Genetic inhibition of fibroblast growth factor receptor 1 in knee cartilage attenuates the degeneration of articular cartilage in adult mice

Abstract

Objective: Fibroblast growth factor (FGF) family members are involved in the regulation of articular cartilage homeostasis. The aim of this study was to investigate the function of FGF receptor 1 (FGFR-1) in the development of osteoarthritis (OA) and its underlying mechanisms.

Methods: FGFR-1 was deleted from the articular chondrocytes of adult mice in a cartilage-specific and tamoxifen-inducible manner. Two OA models (aging-associated spontaneous OA, and destabilization-induced OA), as well as an antigen-induced arthritis (AIA) model, were established and tested in Fgfr1-deficient and wild-type (WT) mice. Alterations in cartilage structure and the loss of proteoglycan were assessed in the knee joints of mice of either genotype, using these 3 arthritis models. Primary chondrocytes were isolated and the expression of key regulatory molecules was assessed quantitatively. In addition, the effect of an FGFR-1 inhibitor on human articular chondrocytes was examined.

Results: The gross morphologic features of Fgfr1-deficient mice were comparable with those of WT mice at both the postnatal and adult stages. The articular cartilage of 12-month-old Fgfr1-deficient mice displayed greater aggrecan staining compared to 12-month-old WT mice. Fgfr1 deficiency conferred resistance to the proteoglycan loss induced by AIA and attenuated the development of cartilage destruction after surgically induced destabilization of the knee joint. The chondroprotective effect of FGFR-1 inhibition was largely associated with decreased expression of matrix metalloproteinase 13 (MMP-13) and up-regulation of FGFR-3 in mouse and human articular chondrocytes.

Conclusion: Disruption of FGFR-1 in adult mouse articular chondrocytes inhibits the progression of cartilage degeneration. Down-regulation of MMP-13 expression and up-regulation of FGFR-3 levels may contribute to the phenotypic changes observed in Fgfr1-deficient mice.

Figure 1

Histologic changes related to aging in the articular cartilage of Fgfr1 conditional knockout (cKO) mice compared to wild-type (WT) (Cre-negative) littermate control mice. A, Sagittal sections of whole knee joints from mice at ages 3, 6, and 12 months were stained with Safranin O–fast green to visualize gross morphologic differences in knee joint degeneration and loss of proteoglycan (arrows). All mice received treatment with the same amounts of tamoxifen. Representative images are shown (n = 5 samples per group). In WT mice at age 12 months, tidemark (open arrowhead) was still well preserved. Bar = 200 μm. B, Summed scores (mean ± SD) for proteoglycan loss in the medial femoral condyle (MFC) and medial tibial plateau (MTP) were determined in the knee joints of Fgfr1 cKO and WT mice at age 12 months. ** = P < 0.001 versus WT.

Figure 2

Characteristics of antigen-induced arthritis (AIA) in Fgfr1-deficient mice compared to wild-type (WT) control mice. A and B, The extent of proteoglycan loss (arrows) was examined in Safranin O–fast green–stained sections from WT mice (A) and Fgfr1 conditional knockout (cKO) mice (B) after intraarticular injection of the knee joints with methylated bovine serum albumin (mBSA) (right) compared to saline control (left). In knee joints injected with mBSA, Fgfr1 deficiency protected the articular cartilage against degeneration induced by AIA. Boxed areas in top panels (bars = 400 μm) are shown at higher magnification in bottom panels (bars = 100 μm). Representative images are shown (n = 7 samples per group). GP = growth plate. C, Summed and maximal scores (mean ± SD) for proteoglycan loss in the medial femoral condyle (MFC) and medial tibial plateau (MTP) were determined in the knee joints of Fgfr1 cKO and WT mice. * = P < 0.05 versus WT.

