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Endocrinology Vol. 138, No. 4 1673-1682
Copyright © 1997 by The Endocrine Society

ARTICLES

Mechanism of Mitogen-Activated Protein Kinase Activation by Gonadotropin-Releasing Hormone in the Pituitary {alpha}T3–1 Cell Line: Differential Roles of Calcium and Protein Kinase C1

Nachum Reiss, Linet N. Llevi, Sharon Shacham, Dagan Harris, Rony Seger2 and Zvi Naor

Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University (N.R., S.S., D.H., Z.N.), Ramat Aviv 69978; and the Department of Membrane Research and Biophysics, The Weizmann Institute of Science (L.N.-L., R.S.), Rehovot 76100, Israel

Address all correspondence and requests for reprints to: Zvi Naor, Ph.D., Department of Biochemistry, Tel Aviv University, Tel-Aviv 69978, Israel. E-mail: NAORZVI{at}CCSG.TAU.AC.IL

The mechanism of mitogen-activated protein kinase (MAPK, ERK) stimulation by the GnRH analog [D-Trp6]GnRH (GnRH-a) was investigated in the gonadotroph-derived {alpha}T3–1 cell line. GnRH-a as well as the protein kinase C (PKC) activator 12-O-tetradecanoyl phorbol-13-acetate (TPA) stimulated a sustained response of MAPK activity, whereas epidermal growth factor (EGF) stimulated a transient response. MAPK kinase (MEK) is also activated by GnRH-a, but in a transient manner. GnRH-a and TPA apparently activated mainly the MAPK isoform ERK1, as revealed by Mono-Q fast protein liquid chromatography followed by Western blotting as well as by gel kinase assay. GnRH-a and TPA stimulated the tyrosine phosphorylation of several proteins, and this effect as well as the stimulation of MAPK activity were inhibited by the PKC inhibitor GF 109203X. Similarly, down-regulation of TPA-sensitive PKC subspecies nearly abolished the effect of GnRH-a and TPA on MAPK activity. Furthermore, the protein tyrosine kinase (PTK) inhibitor genistein inhibited protein tyrosine phosphorylation and reduced GnRH-a-stimulated MAPK activity by 50%, suggesting the participation of genistein-sensitive and insensitive pathways in GnRH-a action. Although Ca2+ ionophores have only a marginal stimulatory effect, the removal of Ca2+ markedly reduced MAPK activation by GnRH-a and TPA, but had no effect on GnRH-a and TPA stimulation of protein tyrosine phosphorylation. Interestingly, the removal of Ca2+ also partly inhibited the activation of MAPK by EGF and vanadate/H2O2. Thus, a calcium-dependent component(s) downstream of PKC and PTK might also participate in MAPK activation. Elevation of cAMP by forskolin exerted partial inhibition on EGF, but not on TPA or GnRH-a action, suggesting that MEK activators other than Raf-1 might be involved in GnRH action. We conclude that Ca2+, PTK, and PKC participate in the activation of MAPK by GnRH-a, with Ca2+ being necessary downstream to PKC and PTK.




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B. R. White, D. L. Duval, J. M. Mulvaney, M. S. Roberson, and C. M. Clay
Homologous Regulation of the Gonadotropin-Releasing Hormone Receptor Gene Is Partially Mediated by Protein Kinase C Activation of an Activator Protein-1 Element
Mol. Endocrinol., April 1, 1999; 13(4): 566 - 577.
[Abstract] [Full Text]

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 EndocrinologyHome page
M. S. Roberson, T. Zhang, H. L. Li, and J. M. Mulvaney
Activation of the p38 Mitogen-Activated Protein Kinase Pathway by Gonadotropin-Releasing Hormone
Endocrinology, March 1, 1999; 140(3): 1310 - 1318.
[Abstract] [Full Text]

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J. J. Evans
Modulation of Gonadotropin Levels by Peptides Acting at the Anterior Pituitary Gland
Endocr. Rev., February 1, 1999; 20(1): 46 - 67.
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X. Lin and P. M. Conn
Transcriptional Activation of Gonadotropin-Releasing Hormone (GnRH) Receptor Gene by GnRH: Involvement of Multiple Signal Transduction Pathways
Endocrinology, January 1, 1999; 140(1): 358 - 364.
[Abstract] [Full Text]

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I. M. Colin and J. L. Jameson
Estradiol Sensitization of Rat Pituitary Cells to Gonadotropin-Releasing Hormone: Involvement of Protein Kinase C- and Calcium-Dependent Signaling Pathways
Endocrinology, September 1, 1998; 139(9): 3796 - 3802.
[Abstract] [Full Text] [PDF]

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D. J. Haisenleder, M. E. Cox, S. J. Parsons, and J. C. Marshall
Gonadotropin-Releasing Hormone Pulses Are Required to Maintain Activation of Mitogen-Activated Protein Kinase: Role in Stimulation of Gonadotrope Gene Expression
Endocrinology, July 1, 1998; 139(7): 3104 - 3111.
[Abstract] [Full Text] [PDF]

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N. L. Levi, T. Hanoch, O. Benard, M. Rozenblat, D. Harris, N. Reiss, Z. Naor, and R. Seger
Stimulation of Jun N-Terminal Kinase (JNK) by Gonadotropin-Releasing Hormone in Pituitary {alpha}T3-1 Cell Line Is Mediated by Protein Kinase C, c-Src, and CDC42
Mol. Endocrinol., June 1, 1998; 12(6): 815 - 824.
[Abstract] [Full Text]

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R. E. Schweppe, A. A. Frazer-Abel, A. Gutierrez-Hartmann, and A. P. Bradford
Functional Components of Fibroblast Growth Factor (FGF) Signal Transduction in Pituitary Cells. IDENTIFICATION OF FGF RESPONSE ELEMENTS IN THE PROLACTIN GENE
J. Biol. Chem., December 5, 1997; 272(49): 30852 - 30859.
[Abstract] [Full Text] [PDF]

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O. Benard, Z. Naor, and R. Seger
Role of Dynamin, Src, and Ras in the Protein Kinase C-mediated Activation of ERK by Gonadotropin-releasing Hormone
J. Biol. Chem., February 9, 2001; 276(7): 4554 - 4563.
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T. Yokoi, M. Ohmichi, K. Tasaka, A. Kimura, Y. Kanda, J. Hayakawa, M. Tahara, K. Hisamoto, H. Kurachi, and Y. Murata
Activation of the Luteinizing Hormone beta Promoter by Gonadotropin-releasing Hormone Requires c-Jun NH2-terminal Protein Kinase
J. Biol. Chem., July 7, 2000; 275(28): 21639 - 21647.
[Abstract] [Full Text] [PDF]


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