Effects of flurbiprofen on the functional regulation of serotonin transporter and its misfolded mutant.

Flurbiprofen, a nonsteroidal anti-inflammatory drug, reportedly exhibits chemical chaperone activity. Herein, we investigated the role of flurbiprofen in regulating serotonin transporter (SERT) function via membrane trafficking. We used COS-7 cells transiently expressing wild-type (WT) SERT or a C-terminus-deleted mutant of SERT (SERTΔCT), a misfolded protein. Flurbiprofen treatment reduced the expression of immaturely glycosylated SERT and enhanced the expression of maturely glycosylated SERT. In addition, we observed increased serotonin uptake in SERT-expressing cells. These results suggest that flurbiprofen modulates SERT function by promoting membrane trafficking. In SERTΔCT-expressing cells, flurbiprofen reduced the protein expression and uptake activity of SERTΔCT. Furthermore, flurbiprofen inhibited the formation of SERTΔCT aggregates. Studies using flurbiprofen enantiomers suggested that these effects of flurbiprofen on SERT were not mediated via cyclooxygenase inhibition. The levels of GRP78/BiP, an endoplasmic reticulum (ER) stress marker, were assessed to elucidate whether flurbiprofen can ameliorate SERTΔCT-induced ER stress. Interestingly, flurbiprofen induced GRP78/BiP expression only under ER stress conditions and not under steady-state conditions. In HRD1 E3 ubiquitin ligase knockdown cells, flurbiprofen affected the ER-associated degradation system. Collectively, the findings suggest that flurbiprofen may function as an inducer of molecular chaperones, in addition to functioning as a chemical chaperone.

The Development of Screening Methods to Identify Drugs to Limit ER Stress Using Wild-type and Mutant Serotonin Transporter.

The function of the serotonin transporter (SERT) is regulated by its membrane trafficking. Previously, we showed that the C-terminus-deleted mutant of SERT (SERTΔCT) exhibited an aberrant membrane trafficking and subsequent retention at the endoplasmic reticulum (ER). In addition, we found that proteasome inhibitor-induced ER stress resulted in the impairment of SERT membrane trafficking and retention of SERT at the ER, an impairment very similar to that of SERTΔCT. Based on the result that the chemical chaperone 4-phnylbutulic acid (4-PBA), which relieves ER stress, accelerated the membrane trafficking and upregulated SERT activity, we hypothesized that drugs that facilitate the membrane trafficking of SERT would have potential therapeutic effects on an ER stress-related disease. In this study, we aimed to develop simple screening methods for such drugs using SERT. We first validated the serotonin uptake assay using fluorescent substrates. This simple and reliable assay method was useful for screening for drugs that affected the wild-type SERT but not SERTΔCT. In addition, we verified an assay focusing on the formation of SERTΔCT aggregates. The drugs 4-PBA and SKF-10047 facilitated the trafficking of SERT to the membrane and reduced SERTΔCT aggregates, indicating that the drugs with such characters could be potential candidates for ER stress relief. For both assays, we clarified the usefulness of a high-content screening microscope. These results could pave the way for high-throughput screening for such drugs.