Deletion of the scavenger receptor Scarb1 in osteoblast progenitors and myeloid cells does not affect bone mass
Michela Palmieri, Teenamol E. Joseph, Horacio Gomez-Acevedo, Ha-Neui Kim, Stavros C. Manolagas, Charles A. O’Brien, Elena Ambrogini, Joseph Roberts, Joseph Roberts, Joseph Roberts
TL;DR
Deleting the Scarb1 gene in bone-forming and immune cells does not change bone mass in mice.
Contribution
Shows that Scarb1 in osteoblast and myeloid cells does not mediate the negative effects of PC-OxPLs on bone.
Findings
Deleting Scarb1 in osteoblast lineage cells had no effect on bone mineral density or bone structure.
Deleting Scarb1 in myeloid cells also showed no changes in bone mass or density.
PC-OxPLs' negative effects on bone are likely mediated by other receptors or pathways.
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.…
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Fig 5- —http://dx.doi.org/10.13039/100000738U.S. Department of Veterans Affairs
- —NIGMS
- —http://dx.doi.org/10.13039/100008519University of Arkansas for Medical Sciences
- —http://dx.doi.org/10.13039/100008519University of Arkansas for Medical Sciences
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Taxonomy
TopicsAtherosclerosis and Cardiovascular Diseases · Antioxidant Activity and Oxidative Stress · Bone Metabolism and Diseases
Introduction
The scavenger receptor class B member 1 (SCARB1), encoded by Scarb1 in mice, is a glycosylated cell surface receptor for high density lipoproteins (HDL), most abundantly expressed in the liver and in steroidogenic tissues such as adrenal glands, ovaries, and testis [1]. SCARB1 is responsible for the uptake of cholesteryl esters from HDL, as well as efflux of cellular cholesterol to HDL [2,3]. In the liver, clearance of HDL cholesteryl esters is essential for anti-atherogenic reverse cholesterol transport [4] and bile acid production, whereas in the adrenal glands it is essential for optimal glucocorticoid generation [5]. Consistent with this, patients with heterozygous loss-of-function mutation of SCARB1 have increased risk of adrenal insufficiency [6] and cardiovascular diseases, despite a significant increase in HDL-cholesterol concentration in the serum [7,8]. SCARB1 is expressed in many other tissues and cell types including adipocytes, endothelial and epithelial cells, monocytes/macrophages, osteoblasts [9], and has been found to be important in many other processes such as regulation of platelet physiology [5] inflammation [7], regulation of bacterial invasion into cells [10], and cancer growth and metastasis [7].
In addition to HDL, SCARB1 can bind, albeit with lower affinity, to low-density lipoproteins (LDL). In endothelial cells, SCARB1 mediates LDL transcytosis and promotes atherosclerosis [11]. Moreover, SCARB1 is a receptor for very-low-density lipoproteins (VLDL), lipoprotein(a) – Lp(a)-, which is primary carrier of oxidized phospholipids [1,12,13], oxidized low-density lipoproteins (OxLDL) and phosphocholine-containing oxidized phospholipids (PC-OxPLs) [1,14,15]. In osteoblasts, SCARB1 has been implicated in the uptake of OxLDL, cholesteryl ester, and estradiol [12,16].
We have previously shown that physiological levels of PC-OxPLs reduce bone mass. Specifically, we found that both male and female transgenic mice expressing a single-chain variable fragment (scFv) of the antigen-binding domain of the natural E06 IgM antibody (E06-scFv), which binds and neutralizes PC-OxPLs, exhibited increased cancellous and cortical bone mass at 6 months of age [9], and were protected from the bone loss caused by a high fat diet or aging [17,18]. These mice display increased osteoblast number and bone formation rate at both cancellous and cortical sites, reduced osteoblast apoptosis in vivo, and decreased osteoclast number in vertebral bone throughout life [9,17,18].
In addition to SCARB1, PC-OxPL is recognized by the scavenger receptor CD36 and by the toll-like receptors 2, 4 and 6 [14], all of which are expressed in osteoblastic cells [12,19,20], as well as in osteoclasts and bone marrow macrophages [14,21]. We have previously shown that Scarb1 is the most abundant scavenger receptor for PC-OxPLs in calvaria-derived osteoblastic cells, as determined by qPCR, and silencing Scarb1 protects calvaria-derived osteoblastic cells from OxLDL-induced apoptosis [9]. Importantly, our studies indicated that both marrow-derived and calvaria-derived osteoblasts from mice with germline deletion of Scarb1 (Scarb1 KO mice) are protected from the pro-apoptotic and anti-differentiating effects of OxLDL [9]. Some studies, but not all, have shown that Scarb1 KO mice have increased osteoblast number, bone formation rate, and high bone mass as well as histomorphometric similarities with E06-scFv transgenic mice [19,22,23]. However, the mechanisms by which PC-OxPLs affect bone mass remain unclear. It is unknown if PC-OxPLs exert their deleterious effects on osteoblasts directly, by binding to scavenger receptors such as SCARB1, or indirectly by acting on other cells.
We hypothesized that SCARB1 is an essential mediator of the pro-apoptotic effects of PC-OxPLs on osteoblasts and that E06-scFv prevents the binding of PC-OxPLs to SCARB1 on these cells thus reducing the pro-inflammatory actions of OxPLs [14]. Therefore, we postulated that deleting Scarb1 in cells of the osteoblast lineage would increase osteoblastogenesis and recapitulate the bone phenotype seen in both female and male E06-scFv transgenic mice at 6 months of age, fed a chow diet [9]. To this end, we deleted Scarb1in cells of the osteoblast lineage using an Osx1-Cre transgene and analyzed the bone phenotype in adult female and male mice. We show that deletion of Scarb1 using Osx1-Cre mice did not affect bone mass or architecture, suggesting that SCARB1 is dispensable for osteoblast differentiation and function and does not mediate the deleterious effects of PC-OxPLs on osteoblasts or bone.
