| Aim:Apert syndrome (AS) is a type of autosomal dominant disease characterized by premature fusion of the cranial sutures, severe syndactyly, and other abnormalities in internal organs. Approximately 70% of AS cases are caused by a single mutation, S252W, in fibroblast growth factor receptor 2 (FGFR2). Two groups have generated FGFR2 knock-in mice Fgfr2S252W/+ that exhibit features of AS. During the present study of AS using the Fgfr2S25W/+ mouse model, an age-related phenotype of bone homeostasis was discovered. The long bone mass was lower in 2 month old mutant mice than in age-matched controls but higher in 5 month old mutant mice. This unusual phenotype suggested that bone marrow-derived mesenchymal stem cells (BMSCs), which are vital to maintain bone homeostasis, might be involved. BMSCs were isolated from Fgfr252W/+ mice and found that S252W mutation could impair osteogenic differentiation BMSCs but enhance mineralization of more mature osteoblasts. A microarray analysis revealed that Wnt/p-catenin pathway inhibitors SRFP1/2/4 were up-regulated in mutant BMSCs. This work provides evidence to show that the Wnt/β-catenin pathway is inhibited in both mutant BMSCs and osteoblasts, and differentiation defects of these cells can be ameliorated by Wnt3a treatment. The present study suggested that the bone abnormalities caused by deregulation of Wnt/(3-catenin pathway may underlie the symptoms of AS.Materials and Methods:Materials:Heterozygous mutant mice generated with Fgfr2NeoS252W and EⅡaCre are here referred to as Fgfr2S252w/+. Genotyping of tail DNA was used to distinguish mutant from wild-type progeny by PCR analysis. To determine the mass of trabecular bone, micro-computed tomography (Micro-CT) analysis of the distal metaphysis of femurs derived from 2-month-old and 5-month-old mice was performed. The analysis of the specimens involved the following bone measurements:trabecular and cortical bone volume fraction (Tb.BV/TV, Ct.BV/TV,%), trabecular number (Tb.N), trabecular and cortical thickness (Tb.Th, Ct.Th), trabecular separation (Tb.Sp), trabecular structure model index (Tb.SMI), trabecular and cortical bone mineral density (Tb.BMD, Ct. BMD). To determine whether the changed bone mass in Fgfr2S252W/+ mice was due to altered bone formation, a histologic analysis of decalcified and undecalcified tibiae was performed by H&E staining and Von Kossa staining. The Tb.BV/TV and Tb.Sp of tibiae were analyzed using OsteoMeasure system. To determine whether bone defects observed in Fgfr2S252W/+ mice are related to systemic alterations in mineral homeostasis, levels of total Ca and phosphate in mouse serum were measured. Procollagen I N-Terminal Propeptide (PINP) is a sensitive and specific marker of osteoblast activity. Serum levels of PINP were detected using ELISA.BMSCs were harvested from the ends of the femurs and tibiae of 6-to 8-week-old mice. We tested the expression of mesenchymal stem-cell markers by FCM of W-BMSCs and M-BMSCs. Cell proliferation was detected using Cell Counting Kit-8 and. To investigate the potential of BMSCs to undergo osteoblastic/adipogenic differentiation, W-BMSCs and M-BMSCs were cultured in osteogenic-inducing or adipogenesis-inducing medium for 21 days. RT-PCR was used to detect the expression of the adipogenesis-related and osteogenic-related genes, PPARy, LPL and Oc, Runx2. Lipid droplets and mineralized nodules were detected using oil red O staining and Alizarin red staining in cells from both wild-type and mutant mice.To assess the effects of S252W mutation on the osteogenic differentiation of BMSCs, the ALP activity and numbers of mineralized bone nodules (Alizarin red staining) were compared in BMSCs isolated from both wild-type and Fgfr2S252W/+ mice. immunofluorescence staining and real time-PCR were used for determining the protein levels and gene expression of Col-I, Op, Oc, Runx2 and osterix in BMSCs. Mineralization were detected using Alizarin red staining.In order to investigate the mechanism underlying the alterations