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Osteoblast-specific Krm2 overexpression and Lrp5 deficiency have different effects on fracture healing in mice.

ABSTRACT: The canonical Wnt/?-catenin pathway plays a key role in the regulation of bone remodeling in mice and humans. Two transmembrane proteins that are involved in decreasing the activity of this pathway by binding to extracellular antagonists, such as Dickkopf 1 (Dkk1), are the low-density lipoprotein receptor related protein 5 (Lrp5) and Kremen 2 (Krm2). Lrp 5 deficiency (Lrp5-/-) as well as osteoblast-specific overexpression of Krm2 in mice (Col1a1-Krm2) result in severe osteoporosis occurring at young age. In this study, we analyzed the influence of Lrp5 deficiency and osteoblast-specific overexpression of Krm2 on fracture healing in mice using flexible and semi-rigid fracture fixation. We demonstrated that fracture healing was highly impaired in both mouse genotypes, but that impairment was more severe in Col1a1-Krm2 than in Lrp5-/- mice and particularly evident in mice in which the more flexible fixation was used. Bone formation was more reduced in Col1a1-Krm2 than in Lrp5-/- mice, whereas osteoclast number was similarly increased in both genotypes in comparison with wild-type mice. Using microarray analysis we identified reduced expression of genes mainly involved in osteogenesis that seemed to be responsible for the observed stronger impairment of healing in Col1a1-Krm2 mice. In line with these findings, we detected decreased expression of sphingomyelin phosphodiesterase 3 (Smpd3) and less active ?-catenin in the calli of Col1a1-Krm2 mice. Since Krm2 seems to play a significant role in regulating bone formation during fracture healing, antagonizing KRM2 might be a therapeutic option to improve fracture healing under compromised conditions, such as osteoporosis.

SUBMITTER: Liedert A

PROVIDER: S-EPMC4111586 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Osteoblast-specific Krm2 overexpression and Lrp5 deficiency have different effects on fracture healing in mice.

Liedert Astrid A Röntgen Viktoria V Schinke Thorsten T Benisch Peggy P Ebert Regina R Jakob Franz F Klein-Hitpass Ludger L Lennerz Jochen K JK Amling Michael M Ignatius Anita A

PloS one 20140725 7

The canonical Wnt/β-catenin pathway plays a key role in the regulation of bone remodeling in mice and humans. Two transmembrane proteins that are involved in decreasing the activity of this pathway by binding to extracellular antagonists, such as Dickkopf 1 (Dkk1), are the low-density lipoprotein receptor related protein 5 (Lrp5) and Kremen 2 (Krm2). Lrp 5 deficiency (Lrp5-/-) as well as osteoblast-specific overexpression of Krm2 in mice (Col1a1-Krm2) result in severe osteoporosis occurring at y ...[more]

PMID: 25061805

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Project description:The aim of the present study was to screen potential key genes associated with osteoporotic fracture healing. The microarray data from the Gene Expression Omnibus database accession number GSE51686, were downloaded and used to identify differentially expressed genes (DEGs) in fracture callus tissue samples obtained from the femora of type I collagen (Col1a1)‑kringle containing transmembrane protein 2 (Krm2) mice and low density lipoprotein receptor‑related protein 5‑/‑ (Lrp5‑/‑) transgenic mice of osteoporosis compared with those in wild‑type (WT) mice. Enrichment analysis was performed to reveal the DEG function. In addition, protein‑protein interactions (PPIs) of DEGs were analyzed using the Search Tool for the Retrieval of Interacting Genes database. The coexpression associations between hub genes in the PPI network were investigated, and a coexpression network was constructed. A total of 841 DEGs (335 upregulated and 506 downregulated) were identified in the Col1a1‑Krm2 vs. the WT group, and 50 DEGs (16 upregulated and 34 downregulated) were identified in the Lrp5‑/‑ vs. the WT group. The DEGs in Col1a1‑Krm2 mice were primarily associated with immunity and cell adhesion (GO: 0007155) functions. By contrast, the DEGs in Lrp5‑/‑ mice were significantly associated with muscle system process (GO: 0003012) and regulation of transcription (GO: 0006355). In addition, a series of DEGs demonstrated a higher score in the PPI network, and were observed to be coexpressed in the coexpression network, and included thrombospondin 2 (Thbs2), syndecan 2 (Sdc2), FK506 binding protein 10 (Fkbp10), 2'‑5'-oligoadneylate synthase‑like protein 2 (Oasl2), interferon induced protein with tetratricopeptide repeats (Ifit) 1 and Ifit2. Thbs2 and Sdc2 were significantly correlated with extracellular matrix‑receptor interactions. The results suggest that Thbs2, Sdc2, Fkbp10, Oasl2, Ifit1 and Ifit2 may serve important roles during the fracture healing process in osteoporosis. In addition, this is the first study to demonstrate that Sdc2, Fkbp10, Oasl2, Ifit1 and Ifit2 may be associated with osteoporotic fracture healing.

