Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Oct;103:270-280.
doi: 10.1016/j.bone.2017.07.018. Epub 2017 Jul 18.

Rad GTPase Is Essential for the Regulation of Bone Density and Bone Marrow Adipose Tissue in Mice

Affiliations
Free PMC article

Rad GTPase Is Essential for the Regulation of Bone Density and Bone Marrow Adipose Tissue in Mice

Catherine N Withers et al. Bone. .
Free PMC article

Abstract

The small GTP-binding protein Rad (RRAD, Ras associated with diabetes) is the founding member of the RGK (Rad, Rem, Rem2, and Gem/Kir) family that regulates cardiac voltage-gated Ca2+ channel function. However, its cellular and physiological functions outside of the heart remain to be elucidated. Here we report that Rad GTPase function is required for normal bone homeostasis in mice, as Rad deletion results in significantly lower bone mass and higher bone marrow adipose tissue (BMAT) levels. Dynamic histomorphometry in vivo and primary calvarial osteoblast assays in vitro demonstrate that bone formation and osteoblast mineralization rates are depressed, while in vitro osteoclast differentiation is increased, in the absence of Rad. Microarray analysis revealed that canonical osteogenic gene expression (Runx2, osterix, etc.) is not altered in Rad-/- calvarial osteoblasts; instead robust up-regulation of matrix Gla protein (MGP, +11-fold), an inhibitor of extracellular matrix mineralization and a protein secreted during adipocyte differentiation, was observed. Strikingly, Rad deficiency also resulted in significantly higher marrow adipose tissue levels in vivo and promoted spontaneous in vitro adipogenesis of primary calvarial osteoblasts. Adipogenic differentiation of wildtype calvarial osteoblasts resulted in the loss of endogenous Rad protein, further supporting a role for Rad in the control of BMAT levels. These findings reveal a novel in vivo function for Rad and establish a role for Rad signaling in the complex physiological control of skeletal homeostasis and bone marrow adiposity.

Keywords: Adipogenesis; Bone marrow adipose tissue; Matrix Gla protein; Osteoblasts; Osteogenesis; Ras GTPase.

Conflict of interest statement

Conflicts of interest: none

Figures

Figure 1.
Figure 1.. Lower trabecular and cortical bone volume in the femora of Rad−/− mice
A) Representative images from μCT analysis of trabecular bone at the distal femora of WT and RadKO mice with accompanying quantification of the trabecular bone volume fraction (BV/TV). B) Representative images from μCT analysis of cortical bone at the midshaft of WT and RadKO mouse femora with corresponding quantification of the cortical bone area fraction (Ct.Ar/Tt.Ar). N=5–15 mice per group, 4 months of age. ** p<0.01, *** p<0.001 compared to WT by Student’s t test.
Figure 2.
Figure 2.. Rad deletion results in altered mechanical properties
Quantification of mechanical properties from four-point bending analysis of mouse femora. N=13–15 mice per genotype, 4 months of age. *** p<0.001 compared to WT by Student’s t test.
Figure 3.
Figure 3.. In vitro osteoclast differentiation is enhanced in the absence of Rad
Representative images and quantification of tartrate-resistant acid phosphatase (TRAP) stained osteoclasts derived from spleen cells. The number of TRAP-positive multinucleated cells (MNCs, at least 3 nuclei) was counted in each well of a 24-well plate. N=3 animals per genotype, male, 2 months of age, *p<0.05 compared to WT by Student’s t test.
Figure 4.
Figure 4.. Modest decrease in osteoclast surface in the absence of Rad
Tartrate-resistant acid phosphatase (TRAP) staining of WT and RadKO distal femur thin sections and corresponding quantification of the percentage of the bone surface occupied by osteoclasts (Oc.S/BS). N=5–8 mice per group, 4 months of age. * p<0.05 compared to WT by Student’s t test.
Figure 5.
Figure 5.. Lower bone formation rate in Rad−/− femora
A) Representative images of calcein double labeling in distal femur trabecular bone of WT and RadKO mice (10X). B) Mineral apposition rate (MAR), mineralizing surface (MS/BS), and bone formation rate (BFR/BS) in the cortical bone (periosteal surface) and trabecular bone of WT and RadKO mice. N=5 mice per genotype, female, 4 months of age * p<0.05, ** p<0.01 compared to WT by Student’s t test.
Figure 6.
Figure 6.. Less mineralization in Rad−/− osteoblasts in vitro.
A) Representative immunoblot of WT and RadKO calvarial osteoblast lysates confirms Rad expression in these cells. N=3 isolations per genotype. B) Representative images of WT and RadKO primary calvarial osteoblasts stained for alkaline phosphatase activity after 7 days in osteogenic media. N=3 isolations per genotype. C) Representative images of Alizarin Red S staining of WT and RadKO primary osteoblast monolayers after 28 days in osteogenic media. Staining was quantified by solubilization of the stain in acetic acid, neutralization, and optical density measurement at 405 nm. N=3–4 isolations per genotype. ** p<0.01 compared to WT by Student’s t test. D) Osteoblast marker gene expression from microarray analysis of WT and RadKO primary osteoblasts. N=2 isolations per genotype. E) RT-PCR analysis of MGP expression confirms microarray result. N=3 isolations per genotype.
Figure 7.
Figure 7.. Loss of Rad confers an adipogenic phenotype
A) Representative images of WT and RadKO primary calvarial osteoblasts stained with Oil Red O after 14 days in mineralizing conditions. The number of ORO-positive cells was counted for 3 random fields per isolation. N=3–4 isolations per genotype. B) Von Kossa/MacNeal’s staining of thin sections from WT and RadKO distal femora. Number of adipocytes per 20X field and the average adipocyte diameter in pixels were quantified. N=5 mice per genotype, female, 4 months of age. * p<0.05, ** p<0.01 by Student’s t test.
Figure 8.
Figure 8.. No change in lean or fat mass percentage in Rad−/− mice
EchoMRI analysis of body composition of WT and RadKO mice indicates no change in lean or fat mass percentage. N=8 WT male, 9 RadKO male, 21 WT female, 11 RadKO female mice, 4 months of age.
Figure 9.
Figure 9.. Adipogenic induction results in lower endogenous Rad levels
A) Representative images of alkaline phosphatase activity in WT and RadKO primary osteoblast monolayers following 7 days in osteogenic (OM) or adipogenic (AM) media. N=3 isolations per genotype. B) RT-PCR analysis of MGP expression levels normalized to 18S RNA. RNA was isolated from WT primary osteoblasts after 3 days in growth (GM) or adipogenic (AM) media. N=3 isolations. C) Western blotting of WT osteoblast lysates after 7 days in growth (−) media or adipogenic (+) media. N=3 isolations. * p<0.05 by Student’s t test.

Similar articles

See all similar articles

Cited by 3 articles

Feedback