Abstract
Interleukin-12 (IL-12), p80, and IL-23 are structurally related cytokines sharing a p40 subunit. We have recently demonstrated that celecoxib and its COX-2-independent analogue 4-trifluoromethyl-celecoxib (TFM-C) inhibit secretion but not transcription of IL-12 (p35/p40) and p80 (p40/p40). This is associated with a mechanism involving altered cytokine-chaperone interaction in the endoplasmic reticulum (ER). In the present study, we found that celecoxib and TFM-C also block secretion of IL-23 (p40/p19 heterodimers). Given the putative ER-centric mode of these compounds, we performed a comprehensive RT-PCR analysis of 23 ER-resident chaperones/foldases and associated co-factors. This revealed that TFM-C induced 1.5-3-fold transcriptional up-regulation of calreticulin, GRP78, GRP94, GRP170, ERp72, ERp57, ERdj4, and ERp29. However, more significantly, a 7-fold up-regulation of homocysteine-inducible ER protein (HERP) was observed. HERP is part of a high molecular mass protein complex involved in ER-associated protein degradation (ERAD). Using co-immunoprecipitation assays, we show that TFM-C induces protein interaction of p80 and IL-23 with HERP. Both HERP siRNA knockdown and HERP overexpression coupled to cycloheximide chase assays revealed that HERP is necessary for degradation of intracellularly retained p80 by TFM-C. Thus, our data suggest that targeting cytokine folding in the ER by small molecule drugs could be therapeutically exploited to alleviate inappropriate inflammation in autoimmune conditions.
Highlights
Celecoxib is a non-steroidal anti-inflammatory drug originally designed to inhibit cyclooxygenase-2 (COX2), up-regulated in cancer and sites of inflammation [13]
Intracellular Cytokine Retention—We have previously shown similar to thapsigargin, TFM-C appears to display a certain that secretion of IL-12 and p80 is blocked by both celecoxib and level of selectivity in the type of oligomeric proteins of which it TFM-C [12]
In this study we have demonstrated the ability of celecoxib and the non-coxib analogue TFM-C to inhibit the secretion of
Summary
Cell Culture Conditions—All tissue culture reagents were purchased from Invitrogen (Paisley, UK) unless otherwise stated. Medium was removed, cells were washed with PBS, and HERP was solubilized using a modified version of protocol 2 described above. One induced flask was treated with 50 M TFM-C and all were left for 24 h After this time, culture medium was discarded and cells washed with ice-cold PBS before being scraped and pelleted at 1000 rpm for 5 min. Cross-linked cell pellets were lysed using a modified version of protocol 2 based on membrane protein solubilization in 8 M urea and 1% protease inhibitors, as outlined earlier. After 20 h, intracellular p19FLAG in cell lysates was purified by ␣-M2 FLAG protein A-Sepharose immunoprecipitation and resolved on reducing-PAGE and Western blot. After 8 h, medium was subjected to reducing SDS-PAGE and probed for IgG heavy chain secretion by Western blot. Data are plotted as mean Ϯ S.E. both cases, 50 M celecoxib or TFM-C had no effect on IgG with significance indicated as p Ͻ 0.05
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