Transcription factor ZBTB20 is activated in early-OA

To elucidate the biological processes of inflammation in osteoarthritis, an in vitro inflammatory model was established via subjecting the cultured primary chondrocytes to interleukin-1β (IL-1β), the most approved approach to mimic the process of inflammation.11 RNA-seq was performed on chondrocytes under IL-1β stimulation for 0/6/48 h, with 6 h considered as times point for rapid response and 48 h for long-term response. The DEGs were categorized into clusters according to their expression profiles. Besides the gradually upregulated or downregulated genes in C1, C3 and C4, a batch of genes exhibited elevated or inhibited expression especially for stimulation of 6 h, which enriched for biological process related to cytokines and cell cycle regulation (Fig. S1A). Subsequently, ATAC-seq was employed to investigate the chromatin accessibility profiling in chondrocytes (3 replicates for each, Fig. S1B). Consisting with the RNA-seq results, a gradual decrease was observed in regions near the gene promoter associated with extracellular matrix organization (Acan, Col2α1), while a significant increase was noted in genes relevant to immune responses (Cxcl1, Cxcl2, Cxcl3) (Fig. S1C). Analysis of TF binding motifs in each group indicated an enrichment of Zinc Finger-containing transcription factors in the 6 h samples, signifying the activation of ZF proteins (Figs. 1a–c and S1D). The higher proportion of down-regulating genes in 6 h compared to the 48 h samples suggested a suppressive role of these ZF transcription factors specifically activated at 6 h (Fig. S1E, F).

Fig. 1

Transcription factor ZBTB20 is activated in early-OA. a Heatmap showing density of annotated ATAC-seq reads 1 kb around the transcription start sites (TSS). b Representative enriched TF binding motifs in chondrocytes treated by IL-1β for 6 h. c Heatmap of enriched motifs in chondrocytes under IL-1β stimulation for 0/6/48 h. a–c n = 3 replicates. d Dot plot graph showing the relative expression level and percentage of ZBTB-containing sub-family members in cell populations. e Representative images of immunofluorescence staining of ZBTB20 in articular cartilage of mice underwent DMM or Sham surgery for 2/4/8 weeks. Panels on the right are high magnifications of the boxed regions in the left panels. The dashed circles marked the edge of nuclei. n = 8, 10, 8, 7, 7, 5 mice. f Statistical analysis of the expressions of ZBTB20 in indicated groups of mice in (e). The top panel is quantification of relative fluorescence intensity of ZBTB20. The bottom panel is the statistical analysis for ZBTB20 cellular distribution. g Western blot of COL II, MMP13 and ZBTB20 in chondrocytes treated with IL-1β for 0-72 h. n = 3 biological independent experiments. h Curves of COL II, MMP13 and ZBTB20 expressions in (g). i Schematic diagram of cartilage tissues of the tibial plateau from patients. j Representative images of Safranin O/Fast Green and immunohistochemistry (IHC) staining for COL II and ZBTB20 in the relative damaged and undamaged cartilage from OA patients

Among the ZF family, members of the ZBTB sub-family are mostly known for their transcriptional repressive functions.12 A single-cell sequencing outcome13 revealed that within the ZBTB-containing sub-family, ZBTB20 exhibits significant expression in clusters of chondrocytes (Fig. 1d). Given its role in modulating the inflammation response, we postulate that ZBTB20 could have a key role in the early pathogenesis of OA. Then we monitored the expression changes of ZBTB20 in OA progress. As shown in Fig. 1e and f, ZBTB20 in cartilage chondrocytes translocated into the nucleus at 2 weeks after DMM surgery, gradually diminished as OA advanced. In cultured chondrocytes, the protein level of ZBTB20 exhibited a downregulation trend upon IL-1β stimulation, correlated with COL II (Fig. 1g, h). To minimize variations in OA patients, cartilage samples from the lateral and medial regions of the tibial plateau were assessed as relatively damaged or undamaged areas. The reduction of ZBTB20 expression was observed in damaged region compared with undamaged region (Figs. 1i, S2 B–D). In conclusion, the integration of RNA-seq, ATAC-seq findings with the results of experiments on chondrocytes, mice, and clinical tissues indicates a strong association between the activation of ZBTB20 and the pathogenesis of OA in the early stages.

