title: Regulation of the laminin beta 1 (LAMB1), retinoic acid receptor beta, and bone morphogenetic protein 2 genes in mutant F9 teratocarcinoma cell lines partially deficient in cyclic AMP-dependent protein kinase activity.
abstract: We stably transfected a gene encoding a dominant negative regulatory subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA) into F9 cells and generated cell lines partially deficient in PKA activity (DN16 and DN19). In these cell lines, the retinoic acid (RA) receptor beta and laminin beta(1) chain (LAMB1) genes were regulated normally by RA alone, indicating that in the absence of exogenous modulation of cAMP levels, the PKA signaling pathway does not seem to play a major role in the RA-associated regulation of these genes. However, alterations in gene regulation were observed when the mutant cell lines were treated with a combination of RA and cAMP analogues. Moreover, in the DN16 cell line, which exhibits the lowest PKA activity among the mutant cell lines [22% of wild type (WT) at 1 microM cAMP], there was a significant decrease in the cAMP-associated activation of the LAMB1 gene DNase I hypersensitivity site 2 enhancer, as measured by chloramphenicol acetyl transferase assays. Using electrophoretic mobility shift assays, less protein binding was observed at one of the motifs (C2) within this enhancer region in the DN16 cells as compared to the F9 WT cells after treatment of the cells with RA and cAMP analogues for 24 h. Furthermore, no increase in C2 binding was observed when extracts from RA-treated F9 ST or DN16 cells were subjected to in vitro phosphorylation, suggesting that PKA is involved in the induction of the C2-binding protein in RA-treated cells. In contrast to the results with RA receptor beta and LAMB1, the effects of cAMP analogues on the RA-associated regulation of the bone morphogenetic protein 2 gene were not altered in the cell lines that exhibited reduced PKA activity. These results suggest that a partial reduction in PKA activity is not sufficient to abrogate the effects of cAMP analogues on all of the genes regulated by RA.
authors.affiliation: Department of Pharmacology, Cornell University Medical College, New York, New York 10021, USA.
authors.forename: J
authors.initials: J
authors.lastname: Shen
authors.forename: C
authors.initials: C
authors.lastname: Li
authors.forename: L J
authors.initials: LJ
authors.lastname: Gudas
mesh_terms: D000818:Animals; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000187:drug effects / Q000235:genetics*; D002454:Cell Differentiation; D000242:Cyclic AMP / Q000031:analogs & derivatives; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D007797:Laminin / Q000187:drug effects / Q000235:genetics*; D051379:Mice; D018168:Receptors, Retinoic Acid / Q000187:drug effects / Q000235:genetics*; D015398:Signal Transduction; D018243:Teratocarcinoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D014162:Transfection; D016212:Transforming Growth Factor beta; D014212:Tretinoin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
chemical_list: C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D007797:Laminin; D018168:Receptors, Retinoic Acid; D016212:Transforming Growth Factor beta; C082925:retinoic acid receptor beta; D014212:Tretinoin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases