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CYCLIC AMP-DEPENDENT PROTEIN KINASE INDUCTION BY POLYCHLORINATED BIPHENYLS (PCBS) STIMULATES CREB PHOSPHORYLATION VIA A CALCIUM-DEPENDENT, PKC-INDEPENDENT PATHWAY IN CORTICAL NEURONS.
Citation:
Inglefield, J R., W R. Mundy, C A. Meacham, AND T J. Shafer. CYCLIC AMP-DEPENDENT PROTEIN KINASE INDUCTION BY POLYCHLORINATED BIPHENYLS (PCBS) STIMULATES CREB PHOSPHORYLATION VIA A CALCIUM-DEPENDENT, PKC-INDEPENDENT PATHWAY IN CORTICAL NEURONS. Presented at Society of Toxicology, Nashville, TN, March 17-21, 2002.
Description:
We have previously demonstrated that the PCB mixture, Aroclor 1254 (A1254), increases the phosphorylated form of CREB (pCREB), the cAMP-responsive element binding protein. This transcription factor is important in nervous system development and plasticity. Phosphorylation
of CREB results in its activation and is mediated by a variety of stimuli, including activation of protein kinases A and C and increases in intracellular calcium. As such, the present experiments were designed to determine the mechanism(s) by which PCBs increase pCREB. Using digital microscopy, semi-quantitative fluorescent immunocytochemistry of serine-133 phosphorylated CREB was performed in primary cultures of cortical neurons in the presence and absence of A1254 and agonists and antagonists of PKA, PKC, and intracellular Ca2+. Cellular pCREB flourescence intensity was analyzed with the aid of image analysis software. The pCREB responses were represented as Gaussian populations where the half-maximal fluorescence (EC50) of the population was the comparator across treatment groups. In controls, pCREB was constitutively and non-uniformly expressed. A1254 (2-20 M) increased pCREB immunoreactivity in a dose-dependent manner. A1254 (10 M) or the agonists glutamate, forskolin and phorbol myristate acetate increased the EC50 values for pCREB fluorescence by 36?5, 51?15, 47?9 and 40?10%, respectively. PKA inhibitor peptide (30 min preincubation) completely inhibited, whereas BAPTA-AM or tetrodotoxin partially inhibited, and the PKC inhibitor, bisindoylmaleimide, did not inhibit pCREB induction by A1254 (10 M, 40 min). We also examined whether cAMP is involved in the activation of PKA by PCBs. The adenylate cylcase activator, forskolin, but not A1254 (2-20 M) elevated intracellular cAMP levels. These data support a role of a PKA and intracellular Ca2+ pathways involved in PCB-induced CREB phosphorylation. (This is an abstract of a proposed presentation and does not necessarily reflect EPA policy).