We then tested the alternative hypothesis that PC-OxPLs may exert their anti-osteogenic effects via activation of SCARB1 in macrophages, possibly leading to increased production of anti-osteoblastogenic cytokines, such as TNF-α [24]. To this end, we deleted Scarb1 specifically in myeloid cells using LysM-Cre mice. Lack of Scarb1 in myeloid progenitors and their descendants also did not alter bone mass in vivo.
We conclude that Scarb1 expression in either osteoblasts or osteoclasts is not involved in bone homeostasis and, therefore, Scarb1 expressed by these cells is not a major mediator of the deleterious effects of PC-OxPLs on bone.
Materials and methods
Animals
C57BL/6J (stock number 000664) and Scarb1 KO mice (stock number 003379) were obtained from the Jackson Laboratories. The mouse line harboring the Scarb1 conditional allele was kindly provided by Philip Shaul, University of Texas Southwestern Medical Center, Dallas, TX [11]. To delete Scarb1 in the entire osteoblast lineage, we crossed Scarb1 floxed mice with Osx1-Cre transgenic mice obtained from the Jackson laboratories (stock number 006361) [25]. The Osx1-Cre transgene becomes active at the earliest stages of osteoblast differentiation [25] and lineage-tracing studies have established that all osteoblasts and osteocytes are derived from progenitors labeled by the Osx1-Cre transgene [26]. We have used this strain extensively for conditional gene inactivation in osteoblast-lineage cells [26–36].
We used a two-step breeding strategy to obtain experimental animals. To delete Scarb1 in the osteoblast lineage we initially crossed hemizygous Osx1-Cre transgenic mice with homozygous Scarb1 floxed mice to generate heterozygous Scarb1 floxed mice with and without a Cre allele. Those mice were used in a second cross to generate the three control groups and the experimental mice: WT, Osx1-Cre, mice homozygous for the Scarb1-floxed allele, hereafter referred to as Scarb1^fl/fl^, and the experimental mice Osx1-Cre; Scarb1^fl/fl^ hereafter referred as Scarb1^ΔOSX1^). All mice from the first cross were fed a doxycycline-containing diet (BioServ doxycycline diet (S3888) 200 mg/kg (Bio Serv, Flemington, NJ, USA). Mice of the second cross were fed a doxycycline-containing diet from conception until weaning, at which time they were switched to regular chow [LabDiet 5K67 Mouse/Auto6F Diet (LabDiet, St. Louis, MO, USA)] to activate the Cre transgene.
To delete Scarb1 in the entire myeloid lineage, Scarb1^fl/fl^ were crossed with LysM-Cre mice, purchased from Jackson Laboratories (stock number 004781) [37]. All mice were in the C57BL/6 background. We used the same two-step breeding strategy described above to obtain mice lacking Scarb1 in the myeloid lineage. This generated the three control groups: WT, LysM-Cre, and Scarb1^fl/fl^ and the experimental mice: LysM-Cre; Scarb1^fl/fl^, hereafter referred as Scarb1^ΔLysM^). All mice were fed regular chow diet (LabDiet 5K67 Mouse/Auto6F Diet).
Mice were group housed under specific pathogen-free conditions and maintained at a constant temperature of 23°C, in a 12:12-hour light-dark cycle; they had ad libitum access to diet and water.
We genotyped the offspring of both lines by PCR using the following primer sequences: Cre-UP2: 5’-GCTAAACATGCTTCATCGTCGG-3’, Cre-DN2: 5’-GATCTCCGGTATTGAAACTCCAGC-3’, product size 650 bp; Scarb1-Fwd: 5’-GCACAGAGGACCCAACAGCGCACAAAATGG-3’, Scarb1-Rev: 5’-GCTGGGATTCAAGGTGTGTGCCACCACTAC-3’, primer for detection of Cre recombination: 5’-AGACCAATGGACCCTGTGCTTGGAGTGAGC-3’, product size wild type 149 bp; floxed allele before recombination 188 bp, floxed allele after recombination 315 bp.
Imaging
Bone mineral density (BMD) measurements and percentages of lean and fat body mass were calculated by dual-energy X-ray absorptiometry (DXA) of sedated mice (2% isoflurane) using a PIXImus densitometer (GE Lunar) [9,38]. The mean coefficient of variation, calculated using a proprietary phantom scanned at the beginning of each session for the mice used in this study, was 0.38% for BMD and 0.15% for the percentage of body fat.
Bone microarchitecture was measured using VivaCT 80 scanner (Scanco Medical AG, Bruettisellen, Switzerland). We measured cancellous bone at the fifth lumbar vertebra (L5) and left femur and the cortical bone at the left femoral diaphysis (midpoint of the bone length as determined at scout view). Briefly, bones were dissected and cleaned from soft tissues. L5 and the left femur were fixed using 10% Millonig’s Neutral Buffered Formalin (Leica Biosystems Inc., Buffalo Grove, IL, USA) with 0.5% sucrose. After fixation, bones were dehydrated in solutions containing progressively increasing ethanol concentrations and kept in 100% ethanol until analysis. For the scan, vertebrae and femora were loaded into scanning tubes and scanned at 10um nominal isotropic voxel size, 500 projections (FOV/Diameter 31.9 mm E = 70 kVp, I = 114 µA, 8W, Integration time 200ms and threshold 200 mg/cm^3^), and integrated into 3-D voxel images (1024x1024-pixel matrices for each individual planar stack). During the conduct of these studies, the mean coefficient of variation of the micro-CT phantom was performed weekly and was 0.16%. The entire vertebral body was scanned to obtain a number of slices ranging between 310 and 330. Femora were scanned from the distal epiphysis to the mid-diaphysis to obtain a number of slices ranging between 750 and 810. For the analysis, two-dimensional evaluation of cancellous bones was performed on contours of the cross sectional acquired images; primary spongiosa and cortex were excluded. On the vertebral body, contours were drawn from the rostral to the caudal growth plate to obtain 220–250 slices (10µm/slice) and bone outside the vertebral body plate was excluded. The evaluation of the cancellous bone at the distal femur was performed on contours drawn from the distal metaphysis to the diaphysis to obtain 151 slices (10 µm/slice). All cancellous bone measurements were made by drawing contours every 10 to 20 slices; voxel counting was used for bone volume per tissue volume measurements and sphere filling distance transformation indices were used for cancellous microarchitecture with a threshold value of 285, without pre-assumptions about the bone shape as a rod or plate.
