Delayed activation of phospholipase D by gonadotropin-releasing hormone in a clonal pituitary gonadotrope cell line (αT3-1)

AbstractStimulation of cultured pituitary cells from a gonadotrope lineage (αT3-1) by the gonadotropin-releasing hormone agonist analog [D-Trp6]GnRH (GnRH-A) resulted in a manifold increase in accumulation of phosphatidylethanol, a specific product of phospholipase D phosphatidyl transferase activity when ethanol is the phosphatidyl group acceptor. Levels of the natural lipid product of phospholipase D, phosphatidic acid, were increased 2–3-fold. Activation of phospholipase D by GnRH-A was dose- and time-dependent and was blocked by a GnRH receptor antagonist [D-pClPhe2,D-Trp3.6]GnRH. GnRH-A stimulated phospholipase D activity after a lag of 1–2 min. We conclude that in αT3-1 gonadotropes GnRH receptor occupancy results in delayed activation of phospholipase D which could participate in late phases of gonadotrope regulation by the neurohormone

Gq-Induced Apoptosis is Mediated by AKT Inhibition That Leads to PKC-Induced JNK Activation

Background/Aims: Gq protein-coupled receptors (GqPCRs) regulate various cellular processes including mainly proliferation and differentiation. In a previous study, we found that in prostate cancer cells, the GqPCR of GnRH induces apoptosis by reducing the PKC-dependent AKT activity and elevating JNK phosphorylation. Since it was thought that GqPCR induces mainly activation of AKT, we undertook to examine how general is this phenomenon and understand its signaling. Methods: We used various cells to follow the phosphorylation of signaling components using western blotting. Results: In a screen of 21 cell lines, we found that PKC activation results in the reduction of AKT activity, which correlates nicely to JNK activation and in some cases to apoptosis. To further understand the signaling pathways involved in this stimulation, we studied in detail the SVOG-4O and αT3-1 cells. We found that PGF2α and GnRH agonist (GnRH-a) indeed induce significant Gq- and PKC- dependent apoptosis in these cells. This is mediated by two signaling branches downstream of PKC, which converge at the level of MLK3 upstream of JNK. One branch consists on c-Src activation of the JNK cascade and the second involves reduction of AKT activity that alleviates its inhibitory effect on MLK3, to allow the flow of the c-Src signal to JNK. At the MAPKK level, we found that the signal is transmitted by MKK7 and not MKK4. Conclusion: Our results present a general mechanism that mediates a GqPCR-induced, death receptors-independent, apoptosis in physiological, as well as cancer-related systems

Proline-rich tyrosine kinase 2 mediates gonadotropin-releasing hormone signaling to a specific extracellularly regulated kinase-sensitive transcriptional locus in the luteinizing hormone beta-subunit gene

G protein-coupled receptor regulation of gene transcription primarily occurs through the phosphorylation of transcription factors by MAPKs. This requires transduction of an activating signal via scaffold proteins that can ultimately determine the outcome by binding signaling kinases and adapter proteins with effects on the target transcription factor and locus of activation. By investigating these mechanisms, we have elucidated how pituitary gonadotrope cells decode an input GnRH signal into coherent transcriptional output from the LH β-subunit gene promoter. We show that GnRH activates c-Src and multiple members of the MAPK family, c-Jun NH(2)-terminal kinase 1/2, p38MAPK, and ERK1/2. Using dominant-negative point mutations and chemical inhibitors, we identified that calcium-dependent proline-rich tyrosine kinase 2 specifically acts as a scaffold for a focal adhesion/cytoskeleton-dependent complex comprised of c-Src, Grb2, and mSos that translocates an ERK-activating signal to the nucleus. The locus of action of ERK was specifically mapped to early growth response-1 (Egr-1) DNA binding sites within the LH β-subunit gene proximal promoter, which was also activated by p38MAPK, but not c-Jun NH(2)-terminal kinase 1/2. Egr-1 was confirmed as the transcription factor target of ERK and p38MAPK by blockade of protein expression, transcriptional activity, and DNA binding. We have identified a novel GnRH-activated proline-rich tyrosine kinase 2-dependent ERK-mediated signal transduction pathway that specifically regulates Egr-1 activation of the LH β-subunit proximal gene promoter, and thus provide insight into the molecular mechanisms required for differential regulation of gonadotropin gene expression

A novel angiogenic role for prostaglandin F2alpha-FP receptor interaction in human endometrial adenocarcinomas

Prostaglandins have been implicated in several neovascular diseases. In the present study, we found elevated FP receptor and vascular endothelial growth factor (VEGF) expression colocalized in glandular epithelial and vascular cells lining the blood vessels in endometrial adenocarcinomas. We investigated the signaling pathways activated by the FP receptor and their role in modulating VEGF expression in endometrial adenocarcinoma (Ishikawa) cells. Ishikawa cells were stably transfected with FP receptor cDNA in the sense or antisense orientations. Treatment of Ishikawa cells with prostaglandin F(2α) (PGF(2α)) rapidly induced transphosphorylation of the epidermal growth factor receptor (EGFR) and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 via the FP receptor. Activation of EGFR-Ras-mitogen-activated protein kinase/ERK kinase (MEK) signaling via the FP receptor resulted in an increase in VEGF promoter activity, expression of VEGF mRNA, and secretion of VEGF protein. These effects of PGF(2α) on the FP receptor could be abolished by treatment of cells with a specific FP receptor antagonist, chemical inhibitors of c-Src, matrix metalloproteinase, and EGFR kinase or by inactivation of signaling with dominant-negative mutant isoforms of EGFR, Ras, or MEK or with small inhibitory RNA oligonucleotides targeted against the EGFR. Finally, we confirmed that PGF(2α) could potentiate angiogenesis in endometrial adenocarcinoma explants by transactivation of the EGFR and induction of VEGF mRNA expression

