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

Michela Palmieri; Teenamol E. Joseph; Charles A. O’Brien; Horacio Gomez-Acevedo; Stavros C. Manolagas; Elena Ambrogini

doi:10.1371/journal.pone.0265893

Abstract

The scavenger receptor class B member 1 (SR-B1 or Scarb1) is a cell surface receptor for high density lipoproteins. It also binds oxidized low density lipoproteins and phosphocholine-containing oxidized phospholipids (PC-OxPL), which adversely affect bone homeostasis. Overexpression of a single chain form of the antigen-binding domain of E06 IgM–a natural antibody that recognizes PC-OxPL–increases trabecular and cortical bone mass in female and male mice by stimulating bone formation. We have previously reported that Scarb1 is the most abundant scavenger receptor for PC-OxPL in calvaria-derived osteoblastic cells. Additionally, bone marrow- and calvaria-derived osteoblasts from Scarb1 knockout mice (Scarb1 KO) are protected from the pro-apoptotic and anti-differentiating effects of OxPL. Previous skeletal analysis of Scarb1 KO mice has produced contradictory results, with some studies reporting elevated bone mass but another study reporting low bone mass. To clarify the role of Scarb1 in osteoblasts, we deleted Scarb1 specifically in cells of the osteoblast lineage using Osx1-Cre transgenic mice. We observed no difference in bone mineral density measured by DXA in either female or male Osx1-Cre;Scarb1^fl/fl mice compared to wild type (WT), Osx1-Cre, or Scarb1^fl/fl littermate controls. Additionally, microCT analysis of 6-month-old females and 7-month-old males did not detect any difference in trabecular or cortical bone mass between genotypes. These results indicate that expression of Scarb1 in cells of the osteoblast lineage does not play an important role in bone homeostasis and, therefore, it is not essential for the effects of PC-OxPL on these cells.

Citation: Palmieri M, Joseph TE, O’Brien CA, Gomez-Acevedo H, Manolagas SC, Ambrogini E (2022) Deletion of the scavenger receptor Scarb1 in osteoblast progenitors does not affect bone mass. PLoS ONE 17(3): e0265893. https://doi.org/10.1371/journal.pone.0265893

Editor: Xing-Ming Shi, Augusta University, UNITED STATES

Received: October 12, 2021; Accepted: March 9, 2022; Published: March 29, 2022

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This work was supported by the Biomedical Laboratory Research and Development Service of the Veterans Administration Office of Research and Development (1I01BX003901-01A2 to EA, 2I01BX001405-05A2 to SCM), the National Institutes of Health (P20 GM125503 to CAO, P01 AG13918 to SCM), the University of Arkansas for Medical Sciences Tobacco Funds and Translational Research Institute (239 G1-50893-01; 1UL1 RR-029884 to EA) and the University of Arkansas for Medical Sciences Barton Endowment funding (271 G1-51451-99 to EA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

The scavenger receptor class B member 1 (SR-B1 or Scarb1) is a glycosylated cell surface receptor for high density lipoproteins (HDL), most abudantly 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 them [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 mutations of Scarb1 have increased risk of adrenal insufficiency [6] and cardiovacular diseases, despite a significant increase in HDL-cholesterol concentration in the serum [7, 8]. In addition to the liver and steroidogenic organs, Scarb1 is expressed in many other tissues and cell types including monocytes/macrophages, adipocytes, endothelial and epithelial cells, 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 [9], and cancer growth and metastasis [7].

In addition to HDL, Scarb1 can bind, albeit with much lower affinity, to bovine serum albumin and advanced glycation end-product modified proteins, bacterial cell components such as lipopolysaccharides (LPS), apoptotic cells, and other lipoproteins including VLDL and LDL [1]. Moreover, Scarb1 is a receptor for oxidized low density lipoproteins (OxLDL) and phosphocholine-containing oxidized phospholipids (PC-OxPL) [1, 10, 11]. In osteoblasts, Scarb1 has been implicated in the uptake of OxLDL, cholesteryl ester, and estradiol [12, 13].

