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.     

Pulse sensitivity of the luteinizing hormone beta promoter is determined by a negative feedback loop Involving early growth response-1 and Ngfi-A binding protein 1 and 2

The hypothalamic-pituitary-gonadal endocrine axis regulates reproduction through estrous phase-dependent release of the heterodimeric gonadotropic glycoprotein hormones, LH and FSH, from the gonadotropes of the anterior pituitary. Gonadotropin synthesis and release is dependent upon pulsatile stimulation by the hypothalamic neuropeptide GnRH. Alterations in pulse frequency and amplitude alter the relative levels of gonadotropin synthesis and release. The mechanism of interpretation of GnRH pulse frequency and amplitude by gonadotropes is not understood. We have examined gene expression in LbetaT2 gonadotropes under various pulse regimes in a cell perifusion system by microarray and identified 1127 genes activated by tonic or pulsatile GnRH. Distinct patterns of expression are associated with each pulse frequency, but the greatest changes occur at a 60-min or less interpulse interval. The immediate early gene mRNAs encoding early growth response (Egr)1 and Egr2, which activate the gonadotropin LH beta-subunit gene promoter, are stably induced at high pulse frequency. In contrast, mRNAs for the Egr corepressor genes Ngfi-A binding protein Nab1 and Nab2 are stably induced at low pulse frequency. We show that Ngfi-A binding protein members inhibit Egr-mediated frequency-dependent induction of the LH beta-subunit promoter. This pattern of expression suggests a model of pulse frequency detection that acts by suppressing activation by Egr family members at low frequency and allowing activation at sustained high-frequency pulses.     

The equine luteinizing hormone beta-subunit promoter contains two functional steroidogenic factor-1 response elements.

The requirements for basal expression of the LH beta-subunit promoter in pituitary gonadotropes are largely unknown. We have used the equine (e) LHbeta subunit promoter as a model to unravel the combinatorial code required for gonadotrope expression. Through the use of 5'-deletion mutagenesis, a region between -185 and -100 of the eLHbeta promoter was shown to play a critical role in maintaining basal promoter activity in alphaT3-1 and LbetaT2 cells. This region encompasses the steroidogenic factor-1 (SF-1) binding site that has been reported to have a functional role in expression of the LHbeta promoter in other species. We have also identified an additional SF-1 site at -55 to -48. Binding of SF-1 to both sites was confirmed by electrophoretic mobility shift assays. Mutations within these sites, either individually or in combination, did not attenuate basal activity of the eLHbeta promoter in alphaT3-1 cells, but did diminish promoter activity in LbetaT2 cells. Interestingly, cotransfection with an expression vector encoding SF-1 induced eLHbeta promoter activity, and this induction was abrogated by mutations within the SF-1 sites in alphaT3-1 cells. Block replacement mutagenesis was performed on the -185/-100 region of the eLHbeta promoter to identify DNA response elements responsible for maintaining basal promoter activity. From this analysis, two regions emerged as being important: a distal 31-bp segment (-181 to -150) and an element located immediately 3' to the distal SF-1 site (-119 to -106). It is hypothesized that these two regions as well as the SF-1 sites represent regulatory elements that contribute to a combinatorial code involved in targeting expression of the eLHbeta promoter to gonadotropes.     

Effects of gonadotropin-releasing hormone analog (GnRHa) on steroidogenic factor-1 (SF-1) and estrogen receptor beta (ERbeta) gene expression in the black porgy (Acanthopagrus schlegeli)

We examined effects of GnRHa on expression of steroidogenic factor-1 (SF-1) and estrogen receptor beta (ERbeta) in the pituitary and gonad of protandrous black porgy (Acanthopagrus schlegeli). Fish were intraperitoneally injected with 0.2 microg GnRHa/g fish and then pituitary, gonad and plasma were sampled at 0, 6, 12, 24 and 48 h after injection. In gonad, the mRNA levels of the SF-1 were high at 6 h post injection, and then continuously decreased until 24 h; high expression of ERbeta mRNA levels was only observed at 12 h. In contrast, pituitary SF-1 mRNA levels were very low during the experimental period. GnRHa stimulation caused a significant increase of plasma testosterone (T) and estradiol-17beta (E(2)) after 24 h. We suggest that SF-1 and ERbeta play an important role in the development of gonad and these genes are involved with sex change in fish.     

