Mechanical stress determines the configuration of TGFβ activation in articular cartilage

Abstract

Our incomplete understanding of osteoarthritis (OA) pathogenesis has significantly hindered the development of disease-modifying therapy. The functional relationship between subchondral bone (SB) and articular cartilage (AC) is unclear. Here, we found that the changes of SB architecture altered the distribution of mechanical stress on AC. Importantly, the latter is well aligned with the pattern of transforming growth factor beta (TGFβ) activity in AC, which is essential in the regulation of AC homeostasis. Specifically, TGFβ activity is concentrated in the areas of AC with high mechanical stress. A high level of TGFβ disrupts the cartilage homeostasis and impairs the metabolic activity of chondrocytes. Mechanical stress stimulates talin-centered cytoskeletal reorganization and the consequent increase of cell contractile forces and cell stiffness of chondrocytes, which triggers αV integrin-mediated TGFβ activation. Knockout of αV integrin in chondrocytes reversed the alteration of TGFβ activation and subsequent metabolic abnormalities in AC and attenuated cartilage degeneration in an OA mouse model. Thus, SB structure determines the patterns of mechanical stress and the configuration of TGFβ activation in AC, which subsequently regulates chondrocyte metabolism and AC homeostasis.

Fig. 1. Excessive activation of TGFβ in AC was correlated with the structural alteration of SB in OA.

a Immunohistological staining (first row, pSmad2/3: brown), immunofluorescence staining (second row, pSmad2/3: green, DAPI: blue) staining of pSmad2/3 in AC, and representative µCT images of SB (third row) of human knee joint specimens. HC healthy control, OA-M OA specimen with minimal cartilage degeneration, OA-S OA specimen with severe cartilage degeneration. b Quantitative analysis of the ratio of pSmad2/3⁺ cells in total chondrocytes in AC, n = 5 or 25 biologically independent joint samples in HC or OA-M and OA-S group, respectively. c–e Quantitative analysis of µCT scanning: BV/TV: percent bone volume, BS/BV: bone surface/volume ratio, SMI: structure model index, n = 6, 10, and 9 biologically independent joint samples in HC, OA-M or OA-S group, respectively. In (b–e), data were analyzed using one-way ANOVA followed by LSD post hoc test. Data are presented as mean values + /− SEM. Source data are provided as a Source data file. f, g The region-defining strategy for medial compartment of mouse tibia. f Top view of tibia plateau for illustration. The gold dotted line represents the location of the sagittal images in g, the black dotted lines represent the locations of the coronal images in h. P posterior, M middle, A anterior. h Representative 3D reconstructed µCT images of coronal sections of the medial compartment of mice tibia (first and fourth columns), and immunohistological staining of pSmad2/3 (brown) in AC at the corresponding locations (second, third, fifth, and sixth columns). The third and sixth columns are the high-magnification images of the box region in columns 2 and 5, respectively. The mice were sacrificed 2 months after sham surgery or ACL-T. i The ratio of pSmad2/3⁺ cells to total chondrocytes in different regions of AC in h. Data were analyzed using two-way ANOVA Tukey’s post hoc test. j The correlation of trabecular pattern factor (Tb. Pf) of the SB and the percentage of pSmad2/3⁺ cells in the above cartilage at the same location. h–j n = 6 biologically independent animals, data are presented as mean values + /− SEM.

Fig. 2. TGFβ activation pattern in AC was aligned with mechanical stress distribution.

a Representative 3D reconstructed images of the tibia medial compartment from mice 1 month after ACL-T or sham surgery. The left images in each panel: top view; right images in each panel: transaxial view of the tibial plateau sectioned at the same horizontal level. b FEA simulation of the maximum principle strain at the tibia plateau based on the SB structure. The numbered boxes represent the individual areas in c. Source data are provided as a Source data file. c Immunofluorescence images of pSmad2/3 staining in full layer of AC of mouse tibia medial plateau at regions with different mechanical stress. The location of the image was indicated in b, pSmad2/3 (red), DAPI (gray). d Quantitative analysis of pSmad2/3 fluorescent intensity of c. n = 3 biologically independent animals, data were analyzed using two-way ANOVA Tukey’s post hoc test. Data are presented as mean values + /− SEM. e–h SV40 chondrocytes were cultured in low (2 ng/ml) or high (10 ng/ml) concentration of recombinant TGFβ1 for 48 h. n = 3 or 6 independent experiments in e–g or h, respectively. Data were analyzed using one-way ANOVA followed by LSD post hoc test. Data are presented as mean values + /− SEM. Source data are provided as a Source data file for e–g. e Glucose uptake assay using glucose analog 2-deoxyglucose (2-DG) to detect and quantify glucose uptake in chondrocytes. f Detection of total levels of cellular ATP based on the production of light caused by the reaction of ATP with added firefly luciferin. g Quantitative analysis of the ratio of pHrodo⁺ cells to total chondrocytes of pHrodo fluorescence staining in chondrocytes treated with low or high concentrations of recombinant TGFβ1. h Quantitative analysis of the ratio of DHE-positive cells to total chondrocytes treated with low or high concentration of recombinant TGFβ1.

