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Cbfa1-independent decrease in osteoblast proliferation, osteopenia, and persistent embryonic eye vascularization in mice deficient in Lrp5, a Wnt coreceptor.

Kato M, Patel MS, Levasseur R, Lobov I, Chang BH, Glass DA, Hartmann C, Li L, Hwang TH, Brayton CF, Lang RA, Karsenty G, Chan L - J. Cell Biol. (2002)

Bottom Line: In vivo and in vitro analyses indicate that this phenotype becomes evident postnatally, and demonstrate that it is secondary to decreased osteoblast proliferation and function in a Cbfa1-independent manner.Lrp5 is expressed in osteoblasts and is required for optimal Wnt signaling in osteoblasts.Moreover, these features recapitulate human osteoporosis-pseudoglioma syndrome, caused by LRP5 inactivation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology and Medicine, Baylor College of Medicine, Houston, TX 77030, USA.

ABSTRACT
The low-density lipoprotein receptor-related protein (Lrp)-5 functions as a Wnt coreceptor. Here we show that mice with a targeted disruption of Lrp5 develop a low bone mass phenotype. In vivo and in vitro analyses indicate that this phenotype becomes evident postnatally, and demonstrate that it is secondary to decreased osteoblast proliferation and function in a Cbfa1-independent manner. Lrp5 is expressed in osteoblasts and is required for optimal Wnt signaling in osteoblasts. In addition, Lrp5-deficient mice display persistent embryonic eye vascularization due to a failure of macrophage-induced endothelial cell apoptosis. These results implicate Wnt proteins in the postnatal control of vascular regression and bone formation, two functions affected in many diseases. Moreover, these features recapitulate human osteoporosis-pseudoglioma syndrome, caused by LRP5 inactivation.

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LRP5-dependent activation of gene expression by Wnt proteins. (A) Wnt1 activates Lef1-dependent gene expression in wild-type but poorly in Lrp5−/− primary osteoblasts. (B) Cotransfection of LRP5 in Lrp5−/− osteoblasts allows Wnt1 to activate Lef1-dependent gene expression to wild-type levels. (C) Cotransfection of LRP5 with Wnt3a-conditioned medium or an expression vector for Wnt4 in wild-type (white bars) and Lrp5−/− primary osteoblasts (black bars). (D) Western blot analysis to confirm expression of transfected plasmids and to demonstrate the ability of Wnt1-LRP5 cotransfection to stimulate cytoplasmic β-catenin accumulation. (E) Direct binding of LRP5 to Wnt1 or Wnt4. COS-7 cells were transfected with Flag-LRP5 and HA-Wnt1 or HA-Wnt4 as indicated. LRP5 or Wnts were immunoprecipitated from the cell lysate using anti-Flag or anti-HA antibody, subjected to SDS-PAGE and immunoblotted with anti-HA and anti-Flag antibody, respectively.
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fig6: LRP5-dependent activation of gene expression by Wnt proteins. (A) Wnt1 activates Lef1-dependent gene expression in wild-type but poorly in Lrp5−/− primary osteoblasts. (B) Cotransfection of LRP5 in Lrp5−/− osteoblasts allows Wnt1 to activate Lef1-dependent gene expression to wild-type levels. (C) Cotransfection of LRP5 with Wnt3a-conditioned medium or an expression vector for Wnt4 in wild-type (white bars) and Lrp5−/− primary osteoblasts (black bars). (D) Western blot analysis to confirm expression of transfected plasmids and to demonstrate the ability of Wnt1-LRP5 cotransfection to stimulate cytoplasmic β-catenin accumulation. (E) Direct binding of LRP5 to Wnt1 or Wnt4. COS-7 cells were transfected with Flag-LRP5 and HA-Wnt1 or HA-Wnt4 as indicated. LRP5 or Wnts were immunoprecipitated from the cell lysate using anti-Flag or anti-HA antibody, subjected to SDS-PAGE and immunoblotted with anti-HA and anti-Flag antibody, respectively.

Mentions: To test this hypothesis, we first performed DNA cotransfection experiments in primary calvarial osteoblasts obtained from wild-type or Lrp5−/− 4-d-old mice. Cells were transfected with a luciferase reporter construct containing multiple copies of an oligonucleotide containing the Lef1 binding site (TOPtkluc) (Korinek et al., 1997), as well as a Lef1 expression vector. Using this assay, cotransfection of a Wnt1 expression vector consistently enhanced Lef1-dependent transcription to a higher degree in wild-type than in Lrp5−/− primary osteoblasts (Fig. 6 A). Importantly, cotransfection of an LRP5 expression vector into Lrp5−/− osteoblasts restored the ability of Wnt1 to induce Lef1-dependent gene expression to wild-type levels (Fig. 6 B).


