okadaic-acid and Teratocarcinoma

In previous work we suggested that a kidney-specific transcription factor LFB3 cooperates with cAMP-response element (CRE)-binding proteins within a cAMP regulatory unit comprised of three protein-binding domains and located 3.4 kilobase pairs upstream of the urokinase-type plasminogen activator (uPA) gene in LLC-PK1 cells (Menoud, P.-A., Matthies, R., Hofsteenge, J., and Nagamine, Y. (1993) Nucleic Acids Res. 21, 1845-1852). The two domains contain a CRE-like sequence, and the third domain is recognized by LFB3. The absolute requirement of LFB3 as well as the cooperation among the three domains for cAMP regulation were confirmed by transient transfection assays in F9 teratocarcinoma cells, in which the level of LFB3 was negligible. Suspecting a possible feedback regulation of LFB3 mRNA expression during cAMP-dependent uPA gene induction in LLC-PK1 cells, we measured LFB3 mRNA levels after cAMP treatment and found a strong reduction. This reduction was not due to a change in template activity of the LFB3 gene because run-on transcription showed no significant change in LFB3 gene transcription. RNA synthesis inhibitor-chase experiments indicated that the down-regulation was post-transcriptional. Interestingly, when the inhibitor was added at the same time as cAMP, the cAMP-induced decrease in LFB3 mRNA levels was abrogated, suggesting that ongoing RNA synthesis is required for the decrease. Similar effects on LFB3 mRNA metabolism were observed with all agents that induce uPA mRNA in LLC-PK1 cells, including 12-O-tetradecanoylphorbol-13-acetate, okadaic acid, colchicine, and cytochalasin. We discuss the significance of this regulation in uPA gene expression.

Topics: Animals; Base Sequence; Binding Sites; Cell Line; Colchicine; Cyclic AMP; Cytochalasin B; DNA-Binding Proteins; Enzyme Induction; Ethers, Cyclic; Feedback; Gene Expression Regulation, Enzymologic; Hepatocyte Nuclear Factor 1-beta; Kidney; Luciferases; Mice; Molecular Sequence Data; Okadaic Acid; Oligodeoxyribonucleotides; Promoter Regions, Genetic; Recombinant Proteins; RNA, Messenger; Signal Transduction; TATA Box; Templates, Genetic; Teratocarcinoma; Tetradecanoylphorbol Acetate; Thymidine Kinase; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transfection; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

Okadaic acid, a protein phosphatase inhibitor, is a strong tumor promoter which apparently activates protein phosphorylation. To examine the role of protein phosphatases in stem cell growth and differentiation, embryonal carcinoma F9 cells were treated with okadaic acid. In the presence of this agent, the cells showed rapid morphological changes and arrest of proliferation at the M phase of the cell cycle, accompanied by a marked increase in the mRNA expression of various differentiation markers. Okadaic acid induced rapid increase in the mRNA levels of both c-jun and junB and results indicate that the inhibition of phosphatase by okadaic acid induces apparent activation of protein phosphorylation and may cause the expression of differentiation marker genes in F9 cells via the activation of AP-1.

Topics: Animals; Biomarkers; Cell Differentiation; Cell Division; Embryonal Carcinoma Stem Cells; Enhancer Elements, Genetic; Ethers, Cyclic; Genes, jun; Mice; Neoplastic Stem Cells; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Plasminogen Activators; Teratocarcinoma; Transcription Factor AP-1