Effects of WNT1 c.110 T>C and c.505G>T mutations on osteoblast differentiation via the WNT1/β-catenin signaling pathway
Abstract Background WNT1 c.110 T>C and c.505G>T missense mutations have been identified in patients with osteogenesis imperfecta (OI). Whether these mutations affect osteoblast differentiation remains to be determined. This study aimed to investigate the effects of WNT1 c.110 T>C and c.505G...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2021-06-01
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| Series: | Journal of Orthopaedic Surgery and Research |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s13018-021-02495-2 |
| _version_ | 1818474220502384640 |
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| author | Bashan Zhang Rong Li Wenfeng Wang Xueming Zhou Beijing Luo Zinian Zhu Xibo Zhang Aijiao Ding |
| author_facet | Bashan Zhang Rong Li Wenfeng Wang Xueming Zhou Beijing Luo Zinian Zhu Xibo Zhang Aijiao Ding |
| author_sort | Bashan Zhang |
| collection | DOAJ |
| description | Abstract Background WNT1 c.110 T>C and c.505G>T missense mutations have been identified in patients with osteogenesis imperfecta (OI). Whether these mutations affect osteoblast differentiation remains to be determined. This study aimed to investigate the effects of WNT1 c.110 T>C and c.505G>T mutations on osteoblast function, gene expression, and pathways involved in OI. Methods Empty vector (negative control), wild-type WNT1, WNT1 c.110 T>C, WNT1 c.505G>T, and WNT1 c.884C>A (positive control) mutant plasmids were constructed and transfected into preosteoblast (MC3T3-E1) cells to investigate their effect on osteoblast differentiation. The expressions of osteoblast markers, including BMP2, RANKL, osteocalcin, and alkaline phosphatase (ALP), were determined using quantitative real-time polymerase chain reaction (RT-qPCR), western blotting (WB), enzyme-linked immunosorbent assay, and ALP staining assay, respectively. The mRNA and protein expression levels of WNT1 or the expression levels of the relevant proteins involved in the WNT1/β-catenin signaling pathway were also determined using RT-qPCR, WB, and immunofluorescence (IF) assays after the different plasmids were transfected into MC3T3-E1 cells. Results Compared with those in the wild-type group, in the mutation groups, the mRNA and protein expression levels of BMP2 were suppressed, the expressions of osteocalcin and ALP were inhibited, and the mRNA and protein expression levels of RANKL were enhanced in MC3T3-E1 cells. WB and IF assays revealed that the protein expression levels of WNT1 in MC3T3-E1 cells were downregulated in the mutation groups compared with those in the wild-type WNT1 group. Furthermore, the expression levels of nonphosphorylated β-catenin (non-p-β-catenin) and phosphorylated GSK-3β (p-GSK-3β) were downregulated in the mutation groups compared with those in the wild-type group. However, no significant changes in the expression level of non-p-β-catenin or p-GSK-3β were observed in the mutation groups. Conclusions WNT1 c.110 T>C and c.505G>T mutations may alter the proliferation and osteogenic phenotype of MC3T3-E1 linked to the progression of OI via the inhibition of the WNT1/β-catenin signaling pathway. This is the first study to confirm the effect of WNT1 c.110 T>C and c.505G>T missense mutations on osteoblast differentiation and propose a new molecular mechanism for OI development. |
| first_indexed | 2024-04-14T04:35:05Z |
| format | Article |
| id | doaj.art-7bd3e1acdf9c4ea8a40fceb51a7ffe67 |
| institution | Directory Open Access Journal |
| issn | 1749-799X |
| language | English |
| last_indexed | 2024-04-14T04:35:05Z |
| publishDate | 2021-06-01 |
| publisher | BMC |
| record_format | Article |
| series | Journal of Orthopaedic Surgery and Research |
