cAMP stimulates proliferation of numerous cell types, particularly of differentiated endocrine origin, by acting synergistically with other growth factors. Thus, downstream targets of cAMP represent attractive candidates for new oncogenes. We have implicated the G-protein Rap1b as a critical target of cAMP mitogenic signaling by showing that Rap1b-expressing cells display enhanced DNA synthesis and are endowed with tumorigenic properties. Additionally, in thyroid follicular cells, which depends on increases in cellular cAMP by thyrotropin (TSH) for mitogenesis, DNA synthesis stimulated by TSH or cAMP requires Rap1b-GTP binding (activation) and its phosphorylation by the cAMP-dependent protein kinase PKA. Thus, we have proposed that Rap1b can be viewed as conditional oncogene--its role in cell proliferation is linked to the mitogenic signaling program elicited by cAMP. We now present new results showing that a GTPase-deficient (G12V) and phosphomimetic (S179D) Rap1b mutant, Rap1b-G12V-S179D, promotes mitogenesis independently of cAMP. Moreover, a dominant negative version of the Rap1b activator Epac, N-Epac, inhibits the mitogenic effect of cAMP but it has no effect on the mitogenic action of Rap1b-G12V-S179D. These results prove conditionality of the cAMP mitogenic signal on cAMP/Epac activation and cAMP/PKA phosphorylation of Rap1b, which is bypassed by Rap1b- G12V-S179D; they implicate activation and phosphorylation of Rab1b as the main biochemical events in the transduction of the cAMP mitogenic signal. However, it is necessary to verify whether Rap1b behaves as a conditional oncogene in vivo, linked to pathophysiological conditions with increased TSH levels. We have generated mice with thyroid-specific expression of an active Raplb mutant, """"""""floxed"""""""" by LoxP sites, which, upon stimulation of CRE activity, will cease to express the active Rap1b mutant and produce a dominant negative Rap1b. This is the first example of a mouse model containing an """"""""in vivo/in lession"""""""" built-in control system permitting the switch of expression of a stimulatory to an inhibitory G protein, encoded by a single gene. Mice expressing an active Rap1b mutant show no discernable alteration of thyroid morphology and function under physiological conditions, but, under pathologically sustained cAMP signaling, their thyroids are enlarged, display enhanced DNA synthesis, and present multiple adenomas, and, even carcinomas. These results suggest that Rap1b also behaves as a conditional oncogene in vivo. We propose to exploit these mice to understand the multi-step tumorigenic process in thyroid.
The specific aims of this proposal are: 1: To establish whether thyroid-specific expression of Rap1b relays a cAMP-dependent mitogenic response. 2: To determine the effect of Rap1b in the establishment and/or progression of thyroid tumorigenesis. 3: To determine whether Rap1b action is required for cAMP-mediated hyperplasia. The long-term goal of this proposal is the identification of mitogenic signals relayed by Rap1b involved in cAMP-dependent cell proliferation in endocrine systems.
