Vías de señalización en la respuesta mitogénica a PGF 2 alfa, lif y citoquinas relacionadas, en células Swiss 3T3 que involucran la expresión de ciclinas D y CDKs

• Autores: Sauane, Moira
• Año de publicación: 2000
• Idioma: español castellano
• Tipo de recurso: tesis doctoral
• Colaborador/a o director/a de tesis: Jiménez de Asúa, Luis; Wolosiuk, Ricardo Alejandro
• Materia: CELULAS DE RATON SWISS 3T3; FACTORES DE CRECIMIENTO; MECANISMOS DE TRANSDUCCION DE SEÑAES; MOLECULAS REGULATORIAS DEL CICLO CELULAR; FACTOR INHIBIDOR DE LA LEUCEMIA (LIF); ONCOSTATINA M (OSM); CITOQUINAS DE TIPO IL-6; TGFBETA1; PGF2ALFA; FASE G1; PROTEINAS QUINASAS DE TIROSINAS (PKT); PROTEINA QUINASA C (PKC); CALCIO; AMPC; PROTEINAS QUINASAS DEPENDIENTES DE AMPC (PTK); CICLINAS D; QUINASAS DEPENDIENTES DE CICLINAS (CDK); TRANSDUCTORES DE SEÑALES Y FACTORES DE TRANSCRIPCION (STATS); SWISS 3T3 CELLS; GROWTH FACTORS; SIGNALLING MECHANISMS; CELL CYCLE; LEUKAEMIA INHIBITORY FACTOR (LIF); ONCOSTATIN M (OSM); CILIARY NEUROTROPHIC FACTOR (CNTF); INTERLEUKIN-6 (IL-6); TRANSFORMING GROWTH FACTOR BETA1 (TGFBETA1); PROSTAGLANDIN 2ALPHA (PGF 2ALPHA); G1 PHASE; PROTEIN TYROSINE KINASE (PKT); CALCIUM; MITOGEN-ACTIVATED PROTEIN KINASE (MAPK); AMPC; PROTEIN KINASE A (PKA); CYCLIN-D; CYCLIN-DEPENDANT KINASE (CDK); SIGNAL TRANSDUCERS AND ACTIVATORS OF TRANSCRIPTION (STATS)
• Institución: Universidad Nacional de Buenos Aires. Facultad de Ciencias Exactas y Naturales

Mammalian cell division, is a highly complex process, regulated and coordinated bymechanisms that are conserved through most species. The physiological control of eucarioticcell proliferation initiation is external, and it is excerted by humoral factors, made by the same orother cells, under certain requirements of the organism. Progression through the differentphases of the cell cycle, is governed by a regulatory machinery conserved through mostspecies, that not only coordinates the various events that made up the cell cycle, but alsoconnects the cell cycle with extracellular signals, that regulates cell proliferation. Beginning witha given mitogenic stimulus acting through a specific receptor in a target cell, signallingmechanisms cascades are generated in the membrane and in the citosol of that cell. Theseearly events, act on the cell cycle machinery, finally leading to cell division. The expression ofproteins that regulate the cell cycle is in part induced by mitogen-stimulated signallingmechanisms. The passage from G0 to S phase, depends on the activity of cyclin-dependent kinases (CDKs). These kinases are CDK4 and CDK6, and they are activated when they form complexes withcyclins D (D1, D2 and D3), induced in the G1 phase. Cyclins D are considered as "sensors" ofthe extracellular medium, since their induction is triggered by mitogenic stimuli. The activatedcomplexes cyclin D-CDK4 and cyclin D-CDKG catalyse the phosphorilation of the Rb protein. In Swiss 3T3 cells, PGF2α is capable of inducing DNA synthesis, by means of multiple signallingmechanisms, in the absence of other factors. However its mitogenic effect is potentiated by TGFβ1 addition. We have shown that PGF2α triggers cyclin D1 mRNA/protein expression prior tocellular entry into the S phase, but fails to raise CDK4 or cyclin D3 levels, while 1-oleoyl-2acetyllglycerol (OAG), a protein kinase C (PKC) and tyrosine kinase (TK) activator, induces onlycyclin D1 expression with no mitogenic response. In contrast, in PKC-depleted or -inhibited cells, PGF2α, but not OAG, increases cyclin D1 expression with no mitogenic response. Finally, OAG,in the presence of orthovanadate (Na3VO4)or TGFβ1, induces DNA synthesis. Thus, it