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Cox-2 Promotes Chromogranin A Expression and Bioactivity: Evidence for a Prostaglandin E₂-Dependent Mechanism and the Involvement of a Proximal Cyclic Adenosine 5'-Monophosphate-Responsive Element

Department of Medicine (R.C., D.G., D.M., B.J., J.A., A.M.), Queen’s University Belfast, Royal Group of Hospitals, Belfast BT12 6BJ, Northern Ireland, United Kingdom; Academic Endocrine Unit (N.L., R.T.), Nuffield Department of Medicine, Oxford Centre for Diabetes, Endocrinology, and Metabolism, Churchill Hospital and Medical Research Council Functional Genomics Unit (D.B.), Department of Human Anatomy and Genetics, University of Oxford, Headington, Oxford OX3 0BP, United Kingdom; and Department of Medicine (D.T.O., L.T.), University of California at San Diego, La Jolla, California 92093

Address all correspondence and requests for reprints to: Ann McGinty, Ph.D., Department of Medicine, Mulhouse Building, RGH Grosvenor Road, Belfast BT12 6BJ, Northern Ireland, United Kingdom. E-mail: a.mcginty{at}qub.ac.uk.

The prostanoid biosynthetic enzyme cyclooxygenase-2 (Cox-2) is up-regulated in several neuroendocrine tumors. The aim of the current study was to employ a neuroendocrine cell (PC12) model of Cox-2 overexpression to identify gene products that might be implicated in the oncogenic and/or inflammatory actions of this enzyme in the setting of neuroendocrine neoplasia. Expression array and real-time PCR analysis demonstrated that levels of the neuroendocrine marker chromogranin A (CGA) were 2- and 3.2-fold higher, respectively, in Cox-2 overexpressing cells (PCXII) vs. their control (PCMT) counterparts. Immunocytochemical and immunoblotting analyses confirmed that both intracellular and secreted levels of CGA were elevated in response to Cox-2 induction. Moreover, exogenous addition of prostaglandin E₂ (1 µM) mimicked this effect in PCMT cells, whereas treatment of PCXII cells with the Cox-2 selective inhibitor NS-398 (100 nM) reduced CGA expression levels, thereby confirming the biospecificity of this finding. Levels of neuron-specific enolase were similar in the two cell lines, suggesting that the effect of Cox-2 on CGA expression was specific and not due to a global enhancement of neuroendocrine marker expression/differentiation. Cox-2-dependent CGA up-regulation was associated with significantly increased chromaffin granule number and intracellular and secreted levels of dopamine. CGA promoter-driven reporter gene expression studies provided evidence that prostaglandin E₂-dependent up-regulation required a proximal cAMP-responsive element (–71 to –64 bp). This study is the first to demonstrate that Cox-2 up-regulates both CGA expression and bioactivity in a neuroendocrine cell line and has major implications for the role of this polypeptide in the pathogenesis of neuroendocrine cancers in which Cox-2 is up-regulated.