Depletion of ZBTB20 attenuates OA progress

Due to ZBTB20’s function of regulating hypertrophic chondrocyte differentiation in growth plate cartilage,10 we employed the TAM-inducible knockout strategy using Col2a1-CreERT2 mice to deplete ZBTB20 at 10 weeks old, to avoid the possible impacts from development defects of Zbtb20-deletion in chondrocyte. The DMM surgery to induce OA progress was carried out at 12 weeks old of mice, 2 weeks post the injection of TAM (Fig. S3A, B). OA manifestations observed previously included erosion of cartilage, increased ratio of hyaline to calcium cartilage thickness, formation of osteophyte, and heightened subchondral bone plate thickness.14,15,16 As shown in Fig. 2a and b, Zbtb20 deficiency alleviated cartilage destruction post DMM-surgery at 8 and 12 weeks. The increased volume of osteophytes and thickness of subchondral bone plate were hindered by Zbtb20 knockout (Fig. 2c, d). Hypertrophic differentiation has been reported to be one of the key factors promoting the initiation and progression of OA.17 As shown in Fig. 2a, the increased number of hypertrophic chondrocytes in articular cartilage was suppressed in Zbtb20-icKO mice. These findings highlight that Zbtb20 depletion in chondrocytes decelerates OA progression.

Fig. 2

ZBTB20 icKO in chondrocytes alleviates OA. a Safranin O/Fast Green staining of articular cartilage from Zbtb20-icKO or corresponding control mice underwent DMM or Sham surgery for 8/12 weeks. Panels in the upper right corner are high magnifications of images. The arrows indicated the hypertrophy-like chondrocytes. n = 8, 9, 9, 9, 4, 3, 4, 3 mice. b Statistical analysis of OARSI scores, thickness of hyaline cartilage (HC), calcified cartilage (CC) and ratio of HC to CC in indicated groups of mice in (a). c Representative images of knee joint osteophytes (top panel) and subchondral bone plate (bottom panel) reconstructed by MicroCT analysis from indicated group of mice. n = 4, 3, 4, 3 mice. d Statistical analysis of volume of osteophytes (panel on the left) and SBP thickness (panel on the right) in indicated groups of mice in (c). e IF staining of COL II, ACAN, MMP13, and ADAMTS5 in the tibial cartilage from indicated group of mice. The dashed lines mark the edge of cartilage or the tidemark. The arrows mark the MMP13+ or ADAMTS5+ chondrocytes. f Statistical analysis of IF signal in indicated groups of cells in (e). n = 3, 3, 3, 3 mice. g Western blot of ZBTB20, COL II, MMP13, and ADAMTS5 in IL-1B treated zbtb20f/f primary chondrocytes transduced with Ad-GFP or Ad-Cre-GFP. h Relative mRNA levels of Zbtb20, Col2α1, Mmp3 and Adamts5 in chondrocytes described in (g). i Western blot of ZBTB20, COL II, MMP13, and ADAMTS5 in IL-1B treated primary chondrocytes transfected with Ad-GFP or Ad-mZbtb20-GFP. j Relative mRNA levels of Zbtb20, Col2α1, Mmp3, and Adamts5 in chondrocytes described in (i). g–j n = 3 biological independent experiments

The imbalance of ECM anabolism and catabolism is considered as the main cause of cartilage degradation, one of the most identical OA features.18 Therefore, the expression of proteins involved in ECM homeostasis was examined. The reduced expression of ECM components (COL II and ACAN) in OA cartilage was restored in Zbtb20 icKO mice, while the abnormal expression of ECM degradation enzymes (MMP13 and ADAMTS5) was blocked by Zbtb20 knockout (Fig. 2e, f). In cultured chondrocytes, manipulation of Zbtb20 expression through transduction of adenovirus or siRNAs, followed by IL-1β stimulation, resulted in similar outcomes as observed in vivo. Specifically, upregulation of MMP3, MMP13 and ADAMTS5 induced by IL-1β treatment was suppressed by Zbtb20 knockout, and the downregulated expression of COL II was recovered in Zbtb20 knockout chondrocytes (Figs. 2g, h and S4A–D). Moreover, overexpression of Zbtb20 exacerbated the disordered ECM homeostasis, resulting in enhanced ECM degradation and impaired ECM synthesis (Figs. 2i, j and S4E). As shown in Fig. S1A, the NF-κB signaling is enriched in the DEGs specifically upregulated in chondrocytes treated by IL-1β for 6 h, when the translocation of ZBTB20 to nucleus is detected, highlighting the possible correlation of ZBTB20 and NF-κB signaling activation. Activation of this pathway was assessed by examining the expression of the target gene Nos2 and the cellular distribution of p65. As shown in Fig. S5, Zbtb20 deletion suppressed NF-κB signaling activation, and vice versa. Altogether, these findings collectively suggest that ZBTB20 accelerates OA progression by regulating ECM maintenance via triggering NF-κB signaling.

