Identification of calcium-dependent and -independent signaling pathways involved in polychlorinated biphenyl-induced cyclic AMP-responsive element-binding protein phosphorylation in developing cortical neurons.
Cyclic AMP (cAMP)-responsive element-binding protein (CREB) is a transcription factor important in developing nervous system cells and is activated by a variety of signaling molecules. Aroclor 1254 (A1254), a polychlorinated biphenyl mixture, perturbs Ca(2+) homeostasis and increases CREB phosphorylation in rat neonatal cortical cell cultures in a time- and concentration-dependent manner. The present experiments determined that the cell type responding to A1254 with Ca(2+) increases and phosphorylated CREB (phospho-CREB) was predominantly of neuronal morphology and microtubule-associated protein (MAP2)-positive phenotype. Similarly, glutamate (100 microM) increased phospho-CREB immunoreactivity selectively in MAP2-immunopositive cells. Using Western blotting and immunocytochemical techniques, we identified key signal transduction pathways operative in phosphorylating CREB in cortical cell cultures and examined their participation in 3 ppm A1254-induced CREB activation. Cortical cultures treated with glutamate, forskolin or the phorbol ester phorbol 12-myristate 13-acetate exhibited robust increases in phospho-CREB. Tetrodotoxin (1 microM) completely inhibited CREB phosphorylation by A1254, suggesting that synaptic activity is involved in A1254-induced CREB activation. Buffering [Ca(2+)](i) with bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl) ester in the absence of extracellular Ca(2+) partially inhibited A1254-induced CREB phosphorylation. Inhibition of mitogen-activated protein kinase (10 microM U0126) or protein kinase C (PKC; bisindoylmaleimide, 5 microM) activation did not inhibit A1254-induced CREB phosphorylation. By contrast, inhibition of protein kinase A (PKA) with 100 microM PKA inhibitor peptide, PKI, blocked A1254-induced CREB phosphorylation. Thus, we examined whether A1254 activates PKA by increasing cAMP; 10 microM forskolin, but not A1254, elevated intracellular cAMP levels. These results indicate that in neocortical cells in culture, CREB phosphorylation occurs via Ca(2+)-, PKA-, and PKC-dependent pathways. Furthermore, A1254-induced CREB phosphorylation occurs predominantly in neurons, is dependent on synaptic activity and mediated by Ca(2+)- and PKA-dependent pathways.
Inglefield
J R
JR
Neurotoxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, US Environmental Protection Agency, Research Triangle Park, NC 27711, USA.
Mundy
W R
WR
Meacham
C A
CA
Shafer
T J
TJ
D000818:Animals; D013956:Antithyroid Agents / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002273:Carcinogens / Q000494:pharmacology; D002540:Cerebral Cortex / Q000166:cytology / Q000254:growth & development; D020111:Chlorodiphenyl (54% Chlorine) / Q000494:pharmacology*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D009457:Neuroglia / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D020318:Rats, Long-Evans; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D009435:Synaptic Transmission / Q000187:drug effects / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D013956:Antithyroid Agents; D002273:Carcinogens; D017362:Cyclic AMP Response Element-Binding Protein; D020111:Chlorodiphenyl (54% Chlorine); D017868:Cyclic AMP-Dependent Protein Kinases; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium