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ACTH receptor: Ectopic expression, activity and signaling

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Abstract

Failure in obtaining expression of functional adrenocorticotropic hormone receptor (ACTHR, or melanocortin 2 receptor, MC2R) in non-adrenal cells has hindered molecular analysis of ACTH signaling pathways. Here, we ectopically expressed the mouse ACTHR in Balb/c mouse 3T3 fibroblasts to analyze ACTH signaling pathways involved in induction of fos and jun genes. Natural constitutive expression of the MC2R accessory protein (MRAP) in Balb3T3 and other mouse 3T3 fibroblasts (NIH, Swiss and 3T3-L1) renders these fibroblastic lines suitable for ectopic expression of ACTHR in its active form properly inserted into the plasma membrane at levels similar to those found in mouse Y1 adrenocortical tumor cells. The Y1 cell line is a cultured cell system well known for stably displaying normal adrenal specific metabolic pathways, ACTHR expression and ACTH functional responses. Thirty-nine sub-lines expressing ACTHR (3T3-AR transfectants) were selected for geneticin-resistance and clonally isolated after transfection of ACTHR-cDNA (in the pSVK3 mammalian plasmidial vector) into Balb3T3 fibroblasts. In addition, sixteen clonal sub-lines of Balb3T3 (3T3-0 transfectants) carrying the pSVK3 empty vector were likewise isolated. Fourteen 3T3-AR and four 3T3-0 clones were screened for response to ACTH39 in comparison with Y1 adrenocortical cells. Eight 3T3-AR clones responded to ACTH39 with activation of adenylate cyclase and induction of c-Fos protein, but the levels of, respectively, activation and induction were not strictly correlated. Other fos and jun genes were also induced by ACTH39 in 3T3-AR transfectants, which express levels of ACTHR protein similar to parental Y1 cells. Signaling pathways relevant to c-Fos induction was extensively investigated in 3 clones: 3T3-AR01 and –07 and 3T3-04. In Y1 cells, specific inhibitors (H89/PKA; PD98059/MEK; Go6983/PKC and SP600125/JNK) show that signals initiated in the ACTH/ACTHR-system activate 4 pathways to induce the c-fos gene, namely: (a) cAMP/PKA/CREB; (b) MEK/ERK1/2; (c) PKC and d) JNK1/2. In 3T3-AR transfectants, both inhibitors PD98059 and Go6983 proved completely ineffective to inhibit c-Fos induction by ACTH39, implying that MEK/ERK and PKC pathways are not involved in this process. On the other hand, SP600125 caused 85% inhibition of c-Fos induction by ACTH39 and, in addition, ACTH39 promotes JNK1/2 phosphorylation, suggesting that JNK is a major signaling pathway mediating c-Fos induction by ACTH39 in these cells. In addiction, PKA inhibitor H89 also inhibits c-Fos induction in 3T3-AR7 cells by ACTH39, implicating activation of the cAMP/PKA/CREB pathway in c-Fos induction by ACTH39. However, the cAMP derivatives db-cAMP and 8Br-cAMP, do not promote CREB phosphorylation and c-Fos induction in parental Balb3T3 and 3T3-AR transfectants, confirming previous report by others. In conclusion, expression of active ACTHR in Balb3T3 fibroblasts renders these cells responsive to ACTH with activation of cAMP/PKA/CREB and JNK pathways and, also, induction of genes from the fos and jun families. These results show that Balb 3T3-AR sublines are useful cellular systems for genetic analysis of ACTH-signaling pathways. However, activation of cAMP/PKA/CREB and JNK pathways and induction of fos and jun genes are not yet sufficient to enable ACTH for interference in morphology, migration and proliferation of Balb3T3 fibroblasts as it does in Y1 adrenocortical cells.

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Abbreviations

ACTH:

adrenocorticotropic hormone

ACTHR:

adrenocorticotropic hormone receptor

Gs:

stimulatory G protein

cAMP:

adenosine 3’:5’-cyclic monophosphate

PKA:

protein kinase A

PKC:

protein kinase C

FGF2:

basic fibroblast growth factor

8BrcAMP:

8-bromo-adenosine 3’:5’-cyclic monophosphate

db-cAMP:

dibutyryl adenosine 3’:5’-cyclic monophosphate

ERK:

extracellular signal-regulated kinase

MAPK:

mitogen-activated protein kinase

GPCR:

G protein-coupled receptor

FCS:

fetal calf serum

SDS-PAGE:

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

G protein:

GTP-binding protein

DME:

Dulbecco’s Modified Eagle’s Medium

DTT:

dithiothreitol

POMC:

pro-opiomelanocortin

StAR:

steroidogenic acute regulatory protein

SF-1:

steroidogenic factor-1

DAX-1:

dosage-sensitive sex reversal-adrenal hypoplasia congenital critical region on the X-chromosome

AP-1:

activator protein-1

CRE:

cAMP/Ca+2 responsive element

CREB:

CRE binding protein

CREM:

CRE modulator protein

ATF:

activator transcription factor

MC2R:

melanocortin 2 receptor

MRAP:

melanocortin 2 receptor accessory protein

JNK:

Jun N-terminal kinase

SAPK:

stress activated protein kinase

References

  1. Mountjoy KG, Robbins LS, Mortrud MT, Cone RD: The cloning of a family of genesthat encode melanocortin receptors. Science 257: 1248–1251, 1992

    Article  PubMed  CAS  Google Scholar 

  2. Boston BA, Cone RD: Characterization of melanocortin receptor subtype andexpression in murine adipose tissues and in the 3T3-L1 cell line. Endocrinology137: 2043–2050, 1996

    Article  PubMed  CAS  Google Scholar 

  3. Beuschlein F, Fassnacht M, Klink A, Allolio B, Reincke: ACTH-receptor expression, regulation and role in adrenocortical tumor formation. European JournalEndocrinology 144: 199–206, 2001

    Article  CAS  Google Scholar 

  4. Lamolet B, Pulichino AM, Lamonerie T, Gauthier I, Brue T, Enjalbert A, Drouin J: Apituitary cell-restricted tbox factor, tpit, activates POMC transcription incooperation with pitx homeoproteins. Cell 104: 849–859, 2001

    Article  PubMed  CAS  Google Scholar 

  5. Westphal CH, Muller L, Zhou X, Booner-Weir S, Schambelan M, Steiner DF, Lindberg I, Leder P: The neuroendocrine protein 7B2 is required for peptide hormone processingin vivo and provides a novel mechanism for pituitary cushing's disease. Cell 96: 689–700, 1999

    Article  PubMed  CAS  Google Scholar 

  6. Stocco DM: The role of StAR protein in steroidogenesis: challenges for the future. J Endocrinol 164: 247–253, 2000

    Article  PubMed  CAS  Google Scholar 

  7. New MI: Diagnosis and management of congenital adrenal hyperplasia. Annu Rev Med 49: 311–28, 1998

    Article  PubMed  CAS  Google Scholar 

  8. Hornsby PJ: Regulation of adrenocortical cell proliferation in culture. EndocrineResearch 10: 259–81, 1985

    CAS  Google Scholar 

  9. Rocha KM, Forti FL, Lepique AP, Armelin HA: Deconstructing the molecular mechanismsof cell cycle control in a mouse adrenocortical cell line: roles of ACTH. MicrosRes Tech 61 268–74, 2003

    Article  CAS  Google Scholar 

  10. Yasumura Y, Tashjian AH Jr, Sato G: Establishment of four functional, clonal strainsof animal cells in culture. Science 154: 1186–1189, 1966

    Article  CAS  Google Scholar 

  11. Schimmer BP: In: Functionally differentiated cell lines, pp. 61-92, Alan R. Liss.,Inc., New York, 1981

    Google Scholar 

  12. Masui H, Garren LD: Inhibition of replication in functional mouse adrenal tumorcells by adrenocorticotropic hormone mediated by adenosine 3′:5′-cyclicmonophosphate. Proc Natl Acad Sci U S A. 68: 3206–10, 1971

    Article  PubMed  CAS  Google Scholar 

  13. Rae PA, Gutmann NS, Tsao J, Schimmer BP: Mutations in cAMP dependet protein kinaseand ACTH sensitive adenylate cyclase affect adrenal steroidogenesis. Proceedings ofNational Academy of Science USA 76: 1869–1900, 1979

    Google Scholar 

  14. The roles of cAMP and cAMP-dependent protein kinase in forskolin's actions on Y1adrenocortical tumor cells. Endocr Res 11: 199–209, 1986