We have previously shown that physiological levels of PC-OxPL reduce bone mass. Specifically, we found that male and females transgenic mice expressing a single chain (scFv) form of the antigen-binding domain of E06 IgM (E06-scFv), which binds and neutralizes PC-OxPL, have increased trabecular and cortical bone mass at 6 months of age [14, 15] and are protected from the bone loss caused by a high fat diet or aging [14, 16]. The increase in bone mass in these mice is due mainly to an increase in osteoblast number and bone formation [14, 15]. These results have elucidated that PC-OxPL affect bone physiology throughtout life.

In addition to Scarb1, PC-OxPL is recognized by the scavenger receptor CD36 and by the toll-like receptors 2, 4 and 6 [10], all of which are expressed in osteoblastic cells [12, 17, 18], as well as in osteoclasts and bone marrow macrophages [10, 19]. Scarb1 is the most abundant scavenger receptor for PC-OxPL in calvaria-derived osteoblastic cells, as determined by qPCR, and silencing Scarb1 protects calvaria-derived osteoblastic cells from OxLDL-induced apoptosis [15]. Importantly, 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 [15]. Finally, Scarb1 KO mice have increased osteoblast number, bone formation rate, and high bone mass as well as histomorphometric similarities with E06-scFv transgenic mice [17, 20, 21]. Based on these findings, we hypothesized that Scarb1 is an essential mediator of the pro-apoptotic effects of PC-OxPL on osteoblasts and that E06-scFv prevents binding of PC-OxPL to Scarb1 on these cells, thereby reducing the pro-inflammatory actions of OxPL [10]. To determine the role of Scarb1 on osteoblasts, we deleted this receptor specifically in 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-OxPL on osteoblasts or bone.

Materials and methods

Animals

C57BL/6J (stock number 000664) and Scarb1 KO mice in the C57BL/6J background (stock number 003379) were obtained from the Jackson Laboratories. The mouse line harboring the Scarb1 conditional allele [C57BL/6N-Scrib<tm1c(NCOM)Mfgc>/Tcp] was created as part of the NorCOMM2 project with C57BL/6NScrib<tm1a(NCOM)Mfgc>/Tcp produced from NorCOMM ES cells [22] at the Toronto Centre for Phenogenomics. A neomycin phosphostransferase selection cassette was removed from the Scrib<tm1a(NCOM)Mfgc> allele (MGI:5513817) leaving behind floxed exons 2–8 (S1 Fig). We obtained this model from the Toronto Centre for Phenogenomics as frozen sperm, which was successfully used for in vitro fertilization in the UAMS Genetic Models Core facility. To delete Scarb1 in the entire osteoblast lineage, we crossed Scarb1 floxed mice with Osx1-Cre mice obtained from the Jackson laboratories (stock number 006361) [23]. The Osx1-Cre transgene becomes active at the earliest stages of osteoblast differentiation [23] and lineage-tracing studies have established that all osteoblasts and osteocytes are derived from progenitors labeled by the Osx1-Cre transgene [24].