| spelling | doaj.art-7bd3e1acdf9c4ea8a40fceb51a7ffe672022-12-22T02:11:55ZengBMCJournal of Orthopaedic Surgery and Research1749-799X2021-06-011611910.1186/s13018-021-02495-2Effects of WNT1 c.110 T>C and c.505G>T mutations on osteoblast differentiation via the WNT1/β-catenin signaling pathwayBashan Zhang0Rong Li1Wenfeng Wang2Xueming Zhou3Beijing Luo4Zinian Zhu5Xibo Zhang6Aijiao Ding7Clinical Laboratory, Affiliated Dongguan People’s Hospital, Southern Medical UniversityClinical Laboratory, Affiliated Dongguan People’s Hospital, Southern Medical UniversityClinical Laboratory, Affiliated Dongguan People’s Hospital, Southern Medical UniversityDepartment of Orthopedic, Affiliated Dongguan People’s Hospital, Southern Medical UniversityClinical Laboratory, Affiliated Dongguan People’s Hospital, Southern Medical UniversityClinical Laboratory, Affiliated Dongguan People’s Hospital, Southern Medical UniversityClinical Laboratory, Affiliated Dongguan People’s Hospital, Southern Medical UniversityClinical Laboratory, Affiliated Dongguan People’s Hospital, Southern Medical UniversityAbstract Background WNT1 c.110 T>C and c.505G>T missense mutations have been identified in patients with osteogenesis imperfecta (OI). Whether these mutations affect osteoblast differentiation remains to be determined. This study aimed to investigate the effects of WNT1 c.110 T>C and c.505G>T mutations on osteoblast function, gene expression, and pathways involved in OI. Methods Empty vector (negative control), wild-type WNT1, WNT1 c.110 T>C, WNT1 c.505G>T, and WNT1 c.884C>A (positive control) mutant plasmids were constructed and transfected into preosteoblast (MC3T3-E1) cells to investigate their effect on osteoblast differentiation. The expressions of osteoblast markers, including BMP2, RANKL, osteocalcin, and alkaline phosphatase (ALP), were determined using quantitative real-time polymerase chain reaction (RT-qPCR), western blotting (WB), enzyme-linked immunosorbent assay, and ALP staining assay, respectively. The mRNA and protein expression levels of WNT1 or the expression levels of the relevant proteins involved in the WNT1/β-catenin signaling pathway were also determined using RT-qPCR, WB, and immunofluorescence (IF) assays after the different plasmids were transfected into MC3T3-E1 cells. Results Compared with those in the wild-type group, in the mutation groups, the mRNA and protein expression levels of BMP2 were suppressed, the expressions of osteocalcin and ALP were inhibited, and the mRNA and protein expression levels of RANKL were enhanced in MC3T3-E1 cells. WB and IF assays revealed that the protein expression levels of WNT1 in MC3T3-E1 cells were downregulated in the mutation groups compared with those in the wild-type WNT1 group. Furthermore, the expression levels of nonphosphorylated β-catenin (non-p-β-catenin) and phosphorylated GSK-3β (p-GSK-3β) were downregulated in the mutation groups compared with those in the wild-type group. However, no significant changes in the expression level of non-p-β-catenin or p-GSK-3β were observed in the mutation groups. Conclusions WNT1 c.110 T>C and c.505G>T mutations may alter the proliferation and osteogenic phenotype of MC3T3-E1 linked to the progression of OI via the inhibition of the WNT1/β-catenin signaling pathway. This is the first study to confirm the effect of WNT1 c.110 T>C and c.505G>T missense mutations on osteoblast differentiation and propose a new molecular mechanism for OI development.https://doi.org/10.1186/s13018-021-02495-2Osteogenesis imperfectaOsteoblastWNT1Mutationβ-Catenin |
| spellingShingle | Bashan Zhang Rong Li Wenfeng Wang Xueming Zhou Beijing Luo Zinian Zhu Xibo Zhang Aijiao Ding Effects of WNT1 c.110 T>C and c.505G>T mutations on osteoblast differentiation via the WNT1/β-catenin signaling pathway Journal of Orthopaedic Surgery and Research Osteogenesis imperfecta Osteoblast WNT1 Mutation β-Catenin |
| title | Effects of WNT1 c.110 T>C and c.505G>T mutations on osteoblast differentiation via the WNT1/β-catenin signaling pathway |
| title_full | Effects of WNT1 c.110 T>C and c.505G>T mutations on osteoblast differentiation via the WNT1/β-catenin signaling pathway |
| title_fullStr | Effects of WNT1 c.110 T>C and c.505G>T mutations on osteoblast differentiation via the WNT1/β-catenin signaling pathway |
| title_full_unstemmed | Effects of WNT1 c.110 T>C and c.505G>T mutations on osteoblast differentiation via the WNT1/β-catenin signaling pathway |
| title_short | Effects of WNT1 c.110 T>C and c.505G>T mutations on osteoblast differentiation via the WNT1/β-catenin signaling pathway |
| title_sort | effects of wnt1 c 110 t c and c 505g t mutations on osteoblast differentiation via the wnt1 β catenin signaling pathway |
| topic | Osteogenesis imperfecta Osteoblast WNT1 Mutation β-Catenin |
| url | https://doi.org/10.1186/s13018-021-02495-2 |
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