Age-related matrix stiffening epigenetically regulates α-Klotho expression and compromises chondrocyte integrity

Abstract

Extracellular matrix stiffening is a quintessential feature of cartilage aging, a leading cause of knee osteoarthritis. Yet, the downstream molecular and cellular consequences of age-related biophysical alterations are poorly understood. Here, we show that epigenetic regulation of α-Klotho represents a novel mechanosensitive mechanism by which the aged extracellular matrix influences chondrocyte physiology. Using mass spectrometry proteomics followed by a series of genetic and pharmacological manipulations, we discovered that increased matrix stiffness drove Klotho promoter methylation, downregulated Klotho gene expression, and accelerated chondrocyte senescence in vitro. In contrast, exposing aged chondrocytes to a soft matrix restored a more youthful phenotype in vitro and enhanced cartilage integrity in vivo. Our findings demonstrate that age-related alterations in extracellular matrix biophysical properties initiate pathogenic mechanotransductive signaling that promotes Klotho promoter methylation and compromises cellular health. These findings are likely to have broad implications even beyond cartilage for the field of aging research.

Fig. 1. Male, but not female, mice display age-related cartilage degeneration and disruption of the PI3K-Akt signaling pathway.

A Aging induced progressive cartilage degeneration in murine medial tibial plateaus in a sex-dependent manner. Representative histological sections stained with Safranin O/Fast Green are provided for each group. Black arrow heads indicate loss of cartilage matrix. The Osteoarthritis Research Society International (OARSI) score is provided (0–24 points; higher value indicates more severe cartilage degeneration), as assessed by a blinded assessor (male, n = 10 for young and aged, n = 7 for middle-aged; female, n = 10 for young and aged, n = 9 for middle-aged). Statistical analyses were performed using linear regression. Data are presented as means ± 95% confidence intervals. Scale bar: 50 μm. B Schematic showing the experimental protocol for mass spectrometry. Knee cartilage was microdissected from young, middle-aged, and aged male and female mice (n = 5/sex/age). Individual proteins were identified and used for KEGG pathways analysis. Proteins from PI3K/Akt signaling were then grouped based on GO terms. C Pathway analysis for young vs. aged in male and female mice. pAcc: the Boolean value of total bootstrap permutations accumulation; pORA: the Boolean value of over-representation p value. D Comparison between young vs. aged and young vs. middle-aged pathway analyses in male mice, and display of perturbation calculations. E Molecular hallmarks of upregulated and downregulated genes associated with PI3K/Akt signaling. F Schematic pathway of PI3K/Akt signaling and linking to α-Klotho. Portions of the figures were created with biorender.com. Source data are provided as a Source Data file.

Fig. 2. Age-related declines in α-Klotho are associated with cartilage degeneration in male mice.

A Aging induced a progressive decline of α-Klotho in the murine medial tibia (n = 5/sex/age). White arrows indicate α-Klotho-positive chondrocytes. White dashed lines indicate cartilage surface. AC articular cartilage. Scale bar: 10 μm. α-Klotho expression per cell was quantified by immunofluorescence (50–100 cells per mouse). B Cartilage in older adults (≥65 years old; 71.9 ± 2.91 years; n = 7 [1 female]) displayed reduced α-Klotho expression compared to young adults (<40 years old; 27.6 ± 6.85 years; n = 7 [2 females]). Scale bar: 20 μm. α-Klotho expression per cell was quantified by immunofluorescence (30–70 cells per cartilage sample). C Loss-of function in Klotho (Klotho^+/−) triggered murine cartilage degeneration in a sex-dependent manner (young, n = 7 for wild-type [3 females], n = 10 for Klotho^+/− [5 females]; middle-aged, n = 10 for wild-type [5 females], n = 8 for Klotho^+/− [5 females]). Black arrows indicate cartilage surface disruption. AC articular cartilage. Scale bar: 50 μm. Statistical analyses were performed using linear regression (A), two-way ANOVA (C), and a two-tailed Student t test (B). Age-sex interaction was not significant for  α-Klotho expression in mice (A, p = 0.468). Data are presented as means ± 95% confidence intervals. Source data are provided as a Source Data file.

Fig. 3. Age-related α-Klotho decline is accompanied with nuclear morphological alterations.