Cbfa1-independent decrease in osteoblast proliferation, osteopenia, and persistent embryonic eye vascularization in mice deficient in Lrp5, a Wnt coreceptor.

Kato M, Patel MS, Levasseur R, Lobov I, Chang BH, Glass DA, Hartmann C, Li L, Hwang TH, Brayton CF, Lang RA, Karsenty G, Chan L - J. Cell Biol. (2002)

LRP5-dependent activation of gene expression by Wnt proteins. (A) Wnt1 activates Lef1-dependent gene expression in wild-type but poorly in Lrp5−/− primary osteoblasts. (B) Cotransfection of LRP5 in Lrp5−/− osteoblasts allows Wnt1 to activate Lef1-dependent gene expression to wild-type levels. (C) Cotransfection of LRP5 with Wnt3a-conditioned medium or an expression vector for Wnt4 in wild-type (white bars) and Lrp5−/− primary osteoblasts (black bars). (D) Western blot analysis to confirm expression of transfected plasmids and to demonstrate the ability of Wnt1-LRP5 cotransfection to stimulate cytoplasmic β-catenin accumulation. (E) Direct binding of LRP5 to Wnt1 or Wnt4. COS-7 cells were transfected with Flag-LRP5 and HA-Wnt1 or HA-Wnt4 as indicated. LRP5 or Wnts were immunoprecipitated from the cell lysate using anti-Flag or anti-HA antibody, subjected to SDS-PAGE and immunoblotted with anti-HA and anti-Flag antibody, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199263&req=5

fig6: LRP5-dependent activation of gene expression by Wnt proteins. (A) Wnt1 activates Lef1-dependent gene expression in wild-type but poorly in Lrp5−/− primary osteoblasts. (B) Cotransfection of LRP5 in Lrp5−/− osteoblasts allows Wnt1 to activate Lef1-dependent gene expression to wild-type levels. (C) Cotransfection of LRP5 with Wnt3a-conditioned medium or an expression vector for Wnt4 in wild-type (white bars) and Lrp5−/− primary osteoblasts (black bars). (D) Western blot analysis to confirm expression of transfected plasmids and to demonstrate the ability of Wnt1-LRP5 cotransfection to stimulate cytoplasmic β-catenin accumulation. (E) Direct binding of LRP5 to Wnt1 or Wnt4. COS-7 cells were transfected with Flag-LRP5 and HA-Wnt1 or HA-Wnt4 as indicated. LRP5 or Wnts were immunoprecipitated from the cell lysate using anti-Flag or anti-HA antibody, subjected to SDS-PAGE and immunoblotted with anti-HA and anti-Flag antibody, respectively.
Mentions: To test this hypothesis, we first performed DNA cotransfection experiments in primary calvarial osteoblasts obtained from wild-type or Lrp5−/− 4-d-old mice. Cells were transfected with a luciferase reporter construct containing multiple copies of an oligonucleotide containing the Lef1 binding site (TOPtkluc) (Korinek et al., 1997), as well as a Lef1 expression vector. Using this assay, cotransfection of a Wnt1 expression vector consistently enhanced Lef1-dependent transcription to a higher degree in wild-type than in Lrp5−/− primary osteoblasts (Fig. 6 A). Importantly, cotransfection of an LRP5 expression vector into Lrp5−/− osteoblasts restored the ability of Wnt1 to induce Lef1-dependent gene expression to wild-type levels (Fig. 6 B).

Bottom Line: In vivo and in vitro analyses indicate that this phenotype becomes evident postnatally, and demonstrate that it is secondary to decreased osteoblast proliferation and function in a Cbfa1-independent manner.Lrp5 is expressed in osteoblasts and is required for optimal Wnt signaling in osteoblasts.Moreover, these features recapitulate human osteoporosis-pseudoglioma syndrome, caused by LRP5 inactivation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology and Medicine, Baylor College of Medicine, Houston, TX 77030, USA.

ABSTRACT
The low-density lipoprotein receptor-related protein (Lrp)-5 functions as a Wnt coreceptor. Here we show that mice with a targeted disruption of Lrp5 develop a low bone mass phenotype. In vivo and in vitro analyses indicate that this phenotype becomes evident postnatally, and demonstrate that it is secondary to decreased osteoblast proliferation and function in a Cbfa1-independent manner. Lrp5 is expressed in osteoblasts and is required for optimal Wnt signaling in osteoblasts. In addition, Lrp5-deficient mice display persistent embryonic eye vascularization due to a failure of macrophage-induced endothelial cell apoptosis. These results implicate Wnt proteins in the postnatal control of vascular regression and bone formation, two functions affected in many diseases. Moreover, these features recapitulate human osteoporosis-pseudoglioma syndrome, caused by LRP5 inactivation.

Show MeSH
Related in: MedlinePlus