Open AccessA secretagogue–small interfering RNA conjugate confers resistance to cytotoxicity in a cell model of Sjögren's syndrome
Author(s) -
Pauley Kaleb M.,
Gauna Adrienne E.,
Grichtchenko Irina I.,
Chan Edward K. L.,
Cha Seunghee
Publication year - 2011
Publication title -
arthritis & rheumatism
Language(s) - English
Resource type - Journals
eISSN - 1529-0131
pISSN - 0004-3591
DOI - 10.1002/art.30450
Subject(s) - secretagogue , small interfering rna , endocytosis , carbachol , transfection , endocrinology , microbiology and biotechnology , chemistry , medicine , biology , receptor , cell culture , stimulation , genetics
Objective Sjögren's syndrome (SS) is characterized by xerophthalmia and xerostomia resulting from loss of secretory function due to immune cell infiltration in lacrimal and salivary glands. Current therapeutic strategies for SS use secretagogues to induce secretion via muscarinic receptor stimulation. The purpose of this study was to create a secretagogue–small interfering RNA (siRNA) conjugate to deliver siRNA into cells via receptor‐mediated endocytosis, thereby altering epithelial cell responses to external cues, such as proinflammatory or death signals, while simultaneously stimulating secretion. Methods Based on our expertise with type 3 muscarinic receptor (M 3 ), we used carbachol, a ligand specific for muscarinic receptor, as the secretagogue. Carbachol was synthesized with an active choline group and was conjugated with an siRNA that targets caspase 3. A human salivary gland (HSG) cell line was used to test the efficacy of this secretagogue–siRNA conjugate. Results Lipofectamine transfection of the conjugate into HSG cells resulted in a 78% reduction in the expression of the caspase 3 gene, while external conjugate treatment of HSG cells resulted in intracellular calcium release and induction of endocytosis at levels similar to those of carbachol stimulation, indicating that the siRNA and carbachol portions of the conjugate retained their function after conjugation. HSG cells treated with conjugate (without Lipofectamine transfection) exhibited a 50% reduction in caspase 3 gene and protein expression, indicating that our conjugate was effective in delivering functional siRNA into cells via receptor‐mediated endocytosis. Furthermore, tumor necrosis factor α–induced apoptosis was significantly reduced in conjugate‐treated cells. Conclusion Our secretagogue–siRNA conjugate prevented cytokine‐induced apoptosis in salivary gland epithelial cells, which is critical to maintaining fluid secretion and potentially reversing the clinical hallmark of SS.