Altered expression of the mRNA stability factor HuR promotes cyclooxygenase-2 expression in colon cancer cells
Dan A. Dixon, … , Guy A. Zimmerman, Stephen M. Prescott
Dan A. Dixon, … , Guy A. Zimmerman, Stephen M. Prescott
Published December 1, 2001
Citation Information: J Clin Invest. 2001;108(11):1657-1665. https://doi.org/10.1172/JCI12973.
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Article

Altered expression of the mRNA stability factor HuR promotes cyclooxygenase-2 expression in colon cancer cells

Abstract

Cyclooxygenase-2 (COX-2) expression is normally tightly regulated. However, constitutive overexpression plays a key role in colon carcinogenesis. To understand the molecular nature of enhanced COX-2 expression detected in colon cancer, we examined the ability of the AU-rich element–containing (ARE-containing) 3′ untranslated region (3′UTR) of COX-2 mRNA to regulate rapid mRNA decay in human colon cancer cells. In tumor cells displaying enhanced growth and tumorigenicity that is correlated with elevated COX-2, vascular endothelial growth factor (VEGF), and IL-8 protein levels, the corresponding mRNAs were transcribed constitutively and turned over slowly. The observed mRNA stabilization is owing to defective recognition of class II-type AREs present within the COX-2, VEGF, and IL-8 3′UTRs; c-myc mRNA, containing a class I ARE decayed rapidly in the same cells. Correlating with cellular defects in mRNA stability, the RNA-binding of trans-acting cellular factors was altered. In particular, we found that the RNA-stability factor HuR binds to the COX-2 ARE, and overexpression of HuR, as detected in tumors, results in elevated expression of COX-2, VEGF, and IL-8. These findings demonstrate the functional significance rapid mRNA decay plays in controlling gene expression and show that dysregulation of these trans-acting factors can lead to overexpression of COX-2 and other angiogenic proteins, as detected in neoplasia.

Authors

Dan A. Dixon, Neal D. Tolley, Peter H. King, L. Burt Nabors, Thomas M. McIntyre, Guy A. Zimmerman, Stephen M. Prescott

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Figure 7

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Overexpression of HuR in LoVo cells promotes enhanced expression of COX-...
Overexpression of HuR in LoVo cells promotes enhanced expression of COX-2 and angiogenic factors. (a) Luciferase–reporter gene expression constructs containing the full-length COX-2 3′UTR (+3′UTR) or no 3′UTR (Δ3′UTR) were cotransfected with Flag-tagged HuR (HuR) or control empty expression vector in LoVo cells. Relative activity was assessed as luciferase activity normalized to total protein. All percentages listed are based on expression of luciferase from the transfection of construct containing no 3′UTR with control vector (pcDNA3) and is the average of three experiments done in triplicate. (b) Immunoblot of COX-2 protein in HuR or vector-transfected LoVo cell lysates. Flag-tagged HuR and β-actin were detected on the same blot as a control for transfection and protein loading. The data shown represent three experiments. (c) COX activity in HuR-transfected (filled bars) or vector-transfected (open bars) LoVo cells. PGE2 levels were measured by ELISA in the media containing arachidonic acid and are the average of three experiments. (d) Expression of angiogenic factors VEGF (left panel) and IL-8 (right panel) in the culture media of HuR-transfected (filled bars) or vector-transfected (open bars) LoVo cells were measured by ELISA and are the average of three experiments.