Article Figures & Data
Figures
- Figure 1. Changes in Wnt/β-catenin pathway and CXXC5 expression during growth plate senescence.
(A) Gene set enrichment analysis (GSEA) of microarray transcriptome data from the proliferative zone of growth plates in 3- and 12-wk-old rats (GEO: GSE16981) for Wnt/β-catenin signaling–activated gene signatures (upper, MSigDB: M11722 and lower, MSigDB: M2680) (n = 5). (B) The relative expression changes of Cxxc5 in the proliferative zone 3-, 6-, 9-, and 12-wk-old rat growth plates (GEO: GSE16981) (mean ± SEM, n = 5, ANOVA, P = 1.88 × 10−3; Tukey’s post-hoc test, *P < 0.05 and **P < 0.005). (C) qRT-PCR analyses of relative mRNA expression of Cxxc5 in the growth plate of proximal tibiae of 3-, 6-, 9-, and 12-wk-old mice (mean ± SEM, n = 5, ANOVA, P = 1.57 × 10−4; Tukey’s post-hoc test, *P < 0.05 and ***P < 0.0005 versus 3-wk-old). (D) Immunoblot analyses with the indicated antibodies were performed in the growth plate of proximal tibiae of 3-, 6-, 9-, and 12-wk-old mice. (E) IHC analyses with the indicated antibodies in the growth plate of proximal tibiae of 3-, 6-, 9-, and 12-wk-old mice (left) and quantitative analyses (mean ± SEM, n = 6, ANOVA, P = 8.09 × 10−7 for CXXC5 expression, P = 1.19 × 10−5 for β-catenin expression; Tukey’s post-hoc test, ##P < 0.005, *** or ###P < 0.0005 versus 3-wk-old) (right). Black and white scale bars, 50 μm. Yellow scale bar, 20 μm. NES, normalized enrichment score; ES, enrichment score; FDR, false discovery rate; RZ, resting zone; PZ, proliferative zone; HZ, hypertrophic zone.
- Figure S1. Analysis of mRNA expression levels of Wnt inhibitors.
Analyses of the well-established negative regulators of the Wnt/β-catenin pathway in the proliferative zone of growth plates from 3- to 12-wk-old rats (GEO: GSE16981). P values of the genes up-regulated in 12-wk-old rats are marked as P < 0.05.
- Figure S2. Comparison of mRNA expression levels between CXXC5 and CXXC4 in various species.
(A, B) Analyses of the relative mRNA expression of CXXC5 and CXXC4 in growth plates during the pubertal period from microarray data (GEO: GSE9160) (mean ± SEM, n = 2; t test, *P < 0.05 and **P < 0.005) (A) (GEO: GSE16981) (mean ± SEM, n = 5, t test, ***P < 0.0005) (B).
- Figure S3. The effects of CXXC5 on differentiation of chondrocytes as an inhibitor of Wnt/β-catenin pathway.
qRT-PCR analyses of mRNA levels of Wnt/β-catenin pathway-target genes major signaling molecules for chondrocyte maturation, and chondrogenic markers in ATDC5 cells cultured in alginate beads with 50 ng/ml recombinant WNT3A for 48 h after transfection with pEGFP-N1 or GFP-CXXC5 (mean ± SEM, n = 3; ANOVA and Tukey’s post-hoc test, *P < 0.05, **P < 0.005, and ***P < 0.0005).
- Figure 2. The effects of estrogen on CXXC5 expression and the mediation of growth plate senescence.
(A) Immunoblotting (upper) and quantitative analyses (mean ± SEM, n = 3) (lower) in C28/I2 cells treated with 100 nM E2 (17β-estradiol) for 0, 1, 3, 6, 24, 48, or 72 h. (B) Immunocytochemical staining (left) and quantitative analyses of the fluorescent intensity (mean ± SEM, n = 3; t test, **P < 0.005 and ***P < 0.0005) (right) in C28/I2 cells treated with 100 nM E2 for 40 h. Scale bars, 100 μm. (C–E) Tibial organ cultures (E15.5) incubated with 100 nM E2 for 6 d. Representative images at 6 d (C, left) and quantitative analyses of the growth changes (mean ± SEM, n = 5; t test, ***P < 0.0005) (C, right). Scale bar, 1 mm. H&E staining (D, left) and quantification of each zone height (mean ± SEM, n = 5; t test, *P < 0.05 and **P < 0.005) (D, right) in the growth plate. Scale bar, 200 μm. IHC analyses of β-catenin and CXXC5 (E). Scale bars, 50 μm. (F) 3-wk-old Cxxc5+/+ and Cxxc5−/− mice were treated with E2 cypionate (70 μg/kg) by i.m. injection once a week for 3 wk (n = 3–4). Representative images of H&E staining and IHC analyses for BrdU, β-catenin, and CXXC5 in the growth plates of proximal tibiae are shown. The area within the dashed lines indicates the growth plate zone. Black and white scale bars, 50 μm. Yellow scale bar, 20 μm. RZ, resting zone; PZ, proliferative zone; HZ, hypertrophic zone.
