Dickkopf‑3 and β‑catenin play opposite roles in the Wnt/β‑catenin pathway during the abnormal subchondral bone formation of human knee osteoarthritis

Abstract

Osteoarthritis (OA) is condition which poses a main concern to the aging population and its severity is expected to increase with the increasing life expectancy. In the future, several possible targets for OA treatment need to be defined. Dickkopf‑related protein 3 (DKK3) is an atypical member of the Wnt‑antagonistic dickkopf‑related protein (DKK) family. The availability of research into the role of DKK3 in the abnormal remodeling of subchondral bone in human knee joints is currently limited. Thus, the aim of the present study was the evaluation of DKK3 expression in the abnormal bone remodeling of subchondral bone in human knee OA in order to clarify the role of DKK3 in subchondral bone remodeling and to acknowledge its potential relevance to β‑catenin. In total, 38 specimens were collected from osteotomies of the medial tibial plateau of the human knee. The patient samples were then divided into the normal, mild, moderate and severe symptom groups, according to the Osteoarthritis Research Society International (OARSI) score. Following hematoxylin and eosin (H&E) and Safranin O‑fast green staining for alkaline phosphatase (AZO method), changes in the distribution and number of osteocytes in the subchondral bone and the degree of sclerosis of the subchondral bone were observed. Immunohistochemical staining, immunofluorescence, western blot analysis and reverse‑transcription quantitative PCR (RT‑qPCR) were used for the detection of DKK3 and β‑catenin expression level changes in osteoblasts in the subchondral bone of the medial tibial plateau. H&E and alkaline phosphatase staining revealed that the total number of osteocytes in the subchondral bone increased with the severity of the disease. The samples were also evaluated using Safranin O‑Fast Green staining and were attributed a score according to the OARSI scoring system: The scoring number and cartilage damage increased along with OA severity. Immunohistochemistry and immunofluorescence assays demonstrated that β‑catenin expression in osteocytes increased from mild to moderate, whereas DKK3 expression decreased with the development of arthritis from normal, mild to moderate. According to the results of western blot analysis, β‑catenin expression was higher in moderate OA and then decreased in severe OA. On the other hand, the DKK3 levels decreased along with the progression from normal, mild to moderate OA. The results of RT‑qPCR demonstrated that β‑catenin and DKK3 gene expression differed with the degree of OA. On the whole, the present study demonstrates that DKK3 and β‑catenin may play opposite roles in OA subchondral bone remodeling.

Keywords: Dickkopf‑3; Wnt/β‑catenin signaling; osteoarthritis; osteoblasts; sclerosis; subchondral bone; β‑catenin.

Figure 1

(A) Preoperative radiographs of the patient. (B) Intraoperative gross pathological images. (C) Intraoperative intercepted bone plates of the tibial plateau including cartilage. (D) Subchondral bone H&E staining. (F) Subchondral bone ALP staining of the subchondral bone tissue. The arrows indicate cells positive for ALP staining. (E and G) Results of cell number statistical analysis. In H&E staining and ALP staining, there was a statistically significant difference between the groups (^*P<0.05, ^****P<0.0001, and ^**P<0.01). (E and G) The data were analyzed using one-way ANOVA; mean of the 95% confidence interval: Normal (n=3), mild (n=5), moderate (n=10) and severe (n=20). ALP, alkaline phosphatase; H&E, hematoxylin and eosin.

Figure 2

(A) Safranin O staining: Each image demonstrates the OARSI histological score of the medial tibial plateau cartilage. (B) Statistical analysis results. The difference between the mild and moderate groups was statistically significant (^****P<0.0001). (B) Data were analyzed using the Kruskal-Wallis test. OARSI score equals cartilage degenerative tissue grading times staging. Normal (n=3), mild (n=5), moderate (n=10) and severe (n=20). OARSI, Osteoarthritis Research Society International.

