INTERCELLULAR HETEROGENEITY OF EARLY MITOGENIC EVENTS - CAMP GENERALIZES THE EGF EFFECT ON C-FOS PROTEIN APPEARANCE BUT NOT ON MAP KINASE PHOSPHORYLATION AND NUCLEAR TRANSLOCATION IN DOG THYROID EPITHELIAL-CELLS

When quiescent dog thyroid epithelial cells in primary culture are sti mulated for 48 h with thyrotropin (TSH), forskolin acting through cAMP , or with cAMP-independent mitogens including epidermal growth factor (EGF), hepatocyte growth factor (HGF), and a tumor promoting phorbol e ster (TPA), only 30-60% of cells progress through the cell cycle. A mo re general growth response requires the combination of EGF and TSH or forskolin. In this study we ask whether this intercellular heterogenei ty in mitogen sensitivity could depend on a similar heterogeneity at e arly stages of the mitogenic stimulation process, i.e., at the levels of p42/p44 MAP kinase nuclear translocation and c-Fos protein appearan ce. We used indirect immunofluorescence microscopy with photometric qu antitation and corroborated data using Western blotting. We analyzed t he double staining of c-Fos and p42/p44 MAP kinases, since the nuclear translocation of these MAP kinases has been suggested as a key step f or the stimulation of c-fos transcription. (i) EGF and HGF induced c-F os accumulation and MAP kinase translocation in variable fractions of the cell population that corresponded to their relative potency as mit ogens. c-Fos appearance and MAP kinase translocation poorly correlated in individual cells. Many cells accumulated c-Pos without any detecta ble p42/p44 MAP kinase translocation. The heterogeneity of proliferati ve responses to EGF could be due to the lack of c-Fos or MAP kinase re sponsiveness of many cells. (ii) TPA induced c-Fos accumulation and MA P kinase translocation within the whole cell population, which did not explain the heterogeneity of the growth response to this factor and s howed that these events are not sufficient to elicit DNA synthesis. (i ii) TSH and forskolin induced a weak c-Fos accumulation in only a mino rity of cells but, as previously shown, no p42/p44 MAP kinase phosphor ylation and translocation. An important c-Fos expression was thus disp ensable for the strong DNA synthesis stimulation exerted by cAMP-depen dent mitogens. (iv) Forskolin potentiated the EGF effect on c-Fos expr ession but not on p42/p44 MAP kinase phosphorylation and translocation . This reflected the fact that EGF induced c-Fos accumulation in 90% o f cells in the presence of forskolin but in 30-50% of cells in its abs ence. This kind of potentiation, which specifically implies an increas e in the fraction of responding cells, is termed ''generalization'' in the present study. cAMP might stimulate dog thyroid cell proliferatio n both by activating its own mitogenic cascade and by acting as a comp etence factor allowing more cells to express c-Fos in response to EGF, which might thus contribute to the generalization of the mitogenic re sponse. (C) 1995 Academic Press, Inc.