Activation of NF-κB signaling by ZBTB20 requires suppression of Pten

To elucidate the underlying mechanism of ZBTB20 triggering NF-κB signaling, RNA-seq analysis was carried out in WT and Zbtb20 KO chondrocytes, brought up a series of DEGs through various comparisons (Fig. 3a). By integrating the top 10 enriched KEGG pathways of DEGs from each comparison, PI3K-Akt pathway emerged as a highly significant signaling pathways in ZBTB20-associated mechanisms, ranking high in WTvsWT_IL-1β group while not present in the KO_IL-1βvsKO group, with overlapping appearance in both KOvsWT and KO_IL-1βvsWT_IL-1β groups (Fig. 3b). The modulation of AKT phosphorylation by ZBTB20 reveals the involvement of PI3K-Akt signaling downstream of ZBTB20 indeed (Fig. 3c, d). To uncover the detail mechanism of ZBTB20 on PI3K-Akt signaling, CUT&Tag-seq19 analysis was performed in cultured chondrocytes that identified numerous peaks (Fig. S6A). Given the suppressive role of ZBTB20, the upregulated genes in KOvsWT comparison related to PI3K-Akt signaling, with ZBTB20 occupancy, were categorized based on their impact on signaling transduction, suggesting Pten as a crucial link between ZBTB20 and PI3K-Akt signaling (Fig. S6B). Distinct ZBTB20 peaks were detected in the promoter region of Pten in CUT&Tag-seq profiles, and further verified by ChIP-qPCR (Figs. 3e, f and S6C). Altered expressions of PTEN, inversely correlated with p65 phosphorylation, in Zbtb20-modulated chondrocytes further supported the suppressive role of ZBTB20 (Fig. 3g–j). Experimental validation using compounds targeting PI3K-Akt signaling was performed to confirm the mechanism. Activation of PI3K-Akt signaling by SC79 disrupted ECM homeostasis restored by Zbtb20 knockout, whereas inhibition by MK2206 recovered the Zbtb20 overexpression-induced imbalance (Fig. 3k, l). Taken together, these results indicate that the NF-κB signaling mediated disturbed ECM maintenance by ZBTB20 requires its suppression of Pten and consequent PI3K-Akt signaling activation.

Fig. 3

Activation of NF-κB signaling by ZBTB20 requires suppression of Pten. a Scatter plot graph showing the expression changes of DEGs from each comparison. b Dot plot graph of the top 10 enriched KEGG pathways of DEGs from each comparison. a, b n = 3 replicates. c Western blot of pAKT-T308, pAKT-S473, and AKT in indicated chondrocytes. d Statistical analysis of the bands in (c). e Representative tracks of CUT&Tag-seq analysis showing the enrichment of ZBTB20 around Pten’s promoter in chondrocytes. f Relative enrichment of ZBTB20 on binding regions compared to IgG in chondrocytes treated by IL-1β or not analyzed by ChIP-qPCR. g Western blot of PTEN, pp65-S536 and p65 in IL-1B treated zbtb20f/f primary chondrocytes transduced with Ad-GFP or Ad-Cre-GFP. h Relative mRNA levels of Zbtb20 and Pten in chondrocytes described in (g). i Western blot of PTEN, pp65-S536 and p65 IL-1B treated primary chondrocytes transduced with Ad-GFP or Ad-mZbtb20-GFP. j Relative mRNA levels of Zbtb20 and Pten in chondrocytes described in (i). k, l Relative mRNA levels of Zbtb20, Mmp3, and Col2α1 in indicated groups of chondrocytes treated with IL-1β or/and SC79/ MK2206. c–l n = 3 biological independent experiments

LATS1 regulates the cellular distribution of ZBTB20

Besides these detailed mechanistic findings, our hypothesis that ZBTB20 is activated at early-stage OA was assessed by examining the target genes’ transcriptome. A single-cell sequence analysis of OA clinical cartilage tissues revealed the transitions of proliferative chondrocytes (ProCs), prehypertrophic chondrocytes (preHTCs), and hypertrophic chondrocytes (HTCs) over the course of OA development, with preHTCs indicating an intermediate state between ProCs and HTCs.20 The dramatically reduced expression level of PTEN in preHTCs, as well as these ZBTB20-target-genes, specifically in preHTCs as opposed to other cells indicates the transcription activation of ZBTB20 in these preHTCs (Fig. S6D, E), further supporting our observations of the dynamic ZBTB20 cellular distribution change during OA progression.