in osteogenic differentiation of S252W BMSCs, microarray data using cluster analysis was assessed. Wnt pathway antagonists secreted frizzled-related proteins 1,2, and 4 (SFRP1, SFRP2, and SFRP4) were all up-regulated in BMSCs isolated from Fgfr2S25W/+ mice. This up-regulation was further confirmed using RT-PCR and Western blot using specific antibodies. To determine whether canonical Wnt/β-catenin signaling is affected in S252W BMSCs, P-catenin proteins were separated into cytosolic and nuclear fractions and the protein levels were assessed. To corroborate the conclusion that Wnt/β-catenin pathway was suppressed in mutant cells, the levels of expression of the Wnt target genes cyclinDl, lefl, and fzd4 were assessed using RT-PCR.If osteogenic differentiation defects in Fgfr2S252W/+ BMSCs are caused by impairment of the Wnt/p-catenin pathway, forced increases in Wnt/β-catenin signaling may be able to mitigate the phenotype. To activate the Wnt/β-catenin signaling pathway,100 ng/mL of Wnt3a was added to the cell culture medium. The proliferative, adipogenic, and osteogenic capabilities of Fgfr2S252W/+ BMSCs and wild-type BMSCs were examined after Wnt3a treatment.Results:1. Quantification of the structural parameters revealed that Tb.N, Tb.Th, Ct.Th, Tb.BV/TV, Ct.BV/TV, Tb.BMD, and Ct. BMD were all lower in Fgfr2S252w/+ mice at 2 months. Tb.Sp in mutant mice was increased. These differences in trabecular connectivity of Fgfr2S252W/+ femora were confirmed by an increase in Tb.SMI. Although Ct.BMD was lower in 5 months mutant mice than in controls, Tb.N, Tb.Th, Ct.Th, Tb.BV/TV, Ct.BV/TV, and Tb.BMD all had higher values in mutant mice than in controls at 5 months in mutant mice. Tb.Sp was lower in mutant mice. These differences were confirmed by a significantly decrease in Tb.SMI. Collectively, these observations indicate that gain-of-function mutation in FGFR2 changed bone mass and compromised architecture in adult mice. Von Kossa staining of undecalcified tibiae showed that the trabecular bone of Fgfr2S25W/+ mice was sparser than wild-type at age 2 months but longer and denser than wild-type at 5 months. The Tb.BV/TV in Fgfr2S252W/+ were significantly reduced at 2 months, but were increased at 5 months. Tb.Sp was significantly increased in mutant mice at 2 months, but were reduced at 5 months.These results are consistent with the results obtained by micro-CT examination. Although the osteoblasts lining trabecular bone of Fgfr2S252W/+ mice at 2 months were longer than those in wild-type mice, the osteoblasts were less. The osteoblasts lining trabecular bone of Fgfr2S252W/+ mice at 5 months were plump and cuboidal than those in wild-type mice. In Fgfr2S252W/+ mice, the trabecular bone was lined with thinner osteoids compared with wild-type mice at 2 months, and it was lined with thicker osteoids than wild-type mice at 5 months old. All these bone phenotypes indicate that Fgfr2S252W+ mice have osteomalacia-like skeleton, which need to be further studied. No significant differences in serum levels of either total Ca or phosphate were found between control and mutant mice. Serum levels of PINP were were found to be lower than in controls at 2 months but higher than in controls at 5 months in Fgfr2S252W/+ mice.2. Although the isolated cells from Fgfr2S252W/+ mice retained similarly typical fibroblastic spindle shape, M-BMSCs were less than W-BMSCs. FACS profiling showed that M-BMSCs expressed stem cell markers of BMSCs similar to those of W-BMSCs. Both M-BMSCs and W-BMSCs were positive for CD90, CD105, and CD146 but negative for CD34 and CD45. M-BMSCs were weakly positive for CD90, CD105, and CD146. BMSCs from Fgfr2S252W/+ mice exhibited less proliferative capacity than cells from wild-type mice. The capacity of early apoptotic and late apoptotic of M-BMSCs were found to be Stronger than those of W-BMSCs. After 21 days adipogenic induction, M-BMSCs had fewer lipid droplets than W-BMSCs and the expression levels of PPARy and LPL were significantly lower in M-BMSCs