Response to inflammatory stimuli is under a tight cellular regulation, including post-transcriptional mechanisms, to ensure the accurate response of cells to various environmental stresses. The aim of this study was to investigate the post-transcriptional regulatory effect of NaBt, a well-known HDAC inhibitor, on COX-2 mRNA in colon cancer cell lines via AU rich elements (ARE) in the 3’UTR.
Caco-2 and HT-29 cells were treated with NaBt and mRNA and protein expressions were determined by qPCR and Western blot, respectively. COX-2 enzymatic activity was determined by Prostaglandin E2 (PGE2) ELISA assay. mRNA stability was assessed by an Actinomycin D (ActD) chase assay.
Treatment of both cell lines at short (0-6h) and long (48h) time points with NaBt strongly reduced COX-2 mRNA and protein expression. This was accompanied by a strong suppression of stabilizing ARE-binding proteins at 48h. Interestingly, PGE2 levels remained unchanged most likely due to the robust induction of COX-1 by NaBt. The ActD chase assay indicated that in the absence of new mRNA synthesis, NaBt induced stability of COX-2 mRNA in Caco-2 cells while it enhanced COX-2 mRNA decay in HT-29 cells. In the same experimental setup in Caco-2 cells, NaBt and NaBt+ActD together enhanced phosphorylation of the stress-related protein kinase MAPKAPK2 (MK2) compared to ActD alone, which can induce mRNA stabilization through nucleo-cytoplasmic shuttling of AREBPs like CUGBP2 and HuR. In HT-29 cells, NaBt and NaBt+ActD treatments decreased p-MK2, but increased the phosphorylation of Chk2, which in turn modulates the nucleo-cytoplasmic shuttling of HuR to facilitate COX2 mRNA degradation. Mild reduction in p-Chk2 was observed in Caco-2 cells in the presence of NaBt.
These findings suggest that NaBt can regulate mRNA stability of COX-2 through post-transcriptional mechanisms and in a contextual manner, dependent on upstream signaling. Future studies will indicate how NaBt can affect the dichotomy in Chk2/MK2 activation in the presence of cellular stress.