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Compositions and methods for bone formation and remodeling

A bone-related and adipogenesis technology, which can be used in botanical equipment and methods, drug combinations, active ingredients of heterocyclic compounds, etc., and can solve problems such as weakening antagonism

Active Publication Date: 2010-02-24
ENZO THERAPEUTICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It was previously reported that the LRP5 HBMG171V mutation appears to attenuate Dkk-mediated antagonism of Wnt signaling (5)

Method used

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  • Compositions and methods for bone formation and remodeling
  • Compositions and methods for bone formation and remodeling
  • Compositions and methods for bone formation and remodeling

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0280] Example 1. Deletion mutants of LRP5

[0281] A series of PCR primers were designed, PCR reaction was carried out, and PCR fragments were subcloned into vectors to obtain several deletion mutants of LRP5. Deletion of the third and fourth domains (646-1198 residues) produces LRP5R12; deletion of the first and second domains (1-646 residues) produces LRP5R34; and deletion of the third domain (947-1198 residues) produces LRP5R124 (see figure 1 A).

Embodiment 2

[0282] Example 2. LRP5 first domain is essential for Mesd-mediated function of LRP5

[0283] Example 2.1 The G171V mutation in the first domain of LRP5 disrupts LRP5 trafficking

[0284] Interaction of LRP5 with Mesd

[0285] like figure 2 As shown in A, HEK cells were transfected with expression plasmids. One day later cells were lysed and immunoprecipitated with anti-Flag antibody. Mesd is Flag-tagged, while all LRP5 molecules are HA-tagged. The results showed that the G171V mutation in the first domain of LRP5 disrupted the relationship between LRP5 and Mesd ( figure 2 A, lanes 1 and 3) and between R12 and Mesd ( figure 2 B, the interactions of lanes 1 and 2), but the E721 mutation in the third domain had no effect on these interactions ( figure 2 A, lanes 2 and 3).

[0286] Mutants of LRP5 cannot efficiently present themselves to the cell surface.

[0287] like figure 2 B and figure 2 As shown in C, HEK cells were transfected with Mesd plasmid and expre...

Embodiment 3

[0297] Example 3. Wnt activity requires the second domain of LRP5

[0298] like Figure 5 HEK cells were transfected with LEF activity reporter plasmid and expression plasmid as indicated. Expression plasmids LRP5R494Q and LRP5G479V are LRP5 receptors containing a point mutation in the second domain. Cells were lysed one day later. Lysed cells were then assayed for GFP levels and luciferase activity as described in "Materials and Methods" and normalized to GFP levels. Figure 5 display with LRP5 Wt In contrast, LRP5R494Q and LRP5G479V abrogate Wnt signaling. These results indicate that the second domain is required for Wnt activity.

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Abstract

The mechanism by which the high bone mass (HBM) mutation (G171V) of the Wnt coreceptor LRP5 regulates the canonical Wnt signaling was investigated. The mutation was previously shown to reduce Dkk protein-1-mediated antagonism, suggesting that the first YWTD repeat domain where G171 is located may be responsible for Dkk protein-mediated antagonism. However, we found that the third YWTD repeat, but not the first repeat domain, is required for DKK1-mediated antagonism. Instead, we found that the G171V mutation disrupted the interaction of LRP5 with Mesd, a chaperon protein for LRP5 / 6 that is required for the coreceptors' transport to cell surfaces, resulting in less LRP5 molecules on the cell surface. Although the reduction in the level of cell surface LRP5 molecules led to a reduction in Wnt signaling in a paracrine paradigm, the mutation did not appear to affect the activity of coexpressed Wnt in an autocrine paradigm. Together with the observation that osteoblast cells produce autocrine canonical Wnt, Wnt7b, and that osteocytes produce paracrine Dkk1, we believe that the G171V mutation may cause an increase in Wnt activity in osteoblasts by reducing the number of targets for paracrine Dkk1 to antagonize without affecting the activity of autocrine Wnt.

Description

[0001] Related Patent Applications Referenced [0002] This application claims priority to U.S. Provisional Patent Application No. 60 / 504,860, filed September 22, 2003, entitled "Compositions and Methods for Stimulating Bone Formation." [0003] This application is related to the patent application entitled "Compositions and Methods for Stimulating or Enhancing Bone Formation and Cell Self-Renewal" filed by Wu Dan et al. on May 19, 2004, the entire contents of which are incorporated by reference manner is fully incorporated into this application. [0004] This application is a continuation-in-part of Application Serial No. 11 / 097,518 filed April 1, 2005, which is a continuation-in-part of Application Serial No. 11 / 084,668, filed March 18, 2005, which was filed on May 18, 2004. Continuation-in-Part of Application Serial No. 10 / 849,067 filed on April 19, the contents of all of the above applications are incorporated into this application by reference. field of invention [00...

Claims

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Application Information

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IPC IPC(8): A01N43/00A61K31/33
CPCC07D265/38A61K31/538C09B19/005A61P19/00A61P19/08A61P25/00A61P3/00A61P35/00A61P3/06A61P3/08A61P3/10
Inventor D·吴D·刘J·J·多尼甘
Owner ENZO THERAPEUTICS
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