Deletion of the scavenger receptor Scarb1 in osteoblast progenitors and myeloid cells does not affect bone mass

Abstract

The scavenger receptor class B member 1 (SCARB1), encoded by Scarb1, is a cell surface receptor for high density lipoproteins, low density lipoproteins (LDL), oxidized LDL (OxLDL), and phosphocholine-containing oxidized phospholipids (PC-OxPLs). Scarb1 is expressed in multiple cell types, including osteoblasts and macrophages. PC-OxPLs, present on OxLDL and apoptotic cells, adversely affect bone metabolism. Overexpression of E06 IgM - a natural antibody that recognizes PC-OxPLs- increases cancellous and cortical bone at 6 months of age in both sexes and protects against age- and high fat diet- induced bone loss, by increasing bone formation. We have reported that SCARB1 is the most abundant scavenger receptor for OxPLs in osteoblastic cells, and osteoblasts derived from Scarb1 knockout mice (Scarb1 KO) are protected from the pro- apoptotic and anti-differentiating effects of OxLDL. Skeletal analysis of Scarb1 KO mice produced contradictory results, with some studies reporting elevated bone mass and others reporting low bone mass. To clarify if Scarb1 mediates the negative effects of PC-OxPLs in bone, we deleted it in osteoblast lineage cells using Osx1-Cre transgenic mice. Bone mineral density (BMD) measurements and micro-CT analysis of cancellous and cortical bone at 6 months of age did not reveal any differences between Scarb1 ^ΔOSX-l mice and their wild-type (WT), Osx1-Cre, or Scarb1 ^fl/fl littermate controls. We then investigated whether PC-OxPLs could exert their anti-osteogenic effects via activation of SCARB1 in myeloid cells by deleting Scarb1 in LysM-Cre expressing cells. BMD measurements and micro-CT analysis at 6 months of age did not show any differences between Scarb1 ^ΔLysM mice and their WT, LysM-Cre, or Scarb1 ^fl/fl controls. Based on this evidence, we conclude that Based on this evidence, we conclude that the adverse skeletal effects of PC-OxPLs in adult mice are not mediated by Scarb1 expressed in osteoblast lineage cells or myeloid cells.

Keywords: SCARB1; bone mass; macrophages; osteoblast; osteoclasts; oxidized phospholipids.

Fig 1.. Deletion of Scarb1 increases osteoblast differentiation and protects against the anti-proliferative effects of OxLDL.

(A) The expression of osteocalcin and alkaline phosphatase was quantified with RT-PCR in calvaria-derived osteoblasts from newborn C57BL6/J (n=5) and Scarb1 KO mice (n=2) cultured for 21 days. Transcripts were normalized to the housekeeping gene MRPS2. Data analyzed by Student’s t-test. (B) Proliferation was measured with Bromodeoxyuridine (BrDU) incorporation in bone marrow-derived osteoblasts from 4-5-month-old WT and Scarb1 KO mice (n=3/group) 3 days after direct addition to the cultures of vehicle or OxLDL (50 μg/ml). Data analyzed by ANOVA; p-values were adjusted using the Holm-Sidak multiple comparison procedure. Data are shown as individual values with mean and standard deviation. All measures were performed in triplicate cultures. ALP, Alkaline phosphatase. RLU, relative light units.

Fig 2.. Deletion of Scarb1 in Osx1-Cre expressing cells does not affect bone mass in females.

(A-C) Determinations of spinal, femoral and total BMD by DXA in 2- and 6-month-old mice [WT n=17; Scarb1^fl/fl n=9; Osx1-Cre n=14; Scarb1^ΔOSX1n=10] Data are shown as mean and standard deviation. Adjusted p-values, calculated by repeated measures using two-way ANOVA, are shown. BMD: bone mineral density. (D-F) Micro-CT analysis of cancellous bone in vertebra and femoral metaphysis, and cortical bone at midshaft in 6-month-old mice [WT n=17; Scarb1^fl/fl n=9; Osx1-Cre n=13; Scarb1^ΔOSX1 n=10]. Data analyzed by 2-way ANOVA; the p-values were adjusted using the Tukey’s pairwise comparison procedure. BV/TV, bone volume /total volume. NS: not significant, p > 0.05

Fig 3.. Deletion of Scarb1 in Osx1-Cre expressing cells does not affect bone mass in males.

(A-C) Determinations of spinal, femoral and total BMD by DXA in 2- and 6-month-old mice [WT n=11; Scarb1^fl/fl n=14; Osx1-Cre n=12; Scarb1^ΔOSX1 n=12]. Data are shown as mean and standard deviation. Adjusted p-values, calculated by repeated measures using two-way ANOVA, are shown. BMD: bone mineral density. (D-F) Micro-CT analysis of cancellous bone in vertebra and femoral metaphysis, and cortical bone at midshaft in 6-month-old mice [WT n=11; Scarb1^fl/fl n=14; Osx1-Cre n=12; Scarb1^ΔOSX1n=12]. Data analyzed by 2-way ANOVA; the p-values were adjusted using the Tukey’s pairwise comparison procedure. BV/TV, bone volume /total volume. . NS: not significant, p > 0.05

Fig 4.. Deletion of Scarb1 in LysM-Cre expressing cells does not affect bone mass in females.

(A-C) Determinations of spinal, femoral and total BMD by DXA in 4- and 6-month-old mice [WT n=12; Scarb1^fl/fl n=8; LysM-Cre n=15; Scarb1^ΔLysM n=12]. Data are shown as mean and standard deviation. Adjusted p-values, calculated by repeated measures using two-way ANOVA, are shown. BMD: bone mineral density. (D-F) Micro-CT analysis of cancellous bone in vertebra and femoral metaphysis, and cortical bone at midshaft in 6-month-old mice [WT n=12; Scarb1^fl/fl n=8; LysM-Cre n=15; Scarb1^ΔLysM n=12]. Data analyzed by 2-way ANOVA; the p-values were adjusted using the Tukey’s pairwise comparison procedure. BV/TV, bone volume /total volume. NS: not significant, p > 0.05.

Fig 5.. Deletion of Scarb1 in LysM-Cre expressing cells does not affect bone mass in males.

(A-C) Determinations of spinal, femoral and total BMD by DXA in 4- and 6-month-old mice [WT n=14; Scarb1^fl/fl n=14; LysM-Cre n=11; LysM-Cre; Scarb1^fl/fl n=16]. Data are shown as mean and standard deviation. Adjusted p-values, calculated by repeated measures using two-way ANOVA, are shown. BMD: bone mineral density. (D-F) Micro-CT analysis of cancellous bone in vertebra and femoral metaphysis, and cortical bone at midshaft in 6-month-old males [WT n=14; Scarb1^fl/fl n=14; LysM-Cre n=11; LysM-Cre; Scarb1^fl/fl n=16]. Data analyzed by 2-way ANOVA; the p-values were adjusted using the Tukey’s pairwise comparison procedure. BV/TV, bone volume /total volume. NS: not significant, p > 0.05.