Cbfβ regulates Wnt/β-catenin, Hippo/Yap, and TGFβ signaling pathways in articular cartilage homeostasis and protects from ACLT surgery-induced osteoarthritis

Abstract

As the most common degenerative joint disease, osteoarthritis (OA) contributes significantly to pain and disability during aging. Several genes of interest involved in articular cartilage damage in OA have been identified. However, the direct causes of OA are poorly understood. Evaluating the public human RNA-seq dataset showed that Cbfβ, (subunit of a heterodimeric Cbfβ/Runx1,Runx2, or Runx3 complex) expression is decreased in the cartilage of patients with OA. Here, we found that the chondrocyte-specific deletion of Cbfβ in tamoxifen-induced Cbfβ^f/fCol2α1-CreER^T mice caused a spontaneous OA phenotype, worn articular cartilage, increased inflammation, and osteophytes. RNA-sequencing analysis showed that Cbfβ deficiency in articular cartilage resulted in reduced cartilage regeneration, increased canonical Wnt signaling and inflammatory response, and decreased Hippo/YAP signaling and TGF-β signaling. Immunostaining and western blot validated these RNA-seq analysis results. ACLT surgery-induced OA decreased Cbfβ and Yap expression and increased active β-catenin expression in articular cartilage, while local AAV-mediated Cbfβ overexpression promoted Yap expression and diminished active β-catenin expression in OA lesions. Remarkably, AAV-mediated Cbfβ overexpression in knee joints of mice with OA showed the significant protective effect of Cbfβ on articular cartilage in the ACLT OA mouse model. Overall, this study, using loss-of-function and gain-of-function approaches, uncovered that low expression of Cbfβ may be the cause of OA. Moreover, Local admission of Cbfβ may rescue and protect OA through decreasing Wnt/β-catenin signaling, and increasing Hippo/Yap signaling and TGFβ/Smad2/3 signaling in OA articular cartilage, indicating that local Cbfβ overexpression could be an effective strategy for treatment of OA.

Keywords: AAV mediated treatment of osteoarthritis; Cbfβ; Osteoarthritis; TGF-β signaling; Wnt signaling; YAP signaling.

Figure 1.. Tamoxifen (TMX) induced Cbfβ^f/fCol2α1-CreER^T mice developed spontaneous OA.

(A) Public human RNA-seq dataset (n=8) (GSE114007) showing Cbfβ mRNA expression level in Normal and OA patient cartilage. (B) Public human methyl-seq dataset (n=5) (GSE63695) showing methylation at the Cbfβ promoter region (cg13500388 and cg00487831) in Normal and OA hip tissue. Statistical significance was assessed using Student’s t-test. Values were considered statistically significant at p<0.05. (C) Western blot to examine Cbfβ protein levels in the hip articular cartilage of 3.5-month-old male oil injected Cbfβ^f/fCol2α1-Cre and TMX injected Cbfβ^f/fCol2α1-CreER^T, and 4-month-old male TMX injected Cbfβ^f/fAggrecan-CreER^T mice (n=3). (D) Quantification of (C). (E) X-ray of 3.5-month-old TMX injected female Cbfβ^f/f mouse hip, shoulder, and knee joint (n=15). (F) X-ray of 3.5-month-old oil injected female Cbfβ^f/fCol2α1-CreER^T mouse hip, shoulder, and knee joint (n=15). (G) X-ray of 3.5-month-old TMX injected female Cbfβ^f/fCol2α1-CreER^T mouse hip, shoulder, and knee joint (n=12). (H) X-ray of 3.5-month-old TMX injected male Cbfβ^f/fCol2α1-CreER^T mouse hip, shoulder, and knee joint. Green arrow: osteophytes in shoulder; yellow arrow: hip joint space; white arrow: hyperosteogeny in knee. (I) X-ray image of hips and knee joints of 9-month-old female Cbfβ^f/fCol2α1-CreER^T mice with oil injection and Cbfβ^f/fCol2α1-CreER^T mice with TMX injection (n=9). Red arrow 1,2,3: worn articular cartilage; Red arrow 4,5: osteophytes (spurs); Red arrow head: narrow joint space; Yellow arrow head: healthy hip joint space. Figure 1-Source data. Raw western blot images for Figure 1C.