- Figure 3. Functional characterization of CXXC5 in growth plate senescence as an inhibitor of the Wnt/β-catenin pathway via interaction with DVL.
(A) Representative radiographs of tibiae of 12-wk-old Cxxc5+/+ and Cxxc5−/− mice. (B) Tibial length of 3-, 6-, 9-, and 12-wk-old Cxxc5+/+ and Cxxc5−/− mice were measured (mean ± SEM, n = 4–10 mice per group; ANOVA, P = 2.37 × 10−2; Bonferroni’s post-hoc test, **P < 0.005). (C–E) H&E staining (C) and quantitative analyses of each zone height (D) in the growth plate of proximal tibiae of 3-, 6-, 9-, and 12-wk-old Cxxc5+/+ and Cxxc5−/− mice (mean ± SEM, n = 3–5 mice per group; ANOVA, P = 2.9 × 10−7 for upper panel, P = 1.92 × 10−9 for lower panel; Bonferroni’s post-hoc test, *P < 0.05, **P < 0.005, and ***P < 0.0005). Quantitative analyses of the cell number per column in the growth plates of 9- and 12-wk-old Cxxc5+/+ and Cxxc5−/− mice (mean ± SEM; t test, n = 5, **P < 0.005) (E). (F) IHC analyses with the indicated antibodies or in situ hybridization for Runx2 in the proximal tibial growth plates of 11-wk-old Cxxc5+/+ and Cxxc5−/− mice. (G) qRT-PCR analyses of mRNA levels of Wnt-target genes and chondrogenic markers in the growth plate of proximal tibiae of 9-wk-old Cxxc5+/+ and Cxxc5−/− mice (mean ± SEM, n = 3; t test, *P < 0.05, **P < 0.005, and ***P < 0.0005). (H–J) The PTD-DBMP (1 mg/kg) were administered to 7-wk-old mice by daily i.p. injection for 2 wk (n = 10). In vivo fluorescent imaging shows the presence of the PTD-DBMP in the treated mice (H). White arrowheads indicate the growth plate regions of tibia. H&E staining, IHC analyses for β-catenin and RUNX2 in the growth plates of proximal tibiae were performed (I). Quantitative analyses of the cell number in the RZ, PZ, and HZ of growth plates (mean ± SEM, n = 3; t test, *P < 0.05 and **P < 0.005) (J). Scale bars, 50 μm.
- Figure S4. Screening of small molecules that block CXXC5–DVL interaction.
An in vitro binding assay was performed for 2,280 compounds (30 μM) to identify inhibitors of the CXXC5–DVL interaction. The binding values were calculated by percent ratio of fluorescent intensity normalized to the DMSO-treated control.
- Figure S5. Binding mode of indirubin analogs, BIO and I3O, docked on DVL PDZ.
(A, B) Binding mode of BIO or I3O docked on DVL PDZ (PDB: 2KAW) is shown as a stick model. Structural simulation of the BIO–DVL PDZ complex showed that residues F261, I262, I264, I266, L321, and V325 are involved in binding with BIO: non-bonded interactions (F261, I262, I266, L321, and V325) and hydrogen bonds (I264) (A). Structural simulation of the I3O–DVL PDZ complex revealed that residues H260, I262, and V325 are involved in binding with I3O: non-bonded interactions (I262 and V325) and hydrogen bonds (H260) (B). BE, binding energy.
- Figure S6. A focused synthesis of indirubin derivatives and binding mode of an improved derivative, KY19382, docked on DVL PDZ.
(A) Focused design of indirubin derivatives for the activation of Wnt/β-catenin signaling was directed by modifications of the functional group at the R1 and R2 sites of the indirubin backbone. The synthesized derivatives were analyzed by three assays: (1) in vitro CXXC5–DVL binding assay, (2) in vitro GSK3β kinase assay, and (3) TOPFlash Wnt reporter assay. (B) Binding mode of KY19382 docked on DVL PDZ (PDB: 2KAW) is shown as a stick model (left). In structure-based pharmacophore features of KY19382–DVL PDZ (cyan, hydrophobe; green, hydrogen bond acceptor; and purple, hydrogen bond donor), the electrostatic surface of DVL PDZ is shown as blue, positively charged; red, negatively charged; white, neural resides (right). The model showed that DVL PDZ residues G263, I264, I266, L321, R322, and V325 are involved in binding with KY19382: non-bonded interactions (I266, L321, R322, and V325) and hydrogen bonds (G263, I264, and V325). BE, binding energy.
