Signal transduction pathways in the mitogenic response to PGF 2 alfa LIF and related cytokines in Swiss 3T3 cells, including cyclins Ds and CDKs expression by diverse signalling pathways

Mammalian cell division, is a highly complex process, regulated and coordinated by mechanisms that are conserved through most species. The physiological control of eucariotic cell proliferation initiation is external, and it is excerted by humoral factors, made by the same or other cells, under certain requirements of the organism. Progression through the different phases of the cell cycle, is governed by a regulatory machinery conserved through most species, that not only coordinates the various events that made up the cell cycle, but also connects the cell cycle with extracellular signals, that regulates cell proliferation. Beginning with a given mitogenic stimulus acting through a specific receptor in a target cell, signalling mechanisms cascades are generated in the membrane and in the citosol of that cell. These early events, act on the cell cycle machinery, finally leading to cell division. The expression of proteins that regulate the cell cycle is in part induced by mitogen-stimulated signalling mechanisms. 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 with cyclins D (D1, D2 and D3), induced in the G1 phase. Cyclins D are considered as "sensors" of the extracellular medium, since their induction is triggered by mitogenic stimuli. The activated complexes 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 signalling mechanisms, 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 to cellular 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 only cyclin 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 appears that PGF2α triggers cyclin D1 expression via two independent signalling events that complement with TGFβ1-triggered events to induce DNA synthesis. TGFβ1 cannot trigger cyclin D1 expression, but, stabilise cyclin D1 mRNA, after PGF2α-triggered its expression. Leukaemia inhibitory factor (LIF) was originally described on the basis of its ability to stimulate the differentiation of murine M1 leukemic cells into granulocytes and macrophages. In Swiss 3T3 cells, both LIF and prostaglandin F2α (PGF2α) trigger initiation of DNA synthesis and cell proliferation. LIF appears to exert its action through signals and processes markedly different from those elicited by PGF2α. While pre-treatment the cell culture with either GF 109203 (bysoindolmalemide), a specific PKC inhibitor, or 12-tetradecanoyl-13-phorbolacetate, which causes PKC down modulation, or lovastatin, known to block mevalonic acid synthesis and protein isoprenylation, totally impairs PGF2α mitogenic action. None of these treatments inhibited LIF-induced DNA replication. Agents capable of rising intracellular cAMP, enhanced both LIF and PGF2α ability to cause cellular entry into the S phase. However, H89 and PKI, both PKA inhibitors, prevented cAMP-mediated potentiation, but did not affect LIF induction of cellular entry into S phase. PD98059, a MEK (MAPKK)inhibitor, prevents PGF2α-mitogenic response but does not block LIF-induced initiation of DNA synthesis. Immunofluorescence studies revealed that LIF and PGF2α responses exhibit marked differences in STAT cytoplasmic-nuclear translocation. After 15 to 30 min, LIF causes STAT1 but not STAT3 or STAT5 translocation. In contrast, PGF2α failed to induce translocation of any of those transcriptional factors. Thus, it appears that LIF triggers mitogenic action through independent signalling events such as those involving PKC, PKA, MEK, p38MAPK and protein isoprenilation. In addition, its mitogenic effect is markedly potentiated by PKC, PKA, and probably PTK mediated signalling mechanisms. 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 later increase 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 signals and processes markedly similar to those elicited by LIF. This conclusion is based in the following facts: 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; PGF2α enhances the effect of LIF or OSM on DNA synthesis, both at subsaturant or saturant concentration. Moreover, LIF and OSM added together at subsaturating concentrations had an additive effect on DNA synthesis. LIF and OSM added together at saturating concentration had an 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 calcium dependent mechanisms would be involved in other processes. Finally, the LlF stimulated mitogenesis 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