A novel AP-1 site is critical for maximal induction of the follicle-stimulating hormone beta gene by gonadotropin-releasing hormone

Regulation of follicle-stimulating hormone (FSH) synthesis is a central point of convergence for signals controlling reproduction. The FSHbeta subunit is primarily regulated by gonadotropin-releasing hormone (GnRH), gonadal steroids, and activin. Here, we identify elements in the mouse FSHbeta promoter responsible for GnRH-mediated induction utilizing the LbetaT2 cell line that endogenously expresses FSH. The proximal 398 bp of the mouse FSHbeta promoter is sufficient for response to GnRH. This response localizes primarily to an AP-1 half-site (-72/-69) juxtaposed to a CCAAT box, which binds nuclear factor-Y. Both elements are required for AP-1 binding, creating a novel AP-1 site. Multimers of this site confer GnRH induction, and mutation or internal deletion of this site reduces GnRH induction by 35%. The same reduction was achieved using a dominant negative Fos protein. This is the only functional AP-1 site identified in the proximal 398 bp, since its mutation eliminates FSHbeta induction by c-Fos and c-Jun. GnRH regulation of the FSHbeta gene occurs through induction of multiple Fos and Jun isoforms, forming at least four different AP-1 molecules, all of which bind to this site. Mitogen-activated protein kinase activity is required for induction of FSHbeta and JunB protein. Finally, AP-1 interacts with nuclear factor-Y, which occupies its overlapping site in vivo.     

Activation of myogenesis by the homeobox gene Lbx1 requires cell proliferation.

Myogenic differentiation can be initiated by a limited number of molecules. In this work, we analyzed the function of the homeobox gene Lbx1 in chicken embryos and explant cultures. We demonstrate that overexpression of Lbx1 in vivo and in vitro leads to a strong activation of various muscle markers. We show that cell proliferation, which is strongly stimulated by Lbx1 and Pax3, is required for Lbx1- or Pax3-dependent myogenic activation. Inhibition of cell proliferation prevents expression of muscle differentiation markers, while the activation of other putative downstream targets of Pax3 and Lbx1 is not affected. Our findings imply that a critical function of Pax3 and Lbx1 during muscle cell formation is the enlargement of muscle cell populations. The growth of the muscle precursor cell population may increase the bias for myogenic differentiation and thus enable myogenic cells to respond to environmental cues.     

Activation of neutral sphingomyelinase by IL-1beta requires the type 1 interleukin 1 receptor

The cytokine interleukin 1beta (IL-1beta) plays an important role in host defence reactions and neuro-immune interactions but it is still not clear which of the two interleukin 1 receptor subtypes is coupled to activation of neutral sphingomyelinase (nSMase) by IL-1beta. To investigate involvement of neutral sphingomyelinase (nSMase) in central IL-1beta effects we used P(2)fractions of brain cerebral cortex from wild-type mice and mice deficient in the type 1 IL-1 receptor. IL-1beta (human, recombinant) was shown to activate, in a dose-dependent manner, nSMase in the P(2)brain fraction of the wild-type mice while in the knock-out mice the stimulatory effect of IL-1beta on nSMase was absent. In the presence of an IL-1 receptor antagonist (IL-1ra), IL-1beta did not activate nSMase either in the cortex of wild-type or knock-out mice. These data suggest that nSMase, a key enzyme of the sphingomyelin signal transduction pathway, might be involved in IL-1beta signalling in the brain and that activation of the enzyme requires the IL-1 receptor type 1.     

