Low-density lipoprotein receptor-related protein 5 (LRP5) is known to be a key component in Wnt signaling pathway. Mutations in LRP5 cause alteration in bone mass. During bone development, a deletion of LRP5 leads to a decrease in bone mass . On the other hand, a gain in LRP5 causes an increase in bone mass  . Thus, LRP5 is involved in disease related to bone. It has been known that homeostasis of bone mass is controlled by LRP5 through osteocytes  . However, a controlling process of bone mass by LRP5 is not well established. It is believed that high bone mass mutation occurs in either limb or osteoblast . For treatment of osteoporosis, an osteocyte-specific protein binding to LRP5 has been used to block Wnt signaling pathway .
Mice that carry the same G171V substitution (e.g., Lrp5G171V mice) show an increase in bone mass and bone density . In addition, Lrp5G171V mice exhibited a decrease in the width of periodontal ligament, which is concomitant with an increase in alveolar bone mass . To create a loss of LRP5 function phenotype, we used OCN-Cre; mice . Analyses of the loss of LRP5 function animal models provided new information regarding homeostasis of periodontal complex.
2. Material and Methods
2.1. Generation of Mouse Strains
The generation of OCN-Cre; mice has been previously described. Ten 3 month-old mice were analyzed; 5 were OCN-Cre; mice and 5 were wild-type littermates.
2.2. Micro-CT Analysis
Micro-CT was taken using MicroXCT-200 (SkyScan, Belgium) at 60 kV and 7.98 Watt and a resolution of 2 microns. CT slices were reconstructed using MicroXCT7.0 reconstruction software (SkyScan, Belgium). Inveon Research Workplace (IRW) (Erlangen, Germany) was used for analysis.
2.3. Sample Preparation, Processing and Histology
Harvested maxillae from the wild type and OCN-Cre; mice were fixed in 4% paraformaldehyde for one night at 4˚C and then decalcified in a heat-controlled microwave in 19% EDTA for 14 days. After this process, specimens were dehydrated using ethanol series and then embedded with paraffin. Eight-micron-thick sections were cut and collected for analyses.
Pentachrome staining was performed .
2.5. Cellular Assays and Immunohistochemistry
Alkaline phosphatase staining was performed to investigate osteogenic factors. For immunostaining analyses, tissue sections were deparaffinized and endogenous peroxidase activity was smothered using 3% hydrogen peroxide then washed with PBS. Slides were blocked out using 5% goat serum (Vector S-1000) for 1 hour. The relevant primary antibody was attached and cultured for one night at 4˚C, then washed with PBS. Samples were cultured using relevant biotinylated secondary antibodies (Vector BA-x) for half an hour, and washed in PBS. An advidin/biotinylated enzyme complex (Kit ABC Peroxidase Standard Vectastain PK-4000) was attached and cultured for 30 minutes and a DAB substrate kit (Kit Vector Peroxidase subtrate DAB SK-4100) was utilized to detect the color reaction. Used antibodies include osteocalcin (Origene, dilution 1:100), Osterix (NIH LF 175, dilution 1:4000), Fibromodulin (Santa Cruz Biotech, dilution 1:1000), dentin sialoprotein (DSP, Millipore, dilution 1:2000), CD 68 (Thermo Fisher Scineticif, dilution 1:100) and Receptor activator of nuclear factor kappa-Β ligand (RANKL, (Lab Vision, dilution 1:100)).
3.1. Bone Volume Is Maintained in OCN-Cre; LRP5fl/fl Mice
Micro-CT examination of the craniofacial skeleton of OCN-Cre; mice revealed that similar bone volume of skeletal elements (Figure 1). Deletion of LRP5 did not affect the size of the skeletal elements (Figure 1). The dentition of OCN-Cre; mice was similar compared to wild-type mice. The overall size, shape, and position of the teeth were the same between wild-type and OCN-Cre; mice (Figure 1). The gross morphology of the molars and incisors was similar in wild-type and mutant mice.
Figure 1. Bone volume is maintained in OCN-Cre; mice. ((A), (B)) Wild-type and OCN-Cre; mice incisors appear to have equivalent size and appearance. Bone volume around incisor area appears similar in wild-type and OCN-Cre; mice. ((C), (D)) The same finding was observed in molars of wild-type and OCN-Cre; mice. ((E), (F)) Cross section of molar areas showed the same finding in the wild-type and OCN-Cre; mice.
