Regulation of RAGE by sRAGE in human salivary gland cells

Objectives: The soluble receptor for advanced glycation endproducts (sRAGE) has been implicated as an anti-inflammatory factor in a number of chronic inflammatory conditions such as diabetes and rheumatoid arthritis. The aim of our study was to investigate the inhibitory effect of sRAGE on RAGE expression in the human salivary gland cell line (HSG), which has been utilized in vitro to study the pathogenesis of autoimmune Sjögren Syndrome (SjS) where up-regulated RAGE was observed.

Methods: RAGE and downstream transcription factor NF-кB were analyzed by RT-PCR, Western blot analysis, and In-Cell-Western, following HSG cell stimulation for 24 hours under the following conditions: untreated, S100A4 (an agonist inducing inflammatory responses through RAGE), sRAGE, sRAGE followed by S100A4, and s100A4 followed by sRAGE treatment.

Results: RT-PCR results revealed that agonist S100A4 increases RAGE expression in a dose dependent manner. RAGE expression level was highest at 150 µg/µL with S100A4 and 50 µg/µL with sRAGE. When sRAGE was added to the cells prior to S100A4, the inhibitory effects of sRAGE on RAGE expression were demonstrated at 10 and 50 µg/µL, but not at 100 µg/µL. Comparatively, this inhibitory effect was demonstrated at 10 µg/µL but not at 50 and 100 µg/µL when S100A4 was added prior to sRAGE. Therefore, the sRAGE inhibitory effect on RAGE expression was most efficient when sRAGE was incubated first, followed by S100A4. In-Cell-Western analysis was consistent with the RT-PCR findings. Finally, Western blot analysis indicates that both p50 and p65 of NF-кb, which is induced by RAGE and S100A4 interaction, were down-regulated when sRAGE was added prior to agonist stimulation.

Conclusion: sRAGE inhibits RAGE expression on HSG cells only when optimal concentrations were applied, which can be a critical factor for in vivo application of sRAGE in the future to dampen chronic inflammatory conditions such as SjS (NIH/NIDCR-U24DE016509 & R21DE016705).