appearsthat PGF2α triggers cyclin D1 expression via two independent signalling events that complementwith TGFβ1-triggered events to induce DNA synthesis. TGFβ1 cannot trigger cyclin D1expression, but, stabilise cyclin D1 mRNA, after PGF2α-triggered its expression. Leukaemia inhibitory factor (LIF) was originally described on the basis of its ability to stimulatethe differentiation of murine M1 leukemic cells into granulocytes and macrophages. In Swiss 3T3cells, both LIF and prostaglandin F2α (PGF2α) trigger initiation of DNA synthesis and cellproliferation. LIF appears to exert its action through signals and processes markedly differentfrom those elicited by PGF2α. While pre-treatment the cell culture with either GF 109203 (bysoindolmalemide), a specific PKC inhibitor, or 12-tetradecanoyl-13-phorbolacetate, whichcauses PKC down modulation, or lovastatin, known to block mevalonic acid synthesis andprotein isoprenylation, totally impairs PGF2α mitogenic action. None of these treatments inhibited LIF-induced DNA replication. Agents capable of rising intracellular cAMP, enhanced both LIFand PGF2α ability to cause cellular entry into the S phase. However, H89 and PKI, both PKAinhibitors, prevented cAMP-mediated potentiation, but did not affect LIF induction of cellularentry into S phase. PD98059, a MEK (MAPKK)inhibitor, prevents PGF2α-mitogenic responsebut does not block LIF-induced initiation of DNA synthesis. Immunofluorescence studiesrevealed that LIF and PGF2α responses exhibit marked differences in STAT cytoplasmic-nucleartranslocation. After 15 to 30 min, LIF causes STAT1 but not STAT3 or STAT5 translocation. Incontrast, PGF2α failed to induce translocation of any of those transcriptional factors. Thus, it appears that LIF triggers mitogenic action through independent signalling events suchas those involving PKC, PKA, MEK, p38MAPK and protein isoprenilation. In addition, its mitogeniceffect is markedly potentiated by PKC, PKA, and probably PTK mediated signallingmechanisms. Western blot analyses of cyclin D1, D2 and D3 expression (implicated in most mitogen actions),revealed that PGF2α, after 7-9 h, caused an increase in cyclin D1 protein levels, and a laterincrease in cyclin D2 levels. In contrast, LIF failed to increase either cyclin D1, D2, D3, CDK4 or CDK6 protein levels. Finally, oncostatin M(OSM), a cytokine closely related to LIF, exerts its action through signalsand processes markedly similar to those elicited by LIF. This conclusion is based in the followingfacts: both cytokines causes STAT1 tranlocation; the effect of Prostaglandin E1 and insulin,when added separately or in combination, enhances the effect of either LIF or OSM; PGF2αenhances the effect of LIF or OSM on DNA synthesis, both at subsaturant or saturantconcentration. Moreover, LIF and OSM added together at subsaturating concentrations had anadditive effect on DNA synthesis. LIF and OSM added together at saturating concentration hadan similar effect to that of these same cytokines when added separately. Interleukin -6 and CNTF, fail to cause either cyclin D expression or mitogenic response. The results obtained suggest that the PGF2α-stimulated mitogenesis would occur through cyclin D1 expression, mediated by DAG/PKC and TK dependent mechanisms, while calciumdependent mechanisms would be involved in other processes. Finally, the LlF stimulatedmitogenesis is not depend on signalling mechanisms such as those that act through PKC, PKA, MEK, p38MAPK and isoprenilated proteins, and also independently of the expression of cyclins D, CDK4 and CDK6.
Fil: Sauane, Moira. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.