Fig. 3. αV integrins mediate mechanical stress-induced TGFβ activation in chondrocytes.

a Double immunofluorescence staining of αVβ6 (red) and pSmad2/3 (green) in AC of human knee joints. HC healthy control, OA-M OA specimen with minimal cartilage degeneration, OA-S OA specimen with severe cartilage degeneration. b Quantitative analysis of αVβ6 and pSmad2/3 double staining in a. n = 10 biologically independent specimens, *P < 0.05 in comparison to αVβ6 and pSmad2/3 double-positive cells. Data are presented as mean values + /− SEM. Data were analyzed using one-way ANOVA LSD post hoc test. PS+: pSmad2/3+, AV+: αVβ6+. c Immunofluorescence staining of αVβ6 (red) and pSmad2/3 (green) in mouse AC harvested 1 month after ACL-T or sham surgery. Nuclei were labeled with DAPI (blue). d–f Quantitative analysis of the percentage of pSmad2/3⁺ cells (d) and αVβ6⁺ cells (e) in total chondrocytes and the ratio of αVβ6⁺ cells in pSmad2/3⁺ cells (f). n = 3 biologically independent animals, data are presented as mean values + /− SEM. Data were analyzed by two-tailed unpaired t test. g Western blot of αV integrin and pSmad2 protein in SV40 chondrocyte cell line. The cells were cultured in the free well or subjected to shear stress (6.58 dynes/cm²) for 48 h. The gene expression of αV integrin or α5 was knocked down using siRNA. Source data are provided as a Source data file. h ELISA of TGFβ in the conditional medium of the SV40 chondrocyte culture. The cells were cultured in the free well or subjected to shear stress (6.58 dynes/cm²) for 48 h. n = 3 independent experiments, data are presented as mean values + /− SEM. Data were analyzed using one-way ANOVA LSD post hoc test. Ctr or si-alphaV: cells treated with siRNA of control or αV integrin, respectively. i Immunofluorescence staining of pSmad2/3 and Smad2 in the SV40 cells with or without shear stress. Ctr or si-alphaV cells treated with siRNA of control or αV integrin, respectively. j Western blot of αV integrin and pSmad2 protein in SV40 chondrocyte cell line subjected to shear stress at different speeds. k Western blot of αV integrin and pSmad2 protein in SV40 chondrocyte cell line. Shear shear stress, hTGFβ recombinant human TGFβ1 (2 ng/ml), SB SB505124, TβRI selective inhibitor (1 µM). All the in vitro experiments were repeated three times independently. Source data are provided as a Source data file.

Fig. 4. Talin-centered cytoskeleton reorganization transmitted mechanical stress to αV integrins in activation of TGFβ.

a Immunofluorescence staining of talin (red) in primary chondrocytes isolated from mouse AC. F-actin was labeled by phalloidin (green). Primary chondrocytes were subjected to shear stress (6.58 dynes/cm²) for 48 h. b–d IP experiments. IP was carried out using the lysates of the SV40 cells subjected to 48-h shear stress or cultured in the free well. IP immunoprecipitation, IB immunoblotting, NTC negative control, β6 β6 integrin, αV αV integrin, si-Talin talin siRNA. Source data are provided as a Source data file. e, f Western blot of the SV40 cell lysates. The cells were subjected to shear stress or cultured in the free well for 48 h. CD cytochalasin D. Source data are provided as a Source data file. g, h ELISA of the concentration of active form TGFβ (g) or ratio to total TGFβ (h) in the conditional medium of the SV40 chondrocyte culture. The cells were cultured in the free well or subjected to shear stress for 48 h. n = 2 independent experiments. Data are presented as mean values + /− SEM. Source data are provided as a Source data file. i Representative image of immunohistochemical staining of talin (top, brown) and pSmad2 (bottom, brown) of AC of knee joints harvested from C57BL/6 mice subjected to and intra-articular injection of si-Talin or control siRNA for 1 month post ACLT or sham surgery. Scale bar: 50 µm. j, k Quantitative analysis of talin staining (j) and pSmad2 staining (k). n = 4 biologically independent animals. Data are presented as mean values + /− SEM. Data were analyzed using one-way ANOVA LSD post hoc test.

