MYL3 protects chondrocytes from senescence by inhibiting clathrin-mediated endocytosis and activating of Notch signaling

Abstract

As the unique cell type in articular cartilage, chondrocyte senescence is a crucial cellular event contributing to osteoarthritis development. Here we show that clathrin-mediated endocytosis and activation of Notch signaling promotes chondrocyte senescence and osteoarthritis development, which is negatively regulated by myosin light chain 3. Myosin light chain 3 (MYL3) protein levels decline sharply in senescent chondrocytes of cartilages from model mice and osteoarthritis (OA) patients. Conditional deletion of Myl3 in chondrocytes significantly promoted, whereas intra-articular injection of adeno-associated virus overexpressing MYL3 delayed, OA progression in male mice. MYL3 deficiency led to enhanced clathrin-mediated endocytosis by promoting the interaction between myosin VI and clathrin, further inducing the internalization of Notch and resulting in activation of Notch signaling in chondrocytes. Pharmacologic blockade of clathrin-mediated endocytosis-Notch signaling prevented MYL3 loss-induced chondrocyte senescence and alleviated OA progression in male mice. Our results establish a previously unknown mechanism essential for cellular senescence and provide a potential therapeutic direction for OA.

Fig. 1. CME is enhanced and MYL3 expression is decreased in senescent chondrocytes.

a Images and quantification of SA-β-Gal positivity and IF staining of p16^INK4A in mouse primary chondrocytes at passage 2 and 7. n = 5, bars = 25 μm. b Images and quantification of transferrin endocytosis in mouse primary chondrocytes at passages 2 and 7. n = 5, bars = 25 μm. c Quantification of the relative expression of myosins with a proteome-wide screen for the proteins of articular cartilage from 2- or 12-month-old mice. d Images of Safranin-O and IF staining of MYL3, p16^INK4A, γH2AX and quantification (e) of OARSI scores, MYL3-, p16^INK4A- and γH2AX-positive cells and in articular cartilage from 2-, 12- and 24-month-old mice, blue indicates DAPI staining of nuclei. n = 8, bars = 50 μm. f Images of Safranin-O, IHC of MYL3 and p16^INK4A, and IF staining of γH2AX and quantification of OARSI, MYL3-, p16^INK4A- and γH2AX-positive cells (g) in intact (n = 5) and damaged (n = 9) articular cartilage sections collected from OA patients. Bars = 50 μm. h Images of Safranin-O staining, IHC staining for MYL3 and IF staining of p16^INK4A and γH2AX, and quantification of OARSI scores and MYL3-, p16^INK4A- and γH2AX-positive cells (i) in articular cartilage from mice of control, 6- and 10- weeks post DMM surgery. n = 8, bars = 50 μm. Data are all shown as means ± SD. P values are from two-tailed Mann–Whitney U test (b), two-tailed unpaired t test (a, g), one-way ANOVA test followed by Tukey’s post hoc test (p16^INK4A, γH2AX in e, MYL3, p16^INK4A, γH2AX in i and Kruskal–Wallis test followed by Dunn’s post hoc test (OARSI, MYL3 in e, OARSI in i). n indicates the number of biologically independent samples, mice per group, or human specimens. Source data are provided as a Source Data file.

Fig. 2. MYL3 negatively regulates CME and cellular senescence in chondrocytes.

a Images and quantification of endocytosed transferrin in mouse primary chondrocytes transfected with control, Myl3 shRNA knockdown (Myl3-KD) or overexpression adenovirus particles (Myl3-OE) at passage 4. n = 5, bars = 25 μm. b IP using control IgG and anti-MYL3 antibody in mouse primary chondrocytes to detect the binding of MYL3 with MYO6 and Clathrin. c IP revealed the binding of MYO6 and MYL3 or Clathrin in chondrocytes. d IP revealed the binding of MYO6 and Clathrin in mouse primary chondrocytes transfected with control, Myl3-KD, or Myl3-OE. e IF images and quantification of Clathrin (green) and MYO6 (red) co-localization at passage 7, SA-β-Gal positivity at passage 2 or 7 (f, g) in mouse primary chondrocytes transduced with control, Myl3-KD or Myl3-OE. n = 5, bars = 25 μm. h Images of p16^INK4A IF and protein levels of MYL3, p16^INK4A, P21, γH2AX, and MMP13 (i) in mouse primary chondrocytes transfected with control, Myl3-KD or Myl3-OE. Bars = 25 μm. Data are representative of three independent experiments and are all shown as means ± SD. P values are from a one-way ANOVA test followed by Tukey’s post hoc test (a, e, g). n indicates the number of biologically independent samples or mice per group. Source data are provided as a Source Data file.

Fig. 3. Deletion of MYL3 in chondrocytes promotes chondrocytes senescence and OA progression in mice.

a Schematic illustration of the spontaneous age-related OA model in control and Myl3-KO mice. b Images and quantification of IHC staining of MYL3, Safranin-O staining images (c), OARSI and synovial inflammation scores (d), and IF staining images of p16^INK4A and γH2AX (e) in sagittal sections of joints from control and Myl3-KO mice at 6 months and 18 months. n = 8 independent biological replicates per group, bars = 50 μm. f Schematic illustration of posttraumatic OA model with DMM surgery in control and Myl3-iKO mice. g Images and quantification of IHC staining of MYL3, Safranin-O staining images (h), OARSI and synovial inflammation scores (i), and IF staining images of p16^INK4A and γH2AX (j) in sagittal sections of joints from control and Myl3-iKO mice at 6- and 10- weeks post-surgery. n = 8 independent biological replicates per group, bars = 50 μm. Data are all shown as means ± SD. P values are from two-tailed Mann–Whitney U test (OARSI, synovial inflammation scores in d) and two-tailed unpaired t test (remaining quantification). n indicates the number of biologically independent samples or mice per group. Source data are provided as a Source Data file.

