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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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Functional defect in Lrp5−/− osteoblasts. (A) Calcein double labeling in 6-mo-old wild-type and Lrp5−/− mice. The distance between the two labels (double-headed arrows) represents the MAR which is decreased in Lrp5−/− mice. As a result, the BFR is significantly lower in Lrp5−/− mice. (B) Decreased osteoblast number per bone area in the primary (left) and secondary (right) spongiosa of Lrp5−/− mice compared with wild-type. (C) Delayed mineralization in primary Lrp5−/− osteoblast cultures compared with wild-type cultures at day 10 of mineralization. (D) Normal tibial growth plate in 2-mo-old Lrp5−/− mice (von Kossa stain). Asterisks indicate a statistically significant difference between two groups of mice (P < 0.05). Error bars represent SD.
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fig4: Functional defect in Lrp5−/− osteoblasts. (A) Calcein double labeling in 6-mo-old wild-type and Lrp5−/− mice. The distance between the two labels (double-headed arrows) represents the MAR which is decreased in Lrp5−/− mice. As a result, the BFR is significantly lower in Lrp5−/− mice. (B) Decreased osteoblast number per bone area in the primary (left) and secondary (right) spongiosa of Lrp5−/− mice compared with wild-type. (C) Delayed mineralization in primary Lrp5−/− osteoblast cultures compared with wild-type cultures at day 10 of mineralization. (D) Normal tibial growth plate in 2-mo-old Lrp5−/− mice (von Kossa stain). Asterisks indicate a statistically significant difference between two groups of mice (P < 0.05). Error bars represent SD.

Mentions: The cellular basis of this low bone mass phenotype was studied using histomorphometric analyses and biochemical and cell-based assays. The bone formation aspect of bone remodeling was analyzed by measuring the bone formation rate (BFR), an indicator of osteoblast activity, after in vivo double labeling with calcein, a marker of newly formed bone. In 6-mo-old Lrp5−/− mice there was a twofold decrease in the BFR compared with wild-type littermates (Fig. 4 A). A similar difference was observed in 2- and 4-mo-old Lrp5−/− mice (unpublished data). The decreased BFR was solely due to a decreased matrix apposition rate (MAR) (Fig. 4 A, arrow), an index of the amount of bone matrix deposited per osteoblast cluster (0.75 ± 0.05 vs. 0.45 ± 0.06 μm/d; P < 0.05) (Aaron et al., 1984). This decreased MAR directly demonstrates a functional defect of osteoblasts in vivo in the absence of Lrp5. Consistent with the dominant nature of the osteoporosis-like phenotype, the BFR was also significantly decreased in Lrp5+/− mice (88.6 ± 4.8 vs. 60.5 ± 3.9 μm3/μm2/y; P < 0.05). We also performed osteoblast counts in the primary and secondary spongiosa. The results obtained were identical in both cases: total osteoblast number per bone area was significantly decreased in Lrp5−/− mice (Fig. 4 B). To further study osteoblast function, we assayed the ability of primary osteoblast cultures to mineralize an extracellular matrix (ECM). As shown in Fig. 4 C, mineralization of the ECM surrounding Lrp5−/− osteoblasts was delayed compared with what we observed in wild-type osteoblasts at day 10 of culture. This latter finding supports the notion that Lrp5−/− osteoblasts have a functional defect. However, it may alternatively be explained by the abnormal proliferation of Lrp5−/− osteoblasts (see below). The growth plate of Lrp5−/− mice appeared normal, indicating that chondrogenesis was not overtly affected (Fig. 4 D).


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)

Functional defect in Lrp5−/− osteoblasts. (A) Calcein double labeling in 6-mo-old wild-type and Lrp5−/− mice. The distance between the two labels (double-headed arrows) represents the MAR which is decreased in Lrp5−/− mice. As a result, the BFR is significantly lower in Lrp5−/− mice. (B) Decreased osteoblast number per bone area in the primary (left) and secondary (right) spongiosa of Lrp5−/− mice compared with wild-type. (C) Delayed mineralization in primary Lrp5−/− osteoblast cultures compared with wild-type cultures at day 10 of mineralization. (D) Normal tibial growth plate in 2-mo-old Lrp5−/− mice (von Kossa stain). Asterisks indicate a statistically significant difference between two groups of mice (P < 0.05). Error bars represent SD.
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Related In: Results  -  Collection

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fig4: Functional defect in Lrp5−/− osteoblasts. (A) Calcein double labeling in 6-mo-old wild-type and Lrp5−/− mice. The distance between the two labels (double-headed arrows) represents the MAR which is decreased in Lrp5−/− mice. As a result, the BFR is significantly lower in Lrp5−/− mice. (B) Decreased osteoblast number per bone area in the primary (left) and secondary (right) spongiosa of Lrp5−/− mice compared with wild-type. (C) Delayed mineralization in primary Lrp5−/− osteoblast cultures compared with wild-type cultures at day 10 of mineralization. (D) Normal tibial growth plate in 2-mo-old Lrp5−/− mice (von Kossa stain). Asterisks indicate a statistically significant difference between two groups of mice (P < 0.05). Error bars represent SD.
Mentions: The cellular basis of this low bone mass phenotype was studied using histomorphometric analyses and biochemical and cell-based assays. The bone formation aspect of bone remodeling was analyzed by measuring the bone formation rate (BFR), an indicator of osteoblast activity, after in vivo double labeling with calcein, a marker of newly formed bone. In 6-mo-old Lrp5−/− mice there was a twofold decrease in the BFR compared with wild-type littermates (Fig. 4 A). A similar difference was observed in 2- and 4-mo-old Lrp5−/− mice (unpublished data). The decreased BFR was solely due to a decreased matrix apposition rate (MAR) (Fig. 4 A, arrow), an index of the amount of bone matrix deposited per osteoblast cluster (0.75 ± 0.05 vs. 0.45 ± 0.06 μm/d; P < 0.05) (Aaron et al., 1984). This decreased MAR directly demonstrates a functional defect of osteoblasts in vivo in the absence of Lrp5. Consistent with the dominant nature of the osteoporosis-like phenotype, the BFR was also significantly decreased in Lrp5+/− mice (88.6 ± 4.8 vs. 60.5 ± 3.9 μm3/μm2/y; P < 0.05). We also performed osteoblast counts in the primary and secondary spongiosa. The results obtained were identical in both cases: total osteoblast number per bone area was significantly decreased in Lrp5−/− mice (Fig. 4 B). To further study osteoblast function, we assayed the ability of primary osteoblast cultures to mineralize an extracellular matrix (ECM). As shown in Fig. 4 C, mineralization of the ECM surrounding Lrp5−/− osteoblasts was delayed compared with what we observed in wild-type osteoblasts at day 10 of culture. This latter finding supports the notion that Lrp5−/− osteoblasts have a functional defect. However, it may alternatively be explained by the abnormal proliferation of Lrp5−/− osteoblasts (see below). The growth plate of Lrp5−/− mice appeared normal, indicating that chondrogenesis was not overtly affected (Fig. 4 D).

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