Abstract
BackgroundWNT1 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.MethodsEmpty 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.ResultsCompared 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.ConclusionsWNT1 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.
Highlights
Osteogenesis imperfecta (OI) is a group of heritable connective tissue disorders characterized by increased bone fragility during early childhood, reduced bone mass, and frequent fractures [1,2,3,4]
T>C and c.505G>T mutations may alter the proliferation and osteogenic phenotype of MC3T3-E1 linked to the progression of osteogenesis imperfecta (OI) via the inhibition of the WNT1/β-catenin signaling pathway
The RTqPCR results showed that the mRNA expression levels of WNT1 were upregulated in the mutation and wildtype groups compared with those in the empty vector (Vector) group and that the mRNA levels of WNT1 were downregulated in the WNT1 c
Summary
Osteogenesis imperfecta (OI) is a group of heritable connective tissue disorders characterized by increased bone fragility during early childhood, reduced bone mass, and frequent fractures [1,2,3,4]. Approximately 20–25% of patients with moderate-tosevere OI have pathogenic mutations in other genes [5] Mutations such as SERPINF1, P3H1, CRTAP, PPIB, BMP1, FKBP10, SP7, PLOD2, TMEM38B, PLOD2, P4HB, SPARC, and SEC24D have been considered closely related to autosomal recessive OI cases [6]. Researches have shown that WNT1 mutation affects osteoblast activity, leading to increased bone mass disorder, brittle fractures, and progressive bone abnormality in patients with OI [7, 8]. In OI, studies have shown that biallelic mutations in WNT1 result in recessive OI, whereas heterozygous mutations in WNT1 are associated with early-onset osteoporosis in dominant hereditary families [10, 11]. 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
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