Figure 3

Histologic features and summed and maximal scores for structural damage in mouse articular cartilage at 4 weeks following destabilization of the medial meniscus (DMM) surgery. A, Whole knee joints from Fgfr1 conditional knockout (cKO) and wild-type (WT) control mice were subjected to sham operation (left) or DMM surgery (right). The articular cartilage was stained with Safranin O–fast green at 4 weeks after surgery to assess the extent of articular cartilage degeneration in WT mice (arrow) and Fgfr1 cKO mice (arrowhead). Representative images are shown (n = 10 mice per genotype). Bar = 200 μm. B, Summed (top) and maximal (bottom) histologic scores for cartilage structure damage (mean ± SD) were determined in the medial femur and tibia of Fgfr1 cKO and WT control mice at 4 weeks following sham operation or DMM surgery. * = P < 0.05; ** = P < 0.001 versus WT.

Figure 4

Histologic features and summed and maximal scores for structural damage in the mouse articular cartilage at 8 weeks following destabilization of the medial meniscus (DMM) surgery. A, Whole knee joints from Fgfr1 conditional knockout (cKO) and wild-type (WT) control mice were subjected to sham operation (left) or DMM surgery (right). The articular cartilage was stained with Safranin O–fast green at 8 weeks after surgery to assess the extent of articular cartilage degeneration. After DMM surgery, WT mice exhibited a loss of uncalcified cartilage and exposed subchondral bone (arrow), whereas Fgfr1 cKO mice showed reduced damage in the articular cartilage (arrowhead). Sham-operated knees showed no cartilage damage. Representative images are shown (n = 10 mice per genotype). Bar = 200 μm. B, Summed (top) and maximal (bottom) histologic scores of cartilage structure damage (mean ± SD) were determined in the medial femur and tibia of Fgfr1 cKO and WT mice at 8 weeks following sham operation or DMM surgery. ** = P < 0.001 versus WT.

Figure 5

Effects of Fgfr1 deficiency on gene expression in primary chondrocytes from mouse articular cartilage. A, The fold change in expression levels of Fgfr1, aggrecan, Fgfr3, Mmp13, Mmp3, and Adamts5 was assessed by real-time polymerase chain reaction in primary chondrocytes from Fgfr1 conditional knockout (cKO) mice compared to wild-type (WT) control mice. * = P < 0.05 versus WT. B, Primary chondrocytes were treated with or without 4-hydroxytamoxifen (4OH-TM) (1 μM) for 48 hours, or stimulated with or without recombinant human interleukin-1β (IL-1β) (1 ng/ml) for 24 hours after incubation with 4OH-TM. Western blot results show that disruption of Fgfr1 in the mouse primary chondrocytes can antagonize the IL-1β–induced up-regulation of matrix metalloproteinase 13 (MMP-13); β-actin was used as a loading control. C, ATDC5 cells were transfected with pcDNA3.1 or pcDNA3.1-Fgfr1 plasmids, and after 48 hours of incubation, the fold change in expression of Fgfr1 and Mmp13 was assessed. * = P < 0.05; ** = P < 0.001 versus pcDNA3.1-transfected cells. Bars in A and C show the mean ± SD.

Figure 6

Immunohistochemical staining for fibroblast growth factor 1 (FGFR-1), type X collagen, matrix metalloproteinase 13 (MMP-13), and FGFR-3 in the knee joints of Fgfr1 conditional knockout (cKO) mice compared to wild-type (WT) control mice. A–C, The knee joints of WT mice and Fgfr1 cKO mice at age 12 months were immunostained for FGFR-1 (A), type X collagen (B), and MMP-13 (C). Bar = 100 μm. D, The knee joints of WT mice and Fgfr1 cKO mice were immunostained for FGFR-3 at 4 weeks after either sham operation (left panels) or destabilization of the medial meniscus (DMM) surgery (right panels). Bar = 200 μm. Insets, Higher-magnification views of the articular cartilage chondrocytes (original magnification × 400). GP = growth plate.