LRP 5 Affects Homeostasis of the Periodontal Complex

Purpose: The signals responsible for homeostasis in the periodontal complex are unclear. The purpose of this study was to evaluate the role of Low-density lipoprotein receptor-related protein 5(LRP 5 ) in this process by removing LRP 5 , and observing the effects of LRP 5 depletion on cells of the periodontal structures. Material and Methods: The function of this LRP 5 was evaluated by conditional elimination of the LRP 5 gene using an Osteocalcin Cre driver. The OCN-Cre; 5 LRP fl fl mice were examined using micro-CT and histology, immunohistochemistry to evaluate the periodontal complex. Results: Elimination of LRP 5 in the periodontal complex of OCN-Cre; 5 LRP fl fl mice results in a different expression of Fibromodulin in the periodontal ligament space. A decrease in osteoclastic activity was found in the periodontal ligament. Conclusion: Osteoclastic activities are decreased and expression of fibromodulin is decreased, which implies the involvement of LRP 5 in homeostasis of the periodontal ligament.

lished. It is believed that high bone mass mutation occurs in either limb or osteoblast [6]. For treatment of osteoporosis, an osteocyte-specific protein binding to LRP 5 has been used to block Wnt signaling pathway [7].
Mice that carry the same G171V substitution (e.g., Lrp5G171V mice) show an increase in bone mass and bone density [8]. In addition, Lrp5G171V mice exhibited a decrease in the width of periodontal ligament, which is concomitant with an increase in alveolar bone mass [9]. To create a loss of LRP 5 function phenotype, we used OCN-Cre; 5 LRP fl fl mice [10]. Analyses of the loss of LRP 5 function animal models provided new information regarding homeostasis of periodontal complex.

Generation of Mouse Strains
The generation of OCN-Cre; 5 LRP fl fl mice has been previously described. Ten 3 month-old mice were analyzed; 5 were OCN-Cre; 5 LRP fl fl mice and 5 were wild-type littermates.

Sample Preparation, Processing and Histology
Harvested maxillae from the wild type and OCN-Cre; 5 LRP fl fl 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.

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   (Figure 2(D)). In addition, the width of periodontal ligament showed no difference between wild-type and OCN-Cre; 5 LRP fl fl mice. Higher magnification revealed that there was no difference in the alveolar bone and root surfaces in OCN-Cre; 5 LRP fl fl mice compared to wild-type mice.
Using Fibromodulin immunostaining [12], we found variations in expression. In wild-type mice, Fibromodulin was uniformly dispensed in the PDL space ( Figure 2(E)). In OCN-Cre; 5 LRP fl fl mice, however, Fibromodulin expression was very low in the PDL space (Figure 2(F)).
There was no difference in expression of DSP between wild-type and mutant mice (Figure 2(G) and Figure 2(H)).

Alteration of Osteogenic Markers in Periodontal Ligament Space of OCN-Cre; LRP5 fl/fl Mice
In wild-type mice, osteocalcin was expressed throughout the PDL space ( Figure  3(A)). In OCN-Cre; 5 LRP fl fl 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)).

Alteration of Osteoclastic Activity in OCN-Cre; LRP5 fl/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; 5 LRP fl fl 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; 5 LRP fl fl mice (Figure 4(D)).
No difference in ALP activity was found between the wild-type and OCN-Cre; 5 LRP fl fl mice periodontal ligament (Figure 4(C) and Figure 4(D)).

Discussion
Wnt signaling pathway is involved in homeostasis of periodontal complex [9] [13] [14] [15]. Using gain-and loss-of-Wnt function animal models, reduced Wnt for this comes from the fact that a reduction in bone mass occurs when LRP 5 is removed only in osteocytes [16].
Periodontal cells are reported to be Wnt responsive [17], so PDL cells are affected by Wnt signaling. Expression of fibromodulin in Lrp5 ACT mice is strong in a previous study [9], while expression of fibromodulin in OCN-Cre; 5 LRP fl fl 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 [18].
Bone formation is known to be influenced by LRP 5 . However, it is not clear that bone resorption depends on LRP 5 . 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 Lrp5 ACT mice, while RANKL expression in Lrp5 ACT mice was not altered compared to the wild-type mice [9]. In this study, osteoclast activity indicated by CD 68 expression, and RANKL expression were decreased in OCN-Cre; 5 LRP fl fl mice. Here, bone resorption was influenced by LRP 5 , 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 [9]. 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 LRP 5 during bone resorption.

Conclusion
Using loss-of-LRP 5 function animal model, we show that reduced LRP 5 is involved in altered collagen structure in the periodontal ligament and bone resorption.