than those in W-BMSCs. After 21 days osteogenic induction, M-BMSCs had larger mineralized colonies and more absorbance of alizarin red and the expression levels of Oc and Runx2 were significantly higher in M-BMSCs than those in W-BMSCs.3. After 4,7,14 days of osteogenic induction, the BMSCs from Fgfr2S252W/+ mice showed fewer crystal-violet-stained cells. The ALP activity of BMSCs was significantly lower in Fgfr2S252W/+ mice than in wild-type mice. Immunofluorescence staining showed that both M-BMSCs and W-BMSCs cultured in osteogenic medium were positive for Op and Oc. The levels of expression of Op, Oc, Runx2, osterix of BMSCs were all lower in Fgfr2S252W/+ mice on days 4,7, and 14, but were all increased on day 21 compared with wild-type mice. The expression levels of Col-I of BMSCs was lower in Fgfr2S252W/+ mice than in wild-type mice on day 4 but higher on days 7,14, and 21.4. Microarray data showed that Wnt/p-catenin pathway antagonists secreted frizzled-related proteins 1,2, and 4 (SFRP1, SFRP2, and SFRP4) were all up-regulated in BMSCs isolated from Fgfr2S252W/+ mice. This up-regulation was further confirmed by RT-PCR and Western blot using specific antibodies. The results showed that, after 7 or 21 days of osteogenic induction, the cytosolic and nucleus β-catenin levels in Fgfr2S25W/+ BMSCs were lower than in the wild-type BMSCs. In both mutant and wild-type BMSCs, β-catenin protein levels continued to decrease with as culture continued and the decrease was more pronounced in S252W cells. In keeping with low Wnt signaling in mutant BMSCs, RT-PCR results showed that the levels of expression of the Wnt target genes cyclinDl, lefl, and fzd4 were reduced. Expression levels of these genes on osteogenic induction day 21 were lower than on day 7, consistent with levels of P-catenin.5. Wnt3a treatment significantly enhanced proliferation of Fgfr2S252W/+ BMSCs. The results showed that adding Wnt3a to adipogenic induction medium can markedly enhance expression of PPARy and LPL in Fgfr2S252W/+ BMSCs. Results also showed that adding Wnt3a to osteogenic medium can promote osteogenic capacity of Fgfr2S252W/+ BMSCs as evidenced by ALP staining of cells induced on day 7 and day 14. ALP activity analysis confirmed an increased activity after Wnt3a treatment. However, there was significantly less mineralization after 21 days of Wnt3a treatment in Fgfr2S25W/+ BMSCs. In line with the decrease in mineralization, the expression levels of osteocyte-related genes Col-Ⅰ, oc, Runx2 in Fgfr2S25W/+ BMSCs were significantly lower after 21 days of osteogenic induction in the presence of Wnt3a.Conclusion:1. In this study we found that gain-of-function mutation in FGFR2 changed bone mass and compromised architecture in adult mice in an age-dependent manner.2. BMSCs from Fgfr2S2i2W/+ mice exhibited less proliferative, adipogenic differentiation capacity and more stongly apoptotic than cells from wild-type mice.3. These results indicate that gain-of-function mutation of FGFR2 can inhibit osteogenic differentiation from BMSCs in the early, but can promoted mineralization later.4. Microarray data showed that Wnt/β-catenin pathway antagonists secreted frizzled-related proteins 1,2, and 4 (SFRP1, SFRP2, and SFRP4) were all up-regulated in BMSCs isolated from Fgfr2S25W/+ mice. These results confirmed Wnt/p-catenin signaling is affected in S252W BMSCs.5. Forced increases in Wnt signaling can mitigate the phenotype of Fgfr2S25W/+ BMSCs. Given the critical role of the canonical Wnt/β-catenin pathway in osteogenesis, inhibition of Wnt/p-catenin signaling may underlie the osteogenic defects of Fgfr2S25W/+ BMSCs.Taken together, the present study revealed an age-related bone mass phenotype in Fgfr2S252W/+ mice and provided evidence to show that dysregulation of Wnt signaling in both BMSCs and osteoblasts plays an important role in defective ossification in Fgfr2S252W/+ mice, so shedding light on the mechanism underlying Apert syndrome. |
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