Figure 3

(A and B) Immunohistochemical staining. Arrows indicate the number of positively stained cells. (C) In the β-catenin group, the difference in cell number was statistically significant between the normal and mild, mild and moderate, and moderate and severe symptom groups (^*P<0.01, ^**P<0.01 and ^**P<0.01 respectively). (D) Statistically significant differences between the mild and moderate symptom groups according to the average optical density values (^***P<0.001 and ^****P<0.0001). (E) In the DKK3 group, cell numbers were statistically significant between the normal and mild, mild and moderate, and moderate and severe symptom groups (^*P<0.05, ^**P<0.01 and ^*P<0.05, respectively). (F) Statistically significant differences between the mild and moderate groups according to the average optical density values (^**P<0.01). (C-F) Data were analyzed using one-way ANOVA; mean of the 95% confidence interval: Normal (n=3), mild (n=5), moderate (n=10) and severe (n=20). DKK3, Dickkopf-related protein 3; ns, not significant.

Figure 4

(A) Immunofluorescence staining (β-catenin). Arrows indicate the number of positively stained cells. (B and C) Statistical analysis results. Statistical significant differences in b-catenin expression were revealed between normal and mild, mild and moderate, and moderate and severe OA in the β-catenin group (^*P<0.05, ^***P<0.001 and ^***P<0.001, respectively). (D) Statistical analysis results of cell numbers. The number of cells was statistically significant between the normal and mild, mild and moderate, and moderate and severe symptom groups (^*P<0.05, ^***P<0.001 and ^***P<0.001, respectively). (B-D) Data were analyzed using one-way ANOVA. Mean 95% confidence interval for average score: Normal (n=3), mild (n=5), moderate (n=10) and severe (n=20). OA, osteoarthritis.

Figure 5

(A) Immunofluorescence staining for DKK3. Arrows indicate the number of positively stained cells. (B and C) Statistical analysis results. The difference in DKK3 expression between the normal and mild, mild and moderate, and moderate and severe groups was statistically significant (^*P<0.05, ^***P<0.001 and ^*P<0.05, respectively). (D) Cell count statistical analysis results. The differences in cell numbers was statistically significant between the normal and mild, mild and moderate, and moderate and severe symptom groups (^*P<0.05, ^**P<0.01, and ^**P<0.01, respectively). (B-D) Data were analyzed using one-way ANOVA; mean 95% confidence interval for average score: Normal (n=3), mild (n=5), moderate (n=10) and severe (n=20). DKK3, Dickkopf-related protein 3.

Figure 6

(A-C) The results of western blot analysis revealed that in the β-catenin group, there was a statistically significant difference between the mild and moderate symptom groups and between the moderate and severe symptom groups (^***P<0.001 and ^**P<0.01, respectively). In the DKK3 group, there were statistically significant differences between the mild and moderate symptom groups, as well as between the moderate and severe symptom groups (^**P<0.01 and ^*P<0.05, respectively). (D and E) The results of RT-qPCR revealed significant differences between the mild and moderate groups as and between the moderate and severe symptom groups in the β-catenin group (^****P<0.0001, and ^**P<0.01, respectively). In the DKK3 group, there was a statistically significant difference between the mild and moderate symptom groups and between the moderate and severe symptom groups (^****P<0.0001 and ^*P<0.01, respectively). (B-E) Data were analyzed using one-way ANOVA. 95% confidence interval for average score: Normal (n=3), mild (n=5), moderate (n=10) and severe (n=20). DKK3, Dickkopf-related protein 3; RT-qPCR, reverse transcription quantitative PCR; ns, not significant.

Figure 7

Overview of the Wnt signaling pathway. (A) Through the silencing the Wnt pathway, DKK3 competes with β-catenin in the cytoplasm and promotes its degradation, inhibiting the Wnt pathway and blocking nuclear transcription. (B) Activation of the Wnt pathway β-catenin aggregates in the cytoplasm. Stable β-catenin is then transferred to the nucleus and stimulates the transcription of Wnt target genes. DKK3, Dickkopf-related protein 3.