Thesis Title

Regulation Of CGRP Gene Expression In A Human Neuronal-Like Cell Line, Dms 153

Date of Graduation

Fall 2006

Degree

Master of Science in Biology

Department

Biology

Committee Chair

Paul Durham

Keywords

CGRP, DMS 153, migraine, trigeminal, botulinum neurotoxin

Subject Categories

Biology

Abstract

Calcitonin gene-related peptide (CGRP) is thought to play an important role in the pathology of migraine and temporomandibular joint disorders. This 37 amino acid neuropeptide is involved in neurogenic inflammation and pain transmission. To investigate CGRP gene expression, I utilized DMS 153 (DMS) cells derived from a small cell carcinoma of human lung. This cell line exhibits a neuroendocrine phenotype, and thus was investigated as a potential human neuronal-like cell model to study gene expression. DMS cells expressed CGRP and SNAP-25, and vanilloid type 1 receptor, tumor necrosis factor alpha type 1 receptor, and serotonin 5-HT₁B and 5-HT₁D receptors, as determined by immunocytochemistry. CGRP secretion, as measured by radioimmunoassay, was increased following a one-hour treatment with KC1, capsaicin (CAP), nitric oxide (NO), TNF-, or protons. The stimulatory effects of CAP and NO on CGRP release were significantly inhibited by co-treatment with botulinum neurotoxin type A. Transient transfection of CGRO/luciferase reporter DNA was used to identify regulatory regions within the 1250 bp fragment of the rat CGRP promoter. Basal CGRP promoter activity was localized to a distal 18 bp enhancer region, while activity of the proximal cAMP-responsive element was stimulated by protein kinase A. In addition, specific cell signaling pathways were investigated using the PathDetect in vivo trans-reporting system. Basal levels of CHOP were much greater than those of Elk1, c-Jun, CREB, NF-B, or ATF2. Overexpression of the mitogen activated protein kinase MEKK greatly stimulated Elk-1, c-Jun, ATF-2 and NF-B activity. In conclusion, my results demonstrate that DMS cells can be used as a model of human neuronal cells to study CGRP gene expression in response to inflammatory as well as anti-inflammatory agents.

Copyright

© William George Hill III

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