- Figure 4. Identification of functional properties of KY19382 in activating the Wnt/β-catenin pathway.
(A) Chemical structure of KY19382. (B) In vitro binding assay to analyze the effect of KY19382 on CXXC5–DVL interaction (mean ± SEM, n = 3). The IC50 value was determined from the dose–response curve. (C) In vitro kinase assay to analyze the effect of KY19382 on kinase activity of GSK3β (mean ± SEM, n = 3). The IC50 value was determined from the dose–response curve. (D) Analyses of TOPFlash activity in HEK293 reporter cells grown with the indicated concentrations of KY19382 for 18 h (mean ± SEM, n = 4; t test, **P < 0.005 and ***P < 0.0005 versus DMSO-treated control). (E) Immunoblot analyses with the indicated antibodies in ATDC5 cells treated with I3O or KY19382 for 24 h. (F) Immunoblot analyses of whole cell lysates immunoprecipitated with anti-Myc in ATDC5 cells treated with 0.1 μM KY19382 for 4 h after transfection with pCMV-FLAG-DVL1 and pcDNA3.1-CXXC5-Myc. (G) Immunocytochemical staining (left) and quantitative analyses (mean ± SEM, n = 3; t test, **P < 0.005) (right) for β-catenin in ATDC5 cells treated with 0.1 μM KY19382 for 48 h. Scale bar, 100 μm.
- Figure 5. The effects of KY19382 on growth plate senescence and longitudinal bone growth.
(A–I) KY19382 (0.1 mg/kg) was administered to 7-wk-old mice (A–E) or 3-wk-old mice (F–I) by daily intraperitoneal injection for 2 wk (n = 7). H&E staining, IHC analyses with the indicated antibodies, and TRAP staining in the growth plates of proximal tibiae treated with KY19382 (A, F). Quantitative analyses of the cell number per column (mean ± SEM, n = 7; t test, ***P < 0.0005) (B) or the height (mean ± SEM, n = 7; t test, ***P < 0.0005) (G) of resting zone and proliferative zone (RZ&PZ) and hypertrophic zone (HZ) in the growth plates of proximal tibiae. Quantitative analyses of BrdU-positive cells in the growth plates (mean ± SEM, n = 5; t test, ***P < 0.0005) (C, H). Quantitative analyses of the number of TRAP-positive foci along 250 μm of the cartilage/bone interface (mean ± SEM, n = 3; t test, *P < 0.05) (D, I). Immunoblot analyses with the indicated antibodies in the growth plate of proximal tibiae of mice treated with KY19382 (E). Scale bars, 50 μm. (J) 3-wk-old mice were intraperitoneally injected with KY19382 (0.1 mg/kg) daily for 10 wk. Representative radiographs are shown (left), and tibial length was measured (right) (mean ± SEM, n = 7–15; t test, ***P < 0.0005). The area within the dashed lines indicates the growth plate zone. n.s., no significance; TRAP, tartrate-resistant acid phosphatase.
- Figure S7. The effects of KY19382 on chondrocyte proliferation and differentiation.
(A) ATDC5 cells treated with 0.01 or 0.1 μM concentrations of KY19382 were incubated for 48 h followed by treatment with 50 μM BrdU for 12 h before harvesting. BrdU incorporation was visualized by immunofluorescent staining using a specific BrdU antibody (left). BrdU-positive cells were quantified (mean ± SEM, n = 3; ANOVA, P = 7.8 × 10−5; Tukey’s post-hoc test, *P < 0.05 and ***P < 0.0005) (right). Scale bar, 100 μm. (B) qRT-PCR analyses of mRNA levels of chondrogenic differentiation markers in ATDC5 cells incubated with 0.1 μM KY19382 for 3 d in three-dimensional alginate beads after transfection with control siRNA or Ctnnb1 siRNA (mean ± SEM, n = 3; t test, *P < 0.05, **P < 0.005, and ***P < 0.0005). (C) qRT-PCR analyses of mRNA levels of chondrogenic differentiation markers in C28/I2 cells incubated with 1 μM KY19382 for 3 d in three-dimensional alginate beads (mean ± SEM, n = 3; t test, *P < 0.05 and ***P < 0.0005). n.s., no significance.
