Cbfβ regulates Wnt/β-catenin, Hippo/Yap, and Tgfβ signaling pathways in articular cartilage homeostasis and protects from ACLT surgery-induced osteoarthritis
- Division in Cellular and Molecular Medicine, Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, Tulane University, United States
- Department of Pathology, School of Medicine, University of Alabama at Birmingham, United States
Figures
Figure 1
Tamoxifen (TMX) induced Cbfbf/f;Col2a1-CreERT mice developed spontaneous OA.
(A) Public human RNA-seq dataset (n=8) (GSE114007) showing CBFB mRNA expression level in Normal and OA patient cartilage. (B) Public human methyl-seq dataset (n=5) (GSE63695) showing methylation at the CBFB 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 Cbfbf/f;Col2a1-CreERT and TMX injected Cbfbf/f;Col2a1-CreERT, and 4-month-old male TMX injected Cbfbf/f;Acan-CreERT mice (n=3). (D) Quantification of (C). (E) X-ray of 3.5-month-old TMX injected female Cbfbf/f mouse hip, shoulder, and knee joint (n=15). (F) X-ray of 3.5-month-old oil injected female Cbfbf/f;Col2a1-CreERT mouse hip, shoulder, and knee joint (n=15). (G) X-ray of 3.5-month-old TMX injected female Cbfbf/f;Col2a1-CreERT mouse hip, shoulder, and knee joint (n=12). (H) X-ray of 3.5-month-old TMX injected male Cbfbf/f;Col2a1-CreERT 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 Cbfbf/f;Col2a1-CreERT mice with oil injection and Cbfbf/f;Col2a1-CreERT 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.
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Figure 1—source data 1
Labeled raw western blot data for Figure 1C (anti-Cbfβ and anti-Gapdh).
- https://cdn.elifesciences.org/articles/95640/elife-95640-fig1-data1-v1.zip
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Figure 1—source data 2
Unlabeled raw western blot data for Figure 1C (anti-Cbfβ and anti-Gapdh).
- https://cdn.elifesciences.org/articles/95640/elife-95640-fig1-data2-v1.zip
Figure 2
Cbfb deletion in Col2a1-CreERT 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 Cbfbf/f;Col2a1-CreERT 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, Cbfb 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: 100 μm.
Figure 3
Cbfbf/f;Col2a1-CreERT mice with ACLT surgery developed early onset OA.
(A) X-ray of 5-month-old male WT (ACLT at 8-week-old) mice knees (n=15). (B) X-ray of 5-month-old male Cbfbf/f;Col2a1-CreERT (ACLT at 8-week-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 Cbfbf/f (ACLT at 8-week-old) mice knees (n=7). (D) SO stain of 4.5-month-old male Cbfbf/f;Col2a1-CreERT (ACLT at 8-week-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: 100 μm (C–D).
Figure 4
RNA-seq 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-week-old male Cbfbf/f and Cbfbf/f;Col2a1-CreERT 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 Cbfbf/f;Col2a1-CreERT mice compared to those of Cbfbf/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 Cbfbf/f;Col2a1-CreERT mice hip articular cartilage. (D) GO functional clustering of the top upregulated BP in 6-week-old male Cbfbf/f;Col2a1-CreERT mice hip articular cartilage. (E) GO functional clustering of the top downregulated KEGG signaling pathways in 6-week-old male Cbfbf/f;Col2a1-CreERT mice hip articular cartilage. (F) GO functional clustering of the top upregulated KEGG signaling pathways in 6-week-old male Cbfbf/f;Col2a1-CreERT mice hip articular cartilage.
Figure 5
Heatmap analysis uncovered that deficiency of Cbfβ in cartilage resulted in decreased chondrocyte genes expression and decreased Tgfβ and Hippo signaling, but increased Wnt signaling.