Two-dimensional evaluation of cortical bone in femur was performed at mid-diaphysis. Contours were drawn at mid-diaphysis to obtain 40 slices (10µm/slice) with a threshold unit of 260 for cortical thickness. A Gaussian filter (sigma = 1.2, sigma = 0.8, support = 1) was applied to all analyzed scans (cancellous and cortical bone respectively) to reduce signal noise.
Culture of osteoblastic cells
Calvaria cells were isolated from C57BL/6J and Scarb1 KO littermate neonatal mice by sequential digestion with collagenase type 2 (Worthington, Columbus OH, USA, cat. CLS-2, lot 47E17554B) as previously described [39]. The cells were cultured in α-MEM medium (Invitrogen, Carlsbad, CA, USA, cat. 11900–0.24) containing 10% Premium Select fetal bovine serum (FBS) (Atlanta Biologicals, Flowery Branch, GA, USA), 1% penicillin/streptomycin/glutamine (PSG) and L-Ascorbic Acid Phosphate (Wako, Richmond, VA, USA cat. 013–12061) for twenty-one days. Gene expression was quantified by PCR as indicated below. Bone marrow cells were obtained by flushing cells from the femoral diaphysis (after removing the proximal and distal ends) and culturing them with α-MEM medium containing 10% Premium FBS, 1% PSG and 1 mM L-Ascorbic Acid Phosphate up to 80% confluence. Proliferation was measured by BrdU incorporation with the Cell Proliferation ELISA kit from Roche Diagnostics (Roche Diagnostics, Indianapolis, IN, USA) following the manufacturer’s instructions. Triplicate cultures were analyzed for all assays. Oxidized LDL (OxLDL) was obtained from Alfa Aesar (Alpha Aesar, Haverhill, MA, USA).
RNA isolation, cDNA synthesis, and Real time Quantitative PCR (RT-qPCR)
Total RNA was extracted from calvaria cells with Trizol (Thermo Fisher Scientific, Waltham, MA, USA) and purified with Direct-zol RNA Miniprep (Zymo Research, Irvine, CA, USA cat. R2050) according to the manufacturer’s instructions. RNA was then quantified using a NanoDrop instrument (Thermo Fisher Scientific), and its integrity was verified by resolution on 0.8% agarose gels. Complementary DNA (cDNA) was reverse transcribed from 0.5 µg of total RNA extract using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA cat. 4368813) according to manufacturer’s instructions. PCR was performed using TaqMan Gene Expression Assays manufactured by Applied Biosystems, as listed in Supporting information S1 Table. Transcript levels were calculated by normalizing to the reference mRNA Mitochondrial Ribosomal Protein S2 (Mrps2) using the ΔCt method [40]. We used Mrps2 as a housekeeping gene because it encodes a mitochondrial ribosomal protein which displays little or no change in expression under a variety of conditions.
Genomic DNA isolation and Taqman assay to quantify gene deletion
To quantify the gene deletion in Scarb1^ΔOSX1^ mice, we obtained genomic DNA from cortical bone of Osx1-Cre and Scarb1^ΔOSX1^. Briefly, after dissection, the distal and proximal ends of femur and tibia were removed, and bone marrow cells were flushed out from the bone cavity with PBS. The surfaces of the bone shafts were scraped with a scalpel to remove the periosteum. Femoral and tibial cortical bones were placed in 14% EDTA solution for 8 days to allow decalcification. After decalcification, the bones were washed twice with water to eliminate EDTA, cut into 2–3 pieces, and placed in an Eppendorf tube to proceed with genomic DNA digestion and purification. Decalcified bone was digested with proteinase K (Cat. P5850, Sigma-Aldrich, St. Louis MO, USA) (0.67 mg/ml proteinase K in 30mM TRIS pH 8.0, 200mM NaCl, 10mM EDTA, and 1% SDS) at 55° C overnight. Genomic DNA was isolated by phenol/chloroform extraction and ethanol precipitation. Spleen was used as negative control and was harvested, cut into 3–4 pieces, and immediately frozen in liquid nitrogen. For genomic DNA extraction, spleen fragments were digested with proteinase K (0.67 mg/ml in 30mM TRIS pH 8.0, 200mM NaCl, 10mM EDTA, and 1% SDS) at 55° C overnight and genomic DNA was isolated by phenol/chloroform extraction and ethanol precipitation.
To quantify gene deletion in Scarb1^ΔLysM^ mice, we obtained genomic DNA from macrophages of LysM-Cre and Scarb1^ΔLysM^. Briefly bone marrow cells were obtained by flushing femur and tibia and cultured in a-MEM with 10% FBS in the presence of 10ng/ml of M-CFS (R&D Systems rhM-CSF cat# 216-MC) for 24 hours. Non-adherent cells (myeloid lineage) were expanded for 3–4 days in the presence of 10ng/ml of M-CSF. At the end of the culture genomic DNA was extracted using the Qiagen QIAamp DNA mini kit (Cat. 51304, Qiagen, Germantown, MD, USA). Spleen was used as negative control as indicated above.
A custom TaqMan assay was obtained from Applied Biosystems to quantify the Scarb1 deletion efficiency in genomic DNA of both strains: Fwd 5’- GGACTGTGTGTGGGTGTGT’; Rev 5’- TTCTGTCTCTGGAGCAATCAATCTC-3’; probe 5’- CTGCCATGCTGAGTTTT-3’. The relative amount of genomic DNA was calculated with the ∆Ct method using an assay for the transferrin receptor gene (Tfrc) as a control, as listed in Supporting information S1 Table [40].
