Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.
EBV-mir-Bart13 Downregulates Stim1, a Protein Involved in Calcium Signaling, in Human Salivary Gland Cells and May Contribute to the Pathogenesis of Sjogren's Syndrome.
Gallo1, Alessia, Tandon1, Mayank, Illei2, Gabor G., Alevizos1, Ilias
Loss of secretory function of salivary glands is one of the most important functional effects of Sjogren's syndrome (SS), a chronic systemic autoimmune disease. Previous studies have reported the presence of Epstein Barr Virus (EBV) DNA in salivary glands of SS patients. MicroRNA profiling studies in our laboratory have identified an EBV microRNA (ebv-mir-BART13) as significantly over-expressed in minor salivary gland biopsies of SS patients, when compared to the expression in minor salivary glands from non-Sjogren's controls.
Using the RNA22 target prediction algorithm, we identified STIM1 3'UTR as a target of ebv-mir-BART13. Store-operated calcium entry (SOCE) and calcium release-activated calcium current (CRAC) are critical events for the replenishment of intracellular calcium stores and have indispensable roles in various cellular functions, including salivary secretion. SOCE is activated by the depletion of calcium in the endoplasmic reticulum (ER). STIM1, is a protein that has recently been identified as a critical molecular component of CRAC channels. STIM1, is suggested to be the ER-Ca2+ sensor protein regulating SOCE in a number of different cell types. Attenuation of SOCE current underlies salivary gland dysfunction in mice lacking transient receptor potential 1 (TRPC1). We hypothesized that ebv-miR-BART13 may contribute to the salivary gland dysfunction by downregulating STIM-1 and decreasing cacium influx in salivary gland epithelial cells.
Summary of the Results:
To test this hypothesis, an expression plasmid was generated containing the 3'UTR of STIM1 downstream of the firefly luciferase coding sequence. The plasmid was co-transfected with ebv-mir-BART13 analogues and antagonists in HSG cells (human submandibular salivary gland cells). A 40% decrease in the intensity of the luciferase activity was observed 48 hrs after transfections, compared to controls, suggesting that ebv-mir-BART13 binds directly to the 3'UTR of STIM1.
HSG cells wer2179ransfected for 24 and 48hrs, with ebv-miR-BART13 analog and the mRNA and protein levels of STIM1 were measured. The mRNA level was not affected by the presence of this viral miRNA. However, the protein level was dramatically decreased (over 70% decrease) both 24 and 48hrs after transfection, suggesting that the ebv-mir-Bart13 exerts its effects on STIM1 not by degrading its transcript, but by repressing STIM1 translation. Functional Ca2+ assays through Thapsigargin-mediated depletion of the ER calcium in the ebv-mir-BART13 transfected cells showed a significant delay in the influx of calcium in the cytosol compared to controls.
Together, these functional measurements suggest that the presence of ebv-miR-BART13 in the salivary glands of SS patients might be, at least partially, responsible for the salivary gland dysfunction in those patients, by reducing the expression of STIM1, a critical member of the saliva secretion mechanism.
To cite this abstract, please use the following information:
Gallo, Alessia, Tandon, Mayank, Illei, Gabor G., Alevizos, Ilias; EBV-mir-Bart13 Downregulates Stim1, a Protein Involved in Calcium Signaling, in Human Salivary Gland Cells and May Contribute to the Pathogenesis of Sjogren's Syndrome. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :2179