Matrilin-3, a Regulator of Chondrocyte Hypertrophy, Bone Mineral Density, and Osteoarthritis, Binds Bone Morphogenetic Protein-2, Vascular Endothelial Growth Factor165 and Insulin-like Growth Factor

INTRODUCTION The matrilins are a family of four non-collagenous extracellular matrix (ECM) proteins which are important regulators of chondrocyte proliferation and differentiation. During development, matrilin-1 (MATN1) and matrilin-3 (MATN3) are expressed in cartilage and/or bone, while matrilin-2 and matrilin-4 have a broader tissue distribution. Mutations in human MATN3 result in a variety of skeletal diseases including multiple epiphyseal dysplasia (characterized by abnormal ossification in the growth plate and early onset osteoarthritis), spondylo-epi-metaphyseal dysplasia (a chondrodystrophy occurring during skeletal development in childhood) and hand osteoarthritis (occurring in adults during aging). MATN3 knockout mice exhibit an expanded hypertrophic zone during embryonic development, increased bone mineral density (BMD) in adulthood, and accelerated joint degeneration during aging. Bone morphogenetic protein-2 (BMP-2) is an essential regulator of bone development and skeletal changes due to aging. BMP-2 also stimulates chondrocyte proliferation and hypertrophy. We have reported that BMP2 signaling is stimulated in chondrocytes lacking MATN3 and inhibited in those over-expressing MATN3. This inhibition of BMP-2 signaling may explain the reported increase in BMD and/or degeneration of articular cartilage observed in MATN3 knockout mice. However, the mechanism by which MATN3 modulates BMP-2 signaling remains unknown. We hypothesize that these in vivo and in vitro effects result from a direct binding interaction between BMP-2 and MATN3. In addition to investigating MATN3’s putative binding interaction with BMP-2, we also screened for binding with other growth factors which regulate bone and cartilage growth and differentiation including insulinlike growth factor (IGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF165 – the most abundant and potent isoform) and transforming growth factor (TGF-β1). METHODS Solid Phase Binding Assay: 96-well plate was coated with 20 nM recombinant human TGF-β1, BMP-2, VEGF165 (R&D Systems), insulin, EGF or IGF (Sigma) in Tris-buffered saline (TBS) (0.9% NaCl, 20 mM Tris, pH 7.4) overnight at 4°C. Non-specific binding sites were blocked with TBS plus 3% (w/v) bovine serum albumin for 2 hours. Following four rinses in wash buffer (TBS plus 0.05% (v/v) Tween-20), recombinant human MATN3 (R&D Systems) in TBS was added to wells for 1 hour under conditions outlined in Table 1. MATN3 was also added to wells without coated protein as a control. Wells were washed four times and then incubated with 0.5 μg/mL mouse monoclonal anti-MATN3 primary antibody (R&D Systems) for 2 hours at room temperature. Wells were then washed four times and incubated with goat anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (diluted 1:5000) (Bio-Rad) for 1 hour. Wells were then washed four times and color was developed with SureBlue Reserve TMB Microwell Peroxidase Substrate (KPL). Color substrate reaction was stopped by TMB Stop Solution (KPL) and absorbance was determined using plate reader (Packard Fusion Universal Microplate Reader) at 450 nm. Non-specific binding of MATN3 to plate was subtracted from corresponding absorbance readings. Each experimental condition was plated in triplicate and all experiments were repeated at least twice.