We used a two-step breeding strategy to obtain the experimental animals. We initially crossed hemizygous Osx1-Cre transgenic mice with heterozygous Scarb1 floxed mice to generate heterozygous Scarb1 floxed mice with and without a Cre allele. Those mice were used in the second cross to generate the 3 control groups and the experimental mice: WT mice, 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 mice. All mice strains were fed a doxycycline-containing diet [Bio Serv doxycycline diet (S3888) 200 mg/kg (Bio Serv, Flemington, NJ, USA)] from conception until one week prior to the start of the second cross, at which time they were switched to regular chow to activate the Cre transgene [LabDiet 5K67 Mouse/Auto6F Diet (LabDiet, St. Louis, MO, USA)]. 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 by PCR using the following primer sequences: Cre-Fwd: 5’-GCTAAACATGCTTCATCGTCGG-3’, Cre-Rev: 5’-GATCTCCGGTATTGAAACTCCAGC-3’, product size 650bp; Scrib-WT-Fwd: 5’-AAAGAGGGCAGGTGCAGTAAGCGAAG-3’, Scrib-WT-Rev: 5’-TTTCAGTGACAGTGGGCTTCTCTGGG-3’, product size wild type 223bp, heterozyous 223 and 346 bp; Scrib_tm1c distal loxp-Fwd: 5’ GCGCAACGCAATTAATGATAAC-3’, Scrib_tm1c distal loxp-Rev: GTCCAAGACTCCCTCCAAACGCACG-3’, product size floxed 222 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) [15, 25]. 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 0.24% for BMD and 0.1% for the percentage of body fat.

Bone microarchitecture was measured using a micro-CT40 scanner (Scanco Medical AG, Bruettisellen, Switzerland) as previously described [14, 15]. We measured trabecular 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. The left femur was fixed using 10% Millonig’s Neutral Buffered Formalin with 5% sucrose (Leica Biosystems Inc., Buffalo Grove, IL, USA) whereas L5 was fixed in B-plus (BBC Biomedical, Mount Vernon, WA, USA). After fixation, bones were dehydrated in solutions containing progressively increasing ethanol concentrations and kept in 100% ethanol until analysis. While performing these studies, the mean coefficient of variation of the micro-CT phantom was monitored weekly and was 0.232%.

Histomorphometry

L1-L3 vertebra were dissected, placed in fixative (10% Millonig’s neutral buffered formalin with 5% sucrose), dehydrated with ethanol, and stored in 100% ethanol until embedding in methyl methacrylate (Sigma-Aldrich, St Louis, MO, USA). Five μm thick longitudinal sections of trabecular bone were stained for tartrate-resistant acid phosphatase (TRAP) with toluidine blue counterstaining to allow the measurement of static indices of osteoclast and osteoblast number. Histomorphometric determinations were made at L2, as previously described in a blinded fashion using Osteomeasure version 7 V4.3.0.0 (OsteoMetrics Inc. Decatur, GA, USA) [14]. Histomorphometric data are reported using the nomenclature recommended by the American Society for Bone and Mineral Research [26]. Osteoblasts were identified as teams of cells (≥ 2) overlying osteoid.

Culture of osteoblastic cells

Calvaria cells were isolated from either C57BL/6J andScarb1 KO or Osx1-Cre and Osx1-Cre;Scarb1^fl/fl littermate neonatal pups mice by sequential digestion with collagenase type 2 (Worthington, Columbus OH, USA, cat. CLS-2, lot 47E17554B) as previously described [27]. 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 the femoral diaphysis (after removing the proximal and distal ends) and culturing 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 manufacturer instructions. Triplicate cultures were analyzed for all assays. Oxidized LDL was obtained from Alfa Aesar (Alpha Aesar, Haverhill, MA, USA).

Genomic DNA isolation and Taqman assay to quantify gene deletion

After dissection, the distal and proximal ends of the 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. The tibial cortical bone was then placed in 14% EDTA solution for 8 days to allow decalcification. After decalcification, the bones were washed twice with water to eliminate EDTA, cut in 2–3 pieces, and placed in an Eppendorf tube to proceed with genomic DNA digestion and purification. Decalcified bone was then 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. Genomic DNA was then isolated by phenol/chloroform extraction and ethanol precipitation.

The spleen was harvested, cut in 3–4 pieces, and immediately frozen in liquid nitrogen. For genomic DNA extraction, the spleen was 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. Genomic DNA was extracted with the same method described above. A custom Taqman assay was obtained from Applied Biosystems to quantify the Scarb1 deletion efficiency in genomic DNA: Fwd 5’- GCACGTGTGCTTCATACAAATAGG-3’; Rev 5’- ACTCACACTAGCACAGACATCTCTA-3’; probe 5’- TCTGGCCCTAGCACTCT-3’. The relative genomic DNA amount was calculated using the ΔCt method using an assay for the transferrin receptor gene (Tfrc) as a control [28].

