Down Syndrome Candidate Region 1 Increases the Stability of the IκBα Protein: IMPLICATIONS FOR ITS ANTI-INFLAMMATORY EFFECTS

Young Sun Kim,Kyung-Ok Cho,Hong Joon Lee,Seong Yun Kim,Yasufumi Sato,Young-Jin Cho

doi:10.1074/jbc.m604659200

Abstract

Down syndrome candidate region 1 (DSCR1), an endogenous inhibitor of calcineurin, inhibits the expression of genes involved in the inflammatory response. To elucidate the molecular basis of these anti-inflammatory effects, we analyzed the role of DSCR1 in the regulation of NF-kappaB transactivation using glioblastoma cells stably transfected with DSCR1.4 or its truncation mutants (DSCR1.4-(1-133) and DSCR1.4-(134-197)). Overexpression of DSCR1.4 significantly attenuated the induction of cyclooxygenase-2 (COX-2) expression by phorbol 12-myristate 13-acetate (PMA) via a calcineurin-independent mechanism. Experiments using inhibitors of the signaling molecules for NF-kappaB activation showed that NF-kappaB is responsible for the induction of COX-2. Full-length and truncated DSCR1.4 decreased the steady-state activity of NF-kappaB as well as PMA-induced activation of NF-kappaB, which correlated with attenuation of COX-2 induction. DSCR1.4 did not affect the PMA-stimulated phosphorylation or degradation kinetics of IkappaBalpha; however, DSCR1.4 significantly decreased the basal turnover rate of IkappaBalpha and consequently up-regulated its steady-state level. In the same context, knockdown of endogenous DSCR1.4 increased the turnover rate of IkappaBalpha as well as COX-2 induction. These results suggest that DSCR1 attenuates NF-kappaB-mediated transcriptional activation by stabilizing its inhibitory protein, IkappaBalpha.

Highlights

Down syndrome candidate region 1 (DSCR1), an endogenous inhibitor of calcineurin, inhibits the expression of genes involved in the inflammatory response
This study shows for the first time that DSCR1.4 decreases the basal turnover rate of I␬B␣ and attenuates the steady-state and stimulus-induced transcriptional activity of Nuclear factor ␬B (NF-␬B) in human U87MG glioblastoma cells
We demonstrate that this inhibition of the NF-␬B pathway by DSCR1.4 is functionally significant by showing that overexpression of DSCR1.4 reduced induction of the NF-␬B target genes cyclooxygenase-2 (COX-2) and interleukin-1␤ (IL-1␤), which were induced by phorbol 12-myristate 13-acetate (PMA) or pro-inflammatory cytokines

Summary

Introduction

Down syndrome candidate region 1 (DSCR1), an endogenous inhibitor of calcineurin, inhibits the expression of genes involved in the inflammatory response. This study shows for the first time that DSCR1.4 decreases the basal turnover rate of I␬B␣ and attenuates the steady-state and stimulus-induced transcriptional activity of NF-␬B in human U87MG glioblastoma cells. DSCR1.4 Inhibits PMA-induced COX-2 Expression via a Calcineurin-independent Mechanism—To examine the effects of elevated DSCR1 levels on COX-2 induction, we generated derivatives of the U87MG cell line by transfection with expression vectors for recombinant DSCR1 proteins with GFP fusion tags at the N termini (ntDSCR1) or C termini (ctDSCR1) or with a vector expressing GFP alone as a control.

Results

Conclusion