    Google Scholar 

  15. Lepique AP, Moraes MS, Rocha KM, Eichler CB, Hajj GN, Schwindt TT, Armelin HA. c-Mycprotein is stabilized by fibroblast growth factor 2 and destabilized by ACTH tocontrol cell cycle in mouse Y1 adrenocortical cells. J Mol Endocrinol 33:623–38,2004

    Article  PubMed  CAS  Google Scholar 

  16. Forti FL, Schwindt TT, Moraes MS, Eichler CB, Armelin HA: ACTH promotion ofp27Kipl induction in mouse Y1 adrenocortical tumor cells is dependent on bothPKA activation and Akt/PKB inactivation. Biochemistry 41:10133–10140, 2002

    Article  PubMed  CAS  Google Scholar 

  17. Metherell LA, Chapple JP, Cooray S, David A, Becker C, Ruschendorf F, Naville D, Begeot M, Khoo B, Nurnberg P, Huebner A, Cheetham ME, Clark AJ: Mutations in MRAP, encoding a new interacting partner of the ACTH receptor, cause familialglucocorticoid deficiency type 2. Nature Genetics 37(2): 166–70, 2005

    Article  PubMed  CAS  Google Scholar 

  18. Naville D, Barjhoux L, Jaillard C, Faury D, Despert F, Esteva B, Durand P, SaezJM, Begeot M: Demonstration by transfection studies that mutations in theadrenocorticotropin receptor gene are one cause of the hereditary syndrome ofglucocorticoid deficiency. J Clin Endocrinol Metab 81: 1442–8, 1986

    Article  Google Scholar 

  19. Forti FL, Armelin HA: ACTH induces c-fos proto-oncogene in fibroblasts expressingthe ACTH receptor. Endocrine Research 24: 433–437, 1998

    Article  PubMed  CAS  Google Scholar 

  20. Aaronson AS, Todaro G: Development of 3T3-like lines from Balb-c mouse embryocultures: transformation susceptibility to SV40. Journal of Cellular Physiology72: 141–148, 1968

    Article  PubMed  CAS  Google Scholar 

  21. Seternes OM, Sorensen R, Johansen B, Moens U: Activation of protein kinase A bydibutyryl cAMP treatment of NIH3T3 cells inhibits proliferation but fails to induceSer-133 phosphorylation and transcriptional activation of CREB. CellularSignalling 11(3): 211–219, 1999

    CAS  Google Scholar 

  22. Schimmer BP: Adenylate cyclase activity in adrenocorticotropic hormone-sensitiveand mutant adrenocortical tumor cell lines. Journal of Biological Chemistry 247:3134–3138, 1972

    PubMed  CAS  Google Scholar 

  23. Buckley DI, Ramachandran J: Characterization of corticotropin receptors onadrenocortical cells. Proceedings of National Academy of Science USA 78:7431–7435, 1981

    Article  CAS  Google Scholar 

  24. Gyles SL, Burns CJ, Whitehouse BJ, Sugden D, Marsh PJ, Persaud SJ, Jones PM: ERKsregulate cyclic AMP induced steroid synthesis through transcription of thesteroidogenic acute regulatory (StAR) gene. Journal of Biological Chemistry 276:34888–34895, 2001

    Article  PubMed  CAS  Google Scholar 

  25. Clark BJ, Ranganathan V, Combs R: Steroidogenic acute regulatory protein expressionis dependent upon post-translational effects of cAMP-dependent protein kinase A. Molecular and Cellular Endocrinology 173: 183–192, 2001

    Article  PubMed  CAS  Google Scholar 

  26. Zazopoulos E, Lalli E, Stocco DM, Sassone-Corsi P: DNA binding and transcriptionalrepression by DAX-1 blocks steroidogenesis. Nature 390: 311–315, 1997.