Statistics
No experimentally derived data were excluded. One of the female Osx1-Cre mice was not harvested with the rest of the mice because it died soon after the DXA measurement at 6 months of age. The micro-CT measurement was not performed in this mouse and therefore, the number of female mice in the Osx1-Cre group was 14 for Fig 2A-2C and in panels A-C in S2 Fig and 13 in Fig 2D-2F, S3 Fig and panels A-C in S5 Fig. In panel F of S5 Fig, the femoral length could not be measured in 1 male Scarb1^fl/fl^ mouse because the head of the femur was damaged during the harvest.
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 using three or six technical replicates. RLU, relative light units. OxLDL, oxidized low density lipoproteins.
Each legend includes the number of mice or samples used in each experiment. Single data points are shown in Figs 1, 2D-2F, 3D-3F, 4D-4F, 5D-5F and S1, S3-S6, S8-S10 Figs with mean ± standard deviation. In Figs 2A-2C, 3A-3C, 4A-4C, 5A-5C and S2 and S7 Figs, the data are shown as mean ± standard deviation.
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; Scarb1fl/fl n = 9; Osx1-Cre n = 14; Scarb1ΔOSX1n=10] Data are shown as mean and standard deviation. Adjusted p-values <0.05, calculated by repeated measures using two-way ANOVA, are shown in S2 Table. 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; Scarb1fl/fl n = 9; Osx1-Cre n = 13; Scarb1ΔOSX1 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.
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; Scarb1fl/fl n = 14; Osx1-Cre n = 12; Scarb1ΔOSX1 n = 12]. Data are shown as mean and standard deviation. Adjusted p-values <0.05, calculated by repeated measures using two-way ANOVA, are shown in S2 Table. 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; Scarb1fl/fl n = 14; Osx1-Cre n = 12; Scarb1ΔOSX1n=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.
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; Scarb1fl/fl n = 8; LysM-Cre n = 15; Scarb1ΔLysM n = 12]. Data are shown as mean and standard deviation. Adjusted p-values <0.05, calculated by repeated measures using two-way ANOVA, are shown in S2 Table. 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; Scarb1fl/fl n = 8; LysM-Cre n = 15; Scarb1ΔLysM 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.
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; Scarb1fl/fl n = 14; LysM-Cre n = 11; Scarb1ΔLysMn = 16]. Data are shown as mean and standard deviation.. 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; Scarb1fl/fl n = 14; LysM-Cre n = 11; Scarb1ΔLysM 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.
Statistical analyses were performed using GraphPad Prism (version 10). Group mean values were compared by Student’s two-tailed t-test or ANOVA as appropriate. When ANOVA indicated a significant effect, pairwise multiple comparisons were performed and the p-values adjusted using the Tukey’s pairwise comparison procedure or the Holm-Sidak method as appropriate. Statistical analysis for the data shown in Figs 2A-2C, 3A-3C, 4A-4C, 5A-5C and S2 and S7 Figs, was performed using ANOVA repeated measures by R (version 4.5). The p-value reported as not significant is > 0.05.
For the in vivo studies, the sample size was adequate to detect a difference of 1.2 standard deviations at a power of 0.8, and p < 0.05 [24]. For in vitro experiments, the number of replicates was sufficient to provide confidence in the measurements.
All data were collected and analyzed by personnel blinded to the identity of the samples. The Raw data are reported in the Supporting information S1 File.
Ethics
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal protocols were approved by the Institutional Animal Care and Use Committees of the University of Arkansas for Medical Sciences (Animal Use Protocol #3809) and the Central Arkansas Veterans Healthcare System (IACUC protocol # 1400199). Anesthesia was provided by inhalation of 2% Isofluorane. Euthanasia was performed by CO_2_ inhalation from a compressed gas tank at a displacement rate of 10% to 30% volume/minute until all movement ceased followed by an additional 1 minute in the chamber. Death was verified by lack of respiration and cervical dislocation or decapitation.
Results
Global deletion of Scarb1 increases osteoblast differentiation and protects against the anti-proliferative effects of OxLDL
The result of our published work strongly suggested that Scarb1 could be mediating the deleterious effect of oxidized phospholipids on osteoblastic cells in vitro [9] Herein, we further expand those studies and show that calvaria-derived osteoblasts obtained from Scarb1 KO mice, cultured for 21 days, expressed more osteocalcin and alkaline phosphatase compared to cells from WT mice (Fig 1A). Moreover, bone marrow-derived osteoblasts obtained from Scarb1 KO mice were protected against the decreased proliferation caused by OxLDL (Fig 1B). These results, together with previous published work [9] suggested that Scarb1 may mediate the effects of OxLDL in osteoblasts by affecting osteoblast proliferation, in addition to apoptosis and differentiation.
Deletion of Scarb1 in Osx1-Cre-targeted cells does not affect body weight and fat mass
To investigate the role of Scarb1 in osteoblasts we deleted this gene in cells targeted by the Osx1-Cre transgene. We crossed Scarb1^fl/fl^ mice with transgenic mice expressing Cre recombinase under the control of Osx1 (Osx1-Cre mice) regulatory elements [25]. The Scarb1^fl/fl^ mice, harboring the Scarb1 allele with loxP sites inserted in intron 1 and intron 3 [11], were provided by Philip Shaul at UT Southwestern. The phenotype of the experimental Scarb1^ΔOSX1^ mice was compared with WT, Osx1-Cre and Scarb1^fl/fl^ littermate controls.
We first quantified the deletion of the Scarb1 gene in cortical bone and found that the levels of Scarb1 floxed exons were 45.5% and 24.3% lower in the cortical bone of femur and tibia of 6-month-old female and male Scarb1^ΔOSX1^, respectively, as compared with Osx1-Cre littermate controls, confirming deletion in bone (S1 Fig). This level of deletion is comparable to that obtained in other experiments where we used Osx1-Cre mice to delete other genes in the osteoblast lineage [26–28,34]. There was no change of Scarb1 levels in the spleen, confirming the specificity of the deletion (S1 Fig).
The Osx1-Cre transgene alone causes mild growth plate/cranial defects during early development. However, most of these effects normalize with age [41,42]. All mice gained weight throughout the observational period and did not present any phenotypic differences.
