The role of translational regulation in ultraviolet C light-induced cyclooxygenase-2 expression

Abstract

Aims: The role of ultraviolet C light (UVC)-induced phosphorylation of the eukaryotic initiation factor 2 (eIF2) in the regulation of cyclooxygenase-2 (COX-2) expression at both transcriptional and translational levels is investigated.

Main methods: Western analysis was used to determine COX expressions. Immunoprecipitation after [(35)S]-Met/Cys metabolic labeling was used to determine the rate for COX-2 synthesis and turnover. Quantitative real-time PCR was used to determine COX-2 mRNA levels. Ingenuity Pathways Analysis 6 was used for mapping COX-2 activation network.

Key findings: UVC induces COX-2 expression in wild-type mouse embryo fibroblasts (MEF(S/S)) and that the inducibility is reduced in MEF(A/A) cells in which the phosphorylation site, Ser-51 in the eIF2alpha, is replaced with a nonphosphorylatable Ala (S51A). UVC-induced transcription of COX-2 is delayed in MEF(A/A) cells, which correlates with NF-kappaB activation as previously reported (Wu, S, Tan, M, Hu, Y, Wang, JL, Scheuner, D, Kaufman, RJ, Ultraviolet light activates NFkappaB through translational inhibition of IkappaBalpha synthesis. The Journal of Biological Chemistry, 279, 34898-34902, 2004). The translational efficiency of COX-2 is higher in MEF(A/A) cells than in MEF(S/S) cells at 4 h, but not at 24 h post-UVC. The translation efficiency is correlated to the ratio of activated COX-2 binding protein HuR/TIAR. In addition, the newly synthesized COX-2 protein is more stable in MEF(A/A) cells than in MEF(S/S) cells. The results demonstrated a complex and dynamic regulation of COX-2 expression.

Significance: UVC induces a prolonged expression of COX-2. While transcriptional regulation of COX-2 expression is intensively studied, the role of translational regulation of COX-2 synthesis upon UVC-irradiation is not yet clear. This study elucidated a novel eIF2alpha phosphorylation-centered network for the regulation of COX-2 expression after UVC-irradiation.

Fig. 1

Effect of translation on UVC-induced COX-2 expression. The MEF^S/S and MEF^A/A cells were irradiated with UVC (30 J/m²) and then were harvested at the indicated time points. (A) The COX-2 and COX-1 protein levels were detected by western blot analysis using anti-COX-2 and anti-COX-1 antibodies respectively. A western blot analysis of β-actin was used to monitor the loading of proteins. (B) The relative intensities of COX-2 protein bands were quantified by ImageJ (v.1.31, NIH). Results represent the means ± SEM for three independent experiments and levels are expressed relative to the COX-2 levels at 0 h post-UVC-irradiation for both cell lines.

Fig. 2

Translational efficiency and stability of COX-2 after UVC-irradiation. (A) MEF^S/S and MEF^A/A cells were irradiated with 30 J/m² of UVC and then metabolically pulse-labeled with [³⁵S]-Met/Cys for 20min at the indicated time points post irradiation. COX-2 protein was immunoprecipitated, subjected to SDS-PAGE. The gel was dried and the newly synthesized COX-2 was detected by autoradiograph. (B) Same cells were metabolically pulse-labeled with [³⁵S]-Met/Cys and then cultured in complete medium without [³⁵S]-Met/Cys for 2 h. COX-2was then immunoprecipitated from equal amounts of proteins and the [³⁵S] labeled COX-2 was detected by autoradiography as described above. (C) MEF^S/S and MEF^A/A cells were treated with UVC (30 J/m²). At the indicated time points, the cells were treated or not treated with CHX (100αg/mL) for 1 h. Total COX-2 protein levels in the treated cells were determined by western blot analysis. (D) Equal amounts of [³⁵S]-labeled proteins prepared in 2A were loaded on SDS-PAGE and dried. Newly synthesized proteins were detected by autoradiography (shown in left panel), and total amount of proteins were visualized by Coomassie Blue R-250 staining (right panel).

Fig. 3

UVC-induced eIF2α phosphorylation up-regulates COX-2 transcription. MEF^S/S and MEF^A/A cells were irradiated with UVC (30 J/m²). At the indicated time points, total mRNA was isolated and q-RT-PCR was used to determine the levels of COX-2 in the cells. Relative amounts of COX-2 transcripts from a triplicate experiment were normalized and expressed relative to the levels of β-actin housekeeping gene in each sample.

Fig. 4

Effect of UVC on post-transcriptional regulators of COX-2. MEF^S/S and MEF^A/A cells were irradiated with 30 J/m² of UVC. At the indicated time points, the cytoplasmic (Panel A) and nuclear (Panel B) proteins were isolated. The proteins were subjected to western blot analysis using antibodies against HuR and TIAR. To monitor the loading and sub-cellular contamination, expression levels of β-actin and nuclear marker hnRNP C1/C2 were also determined using western blot analysis.

Fig. 5

Proposed model for UVC induced COX-2 regulation. The molecular networks were generated by Ingenuity Pathway Analysis (Ingenuity® Systems) using Ingenuity database established on previously published data.