An anchorage-dependent signal distinct from p42/44 MAP kinase activation is required for cell cycle progression

Most normal cells require both mitogens and integrin-mediated attachment for growth. It is generally accepted that the p42/p44 MAP kinase module, which can be activated by both growth factors and adhesion, plays a critical role in G0 to S phase progression of quiescent cells. Studies on various cultured fibroblasts have shown that removal of anchorage leads to cell cycle arrest in G1 and it has been proposed that adhesion-dependent G1 progression requires the joint regulation of p42/p44 MAP kinase by integrins and growth factors. In quiescent CCL39 lung fibroblasts, MAP kinase activation in response to serum becomes compromised when cells are placed in suspension. Under these conditions, serum-stimulated cells arrest their growth in mid-G1 with reduced cyclin D1 expression and increased p21Cip/Waf1 expression, as compared to their attached counterparts. To determine whether a casual link exists between suboptimal activation of MAP kinase in non-adherent cells and the observed G1 block, we used a variant of CCL39 stably expressing an estrogen-inducible activated-Raf-1 construct (deltaRaf-1:ER). We found that even strong and sustained activation of MAP kinase with estradiol, in addition to serum, is not able to boost cyclin D1 expression levels or stimulate hyperphosphorylation of pRb in suspended CCL39-deltaRaf-1:ER cells. These results indicate that p42/p44 MAP kinase activation is not a limiting factor for G1 to S phase transit in absence of anchorage. Thus, at least one adhesion-mediated signalling event, distinct from MAP kinase activation is required for maximal cyclin D1 induction and hyperphosphorylation of pRb.