Fig. 5. Talin mediates the strengthening of αV integrin–RGD bond under mechanical stress.

a The sequence of the dsDNA and the conjugation sites of specific molecules. b Schematic illustration of the TGT principle. A dsDNA tether is immobilized on the PEG surface through an avidin–biotin linker. The location of the biotin determines the tension tolerance (Ttol) of the dsDNA. The integrins expressed by the cell bind to the RGDfk conjugated with the dsDNA at one end. The dsDNA ruptures if the tension applied by the cell through the integrin–RGD bond is greater than its Ttol. The Cy3 fluorescence signals and the adhesion of the cells on the PEG surface are maintained if the tension applied by the cell through the integrin–RGD bond is lower than its Ttol. c Cell density represents the rupture of the DNA tether. n = 13–17 independent cells. Data are presented as mean values + /− SEM. Data were analyzed using two-tailed t test. Source data are provided as a Source data file. d Representative differential interference contrast (DIC) images of the SV40 cells on the PEG surface (upper row) and the direct imaging of RGDfK-dsDNA-Cy3 removal on the PEG surface (bottom row). The cells were prechallenged with or without shear stress. The quantitative analysis is shown in c. Ctr cells were treated with control siRNA, Si-Talin cells were treated with talin siRNA. e Representative phase contrast (left column), the displacement of magnetic beads (middle column), and traction map images (right column) of SV40 cells that were preconditioned with or without shear stress. White lines show the cell boundary; colors show the magnitude of the tractions in Pa (see color scale). f Representative phase contrast (upper row) and traction map images (bottom row) of SV40 cells preconditioned with or without shear stress. Ctr cells were treated with control siRNA, Si-Talin cells were treated with talin siRNA. The quantitative analysis results are shown in g–i. g–i Quantitative analysis of RMS traction force (g), cell stiffness (h), and cytoskeleton remodeling (i) based on the displacement of magnetic beads. n = 10 independent experiments. Data were analyzed using two-way ANOVA Turkey’s post hoc tests. Source data are provided as a Source data file. j Quantitative analysis of cell stiffness of primary chondrocytes isolated from AC of mice tibia medial compartment. The mice were sacrificed 1 month after ACL-T or sham surgery. Ant anterior region, Pos posterior region. n = 3 independent animals. Data were analyzed using two-way ANOVA Turkey’s post hoc test. Source data are provided as a Source data file.

Fig. 6. αV integrin-mediated excessive TGFβ activation in AC degeneration of OA mice.

a Transaxial view of 3D reconstructed images of tibia medial compartment from Col2a-Cre^ERT: αV^fl/fl (αV^−/−) mice at 1 month after ACL-T or sham surgery. b Representative immunofluorescence staining images of pSmad2/3 in AC isolated from mice tibia medial compartment at 1 month after ACL-T or sham surgery (red: pSmad2/3, gray: DAPI). n = 3 biological independent animals in a, b. c Safranin O/fast green staining (left and middle columns) and fluorescence staining of pSmad2/3 (right column) of the sagittal sections of knee joint medial compartment of mice sacrificed at 1 month post sham or ACLT surgery. pSmad2/3: green, DAPI: blue. n = 3 biological independent animals. d–i Double immunofluorescence staining of pSmad2/3 (green) and aggrecan (red) in d and pSmad2/3 (red) and 8-OHdg (green) in e, and the AC were collected from mice at 1 month after ACL-T or sham surgery. f The ratio of pSmad2/3-positive and aggrecan-negative chondrocytes in total chondrocytes. g The ratio of pSmad2/3-negative and aggrecan-positive chondrocytes in total chondrocytes. h The ratio of pSmad2/3-negative and 8-OHdg-positive chondrocytes in total chondrocytes. i The ratio of pSmad2/3 and aggrecan double-positive chondrocytes in total chondrocytes. f–i n = 5 biologically independent animals. Data were analyzed using two-way ANOVA followed by Turkey’s post hoc analysis. Data are presented as mean values + /− SEM. j Schematic illustration of aberrant configuration of the TGFβ activity in OA cartilage due to the redistribution of mechanical stress in AC. k Schematic illustration of abnormal mechanical stress induced aberrant TGFβ activation and subsequent influence on chondrocyte metabolism in OA articular cartilage. Structural alteration of subchondral bone disrupts the mechanical environment in above cartilage. Under low mechanical stress, the αV integrin is in an inactive status and the latent TGFβs are deposited in the extracellular matrix. High mechanical stress induces the activation of αV integrins and the reorganization of cytoskeleton (stress fiber formation). Stress fiber formation increases cell stiffness and ensures chondrocytes exert contractile force higher than the minimal forces required for a conformational change of LAP and release of active TGFβ. Upregulated TGFβ signaling in turn increases cell stiffness and αV integrin expression in chondrocytes.