Female mice carrying the Osx1-Cre transgene (Osx1-Cre and Scarb1^ΔOSX1^) had lower weight compared to WT and Scarb1^fl/fl^ mice (p = 0.0005) and gained less weight with time (p = 0.004). Mice carrying the Scarb1 floxed gene (Scarb1^fl/fl^ and Scarb1^ΔOSX1^) had higher weight (p = 0.047) but gained similar weight with time compared to WT and Osx1-Cre combined (panel A in S2 Fig and S3 Table). In addition, mice carrying the Osx1-Cre transgene (Osx1-Cre and Scarb1^ΔOSX1^) gained less fat mass and lost less lean mass compared to WT and Scarb1^fl/fl^ mice combined (p = 0.030) (panels B-C in S2 Fig and S3 Table). In males, there was no difference in weight between the four groups (panels D in S2 Fig). However, male mice carrying the Osx1-Cre transgene (Osx1-Cre and Scarb1^ΔOSX1^) had higher fat mass and lower lean mass compared to WT and Scarb1^fl/fl^ mice (p = 0.004) (panels E-F in S2 Fig and S3 Table).
We could not detect in either sex and at any time point, any difference between Scarb1^ΔOSX1^ and the other three groups, indicating that, overall, deletion of Scarb1 in osteoblast progenitors does not affect weight, fat mass or lean mass.
Deletion of Scarb1using Osx1-Cre does not affect bone mass
The bone phenotype was analyzed by BMD by DXA at 2 and 6 months of age, and micro-CT of vertebral and femoral cancellous bone, as well as femoral cortical bone, at 6-months of age.
In females, there were no differences in spinal BMD at baseline between the four groups. However, the Scarb1^ΔOSX1^mice gained less spinal BMD with time compared to the control groups (p interaction 0.005) (Fig 2A and S2 Table). At the femur, we found that the mice carrying the Osx1 transgene (Osx1-Cre and Scarb1^ΔOSX1^) had lower BMD compared to WT and Scarb1^fl/fl^ combined (p = 0.0007) but there were no changes in BMD with time between the 4 groups (Fig 2B and S2 Table). Similarly to the femur, the mice carrying the Osx1 transgene (Osx1-Cre and Scarb1^ΔOSX1^) had lower total BMD compared to WT and Scarb1^fl/fl^ mice (p = 0.0012) (Fig 2C and S2 Table). In addition, mice carrying the Scarb1 floxed allele (Scarb1^fl/fl^ and Scarb1^ΔOSX1^) gained less total BMD with time compared to WT and Osx1-Cre mice (p = 0.007) (Fig 2C and S2 Table). However, with the exception of the changes in BMD at the spine, we did not detect any difference in BMD between Scarb1^ΔOSX1^ mice and the other 3 groups, indicating that, deletion of Scarb1 in Osx1-Cre expressing cells does not affect bone mass.
The decrease in BMD at the spine in Scarb1^ΔOSX1^was not confirmed by micro-CT analysis. In fact, micro-CT measurements showed an increase in bone volume/total volume (BV/TV) of the vertebra in the Osx1-Cre mice and Scarb1^ΔOSX1^mice compared to WT, but there was no difference between Osx1-Cre and Scarb1^ΔOSX1^mice, indicating that the increase in cancellous bone was due to the Osx1-Cre transgene and not to the deletion of Scarb1 (Fig 2D). Similarly, in the cancellous bone of the femur there was an increase in BV/TV in the Osx1-Cre mice compared to WT and Scarb1^fl/fl^ and in Scarb1^ΔOSX1^mice compared to WT mice, but there was no difference between Osx1 and Scarb1^ΔOSX1^mice (Fig 2E).
Analysis of the cancellous bone microarchitecture revealed that, in vertebrae, there was an increase in trabecular number and trabecular thickness with decreased trabecular separation in Osx1-Cre mice compared to WT mice (panels A-C in S3 Fig**).** In the femur there was an increased trabecular number and decreased trabecular separation in the Osx1-Cre mice compared to WT and Scarb1^fl/fl^ and in Scarb1^ΔOSX1^ mice compared to WT mice and Scarb1^fl/fl^, but there was no difference between Osx1-Cre and Scarb1^ΔOSX1^mice (panels D-F in S3 Fig). The increase in trabecular number was previously reported in 6-week-old Osx1-Cre mice only when this parameter was corrected by body weight [41].
We have reported previously that Osx1-Cre mice exhibit decreased femoral cortical thickness due to decreased periosteal apposition [27,30]. In females, we observed decreased cortical thickness in the Osx1-Cre mice and in Scarb1^ΔOSX1^mice compared to WT and Scarb1^fl/fl^ but, in analogy to the cancellous bone, but there was no difference between Osx1-Cre and Scarb1^ΔOSX1^ mice (Fig 2F). Total area was decreased in Osx1-Cre and Scarb1^ΔOSX1^mice compared to Scarb1^fl/fl^ (panel A in S5 Fig), and there was no change in medullary area between the four groups (panel B in S5 Fig**).** Femoral length was increased in Scarb1^fl/fl^ compared to WT mice and decreased in Osx1-Cre and Scarb1^ΔOSX1^ compared to WT mice (panel C in S5 Fig**).** These results indicate that deletion of Scarb1 in osteoblast progenitors does not affect bone mass in female mice.
In male mice, there were no differences in spinal BMD between the four groups. (Fig 3A). Similarly to females, the mice carrying the Osx1 transgene (Osx1-Cre and Scarb1^ΔOSX1^) had lower femoral and total BMD compared to WT and Scarb1^fl/fl^ (p = 0.043 and p = 0.037 respectively) (Fig 3B,3C and S2 Table). In addition, mice carrying the Scarb1 floxed allele (Scarb1^fl/fl^ and Scarb1^ΔOSX1^) gained more femoral BMD with time compared to WT and Osx1-Cre mice (p = 0.046) (Fig 3B and S2 Table). We could not detect any change in BMD in Scarb1^ΔOSX1^ compared to the other 3 groups of controls.
