ALX/FPR2 receptor for RvD1 is expressed and functional in salivary glands

Abstract

Sjögren's syndrome (SS) is an autoimmune disorder characterized by chronic inflammation and destruction of salivary and lacrimal glands, leading to dry mouth, dry eyes, and the presence of anti-nuclear antibodies. Despite modern advances, the current therapies for SS have no permanent benefit. A potential treatment could involve the use of resolvins, which are highly potent endogenous lipid mediators that are synthesized during the resolution of inflammation to restore tissue homeostasis. Our previous studies indicate that ALX/FPR2, the receptor for RvD1, is expressed and active in the rat parotid cell line Par-C10. Specifically, activation of ALX/FPR2 with RvD1 blocked inflammatory signals caused by TNF-α and enhanced salivary epithelial integrity. The goal of this study was to investigate RvD1 receptor expression and signaling pathways in primary salivary cells. Additionally, we determined the role of the aspirin-triggered 17R analog (AT-RvD1, a more chemically stable RvD1 epimeric form) in prevention of TNF-α-mediated salivary inflammation in mouse submandibular glands (mSMG). Our results indicate that ALX/FPR2 is expressed in mSMG and is able to elicit intracellular Ca2+ responses and phosphorylation of Erk1/2, as well as Akt. Given that these signaling pathways are linked to cell survival, we investigated whether AT-RvD1 was able to prevent programmed cell death in mSMG. Specifically, we determined that AT-RvD1 prevented TNF-α-mediated caspase-3 activation. Finally, we show that ALX/FPR2 is expressed in human minor salivary glands with and without SS, indicating the potential therapeutic use of AT-RvD1 for this condition.

Keywords: ALX/FPR2; AT-RvD1; GPR32; RvD1; Sjögren's syndrome; resolvins; salivary glands.

Fig. 1.

The resolvin D1 (RvD1) receptor ALX/FPR2 (lipoxin A₄/formyl peptide receptor 2) is expressed in mouse submandibular glands (mSMG). Frozen sections of mSMG were fixed, and localization of ALX/FPR2 was determined using immunofluorescence microscopy: rabbit anti-ALX/FPR2 (green; A, B, and E), phalloidin (red; D and E), and propidium iodide nuclear stain (blue; A, C, and E). All images were obtained and analyzed using a confocal microscope. Arrows and arrowheads indicate acinar and ductal localization of the receptor, respectively. Scale bars, 50 μm.

Fig. 2.

ALX/FPR2 is functional in mSMG. Cells were plated on 8-well coverglasses treated with Cell-Tak and then stimulated with aspirin-triggered RvD1 (AT-RvD1, 100 ng/ml; A), carbachol (100 μM; B), or 1× PBS (C). Changes in fluo 4-AM fluorescence intensity were recorded and analyzed using Leica Application Suite Advanced Fluorescence software. Results are representative of ≥3 experiments for all agonists.

Fig. 3.

Aspirin-triggered RvD1 (AT-RvD1) transiently phosphorylates Erk1/2 and Akt in mSMG. A and B: freshly isolated mSMG cells were incubated with (A) and without (B) AT-RvD1 (100 ng/ml) for 0–60 min. Cells were then lysed with Laemmli buffer, and expression of phosphorylated Erk1/2 (phospho-Erk1/2) was detected by Western blot analysis. Phosphorylated Erk1/2 data were normalized to total protein using a total Erk1/2 antibody. Values are means ± SE of results from ≥3 experiments. Results from a representative experiment are shown at top. p-Erk1/2, phosphorylated Erk1/2; MW, molecular weight. C and D: freshly isolated mSMG cells were incubated with (C) and without (D) AT-RvD1 (100 ng/ml) for 0–60 min. Cells were then lysed with Laemmli buffer, and expression of phosphorylated Akt (phospho-Akt) was detected by Western blot analysis. Phosphorylated Akt data were normalized to total protein using a pan-Akt antibody. Values are means ± SE of results from ≥3 experiments. Results from a representative experiment are shown at top. p-Akt, phosphorylated Akt.

Fig. 4.

AT-RvD1 decreases active caspase-3 activity in mSMG. Freshly isolated mSMG cells were incubated with and without AT-RvD1 (100 ng/ml) for 30 min and then with TNF-α or staurosporine (STP). After 24 h, cells were collected and subjected to an active caspase-3 ELISA. Values are means ± SE of results from ≥3 experiments. *P < 0.05 vs. TNF-α-treated cells.

Fig. 5.

The RvD1 receptor ALX/FPR2 is expressed in human minor salivary glands (hMSG). Frozen hMSG sections without (A–E) and with (F–J) Sjögren's syndrome (SS) were fixed, and expression of ALX/FPR2 was detected using immunofluorescence microscopy with rabbit anti-ALX/FPR2 (green; A, B, E, F, G, and J), phalloidin (red; D, E, I, and J), and propidium iodide nuclear stain (blue; A, C, E, F, H, and J). All images were obtained and analyzed using a confocal microscope. Arrows and arrowheads indicate acinar and ductal localization of ALX/FPR2, respectively. Scale bars, 50 μm.

Fig. 6.

Proposed signaling mechanisms mediated by RvD1 in mSMG. RvD1 binds to ALX/FPR2 expressed on the membrane of acinar and ductal cells. RvD1 receptors signal through a classical 7-transmembrane G protein-coupled receptor, given that they activate intracellular Ca²⁺ ([Ca²⁺]_i) release. After stimulation, AT-RvD1 activates Erk1/2 and Akt signaling, blocking TNF-α signaling and caspase-3 activity, leading to cell survival. TNFR1, TNF receptor type 1; PI3K, phosphatidylinositol 3-kinase.