2002 - Instituto de Química - USP

Hugo Aguirre ArmelinDECONSTRUCTING THE COMPLEXSIGNALING NETWORK UNDERLYINGCELL CYCLE CONTROL INMAMMALIAN CELLSA complexa rede de sinalização subjacente aocontrole do ciclo celular em mamíferos é regulada porum balanço fino entre fatores peptídicos de crescimentoe hormônios clássicos, que são sinais extracelulares. Nósestamos estudando a integração dos sinais antagônicos,mitogênicos e anti-mitogênicos, iniciados nos receptoresde, respectivamente, FGF2 (Fibroblast Growth Factor2), ACTH (Adrenocorticotropic Hormone) e AVP (ArginineVasopressin), das células adrenocorticais tumoraisY1 de camundongo. Nosso objetivo de longo prazo édesconstruir a rede de sinalização que controla o ciclocelular e o crescimento em células de mamífero. FGF2dispara uma robusta resposta mitogênica em célulasY1, que depende de rápida ativação das ERK-MAPquinases (1 e 2). ACTH e AVP, por mecanismosdiferentes, bloqueiam esta ação de FGF2 sem afetar aativação das ERK-MAP quinases. ACTH causa, demaneira concertada: desativação de Akt/PKB;degradação da proteína c-Myc e indução de p27Kip1(3). AVP, por outro lado, bloqueia a indução do geneda ciclina D1 (4).SummaryThe complex signaling network underlying cell cyclecontrol in mammalian cells is regulated by a finely tunedbalance between locally produced growth factors(paracrine, autocrine and intracrine) and classical hormones(distantly produced factors). We have been focusingon characterizing antagonistic mitogenic andanti-mitogenic signals initiated at, respectively, FGF2(Fibroblast Growth Factor 2), ACTH (AdrenocorticotropicHormone) and AVP (Arginine Vasopressin) receptorsin the mouse Y1 adrenocortical tumor cell line,aiming, in the long run, to deconstruct the signalingnetwork that controls cell cycle and growth in adrenocorticalcells. Y1 is a phenotypically stable, ACTH-responsive,steroid secreting, clonal cell line that closelymimics normal cells from the adrenal cortex. Y1 adrenocorticalcells display ERK-MAP kinases under remarkablytight control, whose FGF2 or FCS-dependent stimu-lation is absolutely required for triggering the G0/G1®Stransition of the cell cycle (1 and 2). On the other hand,Y1 cells exhibit high constitutive levels of phosphorylated/activatedAkt/PKB dependent on chronically elevatedc-Ki-Ras·GTP and PI3K activity. Expression of thedominant negative mutant RasN17 in Y1 cells resultsin strong reduction of both c-Ki-Ras·GTP and phosphorylatedAkt/PKB, that are restored by FGF2 treatments.Inhibitors of PI3K lead to rapid dephosphorylation ofAkt/PKB and block phosphorylation of Akt/PKB promotedby FGF2 (3). FGF2 triggers a robust mitogenicresponse in Y1 cells (1 and 2) that is strongly antagonizedby ACTH and AVP, working by two differentmechanisms. ACTH receptors activate the Gas/adenylatecyclase cAMP/PKA pathway to promote dephosphorylationof Akt/PKB, degradation of the c-Myc proteinand induction of the cyclin-dependent kinases’(CDKs) inhibitor p27 Kip1 (3). AVP blocks expression ofthe cyclin D1 gene, very likely via the Gaq11/PLCb/PKCpathway (4).PublicaçõesLotfi CFP & Armelin HA. cfos and cjun antisense oligonucleotides blockmitogenesis triggered by fibroblast growth factor-2 and ACTH in mouseY1 adrenocortical cells. J. Endocrinology (2001) 168, 381-389.Forti FL, Schwindt TT, Moraes MS, Eichler CB & Armelin HA. ACTH promotionof p27Kip1 induction in mouse Y1 adrenocortical tumor cells isdependent on both PKA activation and Akt/PKB inactivation. Biochemistry(2002) 41, 10133-10140.Rocha KM, Forti FL, Lepique AP & Armelin HA. Deconstructing the molecularmechanisms of cell cycle control in a mouse adrenocorticalcell line. Roles of ACTH. Microscopy Research and Techniques (2002), inpress.Schwindt TT, Forti FL, Juliano MA, Juliano L & Armelin HA. Arginine vasopressininhibition of cyclin D1 gene expression blocks cell and proliferationin the mouse Y1 adrenocortical tumor cell line (submitted)Instituto de Química - Pesquisa 2001 - 2002 44 Departamento Departamento de Química de FundamentalBioquímica