The micro-CT analysis did not show any difference in the cancellous bone in either the vertebra or the femur between the four groups (Fig 3D,3E). Analysis of the cancellous bone microarchitecture revealed that, similar to femoral cancellous bone in female, there was an increased trabecular number and decreased trabecular separation in both vertebrae and femur in the Osx1-Cre mice compared to WT and Scarb1^fl/fl^ and in Scarb1^ΔOSX1^ mice compared to WT and Scarb1^fl/fl^ mice, but there was no difference between Osx1-Cre and Scarb1^ΔOSX1^ mice (S4 Fig).
In addition, we did not detect any changes in femoral cortical thickness (Fig 3F) or femoral length (panel F in S5 Fig**).** Total area was decreased in Scarb1^ΔOSX1^ mice compared to Scarb1^fl/fl^ mice (panel D in S5 Fig) and medullary area was decreased in Osx1-Cre and Scarb1^ΔOSX1^mice compared to Scarb1^fl/fl^ mice (panel E in S5 Fig),
Overall, these results indicate that deletion of Scarb1 in cells of the osteoblast lineage does not alter BMD, cancellous and cortical micro-CT parameters in male mice.
Deletion of Scarb1 in LysM-Cre-targeted cells does not affect body weight and fat mass
We then tested the alternative hypothesis that PC-OxPLs may exert their anti-osteogenic effects via activation of SCARB1 in macrophages, possibly leading to increased production of anti-osteoblastogenic cytokines, such as TNF-α [24]. To this end, we deleted Scarb1 specifically in monocytes and macrophages by crossing Scarb1^fl/fl^ mice with transgenic mice expressing the Cre recombinase under the control of LysM-Cre regulatory elements (LysM-Cre mice) [37]. The phenotype of the experimental Scarb1^ΔLysM^ mice was compared with WT, LysM-Cre and Scarb1^fl/fl^ littermate controls.
We quantified deletion of the Scarb1 gene in bone marrow-derived macrophages from 6-month-old female mice. We found that the levels of Scarb1 floxed exons were 88.7% lower in macrophages derived from Scarb1^ΔLysM^ female mice as compared with LysM-Cre littermate controls, confirming deletion of the gene (S6 Fig). This level of deletion is comparable to the one obtained in other experiments in which we used LysM-Cre mice to delete other genes in the myeloid/osteoclast lineage [31,43,44]. There were no changes in Scarb1 DNA levels in the spleen, confirming specificity of the deletion (S6 Fig).
In females, we did not observe any phenotypic differences between the four groups or differences in weight, fat mass, and lean mass at 4 and 6 months of age (panels A-C in S7 Fig). In males, mice carrying the LysM Cre transgene (LysM-Cre and Scarb1^ΔLysM^) had lower weight than WT and Scarb1^fl/fl^ mice (p = 0.021) and gained less weight with time (p = 0.003). Mice carrying the Scarb1 floxed transgene (Scarb1^fl/fl^ and Scarb1^ΔLysM^) had higher weight that WT and LysM-Cre (p = 0.015) but gained similar weight with time (panel D in S7 Fig and S3 Table). Scarb1^fl/fl^ mice alone had higher weight that the other three groups and gained more weight with time (p interaction 0.033) (panel D in S7 Fig ad S3 Table). There was no difference in lean mass and fat mass between the four groups (panels E-F in S7 Fig).
In summary, we could not detect in either sex and at any time point, any difference between Scarb1^ΔLysM^ and the other three groups, indicating that, overall, deletion of Scarb1 in myeloid progenitors does not affect weight, fat mass or lean mass.
Deletion of Scarb1 in myeloid cells does not affect bone mass
Bone mineral density (BMD) by DXA was measured at 4 and 6 months of age. In females, mice carrying the Scarb1 floxed allele (Scarb1^l/fl^ and Scarb1^Δ ΔLysM^) gained spinal BMD with time whereas WT and LysM-Cre mice lost BMD with time (p = 0.036); however, there were no differences in BMD at 6 months. (Fig 4A and S2 Table). No differences were found in femoral BMD between the four groups (Fig 4B and S2 Table). The analysis of total BMD indicated that WT mice lost BMD with time whereas the other three groups gained BMD with time (p interaction 0.014).
Micro-CT analysis, however, did not show any difference in vertebral and femoral cancellous bone and in the femoral cortical bone (Fig 4 D-4F; S8 Fig and panels A-C of S10 Fig).
In males, we did not observe any differences in spinal, femoral or total bone BMD between Scarb1^ΔLysM^ mice and the three groups of littermate controls (WT, LysM-Cre and Scarb1^fl/fl^) at any time point (Fig 5A-5C). Similarly, micro-CT analysis of 6-month-old male mice did not show any differences in vertebral cancellous bone and in femoral cancellous and cortical bone between Scarb1^ΔLysM^ mice and the controls (Fig 5D-5F, S9 Fig and panels D-F of S10 Fig).
Overall, these results indicate that deletion of Scarb1 in cells of the myeloid lineage does not alter bone mass.
Discussion
The results presented herein show that deletion of Scarb1 in either the entire osteoblast lineage or in the myeloid/osteoclast lineage does not affect bone mass in either female or male mice indicating that Scarb1 expression in osteoblasts or osteoclasts does not play a major role in skeletal homeostasis.
Previous reports implicated SCARB1 in bone metabolism. In vitro studies showed that, in osteoblasts, Scarb1 is responsible for selective uptake of cholesteryl esters and estradiol from HDL and LDL [19]. The uptake of these lipoproteins however was similar in osteoblastic cells from WT and Scarb1 KO mice, indicating that this process was not dependent on SCARB1 in these cells [19,23]. Osteoblastic cells derived from Scarb1 KO mice exhibit increased proliferation, increased alkaline phosphatase activity, enhanced matrix mineralization, and higher expression of the osteoblastogenic transcription factors Sp7 and Runx2 [19,23]. Silencing Scarb1 or the absence of Scarb1 in osteoblastic cells attenuates both osteoblast apoptosis and the decrease in differentiation of osteogenic precursors induced by OxLDL (9). Moreover, we show here that the absence of Scarb1 prevents suppression of proliferation by OxLDL. On the other hand, others have reported that Scarb1 is indispensable for HDL-induced proliferation of rat mesenchymal stem cells [45].
