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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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Postnatal onset of the bone phenotype in Lrp5−/− mice. (A) Subtle delay of fifth middle phalanx ossification in newborn Lrp5−/− mice. (B) Delayed ossification of metacarpals. (C) Carpals (bracket). (D) Humeri in 4-d-old Lrp5−/− mice. Arrows in A, B, and D indicate ossification centers. A subtle delay was also evident in 44% (8/18) of heterozygote mice (D, middle). (E) Sections of calvaria from 4-d-old mice showing a decreased number of BrdU-positive cells (arrowhead) in Lrp5−/− mice compared with wild-type littermates. Double-headed arrows indicate the greater thickness of wild-type calvarium. (F) Osteoblast mitotic index (percentage of BrdU-positive cells per total cell number) in wild-type and Lrp5−/− mice (n = 8 per genotype). (G) Quantification of alkaline phosphatase positive stromal cell progenitors (CFU-F) in the bone marrow of wild-type and Lrp5−/− mice (n = 9 mice per genotype). (H) Quantification of apoptotic cells by TUNEL assay in wild-type and Lrp5−/− mice (n = 5 per genotype). (I) Northern blot analysis of Cbfa1 expression in wild-type and Lrp5−/− calvaria, long bones and primary osteoblasts. (J) Northern blot analysis of Osteocalcin expression in wild-type and Lrp5−/− calvaria and long bones. (I and J) Gapdh expression level was used as a loading control; results are representative of three samples (n = 3 mice per sample). Asterisks indicate a statistically significant difference between two groups of mice (P < 0.05). Error bars represent SD.
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fig5: Postnatal onset of the bone phenotype in Lrp5−/− mice. (A) Subtle delay of fifth middle phalanx ossification in newborn Lrp5−/− mice. (B) Delayed ossification of metacarpals. (C) Carpals (bracket). (D) Humeri in 4-d-old Lrp5−/− mice. Arrows in A, B, and D indicate ossification centers. A subtle delay was also evident in 44% (8/18) of heterozygote mice (D, middle). (E) Sections of calvaria from 4-d-old mice showing a decreased number of BrdU-positive cells (arrowhead) in Lrp5−/− mice compared with wild-type littermates. Double-headed arrows indicate the greater thickness of wild-type calvarium. (F) Osteoblast mitotic index (percentage of BrdU-positive cells per total cell number) in wild-type and Lrp5−/− mice (n = 8 per genotype). (G) Quantification of alkaline phosphatase positive stromal cell progenitors (CFU-F) in the bone marrow of wild-type and Lrp5−/− mice (n = 9 mice per genotype). (H) Quantification of apoptotic cells by TUNEL assay in wild-type and Lrp5−/− mice (n = 5 per genotype). (I) Northern blot analysis of Cbfa1 expression in wild-type and Lrp5−/− calvaria, long bones and primary osteoblasts. (J) Northern blot analysis of Osteocalcin expression in wild-type and Lrp5−/− calvaria and long bones. (I and J) Gapdh expression level was used as a loading control; results are representative of three samples (n = 3 mice per sample). Asterisks indicate a statistically significant difference between two groups of mice (P < 0.05). Error bars represent SD.

Mentions: To define the earliest time point when the bone phenotype of the Lrp5−/− mice could be detected we stained skeletal preparations of 17.5 d postcoitum (dpc) embryos, newborn, and 4-d-old (P4) wild-type and Lrp5−/− mice with Alizarin red/Alcian blue. Alcian blue stains proteoglycan-rich cartilage ECM, whereas Alizarin red stains mineralized ECM. There was no ossification defect in any skeletal element in 17.5 dpc Lrp5−/− embryos (unpublished data). In contrast, in newborn Lrp5−/− mice, a subtle delay in osteogenesis could be observed in the digits as shown by the absence of the fifth middle phalangeal ossification center (Fig. 5 A, arrow). In 4-d-old mutant mice, the delay in osteogenesis became more pronounced and ossification centers of the wrist, distal metacarpal bones, femora, humeri, and ulnae that stained red in wild-type mice were absent or smaller in Lrp5−/− mice (Fig. 5, B–D, and unpublished data). Consistent with their osteopenic phenotype, we observed a milder delay of osteogenesis in Lrp5+/− mice (Fig. 5 D). Collectively, these data demonstrate that skeletal development appears to proceed normally until 17.5 dpc, after which osteogenesis and bone formation become hampered.


