 min, and declined thereafter. GnRHa-induced ERK activation was dependent on protein kinase C or extracellular and intracellular Ca(2+), whereas GnRHa-induced JNK activation was not dependent on protein kinase C or on extracellular or intracellular Ca(2+). To determine whether a mitogen-activated protein kinase family cascade regulates rat luteinizing hormone beta (LHbeta) promoter activity, we transfected the rat LHbeta (-156 to +7)-luciferase construct into LbetaT2 cells. GnRH activated the rat LHbeta promoter activity in a time-dependent manner. Neither treatment with a mitogen-activated protein kinase/ERK kinase (MEK) inhibitor, PD98059, nor cotransfection with a catalytically inactive form of a mitogen-activated protein kinase construct inhibited the induction of the rat LHbeta promoter by GnRH. Furthermore, cotransfection with a dominant negative Ets had no effect on the response of the rat LHbeta promoter to GnRH. On the other hand, cotransfection with either dominant negative JNK or dominant negative c-Jun significantly inhibited the induction of the rat LHbeta promoter by GnRH. In addition, GnRH did not induce either the rat LHbeta promoter activity in LbetaT2 cells transfected stably with dominant negative c-Jun. These results suggest that GnRHa differentially activates ERK and JNK, and a JNK cascade is necessary to elicit the rat LHbeta promoter activity in a c-Jun-dependent mechanism in LbetaT2 cells.     

Activation of luteinizing hormone beta gene by gonadotropin-releasing hormone requires the synergy of early growth response-1 and steroidogenic factor-1.

We have previously shown that early growth response (Egr) 1-deficient mice exhibit female infertility, reflecting a luteinizing hormone (LH) beta deficiency. Egr-1 activates the LHbeta gene in vitro through synergy with steroidogenic factor-1 (SF-1), a protein required for gonadotrope function. To test if this synergy is essential for gonadotropin-releasing hormone (GnRH) stimulation of LHbeta, we examined the activity of the LHbeta promoter in the gonadotrope cell line LbetaT2. GnRH markedly stimulated the LHbeta promoter (15-fold). Mutation of either Egr-1 or SF-1 elements within the LHbeta promoter attenuated this stimulation, whereas mutation of both promoter elements abrogated GnRH induction of the LHbeta promoter. Furthermore, GnRH stimulated Egr-1 but not SF-1 expression in LbetaT2 cells. Importantly, overexpression of Egr-1 alone was sufficient to enhance LHbeta expression. Although other Egr proteins are expressed in LbetaT2 cells and are capable of interacting with SF-1, GnRH stimulation of Egr-1 was the most robust. We also found that the nuclear receptor DAX-1, a repressor of SF-1 activity, reduced Egr-1-SF-1 synergy and diminished GnRH stimulation of the LHbeta promoter. We conclude that the synergy between Egr-1 and SF-1 is essential for GnRH stimulation of the LHbeta gene and plays a central role in the dynamic regulation of LHbeta expression.     

GnRH activates ERK1/2 leading to the induction of c-fos and LHbeta protein expression in LbetaT2 cells

GnRH acts on pituitary gonadotropes to stimulate the synthesis and release of LH and FSH. However, the signaling pathways downstream of the GnRH receptor that mediate these effects are not fully understood. In this paper, we demonstrate that GnRH activates ERK, c-Jun N-terminal kinase, and p38MAPK in the LbetaT2 gonadotrope cell line. Phosphorylation of both ERK and p38MAPK are stimulated rapidly, 30- to 50-fold in 5 min, but activation of c-Jun N-terminal kinase has slower kinetics, reaching only 10-fold after 30 min. Activation of ERK by GnRH is blocked by inhibition of MAPK kinase (MEK) and partially blocked by inhibition of PKC and calcium, but not PI3K or p38MAPK signaling. We demonstrate that phosphorylated ERK accumulates in the nucleus in a PKC-dependent manner. We also show that GnRH induces c-fos and LHbeta subunit protein expression in LbetaT2 cells via MEK. Experiments with EGTA or calcium channel antagonists indicated that calcium influx is important for the induction of both genes by GnRH. In conclusion, these results show that GnRH activates all three MAPK subfamilies in LbetaT2 cells and induces c-fos and LHbeta protein expression through calcium and MEK-dependent mechanisms. These results also demonstrate that the nuclear translocation of ERK by GnRH requires PKC signaling.     

Activation of translation in pituitary gonadotrope cells by gonadotropin-releasing hormone

The neuropeptide GnRH is a central regulator of mammalian reproductive function produced by a dispersed population of hypothalamic neurosecretory neurons. The principal action of GnRH is to regulate release of the gonadotropins, LH and FSH, by the gonadotrope cells of the anterior pituitary. Using a cultured cell model of mouse pituitary gonadotrope cells, alphaT3-1 cells, we present evidence that GnRH stimulation of alphaT3-1 cells results in an increase in cap-dependent mRNA translation. GnRH receptor activation results in increased protein synthesis through a regulator of mRNA translation initiation, eukaryotic translation initiation factor 4E-binding protein, known as 4EBP or PHAS (protein, heat, and acid stable). Although the GnRH receptor is a member of the rhodopsin-like family of G protein-linked receptors, we show that activation of translation proceeds through a signaling pathway previously described for receptor tyrosine kinases. Stimulation of translation by GnRH is protein kinase C and Ras dependent and sensitive to rapamycin. Furthermore, GnRH may also regulate the cell cycle in alphaT3-1 cells. The activation of a signaling pathway that regulates both protein synthesis and cell cycle suggests that GnRH may have a significant role in the maintenance of the pituitary gonadotrope population in addition to directing the release of gonadotropins.     

Induction of PER1 mRNA expression in immortalized gonadotropes by gonadotropin-releasing hormone (GnRH): involvement of protein kinase C and MAP kinase signaling

The initiation and maintenance of reproductive function in mammals is critically dependent on the pulsatile secretion of gonadotropin-releasing hormone (GnRH). This peptide drives the pulsatile release of FSH and LH from the pituitary pars distalis via signaling pathways that are activated by the type I GnRH receptor (GnRH-R). Recently, a microarray analysis study reported that a number of genes, including mPer1, are induced by GnRH in immortalized gonadotrope cells. In view of these data, we have begun to analyze in detail the signaling pathways mediating the action of GnRH on mPer1 expression in these cells. Using quantitative real-time polymprose cho read (PCR), we could confirm that exposure of immortalized gonadotropes (LbetaT2 cells) to the GnRH analog, buserelin, markedly induces mPer1 (but not mPer2) expression. Consistent with GnRH receptor signaling via the protein kinase (PK)-C pathway, exposure of the cells to phorbol 12,13-dibutyrate rapidly elevates both mPer1 and LHbeta subunit mRNA levels, while pharmacological inhibition of PKC prevents the mPer1 and LHbeta response to buserelin. As GnRH is known to regulate gonadotropin synthesis via activation of p42/44 mitogen-activated protein kinase (MAPK) signaling pathways, we then examined the involvement of this pathway in regulating mPer1 expression in gonadotropes. Our data reveal that GnRH-induced mPer1 expression is blocked following acute exposure to a MAPK kinase inhibitor. Although the involvement of this signaling mechanism in the regulation of mPer1 is known in neurons, e.g., in the suprachiasmatic nuclei, the induction of mPer1 in gonadotropes represents a novel mechanism of GnRH signaling, whose functional significance is still under investigation.     

Identification of a novel Comamonas testosteroni gene encoding a steroid-inducible extradiol dioxygenase

Insulin-like growth factor-1 (IGF-1) both promotes survival and activates protein synthesis in neurons. In the present paper, we investigate the effect of IGF-1 treatment on cap-dependent translation in primary cultured neuronal cells. IGF-1 treatment increased the phosphorylation of eukaryotic initiation factor (eIF)-4E-binding protein 1 (4E-BP1), exclusively at Thr-36 and Thr-45 residues, and eIF-4G phosphorylation at Ser-1108. In contrast, a significant eIF-4E dephosphorylation was found. In parallel, increased eIF-4E/4G assembly and protein synthesis activation in response to IGF-1 treatment were observed. The phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin and the mammalian target of rapamycin (mTOR) inhibitor rapamycin, but not the mitogen-activated protein kinase (MAPK)-activating kinase (MEK) inhibitor PD98059, reversed the IGF-1-induced effects observed on eIF-4E/4G assembly and phosphorylation status of 4E-BP1, eIF-4E, and eIF-4G. Therefore, our findings show that the IGF-1-induced regulation of cap-dependent translation is largely dependent on the PI-3K and mTOR pathway in neuronal cells.     

Phosphorylation of 4E-BP1 is mediated by the p38/MSK1 pathway in response to UVB irradiation.

In resting cells, eIF4E-binding protein 1 (4E-BP1) binds to the eukaryotic initiation factor-4E (eIF-4E), preventing formation of a functional eIF-4F complex essential for cap-dependent initiation of translation. Phosphorylation of 4E-BP1 dissociates it from eIF-4E, relieving the translation block. Studies suggested that insulin- or growth factor-induced 4E-BP1 phosphorylation is mediated by phosphatidylinositol 3-kinase (PI3-kinase) and its downstream protein kinase, Akt. In the present study we demonstrated that UVB induced 4E-BP1 phosphorylation at multiple sites, Thr-36, Thr-45, Ser-64, and Thr-69, leading to dissociation of 4E-BP1 from eIF-4E. UVB-induced phosphorylation of 4E-BP1 was blocked by p38 kinase inhibitors, PD169316 and SB202190, and MSK1 inhibitor, H89, but not by mitogen-activated protein kinase kinase inhibitors, PD98059 or U0126. The PI3-kinase inhibitor, wortmannin, did not block UVB-induced 4E-BP1 phosphorylation, but blocked both UVB- and insulin-induced activation of PI3-kinase and phosphorylation of Akt. 4E-BP1 phosphorylation was blocked in JB6 Cl 41 cells expressing a dominant negative p38 kinase or dominant negative MSK1, but not in cells expressing dominant negative ERK2, JNK1, or PI3-kinase p85 subunit. Our results suggest that UVB induces phosphorylation of 4E-BP1, leading to the functional dissociation of 4E-BP1 from eIF-4E. The p38/MSK1 pathway, but not PI3-kinase or Akt, is required for mediating the UVB-induced 4E-BP1 phosphorylation.     

Effects of peroxisome proliferator-activated receptor alpha (PPARalpha) agonists on leucine-induced phosphorylation of translational targets in C2C12 cells

Effect of peroxisome proliferator-activated receptor alpha (PPARalpha) agonists, WY-14,643 (WY) and/or clofibrate, on the leucine-induced phosphorylation of translational targets in C2C12 myoblasts was studied. C2C12 cells were treated with WY or clofibrate for 24 h prior to stimulation with leucine. Western blot analyses revealed that the leucine-induced phosphorylation of p70 S6 kinase (p70S6K), a key regulator of translation initiation, was significantly higher in WY-treated cells than in control and clofibrate-treated cells. Phosphorylation of extracellular-regulated kinase (ERK1/2) was higher in WY-treated cells. WY treatment also increased the leucine-induced phosphorylation of ribosomal protein S6 and eukaryotic initiation factor 4B. In contrast, eukaryotic elongation factor 2, a marker for peptide chain elongation process, was significantly activated (dephosphorylated) only in leucine-stimulated control cells. Pre-treatment of the cells with PD98059 (ERK1/2 kinase inhibitor) prevented the phosphorylation of ERK1/2 and decreased the leucine-induced phosphorylation of p70S6K. It is concluded that WY increased the leucine-induced phosphorylation of target proteins involving in translation initiation via ERK/p70S6K pathway, but impaired the signaling for elongation process, suggesting that p70S6K phosphorylation may be essential, but not sufficient for the activation of entire targets for protein translation in WY-treated cells.     

Effects of peroxisome proliferator-activated receptor α (PPARα) agonists on leucine-induced phosphorylation of translational targets in C2C12 cells

Effect of peroxisome proliferator-activated receptor α (PPARα) agonists, WY-14,643 (WY) and/or clofibrate, on the leucine-induced phosphorylation of translational targets in C2C12 myoblasts was studied. C2C12 cells were treated with WY or clofibrate for 24 h prior to stimulation with leucine. Western blot analyses revealed that the leucine-induced phosphorylation of p70 S6 kinase (p70S6K), a key regulator of translation initiation, was significantly higher in WY-treated cells than in control and clofibrate-treated cells. Phosphorylation of extracellular-regulated kinase (ERK1/2) was higher in WY-treated cells. WY treatment also increased the leucine-induced phosphorylation of ribosomal protein S6 and eukaryotic initiation factor 4B. In contrast, eukaryotic elongation factor 2, a marker for peptide chain elongation process, was significantly activated (dephosphorylated) only in leucine-stimulated control cells. Pre-treatment of the cells with PD98059 (ERK1/2 kinase inhibitor) prevented the phosphorylation of ERK1/2 and decreased the leucine-induced phosphorylation of p70S6K. It is concluded that WY increased the leucine-induced phosphorylation of target proteins involving in translation initiation via ERK/p70S6K pathway, but impaired the signaling for elongation process, suggesting that p70S6K phosphorylation may be essential, but not sufficient for the activation of entire targets for protein translation in WY-treated cells.