Chondrocyte-intrinsic Smad3 represses Runx2-inducible matrix metalloproteinase 13 expression to maintain articular cartilage and prevent osteoarthritis

Abstract

Objective: To identify mechanisms by which Smad3 maintains articular cartilage and prevents osteoarthritis.

Methods: A combination of in vivo and in vitro approaches was used to test the hypothesis that Smad3 represses Runx2-inducible gene expression to prevent articular cartilage degeneration. Col2-Cre;Smad3(fl/fl) mice allowed study of the chondrocyte-intrinsic role of Smad3 independently of its role in the perichondrium or other tissues. Primary articular cartilage chondrocytes from Smad3(fl/fl) mice and ATDC5 chondroprogenitor cells were used to evaluate Smad3 and Runx2 regulation of matrix metalloproteinase 13 (MMP-13) messenger RNA (mRNA) and protein expression.

Results: Chondrocyte-specific reduction of Smad3 caused progressive articular cartilage degeneration due to imbalanced cartilage matrix synthesis and degradation. In addition to reduced type II collagen mRNA expression, articular cartilage from Col2-Cre;Smad3(fl/fl) mice was severely deficient in type II collagen and aggrecan protein due to excessive MMP-13-mediated proteolysis of these key cartilage matrix constituents. Normally, transforming growth factor β (TGFβ) signals through Smad3 to confer a rapid and dynamic repression of Runx2-inducible MMP-13 expression. However, we found that in the absence of Smad3, TGFβ signals through p38 and Runx2 to induce MMP-13 expression.

Conclusion: Our findings elucidate a mechanism by which Smad3 mutations in humans and mice cause cartilage degeneration and osteoarthritis. Specifically, Smad3 maintains the balance between cartilage matrix synthesis and degradation by inducing type II collagen expression and repressing Runx2-inducible MMP-13 expression. Selective activation of TGFβ signaling through Smad3, rather than p38, may help to restore the balance between matrix synthesis and proteolysis that is lost in osteoarthritis.

Figure 1. Chondrocyte-intrinsic Smad3 is required to maintain articular cartilage

(A) PCR analysis of genomic DNA reveals that Cre-mediated recombination only occurs in Col2-expressing cartilage. (B) Real-time RT-PCR shows reduction of Smad3 mRNA expression in postnatal day 60 (P60) Col2-Cre;Smad3^fl/fl mouse cartilage (*p-value < 0.02). (C) Immunohistochemistry for Smad3 confirms a reduction of Smad3 protein in articular cartilage of P60 Col2-Cre;Smad3^fl/fl mice relative to Cre-negative Smad3^fl/fl littermates. (D) Safranin-O staining shows no changes in articular cartilage until P60 and later. (E) Magnified safranin-O stained sections of the articular surface of P60 Col2-Cre;Smad3^fl/fl mice show fibrillation and decreased staining compared to Smad3^fl/fl littermates. (F) Col2-Cre;Smad3^fl/fl cartilage displays an increased modified Mankin score for P60 Col2-Cre;Smad3^fl/fl joints, indicative of osteoarthritis-like joint degeneration (* p-value < 0.002). (Bars = 50µm)

Figure 2. Molecular composition of articular cartilage is disrupted in Col2-Cre;Smad3^fl/fl mice

(A) To examine Col2 and Agn expression in costal cartilage of P60 mice, mRNA expression was quantified and normalized to L19 (n=5; *p-value < 0.02). (B) Immunohistochemistry (IHC) negative controls. Primary antibodies were replaced with either IgG (controls for C, D) or preimmune serum (controls for F, G) to confirm specificity of IHC staining. IHC in P60, P90, and P120 Col2-Cre;Smad3^fl/fl mice revealed a progressive reduction in aggrecan (C) and collagen II (D), but an increase in MMP-13 (E) protein expression in articular cartilage of P90 Col2-Cre;Smad3^fl/fl mice. IHC with antibodies to neoepitopes VDIPEN (F) and GPQG (G), MMP-13 cleavage products of Agn and Col2, respectively, shows increased MMP-13 cleavage of cartilage matrix in P90 Col2-Cre;Smad3^fl/fl mice, particularly at the articular surface. (Bars = 100 µm.)

Figure 3. TGF-β regulation of MMP-13 expression in chondrocytes is dynamic

TGF-β (5 ng/ml) rapidly represses and later induces MMP-13 mRNA (A) and protein (B) expression in primary articular chondrocytes harvested from P5 wild type mice. (C) ATDC5 cells display the same trend. The repression of MMP-13 in ATDC5 cells treated for 8 h with TGF-β is sensitive to an inhibitor of the TGF-β type I receptor (TβRI-I, SB431542, 1 µM) but not to an inhibitor of p38 (p38-I, SB203580, 1 µM). (D). However, the TGF-β-mediated induction of MMP-13 at 48 h is sensitive to both the TβRI-I and the p38-I (E). Vehicle treated cells received an equivalent volume of DMSO. (*p < 0.05, **p < 0.001 relative to the vehicle treated controls at each time point.)

Figure 4. TGF-β-mediated repression and induction of MMP-13 is Runx2-dependent

(A) Though TGF-β induces activity of the synthetic Smad3 reporter gene 3TP-Luc transiently transfected with pRK5 in ATDC5 cells, it represses Runx2-inducible p6OSE2-Luc activity, even more with cotransfected pRK5-Smad3 (B). The activity of -370-MMP-13-Luc is increased by co-transfection of Runx2, but decreased with TGF-β addition or with cotransfected Smad3 or constitutively active TβRI (C). Runx2-knockdown was sufficient to nearly abolish both the rapid 8 h repression (D, F) and delayed 24 h induction (E, G) of MMP-13 mRNA (D, E) and protein (F, G) by TGF-β (5 ng/ml). (*p < 0.05, **p < 0.001 relative to the vehicle-treated controls in A, C, E, and F, and relative to the scramble siRNA in D.)

Figure 5. TGF-β-mediated repression of MMP-13 requires Smad3

Smad3-deficient ATDC5 cells (A, C) and primary chondrocytes (E, G) do not show TGF-β mediated repression of MMP-13 mRNA (A, E) or protein (C, G) at 8 h. However, the induction of MMP-13 by TGF-β at 24 h is maintained in Smad3-deficient ATDC5 cells (B, D) and is reduced but present in primary articular chondrocytes (F, H). (*p < 0.05, **p < 0.001 relative to the vehicle-treated controls in A, B, E, and F.)

Figure 6. Proposed schematic of TGF-β signaling in articular cartilage

TGF-β regulation of MMP-13 in articular chondrocytes is dynamic. In vitro, the early response is dominated by Smad3-repression of MMP-13 while the response at later time points is dominated by p38-mediated induction of MMP-13. The mechanisms may involve modulation of lineage-specific transcription factors Sox9 and Runx2, which form transcriptional complexes with CBP and HDACs, as previously reported in chondrocytes (37) and osteoblasts (38). Thus, loss of Smad3 in vivo results in cartilage degradation due to a reduction in Col2 synthesis and an increase in MMP-13-mediated degradation.