- Figure S8. qRT-PCR analyses for target-specificity of KY19382.
qRT-PCR analyses of mRNA levels of pathway-specific target genes in ATDC5 cells treated with 0.01 or 0.1 μM concentrations of KY19382 for 4 h (mean ± SEM, n = 3; ANOVA and Tukey’s post-hoc test, *P < 0.05 and **P < 0.005 versus DMSO-treated control). n.s., no significance versus DMSO-treated control.
- Figure S9. The effects of KY19382 on articular cartilage and liver tissues, or weight.
Data are shown from mice used in Fig 5J. (A, B) H&E staining of articular cartilage (A) and liver (B) tissues. (C) During treatment, the weights of mice were measured every 5–7 d (mean ± SEM, n = 7∼15). Scale bars, 100 μm.
- Figure 6. Schematic representation of the role of CXXC5 and molecular mechanism of KY19382 in longitudinal bone growth.
(A) A proposed model for the role of CXXC5 in the growth plate. With pubertal progression, estrogen, which increases during sexual maturation, induces CXXC5 expression and subsequently inhibits the Wnt/β-catenin pathway, resulting in growth plate senescence. (B) A working model of KY19382 for the stimulation of longitudinal bone growth. In activating Wnt/β-catenin signaling, KY19382 functions as a dual-targeting compound by 1) inactivating GSK3β and 2) inhibiting CXXC5–DVL interaction, which results in the delaying of growth plate senescence and the promotion of longitudinal bone growth. PPI, protein–protein interaction.
Tables
List of primers used.
Gene Strand Primer sequences Human ACTB F 5′-AGAGCTACGAGCTGCCTGAC-3′ R 5′-AGCACTGTGTTGGCGTACA-3′ COL2A1 F 5′-TGGAAAGCCTGGTGATGATGGTG-3′ R 5′-TGACCTTTGACACCAGGAAGGC-3′ MMP13 F 5′-GAAGACCTCCAGTTTGCAGAGC-3′ R 5′-TTCAGGATTCCCGCGAGATTTG-3′ RUNX2 F 5′-CACCTTGACCATAACCGTCTTCAC-3′ R 5′-CATCAAGCTTCTGTCTGTGCCTTC-3′ VEGFA F 5′-AGGGCAGAATCATCACGAAGTGG-3′ R 5′-GTCTCGATTGGATGGCAGTAGC-3′ Mouse Actb F 5′-GGATGCAGAAGGAGATTACT-3′ R 5′-CCGATCCCACACAGAGTACTT-3′ Alp F 5′-GGGACTGGTACTCGGATAAC-3′ R 5′-CTGATATGCGATGTCCTTGC-3′ Col2a1 F 5′-GCCTGTCTGCTTCTTGTAA-3′ R 5′-TGCGGTTGGAAAGTGTTT-3′ Col10a1 F 5′-TCCACTCGTCCTTCTCAG-3′ R 5′-TTTAGCCTACCTCCAAATGC-3′ Ctnnb1 F 5′-ACAAGCCACAAGATTACAAGAA-3′ R 5′-GCACCAATATCAAGTCCAAGA-3′ Fgf18 F 5′-TGGGGAAGCCTGATGGTACT-3′ R 5′-CCCTTGGGGTAACGCTTCAT-3′ Gapdh F 5′-ACCCAGAAGACTGTGGATGG-3′ R 5′-GGATGCAGGGATGATGTTCT-3′ Ihh F 5′-CTCTTGCCTACAAGCAGTTCA-3′ R 5′-CCGTGTTCTCCTCGTCCTT-3′ Mmp9 F 5′-TGAAGTCTCAGAAGGTGGAT-3′ R 5′-ATGGCAGAAATAGGCTTTGT-3′ Mmp13 F 5′-TAAGACACAGCAAGCCAGA-3′ R 5′-CACATCAGTAAGCACCAAGT-3′ Pthlh F 5′-GAGATCCACACAGCCGAAAT-3′ R 5′-CGTCTCCACCTTGTTGGTTT-3′ Runx2 F 5′-AAGGACAGAGTCAGATTACAGA-3′ R 5′-GTGGTGGAGTGGATGGAT-3′ Sox9 F 5′-AACTGGAAACCTGTCTCTCT-3′ R 5′-ACAACACACGCACACATC-3′ Vegfa F 5′-TTATTTATTGGTGCTACTGTTTATCC-3′ R 5′-TCTGTATTTCTTTGTTGCTGTTT-3′
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