hnRNP K-Mediated Regulation of COX-2 Expression in Monocytes by Ligation of the Receptor for Advanced Glycation End Products (RAGE).

Narkunaraja, Phosphatases-Kinases; Ramanatarajan, Shanmugam

Advanced glycation end products (AGEs) act via their receptor, RAGE, and play major roles in diabetic complications. Cycloxygenase (COX)-2 is an early response inflammatory enzyme that is implicated in the pathogenesis of several inflammatory and auto-immune diseases including diabetes. However its expression and functional role under diabetic conditions are not fully clear. We recently showed that AGEs and a specific RAGE ligand, S100b, can induce COX2 in monocytes by transcriptional mechanisms. However, we also noted an increase in mRNA stability and accumulation at earlier time points. The objective of the current study was to examine these mRNA stabilizing mechanisms. S100b treatment of THP-1 monocytes led to a significant 2-3 fold accumulation of COX-2 mRNA at 2 hr which was not blocked by actinomycin-D, nor was there any promoter transcription, suggesting RNA stability as an initiating mechanism rather than transcription. This was further confirmed by showing S100b-induced increased luciferase activity of a COX-2-3'UTR-Luc construct. Heterogenous nuclear ribonucleoprotein-K (hnRNPK) is involved in several cellular functions and gene regulation by orchestrating cross-talk among factors regulating signal transduction, transcription, translation, mRNA stability, and chromatin remodeling. However its role in gene regulation under diabetic conditions is not known. We performed chromatin immunoprecipitation (ChIP) assays using anti-hnRNP K antibody and noted that S100b treatment of THP-1 cells led to a loss of hnRNP K occupancy at the COX-2 promoter at the distal NF-kB binding site, possibly relieving transcriptional repression at this site. At the same time, RNA-IP showed increased binding of hnRNP K to the COX-2 UTR RNA in response to S100b. The latter result was confirmed by RNA-EMSA and super-shift with hnRNP K antibody. Western blot analysis showed S100b treatment demethylates Lys-216 of hnRNP K in monocytes, suggesting that demethylation may be a signal for hnRNP K activation and binding to the COX-2 3'UTR to form RNP complexes that enhance mRNA stability. These new results uncover a novel dual role for hnRNP K in mediating COX-2 mRNA stability as well as preparing the promoter for subsequent transcription in response to S100b in monocytes. These findings show for the first time that diabetic stimuli such as AGEs can increase the stability of inflammatory genes such as COX-2 by modulating the actions of the RNA binding protein hnRNP K ADA-Funded Research

NUCLEOPROTEINS; CYCLOOXYGENASE 2; DIABETES; MONOCYTES; MESSENGER RNA; ACTINOMYCIN; CHROMATIN; RNA-protein interactions

Diabetes, 2007, Vol 56, pA516

0012-1797

Academic Journal