Earlier attempts to determine the bone phenotype of mice with global deletion of Scarb1 (Scarb1 KO) have produced conflicting results. Martineau and colleagues reported that Scarb1 KO mice have increased cancellous bone at 2 and 4 months of age and this increase was associated with increased osteoblast surface, mineralized surface, and bone formation rate with no changes in osteoclasts parameters [19,22,23]. In contrast, Tourkova et al. showed that Scarb1 KO mice have low bone mass at 16 weeks of age with low bone formation and decreased osteoclastogenesis compared to WT mice, suggesting that SCARB1 is required for osteoblast and osteoclast differentiation [46]. All those studies, however, were performed in global knockout mice.
The purpose of our study was to investigate the role of SCARB1 specifically in osteoblasts as a first step in determining whether it mediates the effects of PC-OxPL on this cell type. SCARB1 is the most abundant scavenger receptor in osteoblasts that is known to bind PC-OxPL, which has deleterious effects on bone homeostasis mainly by affecting osteoblasts [9,14]. Male and female mice expressing an antibody fragment (E06-scFv) that neutralizes PC-OxPL, exhibit increased cancellous and cortical bone mass at 6 months of age [9] and are protected from the deleterious effect of aging on bone [18]. E06-scFV increases osteoblast number and activity and decreases osteoblast apoptosis, indicating that PC-OxPL affects osteoblasts under physiological conditions [9]. The results presented herein, however, clearly demonstrate that expression of Scarb1 in osteoblasts is not required for bone mass acquisition and therefore is not an essential mediator of the deleterious effects of PC-OxPL in osteoblasts.
Therefore we tested the alternative possibility that PC-OxPLs may exert their anti-osteogenic effects via activation of SCARB1 in macrophages [24].
The role of SCARB1 in macrophages has been extensively studied in mouse models of atherosclerosis where it has been found to be both pro-atherogenic and anti-atherogenic [47]. Whereas earlier reports indicate that deletion of Scarb1 reduced the development of atherosclerosis [48], another study showed that deletion of this receptor in monocytes and macrophages worsened the extension of atherosclerotic lesions at earlier stages [49]. Specifically, Scarb1 deficiency in LysM-Cre expressing cells increased atherosclerosis by increasing the expression of the apoptosis inhibitor of macrophages (AIM) protein and consequently reducing macrophage apoptosis. Since macrophage apoptosis is associated with attenuation of early atherogenesis, this study suggests that decreased apoptosis is responsible for the increased accumulation of macrophages in the atherosclerotic plaque and expansion of the lesions. In more advanced atherosclerotic lesions SCARB1 present in macrophages binds and mediates the removal of apoptotic cells by efferocytosis; the absence of Scarb1, therefore, increases the numbers of apoptotic cells that did not get removed and increases the necrosis of the atherosclerotic plaques [50]. Moreover, Scarb1 expression in macrophages induces expression of transcription factor EB (TFEB), a master regulator of autophagy, which limits the necrosis and increases stability of atherosclerotic plaques; deletion of Scarb1, therefore, impairs autophagy and worsens the atherosclerosis at later stages [51]. Heretofore, the role of SCARB1 in macrophages has not been studied in bone.
The results presented herein show that deletion of Scarb1 in cells of the myeloid/osteoclast lineage does not affect bone mass in either female or male mice. This evidence indicates that Scarb1 expression in macrophages and osteoclasts does not play a major role in skeletal homeostasis. Therefore, SCARB1 in these cells is not a major mediator of the deleterious effects of PC-OxPLs on bone.
PC-OxPLs bind to other scavenger receptors, such as CD36, and toll-like receptors, such as TLR2, 4 and 6 which may mediate the deleterious effects in bone and compensate for the lack of SCARB1 [14]. Our previous work has indicated that PC-OxPLs decreases Wnt signaling, and this decrease may mediate the negative effects of PC-OxPLs in bone [18]. Thus, identification of the mechanisms by which PC-OxPLs affect bone homeostasis will require further investigation.
An important limitation of this study is that we did not challenge the mice with high fat diet or high cholesterol diet. It remains possible that SCARB1 in myeloid progenitors may play a role in inflammatory conditions with higher levels of oxidized phospholipids. This possibility will be pursued in future studies.
Finally, we acknowledge that the results presented herein do not explain the bone phenotype of the Scarb1 KO mice. Our results suggest that the effect of Scarb1 deletion on bone homeostasis is unlikely to be related to the expression in osteoblasts or osteoclasts alone. It is possible that the effects of Scarb1 are complex and mediated by multiple cell types.
