Interleukin-1 Beta Mediated Regulation of Hyaluronan and Hyaluronan Synthase 2

Author

Ashley Elizabeth Ducale, Virginia Commonwealth University

Defense Date

2005

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Biochemistry

First Advisor

Dr. Dorne R Yager

Abstract

Elevated levels of hyaluronan are associated with numerous inflammatory diseases including ulcerative colitis, Crohn's disease and wound healing. Various proinflammatory cytokines have shown to influence hyaluronan expression in cells originating from connective tissue. The overall purposes of this study included: 1. To determine the effects of IL-1β stimulation on HA and HAS2 steady state transcript levels and the signaling pathways involved in its effects. The signaling pathways utilized by proinflammatory mediators to modulate hyaluronan expression have only begun to be elucidated. In this aim, the effects of IL-1β on hyaluronan and HAS expressions in jejunum-derived mesenchymal cells were determined. Inhibition studies were utilized to determine the signaling pathways involved. The overall hypothesis of this aim was that stimulation of jejunum-derived mesenchymal cells with IL-1β activates the mitogen activated protein kinase pathways resulting in increased HAS2 steady state transcript and hyaluronan levels.Results: The results suggest that IL-1β induction of HAS2 expression involves, in part, the mitogen activated protein kinase signaling pathways that act in concert thus leading to an increase in expression of hyaluronan by jejunum-derived mesenchymal cells.2. To determine the effects of dexamethasone on IL-1β mediated increase in hyaluronan and HAS2 expressions and the mechanisms utilized by this glucocorticoid. Glucocorticoids are a mainstay treatment for the inflammatory component of inflammatory bowel disease. Given the recent evidence demonstrating increased hyaluronan in inflamed tissue from patients affected with inflammatory bowel disease, the objective of this aim was to determine the effect of dexamethasone on IL-1β-mediated induction of hyaluronan. The hypothesis of this aim was that pre-treatment with dexamethasone suppressed the ability of IL-1β to increase HAS2 transcript and hyaluronan levels via inhibition of the p38 MAP kinase pathway. Results: Pre-treatment with dexamethasone inhibited IL-1β-mediated hyaluronan and HAS2 induction by blocking the activation of the p38 MAP kinase pathways. 3. To identify the transcriptional and post-transcriptional mechanisms utilized by IL-1β to upregulate HAS2 steady state transcript levels. Very little is known about transcriptional and post-transcriptional regulation of the hyaluronan synthase 2 gene. In this aim, 5' and 3' mapping, luciferase analyses and actinomycin D studies were used to determine the transcriptional and post-transcriptional mechanisms utilized by IL-1β to regulate HAS2 steady state transcript levels. The hypothesis of this aim was that IL-1β used post-transcriptional mechanisms to regulate the HAS2 gene.Results: Dermal fibroblasts were used to find the 5'- and 3'-termini of the HAS2 message. Promoter constructs extending approximately 1 kb upstream from the transcription start site demonstrated no IL-1β response. Blocking protein synthesis prior to the addition of IL-1β dramatically increased HAS2 steady state transcript levels, while inhibition of transcription suppressed the effect of IL-1β on HAS2. Northern blot analysis revealed that cycloheximide and IL-1β exerted differential effects on the two HAS2 transcripts.

Comments

Part of Retrospective ETD Collection, restricted to VCU only.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

June 2008