    Article  PubMed  CAS  Google Scholar 

  27. Rainey WE, Saner K, Schimmer BP: Adrenocortical cell lines. Mol Cell Endocrinol 228: 23–38, 2004

    Article  PubMed  CAS  Google Scholar 

  28. Kimura E, Sonobe MH, Armelin MCS, Armelin HA: Induction of FOS and JUN proteins byadrenocorticotropin and phorbol ester but not by 3′, 5′-cyclic adenosinemonophosphate derivatives. Molecular Endocrinology 7: 1463–1471, 1993

    Article  PubMed  CAS  Google Scholar 

  29. Viard I, Hall SH, Jaillard C, Berthelon MC, Saez JM: Regulation of c-fos, c-jun andjunB mRNA by angiotensin-II and corticotropin in ovine and bovine adrenocorticalcells. Endocrinology 130: 1193–1200, 1992

    Article  PubMed  CAS  Google Scholar 

  30. Viard I, Penhoat A, Ouali R, Langlois D, Begeot M, Saez JM: Peptide hormone andgrowth factor regulation of nuclear proto-oncogenes and specific functions inadrenal cells. Journal of Steroid Biochemistry and Molecular Biology 50: 219–24,1994

    Article  PubMed  CAS  Google Scholar 

  31. Angel P, Karin M: The role of Jun, fos and the AP-1 complex in cell-proliferationand transformation. Biochimical and Biophysical Acta 1072: 129–157, 1991

    Article  CAS  Google Scholar 

  32. Karin M, Liu Z, Zandi E: AP-1 function and regulation. Current Opinion in CellBiology 9: 240–246, 1997

    Article  CAS  Google Scholar 

  33. Maher P: Phorbol esters inhibit fibroblast growth factor-2-stimulated fibroblastproliferation by a p38 MAP kinase dependent pathway. Oncogene 21(13): 1978–1988,2002

    Article  PubMed  CAS  Google Scholar 

  34. Bravo R: Growth factors inducible genes in fibroblasts. 324–343. In A. Habenicht(Editor), Growth factors, differentiation factors and cytokines. Springer-Verlag, Berlin 1990

    Google Scholar 

  35. Holt JT, Gopal TV, Moulton AD, Nienhuis AW: Inducible production of c-fos antisenseRNA inhibits 3T3 cell proliferation. Proceedings of National Academy of ScienceUSA 83: 4794–4798, 1986

    Article  CAS  Google Scholar 

  36. Riabowol KT, Vosatka RJ, Ziff EB, Lamb NJ and Feramisco JR: Microinjection offos-specific antibodies blocks DNA synthesis in fibroblast cells. Molecular andCellular Biology 8: 1670–1676, 1988

    CAS  Google Scholar 

  37. Simpson ER, Waterman MR: Regulation of the synthesis of steroidogenic enzymes inadrenal cortical cells by ACTH. Annual Review of Physiology 50: 427–440, 1988

    Article  PubMed  CAS  Google Scholar 

  38. Seternes OM, Johansen B, Moens U: A dominant role for the Raf-MEK pathway inforskolin, 12-O-tetradecanoyl-phorbol acetate, and platelet-derived growthfactor-induced CREB (cAMP-responsive element-binding protein) activation, uncoupledfrom srine133 phosphorylation in NIH 3T3 cells. Molecular Endocrinology, 13(7):1071–83, 1999

    Article  PubMed  CAS  Google Scholar 

  39. Manning AM, Davis RJ: Targeting JNK for therapeutic benefit: from junk to gold?.Nature Reviews, 2: 554–565, 2003

    Article  PubMed  CAS  Google Scholar 

  40. Bennett BL, Satoh Y, Lewis AJ: JNK: A new therapeutic target for diabetes. CurrentOpinion in Pharmacology 3: 420–425, 2003

    CAS  Google Scholar 

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Authors and Affiliations

  1. Departamento de Bioquȷmica, Instituto de Quȷmica, Universidade de Sȧo Paulo, Sȧo Paulo-SP, 05508-900, Brasil

    Fȥbio Luȷs Forti, Matheus H. S. Dias & Hugo Aguirre Armelin

  2. Departamento de Bioquȷmica, Instituto de Quȷmica, Universidade de Sȧo Paulo, Avenida Prof. Lineu Prestes, 748 – Bl 09 Inf, SL 926, Cidade Universitȥria, Cep 05508-900, CP 26077, Sȧo Paulo, SP, Brasil

    Fȥbio Luȷs Forti

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  1. Fȥbio Luȷs Forti
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Correspondence to Fȥbio Luȷs Forti.

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Forti, F.L., Dias, M.H.S. & Armelin, H.A. ACTH receptor: Ectopic expression, activity and signaling. Mol Cell Biochem 293, 147–160 (2006). https://doi.org/10.1007/s11010-006-9237-0

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