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)

Postnatal onset of the bone phenotype in Lrp5−/− mice. (A) Subtle delay of fifth middle phalanx ossification in newborn Lrp5−/− mice. (B) Delayed ossification of metacarpals. (C) Carpals (bracket). (D) Humeri in 4-d-old Lrp5−/− mice. Arrows in A, B, and D indicate ossification centers. A subtle delay was also evident in 44% (8/18) of heterozygote mice (D, middle). (E) Sections of calvaria from 4-d-old mice showing a decreased number of BrdU-positive cells (arrowhead) in Lrp5−/− mice compared with wild-type littermates. Double-headed arrows indicate the greater thickness of wild-type calvarium. (F) Osteoblast mitotic index (percentage of BrdU-positive cells per total cell number) in wild-type and Lrp5−/− mice (n = 8 per genotype). (G) Quantification of alkaline phosphatase positive stromal cell progenitors (CFU-F) in the bone marrow of wild-type and Lrp5−/− mice (n = 9 mice per genotype). (H) Quantification of apoptotic cells by TUNEL assay in wild-type and Lrp5−/− mice (n = 5 per genotype). (I) Northern blot analysis of Cbfa1 expression in wild-type and Lrp5−/− calvaria, long bones and primary osteoblasts. (J) Northern blot analysis of Osteocalcin expression in wild-type and Lrp5−/− calvaria and long bones. (I and J) Gapdh expression level was used as a loading control; results are representative of three samples (n = 3 mice per sample). Asterisks indicate a statistically significant difference between two groups of mice (P < 0.05). Error bars represent SD.
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fig5: Postnatal onset of the bone phenotype in Lrp5−/− mice. (A) Subtle delay of fifth middle phalanx ossification in newborn Lrp5−/− mice. (B) Delayed ossification of metacarpals. (C) Carpals (bracket). (D) Humeri in 4-d-old Lrp5−/− mice. Arrows in A, B, and D indicate ossification centers. A subtle delay was also evident in 44% (8/18) of heterozygote mice (D, middle). (E) Sections of calvaria from 4-d-old mice showing a decreased number of BrdU-positive cells (arrowhead) in Lrp5−/− mice compared with wild-type littermates. Double-headed arrows indicate the greater thickness of wild-type calvarium. (F) Osteoblast mitotic index (percentage of BrdU-positive cells per total cell number) in wild-type and Lrp5−/− mice (n = 8 per genotype). (G) Quantification of alkaline phosphatase positive stromal cell progenitors (CFU-F) in the bone marrow of wild-type and Lrp5−/− mice (n = 9 mice per genotype). (H) Quantification of apoptotic cells by TUNEL assay in wild-type and Lrp5−/− mice (n = 5 per genotype). (I) Northern blot analysis of Cbfa1 expression in wild-type and Lrp5−/− calvaria, long bones and primary osteoblasts. (J) Northern blot analysis of Osteocalcin expression in wild-type and Lrp5−/− calvaria and long bones. (I and J) Gapdh expression level was used as a loading control; results are representative of three samples (n = 3 mice per sample). Asterisks indicate a statistically significant difference between two groups of mice (P < 0.05). Error bars represent SD.
Mentions: To define the earliest time point when the bone phenotype of the Lrp5−/− mice could be detected we stained skeletal preparations of 17.5 d postcoitum (dpc) embryos, newborn, and 4-d-old (P4) wild-type and Lrp5−/− mice with Alizarin red/Alcian blue. Alcian blue stains proteoglycan-rich cartilage ECM, whereas Alizarin red stains mineralized ECM. There was no ossification defect in any skeletal element in 17.5 dpc Lrp5−/− embryos (unpublished data). In contrast, in newborn Lrp5−/− mice, a subtle delay in osteogenesis could be observed in the digits as shown by the absence of the fifth middle phalangeal ossification center (Fig. 5 A, arrow). In 4-d-old mutant mice, the delay in osteogenesis became more pronounced and ossification centers of the wrist, distal metacarpal bones, femora, humeri, and ulnae that stained red in wild-type mice were absent or smaller in Lrp5−/− mice (Fig. 5, B–D, and unpublished data). Consistent with their osteopenic phenotype, we observed a milder delay of osteogenesis in Lrp5+/− mice (Fig. 5 D). Collectively, these data demonstrate that skeletal development appears to proceed normally until 17.5 dpc, after which osteogenesis and bone formation become hampered.

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