UP-REGULATION OF ADENYLYL-CYCLASE SIGNALING AND G(S)ALPHA EXPRESSION DURING DIFFERENTIATION OF EMBRYONIC STEM-CELLS

Activation of the cAMP pathway in retinoic acid-treated F9 embryonic c arcinoma cells and pluripotent embryonic stem cells promotes their dif ferentiation to parietal endoderm. In F9 cells, retinoic acid increase s transcription of the stimulatory coupling protein for adenylyl cycla se, G(s) alpha, and enhances the response of adenylyl cyclase to forsk olin and the differentiation factor, parathyroid hormone-related prote in. It is unclear whether post-receptor up-regulation of the adenylyl cyclase signaling pathway is restricted to parietal endoderm different iation, or is a more general feature of exit from the stem cell phenot ype. We therefore determined whether upregulation of the adenylyl cycl ase pathway is associated with differentiation of non-malignant embryo nic stem cells (E14) or pluripotent embryonic carcinoma cells (P19), w hich do not express the parietal endoderm phenotype. Retinoic acid-tre atment increased GTP-stimulated adenylyl cyclase activity in both embr yonic stem and embryonic carcinoma cells (2.6- and 2.2-fold, respectiv ely). In embryonic stem: cells, retinoic acid-treatment also increased the adenylyl cyclase response to a hydrolysis-resistance GTP analog o r forskolin (4.5- and 2.0-fold, respectively). Retinoic acid elicited a small increase in a G(s) alpha protein (Western-blotting), but a sub stantial increase in steady-state levels of G(s) alpha mRNA (70- and f ivefold in embryonic stem and embryonic carcinoma cells, respectively) . Differentiation of embryonic stem and carcinoma cells triggered by a ggregation was also associated with an increase both in adenylyl cycla se activity in response to parathyroid hormone-related protein and in G(s) alpha mRNA and therefore retinoic acid is not required for these events. To determine whether the cAMP signaling pathway may influence the anchorage-independent growth of embryonic carcinoma cells, P19 cel ls were grown as colonies in soft agar in the presence or absence of c AMP analogs. Even in the absence of retinoic acid, dibutyryl cAMP (1 m M) significantly suppressed the growth of P19 cells in soft agar, indi cating loss of the stem cell phenotype. We conclude that RA up-regulat es both receptor (parathyroid hormone-related protein) and post-recept or components of the adenylyl cyclase signaling pathway in pluripotent embryonic carcinoma and embryonic stem cells. Endogenous signals that trigger pluripotent embryonic carcinoma and embryonic stem cell diffe rentiation appear to up-regulate mRNA expression for G(s) alpha and ma y amplify adenylyl cyclase signaling in response to differentiation fa ctors such as parathyroid hormone-related protein.