Proliferative signaling initiated in ACTH receptors

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1 ACTH Brazilian and Journal proliferative of Medical signaling and Biological Research (2000) 33: ISSN X 1133 Proliferative signaling initiated in ACTH receptors C.F.P. Lotfi 1, A.P. Lepique 1, F.L. Forti 1, T.T. Schwindt 1, C.B. Eichler 1, M.O. Santos 1, I.T. Rebustini 1, G.N.M. Hajj 1, L. Juliano 2 and H.A. Armelin 1 1 Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brasil 2 Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal do Estado de São Paulo, São Paulo, SP, Brasil Abstract Correspondence H.A. Armelin Departamento de Bioquímica Instituto de Química, USP São Paulo, SP Brasil haarmeli@quim.iq.usp.br Presented at the First International Meeting on Adrenal Disease: Basic and Clinical Aspects, Ribeirão Preto, SP, Brazil, August 31-September 2, Research supported by FAPESP and CNPq to, respectively, H.A. Armelin and L. Juliano. C.F.P. Lotfi is a post-doctoral and A.P. Lepique, F.L. Forti, T.T. Schwindt, C.B. Eichler, M.O. Santos, I.T. Rebustini and G.N.M. Hajj are recipients of pre-doctoral fellowships from FAPESP. Received December 20, 1999 Accepted March 22, 2000 Introduction Key words ACTH FGF2 Signal transduction MAP kinases Early response genes

2 1134 C.F.P. Lotfi et al.

3 ACTH and proliferative signaling 1135 FGF2 is a strong mitogen for Y1 adrenocortical cells Short pulses of ACTH can elicit a weak mitogenic response in Y1 adrenocortical cells

4 1136 C.F.P. Lotfi et al. ACTH induces transcription of the fos and jun genes, but not of the c-myc gene c-fos and c-jun proteins are mediators of the mitogenic response to both ACTH and FGF2 ACTH antagonizes the mitogenic action of FGF2

ACTH and proliferative signaling 1137 ACTH is a poor activator of ERK- MAPK, but a MEK1 inhibitor blocks ACTH mitogenic activity on Y1 adrenocortical cells A B C 46.

5 ACTH and proliferative signaling 1137 ACTH is a poor activator of ERK- MAPK, but a MEK1 inhibitor blocks ACTH mitogenic activity on Y1 adrenocortical cells A B C 46.5 ERK-1 44 ERK-2 42 ERK-1 44 ERK-2 42 kda ERK-1 44 ERK-2 42 MW U U U U Corticotropin A FGF2 dcamp + FGF min FGF dcamp Cort A Fr4 Fr3 Fr2 Fr Fraction 1 FGF ACTH Fraction Corticotropin A Figure 1 - ERK-MAPK activation in Y1 adrenocortical cells assayed by Western blot. A, Kinetics of activation; 100 mu/ml corticotropin A (Cort A), 1 nm FGF2, 1 mm dibutyryl camp (dcamp). B, Screening of activities of fractions (Fr) 1 to 4 of porcine corticotropin A batch 96H0687 (Sigma) fractionated in an HPLC reverse phase column. C, Dose-response curves for the two most active fractions, namely 1 and 2. Dilution series from 1 to 5, respectively. Corticotropin A: 100, 1, 0.01 and mu/ml; fractions 1 and 2: 10, 0.1, 0.01, and µg/ml. Cell lysates were boiled for 5 min, submitted to 10% SDS-PAGE and electroblotted onto nitrocellulose Hybond-C extramembranes (Amersham Life Science Inc., Cleveland, OH, USA). Membranes were incubated with anti-phospho-mapk rabbit antibody (New England Biolabs Inc., Beverly, MA, USA) and developed by chemiluminescence with HRP-linked anti-rabbit antibody.

6 1138 C.F.P. Lotfi et al. Table 1 - Activities of ACTH 39, ACTH 24 and fractions of porcine corticotropin A regulating functions of Y1 adrenocortical cells. Steroidogenesis was estimated by extracting and assaying the fluorescent steroids liberated by cells into serum-free medium; specific activities were determined from dose-response curves. The unit of activity was arbitrarily defined as the ratio between fluorescence of stimulated cultures over fluorescence of untreated controls. Extracellular signal-regulated kinases-mitogen-activated protein kinases (ERK-MAPK) activation was assayed by Western blot as described in Figure 1. c-fos protein induction was estimated by immunocytochemical staining with anti-c-fos antibodies counting % labeled nuclei under the microscope. All fractions significantly activated adenylate cyclase but specific activities were not determined. ACTH 39 (EPM) and ACTH 24 (EPM) are peptides synthesized in the laboratory of L. Juliano. ACTH 39 (Sigma) is a commercial product. Porcine corticotropin A batch 96H0687 was purchased from Sigma. All peptide fractions were chemically characterized by HPLC and mass spectrometry and also eventually by N-terminal sequencing. ND: Not determined. Porcine corticotropin A Activities Steroidogenesis ERK-MAPK c-fos protein stimulation activation induction (maximal (specific activity (specific activity activity; % labeled arbitrary units) arbitrary units) nuclei) Total Fraction Fraction Fraction 3 1 <1 42 Fraction 4 10 <1 65 ACTH 39 (EPM) >20 <1 25 ACTH 24 (EPM) ND <1 33 ACTH 39 (Sigma) ND <1 55 g