Reciprocal cross talk between gonadotropin-releasing hormone (GnRH) and prostaglandin receptors regulates GnRH receptor expression and differential gonadotropin secretion

The asynchronous secretion of gonadotrope LH and FSH under the control of GnRH is crucial for ovarian cyclicity but the underlying mechanism is not fully resolved. Because prostaglandins (PG) are autocrine regulators in many tissues, we determined whether they have this role in gonadotropes. We first demonstrated that GnRH stimulates PG synthesis by induction of cyclooxygenase-2, via the protein kinase C/c-Src/phosphatidylinositol 3′-kinase/MAPK pathway in the LβT2 gonadotrope cell line. We then demonstrated that PGF(2α) and PGI(2), but not PGE(2) inhibited GnRH receptor expression by inhibition of phosphoinositide turnover. PGF(2α), but not PGI(2) or PGE(2), reduced GnRH-induction of LHβ gene expression, but not the α-gonadotropin subunit or the FSHβ subunit genes. The prostanoid receptors EP1, EP2, FP, and IP were expressed in rat gonadotropes. Incubations of rat pituitaries with PGF(2α), but not PGI(2) or PGE(2), inhibited GnRH-induced LH secretion, whereas the cyclooxygenase inhibitor, indomethacin, stimulated GnRH-induced LH secretion. None of these treatments had any effect on GnRH-induced FSH secretion. The findings have thus elaborated a novel GnRH signaling pathway mediated by PGF(2α)-FP and PGI(2)-IP, which acts through an autocrine/paracrine modality to limit autoregulation of the GnRH receptor and differentially inhibit LH and FSH release. These findings provide a mechanism for asynchronous LH and FSH secretions and suggest the use of combination therapies of GnRH and prostanoid analogs to treat infertility, diseases with unbalanced LH and FSH secretion and in hormone-dependent diseases such as prostatic cancer

Gonadotropin-Releasing Hormone Receptor Signaling and Functions

This eBook provides a comprehensive overview of our current knowledge on Gonadotropin-releasing hormone receptor evolution, structure, signaling and functions. Apart from review articles, it comprises exciting new research, as well as hypotheses and perspectives, all of which are valuable in guiding our further research in this field

Hypothalamic and Thyroidal Regulation of Growth Hormone in Tilapia

A radioimmunoassay (RIA) for recombinant tilapia growth hormone (GH) was established and validated. The ability of various hypothalamic factors to regulate GH secretion in the tilapia hybrid (Oreochromis niloticus x Oreochromis aureus) was studied. Somatostatin1-14 (SRIF1-14; 10-100 micrograms/kg) was found to reduce circulating GH levels in a dose-dependent manner. SRIF1-14 (0.1-1000 nM) inhibited GH release from perifused pituitary fragments (ED50 0.83 nM). Human growth hormone-releasing hormone fragment 1-29 (hGHRH1-29; 100 micrograms/kg) doubled circulating GH levels and modestly stimulated GH secretion in vitro. Carp growth hormone-releasing hormone (cGHRH) stimulated GH secretion in vitro to a similar degree at the same dose (1 microM). Injection of salmon gonadotropin-releasing hormone (sGnRH) superactive analog (10-100 micrograms/kg) increased plasma GH levels sixfold. sGnRH also stimulated GH release in vitro (ED50 142.56 nM). Dopamine (0.1-10 microM) and the D1 DA receptor agonist SKF 38393 increased GH secretion from perifused pituitary fragments dose-relatedly. Thyrotropin-releasing hormone (TRH) had no effect on GH secretion from perifused pituitary fragments, but increased plasma GH levels, as did bovine thyroid stimulating hormone (bTSH). The increased plasma GH in the bTSH-treated fish coincided with a dramatic increase in T4; however, TRH increased GH without changing T4 levels. T3 increased the synthesis of GH by isolated pituitaries (incorporation of [3H]leucine). SRIF1-14 seems to be a most potent hypothalamic regulator of GH secretion in tilapia; sGnRH and DA both increased GH secretion, although sGnRH elicited considerably greater responses at lower doses. Two forms of GHRH increased GH levels, although the unavailability of the homologous peptide prevented an accurate evaluation of its importance in regulating GH secretion. The thyroid axis (TRH, TSH, and T3) stimulates both synthesis and release of GH, although TRH did not appear to have a direct effect on the level of the pituitary

Data for The role of nuclear quantum effects in the relative stability of hexagonal and cubic ice

At atmospheric pressure, hexagonal ice (Ih) is thermodynamically stable relative to cubic ice (Ic), although the magnitude and underlying physical origin of this stability difference are not well defined. Pure Ic crystals are not accessible experimentally, and hence computer simulations have often been used to interrogate the relative stabilities of Ih and Ic; however, these simulations are dominated by molecular interaction models that ignore the intramolecular flexibility of individual water molecules, do not describe intermolecular hydrogen-bonding with sufficient accuracy, or ignore the role of nuclear quantum effects (NQEs) such as zero-point energy. Here, we show that when comparing the relative stability of Ih and Ic using a flexible, anharmonic molecular interaction model, while also accurately accounting for NQEs, a new picture emerges: Ih is stabilized relative to Ic as a result of subtle differences in the intramolecular geometries and intermolecular interactions of water molecules which are modulated by NQEs. Our simulations hence suggest that NQEs are a major contributor to the stabilization of Ih under terrestrial conditions and thus contribute to the well-known hexagonal (sixfold) symmetry of ice crystals