Figure 2.. Cbfβ deletion in Col2α1-CreER^T mice cartilage resulted in more severe OA-like phenotype 3.5-month-old mutant mice with increased osteoclasts and subchondral bone hyperplasia, decreased articular cartilage and osteoblasts

(A-D). H&E staining (A), SO staining (B), TRAP staining (C), and ALP staining (D) of 1-month-old, 2-month-old, and 3.5-month-old male Cbfβ^f/fCol2α1-CreER^T mice hips respectively. (E) Quantification of SO red area of (B). Data was measured by ImageJ. (F) Quantification of TRAP-positive cell numbers of (C). (G) Quantification of ALP-positive cell numbers of (D). TMX=Tamoxifen, Cbfβ deleted group; Oil=Corn Oil, control group. n=7. Data are shown as mean ± SD. NS, no significance; *p<0.05; **p<0.01; ***p<0.001 vs. controls by Student’s t-test. Scale bar: 100μm.

Figure 3.. Cbfβ^f/fCol2α1-CreER^T mice with ACLT surgery developed early onset OA.

(A) X-ray of 5-month-old male WT (ACLT at 8-weeks-old) mice knees (n=15). (B) X-ray of 5-month-old male Cbfβ^f/fCol2α1-CreER^T (ACLT at 8-weeks-old) mice knees. Red arrows indicate subchondral bone; Red arrow heads indicate joint space; Light blue arrows indicate osteophytes; White arrows indicate worn articular cartilage; Purple arrow indicates joint space loss; (n=15). (C) SO stain of 4.5-month-old male Cbfβ^f/f (ACLT at 8-weeks-old) mice knees (n=7). (D) SO stain of 4.5-month-old male Cbfβ^f/fCol2α1-CreER^T (ACLT at 8-weeks-old) mice knees (n=6). (E) Knee joint Osteoarthritis Research Society International (OARSI) score of (C) and (D). Data are shown as mean ± SD. Scale bar: 100μm (C-D).

Figure 4.. RNAseq analysis indicated that deficiency of Cbfβ in cartilage reduces cell fate commitment, cartilage regeneration and repair, and increases canonical Wnt signaling and inflammatory response.

(A) Volcano plot showing differentially regulated gene expression in 6-weeks-old male Cbfβ^f/f and Cbfβ^f/fCol2α1-CreER^T mice hip articular cartilage. (B) Pie chart showing percentage of upregulated and downregulated differentially regulated genes in hip articular cartilage of 6-weeks-old male Cbfβ^f/fCol2α1-CreER^T mice compared to those of Cbfβ^f/f mice. The percentages of genes upregulated and downregulated are shown in red and green, respectively. (C) GO functional clustering of the top downregulated biological process (BP) in 6-week-old male Cbfβ^f/fCol2α1-CreER^T mice hip articular cartilage. (D) GO functional clustering of the top upregulated BP in 6-week-old male Cbfβ^f/fCol2α1-CreER^T mice hip articular cartilage. (E) GO functional clustering of the top downregulated KEGG signaling pathways in 6-week-old male Cbfβ^f/fCol2α1-CreER^T mice hip articular cartilage. (F) GO functional clustering of the top upregulated KEGG signaling pathways in 6-week-old male Cbfβ^f/fCol2α1-CreER^T mice hip articular cartilage.

Figure 5.. Heatmap analysis uncovered that deficiency of Cbfβ in cartilage resulted in decreased chondrocyte genes expression and decreased TGF-β and Hippo signaling, but increased Wnt signaling.

(A) Heatmap for chondrocyte gene expression in (1) 6-weeks-old male Cbfβ^f/f mice hip articular cartilage, (2) 6-weeks-old male Cbfβ^f/fCol2α1-CreER^T mice hip articular cartilage, (3) 12-weeks-old male oil injected Cbfβ^f/fAggrecan-CreER^T mice knee joint articular cartilage, and (4) 12-weeks-old male Cbfβ^f/fAggrecan-CreER^T mice (TMX injected at 6-weeks-old) knee joint articular cartilage. (B) Heatmap showing Wnt signaling-related gene expression. (C) Heatmap showing TGF-β signaling-related gene expression. (D) Heatmap showing Hippo signaling-related gene expression.

Figure 6.. Postnatal Cbfβ deficiency in cartilage resulted in increased Wnt signaling, inflammatory genes expression, decreased cartilage formation genes expression in the knee articulate cartilage.