Quantitative PCR (qPCR)

To quantify gene expression in bone, after dissection, the distal and proximal ends of the right femur were removed, the bone marrow cells were flushed out from the bone cavity with PBS and the surfaces of the bone shafts were scraped with a scalpel to remove the periosteum. Cortical bone was stored at −80°C before RNA extraction. Total RNA was extracted from femoral bone or 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 S1 Table. Transcript levels were calculated by normalizing to the reference mRNA Mitochondrial Ribosomal Protein S2 (MRPS2) using the ΔCt method [28]

Bone remodeling markers

Bone remodeling markers, N-terminal propeptide (P1NP) and Tartrate-resistant acid phosphatase (TRAP) were measured with ELISA kit [cat# AC-33F1 for P1NP and cat # SB-TR103 for TRAP, Immunodiagnostic Systems, Boldon Colliery, UK] according to manufacturer’s instructions.

Statistics

No experimentally derived data were excluded. In Figs 3E, 3F and 4B, BMD, lean and fat mass at 7 months of age could not be measured in 1 mouse in the WT group and 1 mouse in the Osx1-Cre group for issues with imaging acquisition. In Fig 6A one vertebra from the Scarb1^fl/fl group could not be analyzed by microCT because it was damaged during the harvest. In Fig 8A, serum could not be collected for technical reasons from one WT mouse and therefore P1NP and TRAP could not be analyzed. Each figure legend includes the number of mice or samples used in each experiment. All data were collected and analyzed by personnel blinded to the identity of the samples. Single data points are shown in Figs 1, 2, 5–8 with mean ± standard deviation. In Figs 3 and 4 the data are shown as mean ± standard deviation.

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

(A) The expression of osteocalcin and alkaline phosphatase was quantified with RT-PCR in calvaria-derived osteoblasts from new born 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 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 unit.

https://doi.org/10.1371/journal.pone.0265893.g001

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Fig 2. Scarb1 gene was effectively deleted in bone.

Quantitative PCR (qPCR) of loxP-flanked genomic DNA isolated from tibial cortical bone and spleen. (A) 6 month-old female mice [WT (n = 9), Osx1-Cre (n = 10), Scarb1^fl/fl (n = 13), Osx1-Cre;Scarb1^fl/fl (n = 12)]. (B) 7-month-old male mice [WT (n = 6), Osx1-Cre (n = 15), Scarb1^fl/fl (n = 15), Osx1-Cre;Scarb1^fl/fl (n = 8)].

https://doi.org/10.1371/journal.pone.0265893.g002

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Fig 3. Deletion of Scarb1 in Osx1-Cre expressing cells does not affect weight, fat or lean mass in both sexes.

(A) Weight measurements, (B) fat mass and (C) lean mass at 3 and 6 months of age in female mice [WT n = 9; Osx1-Cre n = 10; Scarb1^fl/fl n = 13; Osx1-Cre;Scarb1fl/fl n = 12]. Adjusted p-values, calculated by repeated measures using two-way ANOVA are shown. (D) Weight measurements at 3 and 7 months of age in male mice [WT n = 6; Osx1-Cre n = 15; Scarb1^fl/fl n = 15; Osx1-Cre;Scarb1^fl/fl n = 8]. The measurements of total weight, by ANOVA repeated measures, indicated differences in the rate of weight gain between the genotypes. Further analysis, showed Scarb1^fl/fl mice gained the most weight (average 11.82 ± 3.23 g) between the two time points, while WT, Osx1-Cre and Osx1-Cre; Scarb1 ^fl/fl gained 8.08 ± 3.46 g, 7.76 ± 3.23 g and 9.09 ± 2.22 g respectively. (E) Fat mass and (F) lean mass of the same mice as in (D) [WT n = 5–6; Osx1-Cre n = 14–15; Scarb1^fl/fl n = 15; Osx1-Cre;Scarb1^fl/fl n = 8]. Data are shown as mean and standard deviation.

https://doi.org/10.1371/journal.pone.0265893.g003

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Fig 4. Deletion of Scarb1 in Osx1-Cre expressing cells does not affect BMD measured by DXA.

