Identification of calcium-dependent and -independent signaling pathways involved in polychlorinated biphenyl-induced cyclic AMP-responsive element-binding protein phosphorylation in developing cortical neurons

Abstract

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 μM) 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 μM) 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 μM U0126) or protein kinase C (PKC; bisindoylmaleimide, 5 μM) activation did not inhibit A1254-induced CREB phosphorylation. By contrast, inhibition of protein kinase A (PKA) with 100 μM PKA inhibitor peptide, PKI, blocked A1254-induced CREB phosphorylation. Thus, we examined whether A1254 activates PKA by increasing cAMP; 10 μM 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.