Modulation of luteinizing hormone subunit gene expression by intracerebroventricular microinjection of gonadotropin-releasing hormone or beta-endorphin in female rats

The effects of gonadotropin-releasing hormone (GnRH), beta-endorphin and its antagonist naloxone on the expression of luteinizing hormone (LH) subunit genes and LH secretion were examined in ovariectomized and/or cycling female rats through their direct microinjection into the third cerebral ventricle, in the proximity of the hypothalamus-pituitary complex. GnRH (1 nM) induced a significant augmentation of the pituitary content of alpha mRNA when administered 15, 30 or 60 min intervals over 5 h to ovariectomized rats whereas only the 30 and 60 min intervals were effective in increasing LHbeta mRNA, and the 60 min intervals for LH release. This was in agreement with the established concept of a pulse-dependent regulation of gonadotropin synthesis and release. Hourly pulses of GnRH also increased alpha and LHbeta mRNA levels when microinjected in female cycling rats during proestrus or diestrus II. Using this model we observed a marked negative influence of hourly intracerebral microinjections of beta-endorphin on LH mRNA content and LH release in ovariectomized rats while naloxone had no effect. This suggests that endogenous beta-endorphin was unable to exert its negative action on beta-endorphin receptors that were present and responded to the ligand. The present approach would be valuable for the exploration of the mechanisms of action of beta-endorphin or other substances on the functions of the gonadotrophs.     

Supplementation of equine early spring transitional follicles with luteinizing hormone stimulates follicle growth but does not restore steroidogenic activity

This study was conducted to test the hypothesis that supplementation of growing follicles with LH during the early spring transitional period would promote the development of steroidogenically active, dominant follicles with the ability to respond to an ovulatory dose of hCG. Mares during early transition were randomly assigned to receive a subovulatory dose of equine LH (in the form of a purified equine pituitary fraction) or saline (transitional control; n = 7 mares per group) following ablation of all follicles >15 mm. Treatments were administered intravenously every 12 h from the day the largest follicle of the post-ablation wave reached 20 mm until a follicle reached >32 mm, when an ovulatory dose of hCG (3000 IU) was given. Saline-treated mares during June and July were used as ovulatory controls. In a preliminary study, injection of this pituitary fraction (eLH) to anestrus mares was followed by an increase in circulating levels of LH (P < 0.01) but not FSH (P > 0.6). Administration of eLH during early transition stimulated the growth of the dominant follicle (Group x Day, P < 0.00001), which attained diameters similar to the dominant follicle in ovulatory controls (P > 0.1). In contrast, eLH had no effect on the diameter of the largest subordinate follicle or the number of follicles >10 mm during treatment (P > 0.3). The numbers of mares that ovulated in response to hCG in transitional control, transitional eLH and ovulatory control groups (2 of 2, 3 of 5 and 7 of 7, respectively) were not significantly different (P > 0.1). However, after hCG-induced ovulation, all transitional mares returned to an anovulatory state. Circulating estradiol levels increased during the experimental period in ovulatory controls but not in transitional eLH or transitional control groups (Group x Day, P = 0.013). In addition, although progesterone levels increased after ovulation in transitional control and transitional eLH groups, levels in these two groups were lower than in the ovulatory control group after ovulation (Group, P = 0.045). In conclusion, although LH supplementation of early transitional waves beginning after the largest follicle reached 20 mm promoted growth of ovulatory-size follicles, these follicles were developmentally deficient as indicated by their reduced steroidogenic activity.     

Regulation of the biopotency of primate luteinizing hormone by gonadotropin-releasing hormone in vitro and in vivo

Gonadotropin biological/immunological (B/I) ratios have proven to be valuable indicators of the biopotencies of LH and FSH. Observations of rapidly changing LH B/I have been made which suggest the existence of a readily mobilized pool of highly bioactive pituitary gonadotropins. To test this hypothesis, we have examined the role of GnRH in the regulation of LH B/I in vivo and in vitro. The rhesus monkey was used as a model due to its many physiological similarities with the human. A rapid elevation in circulating LH B/I was observed following GnRH administration to male monkeys that was sustained for at least 2 h (15 min; p less than 0.05). The administration of 1 or 10 nM GnRH to cultured pituitary cells was found to significantly increase the B/I of secreted, but not intracellular, LH (p less than 0.05). In unstimulated controls, the B/I of intracellular LH was higher than that of secreted LH (p less than 0.05). These findings are consistent with the notion that a pool of highly active LH exists within the gonadotrophs in primates. One way that GnRH may regulate the bioactivity of circulating LH is by rapidly mobilizing this gonadotropin pool.