(A-E) IHC staining of (A) anti-Cbfβ, (B) anti-Col2α1, (C) anti-Adamts5, and (D) anti-Mmp13 of hip joint from 2-month-old male Cbfβ^f/fCol2α1-CreER^T mice. (E) Negative control of (A-D). (F) Quantification for (A). (G) Quantification for (B-D). (H-I) IF staining of (H) anti-Cbfβ and (I) Active-β-catenin of knee joint from 3-month-old male Cbfβ^f/fAggrecan-CreER^T mice. (J-K) Quantification of (H) and (I). (L-M) IF staining of (L) anti-Sox9, and (M) anti-Dkk1 of knee joint from 4.5-month-old male Cbfβ^f/fAggrecan-CreER^T mice with oil injection or TMX injection. (N-O) Quantification of (L) and (M). Data are shown as mean ± SD. n= 3. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7.. Locally administrated AAV-mediated Cbfβ overexpression inhibited β-Catenin expression and enhanced Yap expression in knee joints articular cartilage of ACLT-induced OA mice.

(A-C) IF staining of anti-active-β-catenin in the knee joints articular cartilage of 6.5-month-old male (A) Normal WT, (B) AAV-YFP with ACLT surgery, and (C) AAV-Cbfβ mice with ACLT surgery (n=3). (D-F) IF staining of anti-YAP in the knee joints articular cartilage of 6.5-month-old (D), (E) AAV-YFP ACLT surgery, and (F) AAV-Cbfβ mice with ACLT surgery (n=3). (G) Negative control of (A-F). (H) Quantification of (A-C). (I) Quantification of (D-F). Data are shown as mean ± SD.

Figure 8.. Deficiency of Cbfβ protein levels increased β-catenin and articular cartilage degradation markers while also reducing Yap signaling activation and Col2α1.

(A) Western blot showing protein expression level of Yap in ATDC5 cells (n=3). (B) Quantification of Yap protein levels in (A). (C) Western blot of 10-week-old male hip cartilage from Cbfβ^f/fCol2α1-CreER^T mice injected with either oil or TMX showing the expression of Cbfβ, p-Smad2/3, Smad2/3, and Mmp13 (n=5). (D) Western blot of knee joint cartilage from 16-week-old male WT and Cbfβ^f/fCol2α1-CreER^T mice with ACLT surgery and injected with either oil or TMX showing the expression of Cbfβ (n=6). (E) Western blot of WT mice knee joint cartilage from 16-week-old male mice with ACLT surgery, treated with AAV-luc-YFP or AAV-Cbfβ, and injected with either oil or TMX showing the expression of Cbfβ and active β-catenin (n=6). (F) Quantification of (C). (G) Quantification of (D). (H) Quantification of (E). Data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. NS Not Significant. Figure 8-Source data. Raw western blot images for Figure 8A, C–E.

Figure 9.. Adeno-associated virus (AAV)-mediated Cbfβ overexpression protects against ACLT mechanical OA.

(A-B) X-ray images of the knee joints of 22-week-old male WT mice with ACLT surgery at 8-weeks-old with (A) AAV-YFP treatment and (B) AAV-Cbfβ treatment (n=15). Yellow arrows indicates normal joint space; White arrows indicate worn articular cartilage; blue arrows indicate osteophytes; red arrows indicate joint space loss. (C-D) SO staining of knees from 16-week-old male WT mice with (C) AAV-YFP (control) or (D) AAV-Cbfβ treatment in ACLT mediated OA (ACLT surgery at 8-weeks-old) (n=5). (E) Knee joint of OARSI score of (C) and (D). (F-G) X-ray images of mouse knee joints of 16-week-old male mice after sham/DMM surgery with (F) no treatment or (G) AAV-Cbfβ treatment (n=15). White arrows: osteophytes and worn articular cartilage. (H-J) SO staining of knee joints of 16-week-old mice after sham/DMM surgery (DMM surgery at 8-weeks-old) with (H) Sham no treatment, (I) DMM surgery AAV-YFP treatment, or (J) AAV-Cbfβ treatment (n=5). (K) Knee joint OARSI score of (H-J). The results are presented as the mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001. DMM surgery AAV-YFP treatment group shows severe cartilage damage, osteophytes, and delocalized knee joint, while the AAV-Cbfβ treated group shows less cartilage loss and osteophytes than control.