Abstract
Purpose :
We previously reported that treatment of lacrimal gland (LG) myoepithelial cells (MEC) with interleukin-1β (IL-1β) resulted in degradation of contractile proteins: α-smooth muscle actin (SMA) and calponin, inhibiting oxytocin (OXT)-induced MEC contraction. The aim of this study was to investigate, using in vitro treatment of GFP-tagged MEC, the role of c-Jun N-terminal kinase (JNK) and matrix metalloproteinase 2 (MMP-2) in the observed effects on lacrimal gland MEC structure and function after IL-1β treatment.
Methods :
MEC, isolated from a SMA-GFP transgenic mouse, were treated with IL-1β alone (10 ng/ml) or in the presence of the JNK inhibitor, SP600125 (20 µM), or selective MMP-2 inhibitor, ARP100 (10 µM). Still images of MEC cultures were taken on days 0, 2, 4 and 7, using a digital camera mounted on an inverted light microscope. These images were used to measure GFP intensity and cell size/area (n= 37-57) using the ImageJ/Fiji software. On day 7, both contractile protein amounts were assessed by western blot, and OXT-induced MEC contraction was measured. All data from at least 3 independent experiments.
Results :
At day 0, control and treated cells showed no differences in GFP intensity or cell size. By day 7, inhibition of JNK significantly alleviated the effects of IL-1β on MEC GFP intensity (p=0.0033). IL-1β significantly reduced MEC size (p<0.0001) compared to control and this effect was alleviated by inhibition of JNK (61% down in the IL-1β group to just 26% down after JNK inhibition). Inhibition of MMP-2 showed a similar alleviating pattern on MEC GFP intensity and cell area/size. Quantitative western blot showed the amount of SMA and calponin protein levels were lower, although not statistically significant for SMA, in IL-1β treated MEC. Inhibition of JNK completely alleviated the effects of IL-1β on calponin protein levels (p=0.045). Lower expression of SMA and calponin in IL-1β treated samples increased after MMP-2 inhibition.
Conclusions :
Our data suggest that IL-1β uses the JNK/MMP-2 pathways to alter lacrimal gland MEC structure and function, which might account for the diminished tears associated with aqueous-deficient dry eye disease. Targeting these pathways could be a viable option to alleviate chronic inflammation of the lacrimal gland as observed in patients suffering from Sjögren's syndrome.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.