Background/Purpose Aquaporin (AQP) 5 belongs to a family of small integral membrane proteins which function as a water channels in cells. AQP5 plays a critical role in mediating the secretion of fluid in salivary gland. Decrease or no saliva flow is one of the key symptoms in Sjögren’s syndrome patients. Towards understanding the molecular basis of the loss of salivary secretion, here we have studied the regulation of AQP5 expression in primary human salivary gland epithelial cells (phSG). 

Methods cell culture, Western blot, qPCR, siRNAs, site-directed mutagenesis,  chromatin immunoprecipitation 

Results We observed that the phenotype of the cells depended largely on the calcium levels in the culture conditions. AQP5 transcript showed more than 3-fold increase in phSG cells grown in high calcium (0.8 mM) medium compared to that in low calcium (0.05 mM) condition. This was confirmed by both immunofluorescence staining and Western blot studies. Evaluation of expression transcripts of calcium signaling proteins in phSG cells revealed that NFAT1 expression was dependent on calcium in a dose-response manner. Furthermore, when cells were switched to a high calcium medium pGFP-NFAT1 was translocated from the cytoplasm into the nucleus. Expression of AQP5 was also reduced when phSG cells were treated with cyclosporine A confirming the involvement of calcineurin-NFAT1 signaling pathway in regulation of AQP5 expression. Since NFAT activation has been linked to calcium influx, we examined the role of the calcium entry regulatory proteins STIM1 and STIM2 as well as the channel protein Orai1. Knockdown of NFAT1 and STIM1, but not STIM2 or Orai1, resulted in 70% decrease of AQP5 expression. Further analysis revealed that several NFAT binding motifs were identified in the AQP5 promoter and these were validated using AQP5-promoter-luciferase assays. Mutagenesis of putative NFAT-binding regions as well as chromatin immunoprecipitation analyses revealed the presence of functional NFAT binding sites within the proximal AQP5 promoter region.

Conclusion These data demonstrate that AQP5 expression in phSG cells is regulated by a calcineurin-NFAT-dependent signaling pathway. Importantly, STIM1, but not Orai1 is involved in this mechanism. We propose that external Ca²⁺-dependent Ca²⁺ entry triggers activation of AQP5 expression in phSG cells. Thus alterations in calcium signaling could lead to alterations in AQP5 expression and function in pSS. Further studies are being directed towards determining the status of calcium signaling in pSS salivary glands.

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« Back to 2014 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/calcium-calcineurin-nfat-signaling-pathway-regulates-aqp5-expression-in-primary-salivary-gland-acinar-cells/