ABSTRACT
The aim of the present study is to investigate the role of c-Jun N-terminal kinase (JNK) and matrix metalloproteinase-2 (MMP-2) in mediating the effects of interleukin-1ß (IL-1ß) on the function of lacrimal gland myoepithelial cells (MECs). MECs isolated from an α-smooth muscle actin-green fluorescent protein (SMA-GFP) transgenic mouse were treated with IL-1ß alone or in the presence of SP600125, a JNK inhibitor, or ARP100, an MMP-2 inhibitor. The GFP intensity and the cell size/area were measured, and on day 7, the SMA, calponin, and pro-MMP-2 protein levels and the MEC contraction were assessed. At baseline, the control and treated cells showed no differences in GFP intensity or cell size. Starting on day 2 and continuing on days 4 and 7, the GFP intensity and cell size were significantly lower in the IL-1ß-treated samples, and these effects were alleviated following inhibition of either JNK or MMP-2. Compared with the control, the levels of SMA and calponin were lower in the IL-1ß-treated samples, and both the JNK and MMP-2 inhibitors reversed this trend. The pro-MMP-2 protein level was elevated in the IL-1ß-treated samples, and this effect was abolished by the JNK inhibitor. Finally, oxytocin-induced MEC contraction was diminished in the IL-1ß-treated samples, and both the JNK and MMP-2 inhibitors reversed this effect. Our data suggest that IL-1ß uses the JNK/MMP-2 pathways to alter MEC functions, which might account for the diminished tears associated with aqueous-deficient dry eye disease.
ABSTRACT
The aim of these studies was to investigate the involvement of the second messenger 3',5'-cyclic adenosine monophosphate (cAMP) and its downstream effectors in oxytocin (OXT)-mediated lacrimal gland myoepithelial cell (MEC) contraction. Lacrimal gland MEC were isolated and propagated from alpha-smooth muscle actin (SMA)-GFP mice. RNA and protein samples were prepared to analyze G protein expression by RT-PCR and western blotting; respectively. Changes in intracellular cAMP concentration were measured using a competitive ELISA kit. To increase intracellular cAMP concentration, the following agents were used: forskolin (FKN, a direct activator of adenylate cyclase), 3-isobutyl-1-methylxanthine (IBMX, an inhibitor of the phosphodiesterase that hydrolyzes cAMP), or a cell permeant cAMP analog, dibutyryl (db)-cAMP. In addition, inhibitors and selective agonists were used to investigate the role of cAMP effector molecules, protein kinase A (PKA) and exchange protein activated by cAMP (EPAC) in OXT-induced MEC contraction. MEC contraction was monitored in real time and changes in cell size were quantified using ImageJ software. The adenylate cyclase coupling G proteins, Gαs, Gαo, and Gαi, are expressed in lacrimal gland MEC at both the mRNA and protein levels. OXT increased intracellular cAMP in a concentration-dependent manner. FKN, IBMX and db-cAMP significantly stimulated MEC contraction. Preincubation of cells with either Myr-PKI, a specific PKA inhibitor or ESI09, an EPAC inhibitor, resulted in almost complete inhibition of both FKN- as well as OXT-stimulated MEC contraction. Finally, direct activation of PKA or EPAC using selective agonists triggered MEC contraction. We conclude that cAMP agonists modulate lacrimal gland MEC contraction via PKA and EPAC activation which also play a major role in OXT induced MEC contraction.