title: Cyclic AMP-dependent protein kinase in human embryonic palate mesenchymal cells.
abstract: Growth and differentiation of cells derived from the embryonic palate are critically dependent on the intracellular cAMP-mediated signal transduction pathway. Human embryonic palate mesenchymal (HEPM) cells have been widely used to examine the effect of teratogens on palatal tissue growth and differentiation, as well as a prescreen for environmental teratogens. This study examined responsiveness of HEPM cells to agents known to stimulate adenylate cyclase, characterized cAMP-dependent protein kinases (cAMP-dPK) (EC 2.7.1.37) and investigated to what extent HEPM cells reveal adaptational responses to cAMP at the level of cAMP-dependent protein kinase. HEPM cells exhibited a total cell cycle transit time of approximately 22 h and responded maximally, when confluent, to prostacyclin (PGI2), prostaglandin E2 (PGE2), and isoproterenol with time- and dose-dependent increases in intracellular levels of cAMP. The order of sensitivity to hormonal activation of adenylate cyclase was PGE2 > isoproterenol > PGI2. Basal cAMP-dependent protein kinases activity was 0.184 fmol phosphate transferred from ATP to histone per microgram protein per minute under conditions where endogenous phosphatases did not significantly affect protein phosphorylation. Regulatory subunits of cAMP-dPK in HEPM cells were characterized by the binding of [3H]cAMP to cytosolic fractions. Specific binding was saturable at approximately 50 nM indicating the presence of binding sites that are finite in number. Calculation of half-maximal binding yielded an estimated Kd of 25 nM indicating the presence of high affinity binding sites. Cyclic AMP-dPK regulatory subunits were also photoaffinity labeled with 8-N3-[32P]-cAMP, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and radiolabeled bands visualized by autoradiography. Photoactivated incorporation of 8-N3-[32P]cAMP was detected into two proteins of molecular weight (M(r)) 45,000 and M(r) 51,000 representing, respectively, the RI alpha and RII beta subunits of cAMP-dPK. Binding of [32P]8-azido cAMP to proteins of M(r) 45,000 (RI alpha) and M(r) 51,000 (RII beta) was increased in response to elevation of intracellular cAMP via inhibition of its breakdown with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, or by direct activation of adenylate cyclase with forskolin. HEPM cells thus revealed adaptational responses to cAMP at the level of cAMP-dependent protein kinase. Characterization of the cAMP signal transduction pathway in HEPM cells, derived from embryonic palatal tissue which is critically dependent on this pathway for normal development, may provide information fundamental to a clear understanding of cellular events involved in palatal ontogeny. These results highlight several important differences between HEPM cells and murine embryonic palate mesenchymal cells.(ABSTRACT TRUNCATED AT 400 WORDS)
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000378:metabolism; D002454:Cell Differentiation; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology; D015232:Dinoprostone / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D011464:Epoprostenol / Q000494:pharmacology; D005333:Fetus / Q000166:cytology / Q000201:enzymology; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D008648:Mesoderm / Q000166:cytology* / Q000201:enzymology*; D010159:Palate / Q000166:cytology* / Q000196:embryology*; D011494:Protein Kinases / Q000032:analysis*; D015398:Signal Transduction / Q000502:physiology; D013997:Time Factors
chemical_list: D005576:Colforsin; D011464:Epoprostenol; D000242:Cyclic AMP; D011494:Protein Kinases; D000262:Adenylyl Cyclases; D015232:Dinoprostone; D007545:Isoproterenol; D015056:1-Methyl-3-isobutylxanthine