The increased nucleus accumulation of ZBTB20 was detected in chondrocytes upon IL-1β stimulation (Fig. 4a–d), validating our findings of ZBTB20’s activity change in OA pathogenesis, attributing to the ECM degradation via suppressing the expression of Pten and consequent NF-κB signaling activation at early stage of OA. Thus, approaches inhibiting ZBTB20 activity emerge as promising treatments for osteoarthritis. Since multiple challenges exist in modulating the activity of transcription factors, understanding the upstream regulation mechanism can provide insights for addressing these challenges, while the upstream regulating mechanism of ZBTB20 has been much less explored. The research conducted in drosophila revealed that the kinase Warts directly interacts with Lola, the homolog of ZBTB20 in drosophila, providing a clue to solve this question.21 Primarily, increased phosphorylated modification of LATS1 was observed in IL-1β treated chondrocytes as well as DMM-induced OA cartilage, especially 2 weeks after the surgery (Figs. 4e, f and S7A–C), indicating that the elevated phosphorylation of LATS1 may be the cause of ZBTB20 cellular re-distribution. Secondly, Co-IP was carried out using tag-fused proteins (Flag-ZBTB20, Myc-LATS1), turning out a reciprocal interaction of ZBTB20 and LATS1 to be detected, and weakened binding upon IL-1β stimulation (Fig. 4g). To further validate the interaction in situ, proximity labeling using TurboID-fused ZBTB20 was employed (TurboID-Flag-ZBTB20, Fig. S8). Consist with the Co-IP results, the interaction was proved by the detected biotin-labeled LATS1, suppressed by IL-1β treatment (Fig. 4h, i). Finally, supplementation of TRULI, a selective LATS1/2 inhibitor, countered IL-1β-induced nucleus accumulation of ZBTB20, indicating LATS1’s role in ZBTB20’s subcellular distribution (Fig. 4j–k). In summary, these results reveal that the kinase LATS1 controls cellular localization of ZBTB20. Phosphorylation of LATS1 induced by IL-1β treatment abolishes the interaction of ZBTB20 and LATS1, therefore releases ZBTB20 to translocate into nucleus.

Fig. 4

LATS1 regulates the cellular distribution of ZBTB20 in chondrocytes. a Representative images of immunofluorescence staining of ZBTB20 in primary chondrocytes exposed to IL-1β at concentrations ranging from 1 to 5 ng/mL. n = 3 biological independent experiments. b Statistical analysis of the cellular distribution of ZBTB20 in indicated groups of cells in (a). n = 6 views per group for one biological replicate. The left panel is quantification of N/C ratio of ZBTB20. The right panel is the statistical analysis for ZBTB20 cellular distribution. c Western blot of ZBTB20 in nucleus or cytoplasm extraction of chondrocytes treated with IL-1β ranging from 0 to 5 ng/mL. d Curves showing the cellular distribution of ZBTB20 in (c). e Western blot of COL II, LATS1 and pLATS1 in chondrocytes exposed to IL-1β. f Statistical analysis of fluorescence signal of LATS1 and pLATS1 in articular cartilage from mice underwent Sham or DMM surgery for 2/4/8 weeks. n = 3 mice per group. g Co-immunoprecipitation of Fg-ZBTB20 and Myc-LATS1. h Proximity labeling with TurboID and TurboID-ZBTB20 in chondrocytes. i Statistical analysis of bands of LATS1 in (h). j Representative images of immunofluorescence staining of ZBTB20 in primary chondrocytes upon IL-1β or/and TRULI stimulation. n = 3 biological independent experiments. k Statistical analysis of N/C ratio of ZBTB20 in (j). n = 6, 6, 5 views for one biological replicate. c–e, g–i n = 3 biological independent experiments

As an essential element of the Hippo signaling pathway, a mechanical transduction singling, LATS1 undergoes phosphorylation and activation in response to mechanical stimuli.22 Since overloading is considered as one of the causes of OA, we wondered whether ectopic mechanical forces affect ZBTB20’s cellular distribution in chondrocytes in a LATS1-dependent manner. As shown in Fig. S9A–D, the nucleus accumulation of ZBTB20, as well as p65, is evident and reduced upon treatment with TRULI. This suggests that ectopic mechanical loading, which triggers LATS1 phosphorylation, in turn facilitates the translocation of ZBTB20 in chondrocytes.