3.2. Fibromodulin Expression Is Altered in OCN-Cre; LRP5fl/fl Mice
Histologic examination of the maxillary periodontal complex confirmed the finding in micro-CT examination (Figure 2(A) and Figure 2(B)). In wild-type mice (Figure 2(C)), the periodontal ligament consisted of numerous cells and
Figure 2. Fibromodulin expression is altered in OCN-Cre; mice. ((A), (B)) Pentachrome staining showed that intact tooth structure and periodontal complex in both the wild-type and OCN-Cre; mice. ((C), (D)) Higher magnification revealed that intact cementum and well organized periodontal ligament fibers in the wild-type and mutant mice. ((E), (F)) Expression of Fibromodulin in OCN-Cre; mice were reduced compared to the wild-type mice. Black arrows inidicated that expression of Fibromodulin in the periodontal ligament space was significantly reduced. ((G), (H)) Expression of DSP was found in the wild-type and OCN-Cre; mice. Scale bar: 50 μm.
collagen fiber bundles. Histological examination of the periodontal complex demonstrated that, compared to wild-type mice, there was no alteration in the fibrillar structure of the PDL in OCN-Cre; mice (Figure 2(D)). In addition, the width of periodontal ligament showed no difference between wild-type and OCN-Cre; mice. Higher magnification revealed that there was no difference in the alveolar bone and root surfaces in OCN-Cre; mice compared to wild-type mice.
Using Fibromodulin immunostaining , we found variations in expression. In wild-type mice, Fibromodulin was uniformly dispensed in the PDL space (Figure 2(E)). In OCN-Cre; mice, however, Fibromodulin expression was very low in the PDL space (Figure 2(F)).
3.3. Alteration of Osteogenic Markers in Periodontal Ligament Space of OCN-Cre; LRP5fl/fl Mice
In wild-type mice, osteocalcin was expressed throughout the PDL space (Figure 3(A)). In OCN-Cre; mice, osteocalcin was minimally expressed in the periodontal ligament (Figure 3(B)). We also found that Osterix was strongly expressed in the wild-type and mutant periodontal ligament (Figure 3(C) and Figure 3(D)).
Figure 3. Osteogenic markers in periodontal ligament space of OCN-Cre; mice. ((A), (B)) Osteocalcin was clearly expressed throughout the PDL space in wild-type mice while a decrease in Osteocalcin expression was found in OCN-Cre; mice. ((C), (D)) Osterix expression was found in both the wild-type and OCN-Cre; mice. Scale bar: 50 μm.
3.4. Alteration of Osteoclastic Activity in OCN-Cre; LRP5fl/fl Mice
In wild-type mice, CD 68 was expressed throughout the PDL space (Figure 4(A)), while expression of CD 68 was altered in the periodontal ligament of OCN-Cre; mice (Figure 4(B)). RANKL expression in wild-type mice found throughout the PDL space (Figure 4(C)) while expression of RANKL was reduced in the periodontal ligament of OCN-Cre; mice (Figure 4(D)). No difference in ALP activity was found between the wild-type and OCN-Cre; mice periodontal ligament (Figure 4(C) and Figure 4(D)).
Wnt signaling pathway is involved in homeostasis of periodontal complex    . Using gain- and loss-of-Wnt function animal models, reduced Wnt
Figure 4. Alteration of osteoclastic activity in OCN-Cre; mice. ((A), (B)) Altered CD 68 expression was observed in periodontal ligament space of OCN-Cre; mice compared to the wild-type mice. ((C), (D)) RANKL expression was decreased in OCN-Cre; mice periodontal ligament space compared to that in the wild-type mice. ((E), (F)) ALP expression was similar between wild-type and OCN-Cre; mice. Scale bar: 50 μm.
signaling exhibits an increase in the width of the PDL while elevated Wnt signaling reduces the width of the PDL. Elevated Wnt signaling by mutations in the Wnt co-receptor Lrp5 caused an increased osteogenic gene expression and decreased bone resorption, which leaded to alveolar bone accumulation. On the other hand, our CT data and histology showed that OCN-Cre; mice exhibited insignificant changes in alveolar bone mass. One possible explanation for this comes from the fact that a reduction in bone mass occurs when LRP5 is removed only in osteocytes .
Periodontal cells are reported to be Wnt responsive , so PDL cells are affected by Wnt signaling. Expression of fibromodulin in Lrp5ACT mice is strong in a previous study , while expression of fibromodulin in OCN-Cre; mice was dramatically reduced. The reason for this is not known. However, it may implicate that a reduction of fibromodulin leads to a disorganized periodontal collagen and missing its typical extracellular matrix .
Bone formation is known to be influenced by LRP5. However, it is not clear that bone resorption depends on LRP5. Osteoclast activity is known to be influenced by the coupled action of the Osteoprotegerin and RANKL. Osteoclast activity indicated by TRAP staining was significantly decreased in Lrp5ACT mice, while RANKL expression in Lrp5ACT mice was not altered compared to the wild-type mice . In this study, osteoclast activity indicated by CD 68 expression, and RANKL expression were decreased in OCN-Cre; mice. Here, bone resorption was influenced by LRP5, although the mechanism appears elusive. On the other hand, Ad-Dkk1 treated mice showed a significant increase in both TRAP activity and expression of RANKL . In the case where Wnt signaling is particularly lower, both TRAP and RANKL activity are influenced. Ongoing work is in progress to explain the mechanism related to the role of LRP5 during bone resorption.
Using loss-of-LRP5 function animal model, we show that reduced LRP5 is involved in altered collagen structure in the periodontal ligament and bone resorption.
This work was supported by a Gran No. 05-2014-0011 from the Seoul National University Dental Hospital Research Fund.
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