7 ACTH and proliferative signaling 1139 Conclusions References 1. Hornsby PJ (1985). Regulation of adrenocortical cell proliferation in culture. Endocrine Research, 10: Armelin HA, Lotfi CFP & Lepique AP (1996). Regulation of growth by ACTH in the Y-1 line of mouse adrenocortical cells. Endocrine Research, 22: Lotfi CFP, Todorovic Z, Armelin HA & Schimmer BP (1997). Unmasking a growth-promoting effect of the ACTH in Y1 mouse adrenocortical tumor cells. Journal of Biological Chemistry, 272: Gutkind JS (1998). The pathways connecting G protein-coupled receptors to the nucleus through divergent mitogen-activated protein kinase cascades. Journal of Biological Chemistry, 273: Kimura E & Armelin HA (1990). Phorbol ester mimics ACTH action in corticoadrenal cells stimulating steroidogenesis, blocking cell cycle, changing cell shape and inducing c-fos proto-oncogene expression. Journal of Biological Chemistry, 265: Kimura E, Sonobe MHH, Armelin MCS & Armelin HA (1993). Induction of FOS and JUN proteins by adrenocorticotropin and phorbol ester but not by 3',5'-cyclic a- denosine monophosphate derivatives. Molecular Endocrinology, 7: Angel P & Karin M (1991). The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochimica et Biophysica Acta, 1072: Holt JT, Gopal TV, Moulton AD & Nienhuis AW (1986). Inducible production of c-fos antisense RNA inhibits 3T3 cell proliferation. Proceedings of the National Academy of Sciences, USA, 83: Riabowol KT, Vosatka R, Ziff EB, Lamb NJ & Feramisco JR (1988). Microinjection of fos-specific antibodies blocks DNA synthesis in fibroblast cells. Molecular and Cellular Biology, 8: Kovary K & Bravo R (1991). The Jun and Fos protein families are both required for cell cycle progression in fibroblasts. Molecular and Cellular Biology, 11: Burgess WH & Maciag T (1989). The heparin-binding fibroblast growth factor family of proteins. Annual Review of Biochemistry, 58: Nishimura T, Utsunomiya Y, Hoshikawa M, Ohuchi H & Itoh N (1999). Structure and expression of a novel human FGF, FGF19, expressed in fetal brain. Biochimica et Biophysica Acta, 1444: Meyers EN & Martin GR (1999). Differences in left-right axis pathways in mouse and chick: functions of FGF8 and SHH. Science, 285: Jung J, Zheng M, Goldfarb M & Zaret KS (1999). Initiation of mammalian liver development from endoderm by fibroblast growth factors. Science, 284: Metzger RJ & Krasnow MA (1999). Genetic control of branching morphogenesis. Science, 284: Miralles F, Czernichow P, Ozaki K, Itoh N & Scharfman R (1999). Signaling through FGF receptor 2b plays a key role in the development of the exocrine pancreas. Proceedings of the National Academy of Sciences, USA, 96: Mesiano S, Mellon SH, Gospodarowicz D, DiBlasio AM & Jaffe RB (1991). Basic FGF expression is regulated by corticotropin in the human fetal adrenal - a model for adrenal growth regulation. Proceedings of the National Academy of Sciences, USA, 88: Schimmer BP (1981). The adrenocortical tumor cell line, Y-1. In: Sato G (Editor), Functionally Differentiated Cell Lines. Alan R. Liss, Inc., New York, Rae PA, Gutmann NS, Tsao J & Schimmer BP (1979). Mutations in cyclic AMP-dependent protein kinase and corticotropin (ACTH)-sensitive adenylate cyclase affect adrenal steroidogenesis. Proceedings of the National Academy of Sciences, USA, 76: Schimmer BP & Parker KL (1992). Promoter elements of the mouse 21-hydroxylase (Cyp-21) gene involved in cell-selective and camp-dependent gene expression. Journal of Steroid Biochemistry and

8 1140 C.F.P. Lotfi et al. Molecular Biology, 43: Li CH, Chung D, Yamashiro D & Lee CY (1978). Isolation, characterization and synthesis of a corticotropin-inhibiting peptide from human pituitary glands. Proceedings of the National Academy of Sciences, USA, 75: Forti FL & Armelin HA (1998). ACTH induces c-fos proto-oncogene in fibroblasts expressing the ACTH receptor. Endocrine Research, 24: Lotfi CFP & Armelin HA (1998). c-fos protein is a mediator in mitogenic response to ACTH. Endocrine Research, 24: Watanabe G, Pena P, Albanese C, Wilsbacher LD, Young JB & Pestell RG (1997). Adrenocorticotropin induction of stress-activated protein kinase in the adrenal cortex in vivo. Journal of Biological Chemistry, 272: New MI (1998). Diagnosis and management of congenital adrenal hyperplasia. Annual Review of Medicine, 49:

Standardization of a fluorimetric assay for the determination of tissue angiotensin-converting enzyme activity in rats

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