Supporting information
S1 FigScarb1 gene was effectively deleted in bone.Quantitative PCR (qPCR) of genomic DNA isolated from femoral and tibial cortical bone and spleen in 6-month-old females [Osx1-Cre n = 4, Osx1-Cre; Scarb1^ΔOSX1^n=4] (A) and in 6-month-old males [Osx1-Cre n = 4, Scarb1^ΔOSX1^n=4] (B). Data are shown as mean and standard deviation. Data analyzed by unpaired T-test.(TIF)
S2 FigDeletion of Scarb1 in Osx1-Cre expressing cells does not affect weight, fat or lean mass in both sexes.Measurements of weight, fat mass and lean mass in 2- and 6-month-old females [WT n = 17; Scarb1^fl/fl^ n = 9; Osx1-Cre n = 14; Scarb1^ΔOSX1^n=10] (A-C) and 2- and 6-month-old males [WT n = 11; Scarb1^fl/fl^ n = 14; Osx1-Cre n = 12; Scarb1^ΔOSX1^n=12] (D-F). Data are shown as mean and standard deviation. Adjusted p-values <0.05, calculated by repeated measures using two-way ANOVA, are shown in S3 Table.(TIF)
S3 FigDeletion of Scarb1 in Osx1-Cre expressing cells does not affect microarchitecture of cancellous bone in female mice.Micro-CT analysis of cancellous bone architecture in 6-month-old females. (A) Trabecular number, (B) trabecular thickness, and (C) trabecular separation of vertebral cancellous bone. (D) Trabecular number, (E) trabecular thickness, and (F) trabecular separation of femoral cancellous bone [WT n = 17; Scarb1^fl/fl^ n = 9; Osx1-Cre n = 13; Scarb1^ΔOSX1^n=10]. Data are shown as mean and standard deviation. Data analyzed by 2-way ANOVA; the p-values were adjusted using the Tukey’s pairwise comparison procedure. Tb, trabecular.(TIF)
S4 FigDeletion of Scarb1 in Osx1-Cre expressing cells does not affect microarchitecture of cancellous bone in male mice.Micro-CT analysis of cancellous bone architecture in 6-month-old males. (A) Trabecular number, (B) trabecular thickness, and (C) trabecular separation of vertebral cancellous bone. (D) Trabecular number, (E) trabecular thickness, and (F) trabecular separation of femoral cancellous bone [WT n = 11; Scarb1^fl/fl^ n = 14; Osx1-Cre n = 12; Scarb1^ΔOSX1^n=12]. Data are shown as mean and standard deviation. Data analyzed by 2-way ANOVA; the p-values were adjusted using the Tukey’s pairwise comparison procedure. Tb, trabecular.(TIF)
S5 FigDeletion of Scarb1 using Osx1-Cre does not affect cortical bone in female or femoral length in female or male mice.Micro-CT analysis of cortical bone architecture and femoral length. (A) Total area, (B) Medullary area, (C) Femoral length in 6-month-old females [WT n = 17; Scarb1^fl/fl^ n = 9; Osx1-Cre n = 13; Scarb1^ΔOSX1^n=10]. (D) Total area, (E) Medullary area, (F) Femoral length in 6-month-old males [total area and medullary area WT n = 11; Scarb1^fl/fl^ n = 14; Osx1-Cre n = 12; Scarb1^ΔOSX1^n=12], [femoral length WT n = 11; Scarb1^fl/fl^ n = 13, Osx1-Cre n = 12; Scarb1^ΔOSX1^n=12]. Data are shown as mean and standard deviation. Data analyzed by 2-way ANOVA; the p-values were adjusted using the Tukey’s pairwise comparison procedure.(TIF)
S6 FigScarb1 gene was effectively deleted in bone marrow macrophages.Quantitative PCR (qPCR) of genomic DNA isolated from bone marrow-derived macrophages and spleen of 6-month-old female mice [LysM-Cre n = 3, Scarb1^ΔLysM^ n = 3]. Data are shown as mean and standard deviation. Data analyzed by unpaired T-test.(TIF)
S7 FigDeletion of Scarb1 in LysM-Cre expressing cells does not affect weight, fat or lean mass in both sexes.Measurements of weight, fat mass and lean mass in 4- and 6-month-old females [WT n = 12; Scarb1^fl/fl^ n = 8; LysM-Cre n = 15; Scarb1^ΔLysM^ n = 12] (A-C) and 4- and 6-month-old males [WT n = 14; Scarb1^fl/fl^ n = 14; LysM-Cre n = 11; Scarb1^ΔLysM^ n = 16]. Data are shown as mean and standard deviation. Adjusted p-values <0.05, calculated by repeated measures using two-way ANOVA, are shown in S3 Table.(TIF)
S8 FigDeletion of Scarb1 in LysM-Cre expressing cells does not affect microarchitecture of cancellous bone in female mice.Micro-CT analysis of cancellous bone architecture in 6-month-old females. (A) Trabecular number, (B) trabecular thickness, and (C) trabecular separation of vertebral cancellous bone. (D) Trabecular number, (E) trabecular thickness, and (F) trabecular separation of femoral cancellous bone [WT n = 12; Scarb1^fl/fl^ n = 8; LysM-Cre n = 15; Scarb1^ΔLysM^ n = 12]. Data are shown as mean and standard deviation. Data analyzed by 2-way ANOVA; the p-values were adjusted using the Tukey’s pairwise comparison procedure. Tb, trabecular.(TIF)
S9 FigDeletion of Scarb1 in LysM-Cre expressing cells does not affect microarchitecture of cancellous bone in male mice.Micro-CT analysis of cancellous bone architecture in 6-month-old males. (A) Trabecular number, (B) trabecular thickness, and (C) trabecular separation of vertebral cancellous bone. (D) Trabecular number, (E) trabecular thickness, and (F) trabecular separation of femoral cancellous bone [WT n = 14; Scarb1^fl/fl^ n = 14; LysM-Cre n = 11; Scarb1^ΔLysM^ n = 16]. Data are shown as mean and standard deviation. Data analyzed by 2-way ANOVA; the p-values were adjusted using the Tukey’s pairwise comparison procedure. Tb, trabecular.(TIF)
S10 FigDeletion of Scarb1 using LysM-Cre does not affect cortical bone in female or femoral length in female or male mice.Micro-CT analysis of cortical bone architecture and femoral length. (A) Total area, (B) Medullary area, (C) Femoral length in 6-month-old females [WT n = 12; Scarb1^fl/fl^ n = 8; LysM-Cre n = 15; LysM-Cre; Scarb1^fl/fl^ n = 12], (D) Total area, (E) Medullary area, (F) Femoral length in 6-month-old males [WT n = 14; Scarb1^fl/fl^ n = 14; LysM-Cre n = 11; Scarb1^ΔLysM^ n = 16]. Data are shown as mean and standard deviation. Data analyzed by 2-way ANOVA; the p-values were adjusted using the Tukey’s pairwise comparison procedure.(TIF)
S1 TableTaqMan assays.List of the TaqMan primers used for quantification of mRNA and genomic DNA by qPCR.(DOCX)
S2 TableList of adjusted p values <0.05 for the indicated figures.(DOCX)
S3 TableList of adjusted p values <0.05 for the indicated figures.(DOCX)
S1 FileRaw data for all the Figures and Supporting information present in the manuscript.(XLSX)
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