A Age-related changes in nuclear envelope proteins in male mice quantified by mass spectrometry-based proteomics. The heat map represents log10 (p value) of each protein, with >1.3 considered significant. B Aging increased lamin A/C and lamin B2 protein expression (n = 5/age). C Analytical flow of the nuclear morphological analysis (53 variables) using CellProfiler software. D Representative nuclear images in murine medial tibia generated by CellProfiler, which was reproducible in another set of images. Dashed white lines indicate the cartilage surface. Scale bar: 10 μm. E Principal component analysis (PCA) of nuclear morphology characteristics showing separation of clusters between young and aged animals (n = 5/age; 30–60 nuclei per individual mice). F Heat map of the top 10 nuclear morphological variables contributing principal component 1. G Murine (n = 5/age) and human cartilage (n = 7/age) share changes in nuclear eccentricity, with higher age associated with increased nuclear eccentricity (i.e., less roundness). H Age-related increased nuclear eccentricity is associated with decreased α-Klotho expression in murine (n = 5/age) and human cartilage (n = 7/age). I Schematic showing the relationship between chronological age, α-Klotho expression, and nuclear morphology. Linear regression model shows the substantial and independent contribution of nuclear morphology (Nuclear_human) in the prediction of α-Klotho expression level in human cartilage beyond the effect of chronological age (Age_human). Coefficient of determinations are provided for mouse (R²_m) and human cartilage (R²_h). Statistical analysis was performed using linear regression (B, G, H, I). Data are presented as means ±95% confidence intervals. Portions of the figure were created with biorender.com. Source data are provided as a Source Data file.

Fig. 4. Matrix stiffness regulates chondrocyte health via α-Klotho.

A Schematic showing the experimental protocol. Primary chondrocytes isolated from young murine cartilage were seeded onto polyacrylamide gels engineered to mimic a physiological range of cartilage matrix stiffness (5 kPa, 21 kPa, and 100 kPa). B. Stiff substrates reduced α-Klotho, type II collagen (Col2), and aggrecan (Acan) in young chondrocytes, as quantified by immunofluorescence (n = 5/group; 30–70 cells per individual sample). Scale bar: 50 μm. C, D siRNA Klotho treatment inhibited the beneficial effect of soft substrates on type II collagen and aggrecan expression, as quantified by immunofluorescence (n = 3/group; 20–40 cells per individual sample). Scale bar: 50 μm. E Measurement of matrix stiffness (Young’s modulus) for 8% and 20% GelMA (n = 6/group) using atomic force microscopy (AFM). Young’s modulus for young and aged articular cartilage was provided as reference values. F A stiff 3D environment reduced α-Klotho, type II collagen, and aggrecan in chondrocytes quantified by immunofluorescence (n = 3/group; 30–60 cells per individual sample). Scale bar: 20 μm. Statistical analyses were performed using linear mixed effect model (B) or two-tailed paired t test (C, D, F). Data are presented as means ±95% confidence intervals. Source data are provided as a Source Data file.

Fig. 5. Epigenetic regulation of α-Klotho by stiff matrix disrupts chondrocytes health.

A Stiff substrates downregulated Klotho expression and increased Klotho promoter methylation as well as global DNA methylation in young chondrocytes (n = 3/group). B Stiff substrates increased DNMT1 expression in young chondrocytes as quantified by immunofluorescence (n = 5/group; 30–50 cells per individual sample). Scale bar: 20 μm. C Stiff substrates increased binding of DNMT1 at Klotho promoter in young chondrocytes quantified by chromatin immunoprecipitation (ChIP) analyses (n = 4/group). D Stiff substrates increased binding of RNA Polymerase II (Pol II), active chromatin mark H3K4M2, and c-MYC, but not the repressive chromatin mark, H3K9M2, at the Dnmt1 promoter in young chondrocytes, as quantified by ChIP analyses (n = 4/group). E, F siRNA Dnmt1 treatment inhibited the deleterious effect of a stiff microenvironment on α-Klotho, type II collagen (Col2), and aggrecan (Acan) expression, as quantified by immunofluorescence (n = 4/group; 40–50 cells per individual sample). Scale bar: 50 μm. G, H 5-Aza-2’-deoxycytidine (5 Aza) treatment inhibits the deleterious effect of a stiff microenvironment on α-Klotho, Col2, and Acan expression. The effects of 5 Aza treatment were blocked when combined with siRNA Klotho (siRNA KL) treatment. Data were quantified by immunofluorescence (n = 4/group; 30–45 cells per individual sample). Scale bar: 50 μm. Statistical analyses were performed using a linear mixed effect model (A–D), two-tailed paired t test (E–G) or analysis of variance with post-hoc Tukey–Kramer test (H). **p < 0.01, ***p < 0.001. Data are presented as means ±95% confidence intervals. Source data are provided as a Source Data file.

