Regulation of Gonadotropin mRNA Levels in Cultured Rat Pituitary Cells by Gonadotropin-Releasing Hormone (GnRH): Role for Ca2+ and Protein Kinase C

Abstract

Incubation of cultured rat pituitary cells with gonadotropin-releasing hormone (GnRH, 1 nM) resulted in a rapid elevation of gonadotropin subunit steady-state mRNA levels(alpha, 2.2-fold, LH beta, 2.1-fold, and FSH beta 2.2-fold increases at 30 min). Addition of actinomycin D abolished the stimulatory effect of GnRH upon alpha and LH beta and reduced the effect upon FSH beta mRNA levels. The effect of GnRH is biphasic, where the early phase is being observed at 30-60 min, while the late phase is noticed between 12-24 h. A significant decrease in FSH beta mRNA levels was found after 6 h of incubation when using a stable GnRH analog. The unique profile of the time response enabled us to attempt to dissect the signal transduction cascade involved in the neurohormone action. Addition of the protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol 13-acetate (TPA), or the Ca2+ ionophore, ionomycin, mimicked the profile of GnRH-induced alpha and LH beta mRNA elevation. The two phases of FSH beta mRNA elevation induced by GnRH could be mimicked by TPA, while the decrease at 6 h was mimicked by ionomycin. The rapid stimulatory effect of GnRH on gonadotropin subunit mRNA levels was abolished by the PKC inhibitors, staurosporine and GF 109203X. Similarly, the rapid stimulatory effect of GnRH on alpha and LH beta, but not FSH beta, was abolished in Ca(2+)-free medium. While additivity in LH release is obtained upon the combined addition of TPA and ionomycin for 30 min of incubation, LH beta and FSH beta gene expression is inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of PKC in the regulation of gonadotropin subunit mRNA levels: interaction with two native forms of gonadotropin-releasing hormone

“…Interestingly, treatment with TPA or DiC8 did not affect growth hormone mRNA levels in the same experiments (Klausen C, Tsuchiya T, Chang JP, and Habibi HR, unpublished observations), indicating that these stimulatory effects on hormone mRNA levels were cell-type specific. Stimulation of GtH subunit gene expression by TPA has been reported in other mammalian systems (8,9,13,44,47,48). In fish, GtH-␣ and LH-␤ mRNA levels were significantly elevated by TPA in cultured tilapia pituitary cells (18,37).…”

“…In mammals, treatment with GF did not affect basal GtH-␣, FSH-␤, and LH-␤ mRNA levels in cultured rat pituitary cells (8); GtH-␣ mRNA levels in ␣T3-1 cells (9); human GtH-␣-LUC, rat FSH-␤-LUC, and rat LH-␤-LUC expression in GGH 3 -1Ј cells (44); equine LH-␤-LUC expression in ␣T3-1 cells (13); and ovine FSH-␤-LUC and rat LH-␤-LUC expression in L␤T2 or L␤T4 cells (47,48,50). In tilapia pituitary cells, basal GtH-␣ mRNA levels were decreased, whereas LH-␤ mRNA levels were not significantly altered by GF (18,19,37).…”

“…The objective of this study was to investigate whether the PKC pathway is involved in the regulation of GtH subunit mRNA levels and the possible involvement of PKC signaling in sGnRH-and cGnRH-II-induced increases in GtH-␣, FSH-␤ and LH-␤ mRNA levels. 8 ] GnRH (cGnRH-II) from American Peptide (Sunnyvale, CA) were solubilized in 0.1 M acetic acid and stored at Ϫ20°C. Calphostin C (Cal C), GF109203X (GF), and tetradecanoylphorbol (TPA) were purchased from Calbiochem (La Jolla, CA), dissolved in DMSO, and stored at Ϫ20°C, 4°C and Ϫ20°C, respectively.…”

Protein kinase C as a signal for exocytosis

“…On the other hand, these arguments do not detract from clear observations that GnRH does initiate PKC-dependent actions in gonadotropes. Thus GnRH causes translocation of several PKC isoforms to the membrane and, through a mechanism that is sensitive to BIS and mimicked by PMA, raises messenger-RNA levels for LH and follicle stimulating hormone ␣-and ␤-chains and for PKC-␤ within minutes (39)(40)(41).…”

Egr-1 Is a Downstream Effector of GnRH and Synergizes by Direct Interaction with Ptx1 and SF-1 To Enhance Luteinizing Hormone β Gene Transcription

“…GnRH binds the GnRH-R, a seven-transmembrane G-protein-coupled receptor, present at the membrane of pituitary gonadotropes: this triggers the activation of phospholipase C (PLC), which cleaves phosphatidylinositol-4,5-biphosphate (PIP 2 ) to generate triphosphate inositol (IP 3 ) and diacylglycerol (DAG). IP 3 increases intracellular calcium levels, whereas DAG activates protein kinase C (PKC) (1,2,20). Activation of PKC leads to increased mitogen-activated protein kinase kinase (MAPKK or MEK) and mitogen-activated protein kinase (MAPK or ERK) activity and to increased gonadotropin mRNA levels (15,20,51).…”