Fig. 4. Overexpression of MYL3 attenuates age-related and experimental OA development in mice.

a Schematic illustration of Ad-Myl3 or Ad-control delivery schedules in an age-related OA model in mice. b Images and quantification of IHC staining of MYL3, Safranin-O staining images (c), OARSI and synovial inflammation scores (d), and IF staining images of p16^INK4A and γH2AX (e) in sagittal sections of joints from control and Myl3-OE mice at 6 months and 18 months. n = 8 independent biological replicates per group, bars = 50 μm. f Schematic illustration of Ad-Myl3 or Ad-control delivery schedules in a posttraumatic OA model with DMM surgery in mice. g Images and quantification of IHC staining of MYL3, Safranin-O staining images (h), OARSI and synovial inflammation scores (i), and IF staining images of p16^INK4A and γH2AX (j) in sagittal sections of joints from control and Myl3-OE mice at 6- and 10 weeks post surgery; n = 8 independent biological replicates per group, bars = 50 μm. Data are all shown as means ± SD. P values are from two-tailed Mann–Whitney U test (OARSI, synovial inflammation scores in d, i) and two-tailed unpaired t test (remaining quantification). n indicates the number of biologically independent samples or mice per group. Source data are provided as a Source Data file.

Fig. 5. Loss of MYL3 promotes the activation of Notch signaling and induces senescence by enhancing CME.

a Volcano plot of RNA-seq of primary chondrocytes from control and Myl3-KO mice. The dotted line denotes P = 0.05. b Top 10 pathway enrichment of KEGG pathway analysis following Myl3 knockout in chondrocytes. c Relative mRNA expression of Myl3, Hes1, Hey1, p16^INK4A, and p53, and protein levels of MYL3, NICD, HES1, and HEY1 (d) in primary chondrocytes from control and Myl3-KO mice. e Images and quantification of IF staining for NICD (green) and clathrin (red) co-localization in mouse primary chondrocytes from control, jag1, jag1 + RO4929097, jag1+Myl3-OE, or jag1+dynasore group. n = 5, bars = 25 μm. f Images and quantification of IF staining for NICD (green) and clathrin (red) co-localization in primary chondrocytes from control, Myl3-KO, KO + RO4929097, KO+Dynasore, KO+DynK44A-OE, or KO+Clathrin-KD group. n = 5, bars = 25 μm. g Protein levels of p16^INK4A, p53, γH2AX, HES1, and HEY1 in primary chondrocytes from control, jag1, jag1 + RO4929097, jag1+Myl3-OE, or jag1+dynasore group. h Protein levels of, p16^INK4A, p53, γH2AX, HES1, and HEY1 in primary chondrocytes from control, Myl3-KO, KO + RO4929097, KO+Dynasore, KO+DynK44A-OE, or KO+Clathrin-KD group. i Protein levels of p16^INK4A, p53, γH2AX, HES1, and HEY1 in primary chondrocytes from control, RO4929097, Myl3-OE, Clathrin-KD or Dynasore with vehicle or Notch-DeltaE. Data are representative of three independent experiments and are all shown as means ± SD. P values are from two-tailed unpaired t test (c) and one-way ANOVA test followed by Tukey’s post hoc test (e, f). n indicates the number of biologically independent samples or mice per group. Source data are provided as a Source Data file.

Fig. 6. Targeting MYL3-Notch signaling alleviates age-related and experimental OA.

a Schematic illustration of dynasore or PBS delivery schedules in a posttraumatic OA model with control and Myl3-iKO mice. b Safranin-O staining images and quantification of OARSI and synovial inflammation scores (c), images and quantification of IF staining for NICD, p16^INK4A, and γH2AX (d) in sagittal sections of joints from control or Myl3-iKO mice treated with dynasore or vehicle intraarticular injections at 6 weeks post surgery. n = 8 independent biological replicates per group, bars = 50 μm. e Schematic illustration of RO4929097 or vehicle delivery schedules in a posttraumatic OA model with control and Myl3-iKO mice. f Safranin-O staining images and quantification of OARSI and synovial inflammation scores (g), images and quantification of IHC staining for NICD and IF staining for p16^INK4A and γH2AX. h in sagittal sections of joints from control or Myl3-iKO mice treated with RO4929097 or vehicle intraarticular injections at 6 weeks post surgery. n = 8 independent biological replicates per group, bars = 50 μm. Data are all shown as means ± SD. P values are from two-tailed Mann–Whitney U test (synovial inflammation scores in c, g) and the two-tailed unpaired t test (remaining quantification). n indicates the number of biologically independent samples or mice per group. Source data are provided as a Source Data file.

Fig. 7. Schematic of the role of MYL3 in mediating CME of Notch signaling and cellular senescence in chondrocytes.

In the process of aging or osteoarthritis development, a reduction of MYL3 in chondrocytes leads to enhanced CME by promoting the interaction between MYO6 and clathrin, and consequently induces the internalization of Notch receptors and nuclear translocation of NICD, resulting in the activation of Notch signaling. This process triggers chondrocytes senescence, promotes cartilage destruction, and aggravates OA progression. Supplementation of MYL3 and strategically blocking CME-Notch signaling effectively mitigates chondrocyte senescence and OA development in mice.