Document Type
Article
Publication Date
2-19-2026
Abstract
Background
Proteoglycan-4 (PRG4) is a mucinous glycoprotein secreted by synovial fibroblasts and superficial zone chondrocytes. PRG4 inhibits synovial macrophage (SM) activation via xanthine oxidase (XO) and hypoxia inducible factor alpha (HIF-1α) suppression. We aimed to evaluate the contribution of PRG4-CD44 interaction to synovial homeostasis and investigate PRG4’s signaling dysfunction in synovial tissues from patients with osteoarthritis (OA). We hypothesized that CD44 mediates synovitis due to PRG4 loss and that PRG4 signaling dysfunction is associated with high-grade synovitis in OA.
Methods
Prg4FrtloxP/FrtloxP are transgenic mice wherein tamoxifen (TAM) inactivates the Frt allele and creates a knockout state (Prg4FrtKO/FrtKO). TAM (Prg4Off) or corn oil (Prg4On) administration occurred in 4 weeks-old animals (4–6 animals with 2–3 males per group). We crossed this mouse with Cd44−/− mice to generate Cd44+/+ & Prg4On, Cd44+/+ & Prg4Off, Cd44−/− & Prg4On, and Cd44−/− & Prg4Off. XO and HIF-1α immunostaining was conducted. Isolated SMs were activated using LPS + IFNγ and SM glycolytic activation was measured by proton efflux rate (PER). HIF-1α levels were measured by ELISA. Synovial tissues were collected from the medial and lateral joint compartments of OA patients undergoing knee arthroplasty (n = 9; 7 females and 2 males). Specimens were classified by Krenn’s synovitis score as low-grade (Score: 2–4) or high-grade (score: 5–9) synovitis. Isolated CD14 + cells were stimulated with LPS ± febuxostat, and glycolytic activation was measured by PER. Immunohistochemistry (IHC) included PRG4, CD44, XO and HIF-1α.
Results
CD44 deficiency reduced XO and HIF-1α staining in addition to synovial pathology following Prg4 inactivation (p < 0.05). SMs from Cd44−/− & Prg4Off mice were less activated than Cd44+/+ & Prg4Off mice (p < 0.001) and had lower HIF-1α levels (p < 0.0001). High-grade synovitis tissues displayed less PRG4 and greater CD44, XO and HIF-1α (p < 0.001) IHC staining compared to low-grade and normal tissues. Febuxostat reduced CD14 + cell activation from medial (p < 0.0001) and lateral (p < 0.05) joint compartments.
Conclusions
CD44 loss abrogated chronic synovitis observed following PRG4 loss. Dysfunction in PRG4 signaling, demonstrated by lower tissue levels of PRG4 along with higher CD44, XO and HIF-1α, was associated with high-grade synovitis. Targeting the downstream events of PRG4 loss is potentially therapeutic in OA synovitis.
Recommended Citation
Elsaid, K.A., Zhang, L., Zhao, T. et al. Disruption of proteoglycan 4 (PRG4)-CD44 signaling modulates chronic synovitis in conditionally inactivated mice. Arthritis Res Ther 28, 74 (2026). https://doi.org/10.1186/s13075-026-03773-2
Supplementary Material 1: Supplementary Fig. 1. Findings in synovial tissues in proteoglycan-4 (Prg4) conditionally inactivated mice include depletion of claudin-5 expressing TREM2 + anti-inflammatory macrophages, and these changes were suppressed in Cd44−/− mice. Prg4 inactivation (Prg4Off) was performed using intraperitoneal tamoxifen (0.1 mg/gram) daily for 10 days starting at 4 weeks or vehicle corn oil (100 µl) (Prg4On) and histological analyses were performed 6 weeks later. Experimental groups included 4–5 animals with each group including 2 males and 2–3 females. ns: non-significant; *p < 0.05; ***p < 0.001; ****p < 0.0001. (A) TREM2 staining was attenuated to a greater extent in the Cd44+/+ & Prg4Off synovium than the Cd44−/− & Prg4Off synovium. (B) Claudin-5 staining was attenuated to a greater extent in the Cd44+/+ & Prg4Off synovium than the Cd44−/− & Prg4Off synovium.
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Supplementary Material 2: Supplementary Fig. 2. Profile of circulating immune cells in proteoglycan-4 (Prg4) conditionally inactivated mice as a function of Cd44 expression status. Prg4 conditionally inactivated mice were crossed with Cd44−/− mice to generate Cd44−/− & Prg4On/Off and Cd44+/+ & Prg4On/Off mice. Prg4 inactivation (Prg4Off) was performed using intraperitoneal tamoxifen (0.1 mg/gram) daily for 10 days starting at 4 weeks or vehicle corn oil (100 µl) (Prg4On). Complete blood counts (CBC) were determined 6 weeks later. ns: non-significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (A) Prg4 loss increased circulating white blood cells (WBCs) in Cd44+/+ animals. (B) Prg4 loss increased circulating lymphocytes in Cd44+/+ animals. (C) Prg4 loss increased circulating monocytes in Cd44+/+ animals. (D) Prg4 loss increased circulating granulocytes in Cd44+/+ animals.
13075_2026_3773_MOESM3_ESM.tif (8848 kB)
Supplementary Material 3: Supplementary Fig. 3. Expression of proteoglycan 4 (PRG4) signaling axis in synovial tissues from CD44 competent or null & Prg4 conditionally inactivated mice, where Prg4 is normally expressed (On) or is inactivated (Off). Arrows indicate high or low expression and green color indicate homeostasis and red color indicates inflammation.
13075_2026_3773_MOESM4_ESM.tif (6340 kB)
Supplementary Material 4: Supplementary Fig. 4. Expression of proteoglycan 4 (PRG4) signaling axis in synovial tissues from normal, low-grade and high-grade synovitis OA. Arrows indicate high or low expression and green color indicate homeostasis and red color indicates inflammation.
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Comments
This article was originally published in Arthritis Research & Therapy, volume 28, in 2026. https://doi.org/10.1186/s13075-026-03773-2