Fig. 6. Mechanotransductive signals delivered by stiff substrates drive epigenetic repression of α-Klotho.

A Latrunculin A, an inhibitor of actin polymerization, rescued stiff matrix-induced increases in DNMT1 and reduction of α-Klotho, type II collagen (Col2), and aggrecan (Acan) in aged chondrocytes, as quantified by immunofluorescence (n = 3/group except for Col2 [n = 4/group]; 30–60 cells per individual sample). Scale bar: 20 μm. B Inhibition of actin polymerization blocked the stiff substrate-driven increased Klotho promoter and global DNA methylation in aged chondrocytes (n = 3/group). C Inhibition of actin polymerization blocked the stiff substrate-driven increased binding of RNA Polymerase II (Pol II), c-MYC, but not H3K4M2, at Dnmt1 promoter in aged chondrocytes as quantified by chromatin immunoprecipitation (ChIP) analyses (n = 4/group). D Inhibition of actin polymerization blocked the stiff substrate-driven increased binding of DNMT1 at Klotho promoter in aged chondrocytes as quantified by ChIP analyses (n = 4/group). Statistical analyses were performed using a linear mixed effect model (A) or two-tailed paired t test (B–D). Data are presented as means ± 95% confidence intervals. Source data are provided as a Source Data file.

Fig. 7. BAPN injection improves α-Klotho expression and cartilage integrity in aged mice.

A Principal component analysis (PCA) showing the separate clusters in ECM proteins from young and aged cartilage (n = 5/group). B Principal component 2 (PC2) in ECM protein expression is positively correlated with lamin A/C expression. C Heat map of the top 20 ECM proteins contributing to PC2, showing age-related ECM remodeling. Color indicates z score for each variable. Lox, an enzyme that induces collagen cross-linking, was in the top 20 proteins contributing to PC2 and was positively correlated with lamin A/C, supporting a connection between LOX-mediated increased collagen cross-linking and nuclear stiffness. D Schematic showing the experimental protocol for 4 weeks of daily BAPN injections in aged mice (n = 8 for saline, n = 9 for BAPN). E Schematic showing the atomic force microscopy measurement of murine medial tibial cartilage with 10 × 10 µm region of interest. F BAPN treatment significantly reduced Young’s modulus of medial tibial cartilage (n = 5/group). Young’s modulus data were transformed into Log MPa and normalized by Young’s modulus value in naive young group. G. Representative CellProfiler-generated nuclear images in murine medial tibial plateaus after 4-week injection of saline or BAPN. Nuclei were pseudocolored. White dashed lines indicate cartilage surface. Scale bar: 10 μm. BAPN injection in aged mice decreases nuclear eccentricity towards young level (n = 8 for saline, n = 9 for BAPN; 30–40 nuclei per individual mouse). H PCA showing the same cluster of young and aged+BAPN injection. I. BAPN injection in aged mice improves α-Klotho expression in articular cartilage quantified by immunofluorescence (n = 8 for saline, n = 9 for BAPN; 30–65 cells per individual sample). J BAPN injection in aged mice improves cartilage integrity (n = 8 for saline, n = 9 for BAPN). Representative histological sections stained with Safranin O/Fast Green are provided. Black arrows indicate loss of cartilage matrix. OARSI score (0–24 points; higher value indicates more severe cartilage degeneration) assessed by blinded assessor is provided. OARSI score in young cartilage is provided as a reference. K Beneficial effect of BAPN injection was blocked in Klotho^+/− mice (n = 5 each for saline and BAPN). Statistical analyses were performed using linear regression analysis (B, C) or two-tailed Student t test (F, G, I–K). Data are presented as means ±95% confidence intervals. Portions of the figures were created with biorender.com. Source data are provided as a Source Data file.

Fig. 8. Graphical abstract.

Age-related matrix stiffening in articular cartilage initiates pathogenic mechanotransductive signaling, driving chondrocyte dysfunction as well as disrupted cartilage integrity through Klotho promoter hypermethylation. Portions of the figure were created with biorender.com.