Abstract
Purpose :
Cathepsin S (CTSS) activity is increased in tears of Sjögren’s Syndrome patients (SS). Rab27a, a Ras-related small GTPase, has previously been linked to regulated secretion of proteins including CTSS in lacrimal gland acinar cells (LGACs). The lacrimal gland (LG) is innervated by both sympathetic and parasympathetic nerves. Here we compare the role of Rab27a in parasympathetic cholinergic and sympathetic α1-adrenergic stimulation of tear CTSS secretion.
Methods :
Parent C57BL/6 mice (C57) and Rab27a-mutant Ashen mice (Ash) were used. In vitro secretion assays were performed on freshly isolated mouse LGACs treated with the a1-adrenergic agonist phenylephrine (PE,100 μM) or the cholinergic agonist carbachol (CCh, 100 μM). CTSS activity and total protein content of culture media was measured and normalized to cellular protein. In vivo secretion assays used topical stimulation of the LG with CCh (50 μM) or PE (100 μM). Stimulated tear volume, total tear protein and tear CTSS activity were measured. Lysosomes were isolated from LG using Opti-prep gradients and CTSS content assessed by Western blotting. Statistical comparisons used 1- and 2-way ANOVA.
Results :
In vivo tear secretion studies showed that topical stimulation of the LG with PE induced greater secretion of CTSS activity relative to CCh (1-fold) in both C57 and Ash mice (C57: p=0.00179; Ash: p=0.00289, N=15). PE also induced more robust protein secretion compared to CCh stimulation, showing 40% increase in tear protein concentration in C57 mice (p=9.335×10-5) and 69% increase in Ash mice (p=3.146×10-9). Analysis of in vitro secretion from cultured LGAC revealed additional details of PE-induced CTSS secretion, with Ash mouse LGAC releasing 50% more CTSS activity compared to C57 mouse LGAC (p=0.0307, N=3). Preliminary studies evaluating recovery of CTSS in isolated lysosomes by Western blotting showed increased CTSS in lysosomes from Ash mouse LG relative to those from C57 mouse LG, and reduction of both pools by CCH stimulation. However, complete depletion of lysosomal CTSS stores appeared after PE stimulation only in Ash but not C57 mouse LG lysosomes.
Conclusions :
PE stimulates CTSS secretion into tears more effectively than CCH. Dysfunction of Rab27a significantly increases accumulation of CTSS in lysosomes and its resulting increased secretion following PE stimulation.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.