Project description:Bone formation via intramembranous and endochondral ossification is necessary for successful healing after a wide range of bone injuries. The pleiotropic cytokine, vascular endothelial growth factor A (VEGFA) has been shown, via nonspecific pharmacologic inhibition, to be indispensable for angiogenesis and ossification following bone fracture and cortical defect repair. However, the importance of VEGFA expression by different cell types during bone healing is not well understood. We sought to determine the role of VEGFA from different osteoblast cell subsets following clinically relevant models of bone fracture and cortical defect. Ubiquitin C (UBC), Osterix (Osx), or Dentin matrix protein 1 (Dmp1) Cre-ERT2 mice (male and female) containing floxed VEGFA alleles (VEGFAfl/fl ) were either given a femur full fracture, ulna stress fracture, or tibia cortical defect at 12 weeks of age. All mice received tamoxifen continuously starting 2 weeks before bone injury and throughout healing. UBC Cre-ERT2 VEGFAfl/fl (UBC cKO) mice, which were used to mimic nonspecific inhibition, had minimal bone formation and impaired angiogenesis across all bone injury models. UBC cKO mice also exhibited impaired periosteal cell proliferation during full fracture, but not stress fracture repair. Osx Cre-ERT2 VEGFAfl/fl (Osx cKO) mice, but not Dmp1 Cre-ERT2 VEGFAfl/fl (Dmp1 cKO) mice, showed impaired periosteal bone formation and angiogenesis in models of full fracture and stress fracture. Neither Osx cKO nor Dmp1 cKO mice demonstrated significant impairments in intramedullary bone formation and angiogenesis following cortical defect. These data suggest that VEGFA from early osteolineage cells (Osx+), but not mature osteoblasts/osteocytes (Dmp1+), is critical at the time of bone injury for rapid periosteal angiogenesis and woven bone formation during fracture repair. Whereas VEGFA from another cell source, not from the osteoblast cell lineage, is necessary at the time of injury for maximum cortical defect intramedullary angiogenesis and osteogenesis. © 2019 American Society for Bone and Mineral Research.

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Project description:Chronic use of selective serotonin reuptake inhibitors (SSRIs) for the treatment of depression has been linked to osteoporosis. In this study, we investigated the effect of chronic SSRI use on fracture healing in two murine models of bone regeneration. First, we performed a comprehensive analysis of endochondral bone healing in a femur fracture model. C57/BL6 mice treated with fluoxetine, the most commonly prescribed SSRI, developed a normal cartilaginous soft-callus at 14 days after fracture and demonstrated a significantly smaller and biomechanically weaker bony hard-callus at 28 days. In order to further dissect the mechanism that resulted in a smaller bony regenerate, we used an intramembranous model of bone healing and revealed that fluoxetine treatment resulted in a significantly smaller bony callus at 7 and 14 days postinjury. In order to test whether the smaller bony regenerate following fluoxetine treatment was caused by an inhibition of osteogenic differentiation and/or mineralization, we employed in vitro experiments, which established that fluoxetine treatment decreases osteogenic differentiation and mineralization and that this effect is serotonin-independent. Finally, in a translational approach, we tested whether cessation of the medication would result in restoration of the regenerative potential. However, histologic and μCT analysis revealed non-union formation in these animals with fibrous tissue interposition within the callus. In conclusion, fluoxetine exerts a direct, inhibitory effect on osteoblast differentiation and mineralization, shown in two disparate murine models of bone repair. Discontinuation of the drug did not result in restoration of the healing potential, but rather led to complete arrest of the repair process. Besides the well-established effect of SSRIs on bone homeostasis, our study provides strong evidence that fluoxetine use negatively impacts fracture healing. © 2017 American Society for Bone and Mineral Research.

Dataset Information

Osteoblast-specific Krm2 overexpression and Lrp5 deficiency have different effects on fracture healing in mice.

Publications

Osteoblast-specific Krm2 overexpression and Lrp5 deficiency have different effects on fracture healing in mice.

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