Both TRULI and DAPA restore ECM homeostasis in chondrocytes

Our observations that in early-stage OA chondrocytes ZBTB20 translocates into the nucleus in a LATS1-dependent manner, triggers NF-κB signaling and enhances ECM degradation, leads us to consider different approaches to restore ECM balance: either by inhibiting LATS1 to impede ZBTB20 activity or by directly reducing ZBTB20 expression. As mentioned above, the small molecular compound TRULI can block the nucleus accumulation of ZBTB20 via inhibiting LATS1 phosphorylation. Then we evaluated the expressions of proteins associated with ECM homeostasis. The reduced ECM synthesis induced by IL-1β treatment was restored, while the abnormal ECM degradation was blocked by co-treatment of TRULI in a dosage dependent manner (Fig. 5a, b, e, f), indicating the therapeutic role of TRULI against OA. Research has identified Zbtb20 as a potential therapeutic target of dapagliflozin (DAPA) from the RNA-seq analysis for the protective effects in hypertensive nephropathy,23 suggesting us DAPA as a proposing drug targeting Zbtb20. As shown in Fig. 5c, supplement of DAPA further minimized the expression of ZBTB20 indeed, as well as increased the expression of PTEN as a result. The disordered ECM balance, including disrupted expression of ECM components and ectopic expression of ECM degradation enzymes, was recovered by DAPA in a dosage dependent manner as well (Fig. 5c–f), suggesting DAPA’s protective effects in OA chondrocytes. Considering the crucial role of NF-κB signaling linking ZBTB20 and ECM maintenance previously described, its activation was assessed then. The increased nucleus accumulation of p65, reflecting the activation of NF-κB signaling, induced by IL-1β treatment was inhibited by both TRULI and DAPA gradually, further proving the protective role of TRULI and DAPA both (Fig. 5g, h). As described above, two independent approaches to modulating ZBTB20 via utilizing TRULI and DAPA can restore ECM homeostasis through triggering NF-κB signaling.

Fig. 5

Both TRULI and DAPA are capable to restore ECM homeostasis. a Western blot of COL II, ADAMTS5, and MMP13 in chondrocytes treated with IL-1β, supplement with increased dosage of TRULI. b Relative mRNA levels of Mmp3, Mmp13, Col2α1 and Acan in chondrocytes described in (a). c Western blot of COL II, ADAMTS5, and MMP13 in chondrocytes treated with IL-1β, supplement with increased dosage of DAPA. d Relative mRNA levels of Mmp3, Mmp13, Col2α1 and Acan in chondrocytes described in (c). e Representative images of immunofluorescence staining of COL II in chondrocytes treated with IL-1β, supplement with increased dosage of TRULI or DAPA. f Statistical analysis of the relative fluorescence intensity of COL II in indicated groups of cells in (e). g Representative images of immunofluorescence staining of p65 in chondrocytes treated with IL-1β, supplement with increased dosage of TRULI or DAPA. Curves on the top of images showing the fluorescence intensity profile of crop section indicated by the white dashed line. The dashed circles mark the nucleus. h Statistical analysis of the cellular distribution of p65 in indicated groups of cells in (g). a–e, g n = 3 biological independent experiments. f, h n = 12, 12, 6, 6, 5, 5, 6, 5 views for one biological replicate