(A) Heatmap for chondrocyte gene expression in (1) 6-weeks-old male Cbfbf/f mice hip articular cartilage, (2) 6-week-old male Cbfbf/f;Col2a1-CreERT mice hip articular cartilage, (3) 12-week-old male oil injected Cbfbf/f;Acan-CreERT mice knee joint articular cartilage, and (4) 12-week-old male Cbfbf/f;Acan-CreERT mice (TMX injected at 6-week-old) knee joint articular cartilage. (B) Heatmap showing Wnt signaling-related gene expression. (C) Heatmap showing Tgfβ 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 Cbfbf/f;Col2a1-CreERT 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 Cbfbf/f;Acan-CreERT 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 Cbfbf/f;Acan-CreERT 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 with 1 supplement
Locally administrated AAV-mediated Cbfb 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-Cbfb 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-Cbfb 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 7—figure supplement 1
Successful AAV-luc-YFP infection in mice.
Observed by fluorescence microscope. (A) DAPI staining for 8-weeks-old male mouse knee articular cartilage Observed by fluorescence microscope. (B) DAPI staining and expression for YFP in 8-weeks-old male mice knee articular cartilage Observed by fluorescence microscope. Scale bar: 50 μm (A, B). (n=3).
Figure 8 with 1 supplement
Deficiency of Cbfβ protein levels increased β-catenin and articular cartilage degradation markers while also reducing Yap signaling activation.
(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 Cbfbf/f;Col2a1-CreERT 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 Cbfbf/f;Col2a1-CreERT 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-Cbfb, 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.
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Figure 8—source data 1
Labeled raw western blot data for Figure 8A (anti-Yap and anti-Gapdh).
- https://cdn.elifesciences.org/articles/95640/elife-95640-fig8-data1-v1.zip
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Figure 8—source data 2
Unlabeled raw western blot data for Figure 8A (anti-Yap and anti-Gapdh).
- https://cdn.elifesciences.org/articles/95640/elife-95640-fig8-data2-v1.zip
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Figure 8—source data 3
Labeled raw western blot data for Figure 8C (anti-Cbfβ, anti-p-Smad2/3, anti-Smad2/3, anti-Mmp13, and anti-Gapdh).
- https://cdn.elifesciences.org/articles/95640/elife-95640-fig8-data3-v1.zip
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Figure 8—source data 4
Unlabeled raw western blot data for Figure 8C (anti-Cbfβ, anti-p-Smad2/3, anti-Smad2/3, anti-Mmp13, and anti-Gapdh).
- https://cdn.elifesciences.org/articles/95640/elife-95640-fig8-data4-v1.zip
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Figure 8—source data 5
Labeled raw western blot data for Figure 8D (anti-Cbfβ and anti-Gapdh).
- https://cdn.elifesciences.org/articles/95640/elife-95640-fig8-data5-v1.zip
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Figure 8—source data 6
Unlabeled raw western blot data for Figure 8D (anti-Cbfβ and anti-Gapdh).
- https://cdn.elifesciences.org/articles/95640/elife-95640-fig8-data6-v1.zip
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Figure 8—source data 7
Labeled raw western blot data for Figure 8E (anti-Cbfβ, anti-active β-catenin, and anti-Gapdh).
- https://cdn.elifesciences.org/articles/95640/elife-95640-fig8-data7-v1.zip
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Figure 8—source data 8
Unlabeled raw western blot data for Figure 8E (anti-Cbfβ, anti-active β-catenin, and anti-Gapdh).
- https://cdn.elifesciences.org/articles/95640/elife-95640-fig8-data8-v1.zip
Figure 8—figure supplement 1
Alcian Blue staining of primary chondrocytes from Cbfβ deficient newborn mice show reduced matrix deposition.
(A) Alcian Blue staining of newborn WT mouse primary chondrocytes. (B) Alcian Blue staining of newborn (P0) Cbfbf/fCol2a1-Cre mouse primary chondrocytes. (n=5).
Figure 9
Adeno-associated virus (AAV)-mediated Cbfb 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-week-old with (A) AAV-YFP treatment and (B) AAV-Cbfb 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-Cbfb treatment in ACLT mediated OA (ACLT surgery at 8-week-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-Cbfb 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-week-old) with (H) Sham no treatment, (I) DMM surgery AAV-YFP treatment, or (J) AAV-Cbfb 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-Cbfb treated group shows less cartilage loss and osteophytes than control.
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Cbfβ regulates Wnt/β-catenin, Hippo/Yap, and Tgfβ signaling pathways in articular cartilage homeostasis and protects from ACLT surgery-induced osteoarthritis
eLife 13:e95640.
https://doi.org/10.7554/eLife.95640
