Regulation of α5 and αV Integrin Expression by GDF-5 and BMP-7 in Chondrocyte Differentiation and Osteoarthritis

Abstract

The Integrin β1 family is the major receptors of the Extracellular matrix (ECM), and the synthesis and degradation balance of ECM is seriously disrupted during Osteoarthritis (OA). In this scenario, integrins modify their pattern expression and regulate chondrocyte differentiation in the articular cartilage. Members of the Transforming growth factor beta (Tgf-β) Superfamily, such as Growth differentiation factor 5 (Gdf-5) and Bone morphogenetic protein 7 (Bmp-7), play a key role in joint formation and could regulate the integrin expression during chondrocyte differentiation and osteoarthritis progression in an experimental OA rat model. Decrease of α5 integrin expression in articular cartilage was related with chondrocyte dedifferentiation during OA progression, while increase of α1, α2, and α3 integrin expression was related with fibrous areas in articular cartilage during OA. Hypertrophic chondrocytes expressed αV integrin and was increased in the articular cartilage of rats with OA. Integrin expression during chondrocyte differentiation was also analyzed in a micromass culture system of mouse embryo mesenchymal cells, micromass cultures was treated with Gdf-5 or Bmp-7 for 4 and 6 days, respectively. Gdf-5 induced the expression of the α5 sub-unit, while Bmp-7 induced the expression of the αV sub-unit. This suggests a switch in signaling for prehypertrophic chondrocyte differentiation towards hypertrophy, where Gdf-5 could maintain the articular chondrocyte phenotype and Bmp-7 would induce hypertrophy. Decrease of Ihh expression during late stages of OA in rat model suggest that the ossification in OA rat knees and endochondral ossification could be activated by Bmp-7 and αV integrin in absence of Ihh. Thus, chondrocyte phenotype in articular cartilage is similar to prehypetrophic chondrocyte in growth plate, and is preserved due to the presence of Indian hedgehog (Ihh), Gdf-5 and α5 integrin to maintain articular cartilage and prevent hypertrophy.

Fig 1. Extracellular matrix (ECM) re-changes during OA.

Herovici stain (A–D) showed mature collagen in red and young collagen in blue, immunohistochemical staining for α1 integrin (E–H) and IgG control (I-L); exhibited integrin α1 in the fibrotic area; in healthy articular cartilage (A, E, I); during OA at day 5 (B, F, J), at day 10 (C, G, K), and day 20 (D, H, L).

Fig 2. Integrin α5 is expressed in healthy articular cartilage surface, and integrin α V on osteoarthritic articular cartilage recapitulates chondrocyte differentiation, as in the in cartilage growth plate.

Immunohistochemical stain for β1 (A–D), α5 (E–H), αV (J–M) integrins; in healthy articular cartilage (A, E, J); during Osteoarthritis (OA) at day 5 (B, F, K), day 10 (C, G, L), and at day 20 (D, H, M), and in the growth plate cartilage, α5 integrin (I) and αV integrin (N). Fibronectin is localizes in the pericellular area of chondrocytes (R-U).

Fig 3. Gdf-5 is associated with healthy articular cartilage and Bmp-7 is associated with OA.

Immunofluorescence for Gdf-5 (A, B) and in situ hybridization for Bmp-7 (C, D) in healthy rat articular cartilage (A, C) and at day 10 of OA (B, D) showed Gdf-5 expression in prehypertrophic chondrocytes in healthy articular cartilage. Bmp-7 is associated with OA from day 10 post-surgery.

Fig 4. Regulation of Extracellular matrix (ECM) composition by Bone morphogenetic protein (BMP) family growth factors during chondrogenesis.

Mouse micromass cultures without treatment (A–E), Gdf-5 treatment (F–J), and Bmp-7 treatment (K–O); Alcian blue stain (A–K) showed cartilage nodules, Immunohistochemical stain for type 1 collagen (A, F, K), type II collagen (C, H, M), aggrecan (D, I, N), and fibronectin (E, J, O). Statistical analysis for histochemical and immunohistochemical data, (P) = percentage of positive area for Alcian blue, type I collagen, type II collagen, aggrecan, and fibronectin stains in micromasses with different treatments; mean values are shown with Standard deviations (SD) (n = three independent experiments), One-way Analysis of variance (ANOVA). ***P ˂0.0001; **p ˂0.001; *p ˂0.05). Gdf-5 mainly induces aggrecan expression, type II collagen, and fibronectin; aggrecan is an articular cartilage marker; in contrast, Bmp-7 induce type I collagen, an extracellular component of hypertrophic cartilage and bone.

Fig 5. Regulation of integrin expression by Growth differentiation factor (Gdf)-5 and Bone morphogenetic protein (Bmp)-7 during chondrogenesis.

Mouse micromass cultures without treatment (A–E), Gdf-5 treatment (F–J), and Bmp-7 treatment (K–O). Immunohistochemical stain for β1 (A, F, K), α1 (B, G, L), α5 (C, H, M), and αV (D, I, N) integrins, IgG control for Immunohostochemestry (E, J, O). Statistical analysis for integrin immunohistochemistry data (P), percentage of positive area for integrin β1, integrin α1, integrin α5, integrin αV and IgG control stains in micromasses with different treatments (control, GDF-5, and BMP-7); mean values are shown with Standard deviations (SD) (n = three independent experiments). One-way Analysis of variance (ANOVA). ***P ˂0.0001, **p ˂0.001, *p ˂0.05. Micromass cultures with Gdf-5 treatment induce α5 integrin expression and Bmp-7 treatment induces αV integrin expression.

Fig 6. Regulation between α5 integrin and Ihh signaling.

In situ hybridization for Indian hedgehog (Ihh) gene (A–D), in healthy articular cartilage (A), during OA at day 5 (B), at day 10 (C), and at day 20 (D) showed decrease of Ihh gene expression in Osteoarthritis (OA). Immunofluorescence in mouse micromass cultures (E–H), without treatment (E, G) showed expression for Ihh (E) and integrin α5 (F); treatment micromasses with blocking antibodies (G, H) for integrin α5 inhibited Ihh expression (F), and treatment with blocking antibodies for Ihh inhibits integrin α5 expression (H).