Both TRULI and DAPA may serve as anti-OA drugs

The results indicating that both TRULI and DAPA can restore ECM balance in cultured chondrocytes imply their potential therapeutic efficacy in attenuating OA progression. As a result, we conducted DMM surgery followed by drugs administration to assess their protective abilities. Based on our observations, the initial two weeks post-DMM surgery are considered a critical phase for the pathogenesis of OA, during which LATS1 undergoes phosphorylation, leading to the release of ZBTB20 for translocation into the nucleus. Therefore, we compared the impacts of TRULI and DAPA when given either immediately after the operation (TRULI_0 week, DAPA_0 week) or 2 weeks post-surgery (TRULI_2 weeks, DAPA_2 weeks) (Figs. 6a and S10A). A behavior test was performed using gait analysis to evaluate their therapeutic effects at 4/6/8 weeks post the surgery. As osteoarthritis develops, an increase in absolute paw angle, decrease in paw area, shorter stance time, and longer swing time were noted in the legs subjected to DMM surgery compared to the contralateral legs, indicative of OA-related pain. Administration of either TRULI or DAPA at both time points, immediately or 2 weeks post the surgery, improved the abnormal behaviors, particularly in the DAPA_0W group (Figs. 6b and S10B–D). Further exploration of the cartilage destruction in the defined mouse cohorts revealed that all administered medications demonstrated varying degrees of improvement in cartilage matrix loss and erosion at the cartilage periphery, among which TRULI_2 weeks and DAPA_0 week showed superior efficacy compared to the rest on restoring the thickness of hyaline cartilage (Fig. 6c, d). These results demonstrate that both TRULI and DAPA can alleviate OA progression, improving the abnormal behavior and moderating cartilage degeneration.

Fig. 6

Both TRULI and DAPA may serve as anti-OA drugs. a Schematic diagram of administration of TRULI and DAPA in mice in injury induced OA model. b Dot plot graph showing the behavior abnormality indicated by ratios of the parameters between the right hind paw and left hind paw in each group of mice. c Representative images of Safranin O/Fast Green staining of articular cartilage from indicated groups of mice. d Statistical analysis of OARSI scores, thickness of hyaline cartilage (HC), calcified cartilage (CC) and ratio of thickness of HC to CC in indicated groups of mice described in (a). a–d n = 7, 7, 8, 8, 9, 9 mice. e Representative images of Safranin O/Fast Green staining of cultured cartilage explants from patient exposed to IL-1β, supplement with increased dosage of TRULI or DAPA. f Statistical analysis of released GAG into medium by DMMB assay in cultures described in (e). e, f n = 3 biological independent experiments. g Dot plot graph of the top 10 enriched KEGG pathways of DEGs from each comparison in human primary chondrocytes. g n = 4 replicates. h Schematic diagram representing molecular mechanisms modulating inflammation and ECM maintenance via LATS1-ZBTB20-PTEN axis

Next, we examined the beneficial effects of TRULI and DAPA in human cartilage that inhibiting matrix degradation ex vivo. The relative intact explants of the femoral head cartilage from patients receiving hip joint replacement were subjected to IL-1β, followed by supplement of TRULI and DAPA. The IL-1β induced surface erosion and loss of proteoglycan, as indicated by S.O. staining, were significantly and dose-dependently improved in cultures co-treated with TRULI or DAPA (Fig. 6e). Likewise, all groups treated with the compounds exhibited a noteworthy dose-dependent inhibitory influence on the release of glycosaminoglycans (GAGs), quantified via the dimethylmethylene blue (DMMB) assay (Fig. 6f). Summarily, these results showed the advantageous impacts of TRULI and DAPA on human cartilage.

Lastly, to gain a more profound comprehension of TRULI and DAPA mechanisms on ECM balance, we performed RNA-seq analysis using human primary cartilage chondrocytes, characterized by the expression of SOX9 and COL II (Fig. S11A), exposing to IL-1β solely or in conjunction with TRULI or DAPA. Integration of the top 10 enriched KEGG pathways from DEGs of each comparison reflects the feature changes of transcriptomes, indicating that both TRULI and DAPA treatment eliminated the association with various inflammation pathways including Measles, Kaposi sarcoma-associated herpesvirus infection, Influenza A and so on (Fig. 6g), further supporting our observations on ECM phenotypes. Nonetheless, various mechanisms indicated by the enriched KEGG pathways also account for the distinct effects of TRULI and DAPA on OA pathogenesis: TRULI predominantly influences the cell cycle, whereas DAPA primarily targets proteoglycans and ECM-receptor interactions (Figs. 6g and S11B–G). Collectively, these findings show that both TRULI and DAPA exhibit varying degrees of beneficial impacts in alleviating OA progress, attributed to the minor differences in the underlying mechanisms.