INHIBITION OF LOX MEDIATED EXTRACELLULAR MATRIX STIFFENING PREVENTS WNT3A INDUCED CHONDROCYTE DIFFERENTIATION VIA THE YAP/ TAZ PATHWAY

Purpose: During aging the extracellular matrix (ECM) of cartilage gets stiffer due to the action of ECM cross linking enzymes. The lysyl oxidase (LOX) is an important player in this process as it contributes to the crosslinking of collagen fibers. Matrix stiffness is known to regulate Yap/ TAZ signaling, which interferes with canonical WNT signaling and thereby regulates cell differentiation. Increased cytosolic Yap levels increase the degradation of ß-catenin thereby downregulating canonical Wnt signaling. Syndecan-4 (Sdc4) has been shown to bind Lox at the side chains. Sdc4 knockout (Sdc4-/-) mice are protected from OA-like changes in cartilage. We investigated whether the Sdc-4 dependent regulation of Lox activity prevents chondrocyte differentiation via Yap/Taz signaling.

Methods: Atomic force microscopy (AFM) was used on wild type (WT) and Sdc4-/- mouse knee joint sections of neonatal, 8 and 45 weeks old mice to analyze ECM stiffness. Immunohistological Yap and Taz staining was performed on knee joint sections at different ages of the mice, as well as DMM induced knee on joint sections of WT and Sdc4-/- mice undergoing DMM surgery as an model for osteoarthritis. Chondrocytes were isolated from neonatal knees of WT and Sdc4-/- mice. Electron microscopy (EM) of primary chondrocytes monolayer, micromass cultures (MM) and mouse knee joints were applied to visualize differences in ECM architecture and Lox localization. β-aminopropionitrile (BAPN) was used to inhibit Lox and fibril architecture was investigated using EM in WT and Sdc4-/- chondrocytes. Western Blot of BAPN treated MM was performed to investigate Yap content, as well as Wnt3a induced ß-catenin stabilization. Expression of Sox9, Acan, Col2, Col10, CTGF and Lox was analyzed by quantitative Real-time (qRT) PCR with and without Wnt3a stimulation. Western blot of ß-catenin was performed of WT and Sdc4-/- chondrocytes stimulated with Wnt3a. TCF/Lef reporter Assays were performed to investigate the activation of canonical Wnt signaling in WT and Sdc4-/- chondrocytes upon Wnt3a stimulation.

Results: Sdc4-/- chondrocytes and articular knee cartilage exhibit a thinner fibril structure and a finer and softer ECM structure at the age of 8 and 45 weeks. Neonatal cartilage shows the same architecture. The age dependent increase in ECM stiffness for the proteoglycan and collagen network is slower in Sdc4-/- cartilage. We found less LOX staining in ECM of Sdc4-/- cartilage compared to WT at 8 weeks using EM. Furthermore, there was less Lox activity in Sdc4-/- cartilage compared to wt cartilage. Lox inhibition using BAPN changed the ECM structure according to Sdc4-/- phenotype in MM. At the same time the Yap content in WT was increased to the level of Sdc4-/- with BAPN, whereas in Sdc4-/- MM was no change in Yap after treatment with BAPN was observed. We found an increased Yap and Taz staining in Sdc-4 cartilage at 8 and 45 weeks. DMM induction abolished Yap staining in WT, but left some residual Yap in Sdc4-/- cartilage. CTGF expression was significantly increased in Sdc4-/- chondrocytes. Basal expression of ACAN, SOX9, COL2, COL9 and COL11 was increased in Sdc4-/- chondrocytes. As Yap/Taz signaling is known to interfere with canonical Wnt signaling WT and Sdc4-/- chondrocytes were treated with Wnt3a. Western Blot analysis showed less ß-catenin in Sdc4-/- chondrocytes compared to WT, as well as less stabilization of ß-catenin upon stimulation with Wnt3a. The TCF/Lef promoter activity revealed that Sdc4-/- chondrocytes were less responsive to Wnt3a compared to WT. Similar effects could be achieved by treatment with BAPN. Wnt3a decreased the expression of Acan, Sox9 and Col2 in WT and Sdc4-/- chondrocytes, but to a smaller extent in Sdc4-/-.

Conclusions: Sdc4 determines the ECM architecture via binding of LOX and ultimately impacts on mechanical properties of cartilage. Adult Sdc4-/- cartilage is softer and resembles immature, still developing cartilage due to decreased LOX presence in the cartilage. The decreased cartilage stiffness increases cytosolic Yap/Taz presence, thereby inhibiting canonical WNT-signaling by increased degradation of ß-catenin. Less responsiveness to canonical Wnt signaling protects Sdc4-/- chondrocytes from hypertrophic differentiation of chondrocytes and OA like cartilage changes. Similar effects could be achieved by BAPN mediated Lox inhibition.