Determinations of femoral, spinal and total BMD by DXA in: (A) 3 and 6 month old females [WT n = 9; Osx1-Cre n = 10; Scarb1^fl/fl n = 13; Osx1-Cre;Scarb1^fl/fl n = 12] and in (B) 3-and 7-month-old males [WT n = 5–6; Osx1-Cre n = 14–15; Scarb1^fl/fl n = 15; Osx1-Cre;Scarb1^fl/fl n = 8]. Data are shown as mean and standard deviation. Adjusted p-values, calculated by repeated measures using two-way ANOVA, are shown.

https://doi.org/10.1371/journal.pone.0265893.g004

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Fig 5. Deletion of Scarb1 in Osx1-Cre expressing cells does not affect trabecular bone in female mice.

Micro-CT determination of trabecular bone architecture in 6-month-old female mice in the (A) vertebra and (B) femoral metaphysis [WT n = 9; Osx1-Cre n = 10; Scarb1^fl/fl n = 13; Osx1-Cre;Scarb1^fl/fl n = 12]. Data are shown as mean and standard deviation. Data analyzed by ANOVA, the p-values were adjusted using the Tukey’s pairwise comparison procedure. BV/TV, bone volume /total volume. Tb, trabecular.

https://doi.org/10.1371/journal.pone.0265893.g005

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Fig 6. Deletion of Scarb1 in Osx1-Cre expressing cells does not affect trabecular bone in male mice.

Micro-CT determination of trabecular bone architecture in 7-month-old male mice in (A) vertebra [WT n = 6; Osx1-Cre n = 15; Scarb1^fl/fl n = 14; Osx1-Cre;Scarb1^fl/fl n = 8] and (B) femoral metaphysis [WT n = 6; Osx1-Cre n = 15; Scarb1^fl/fl n = 15; Osx1-Cre;Scarb1^fl/fl n = 8]. Data are shown as mean and standard deviation. Data analyzed by ANOVA, the p-values were adjusted using the Tukey’s pairwise comparison procedure. BV/TV, bone volume /total volume. Tb, trabecular.

https://doi.org/10.1371/journal.pone.0265893.g006

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Fig 7. Deletion of Scarb1 using Osx1-Cre does not affect cortical bone in female or male mice.

Micro-CT determination of cortical bone and femoral length in (A) 6-month-old female [WT n = 9; Osx1-Cre n = 10; Scarb1^fl/fl n = 13; Osx1-Cre;Scarb1^fl/fl n = 12], and (B) 7-month-old male mice [WT n = 6; Osx1-Cre n = 15; Scarb1^fl/fl n = 15; Osx1-Cre;Scarb1^fl/fl n = 8]. Data are shown as mean and standard deviation. Data analyzed by ANOVA, the p-values were adjusted using the Tukey’s pairwise comparison procedure. BV/TV, bone volume /total volume. Tb, trabecular.

https://doi.org/10.1371/journal.pone.0265893.g007

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Fig 8. Deletion of Scarb1 using Osx1-Cre increases osteoblasts differentiation in vitro, but does not affect osteoblasts or osteoclasts in vivo.

(A) The expression of osteocalcin and alkaline phosphatase was quantified with RT-PCR in calvaria-derived osteoblasts from new born Osx1-Cre (n = 5) and Osx1-Cre;Scarb1^fl/fl mice (n = 5) cultured for 21 days. Transcripts were normalized to the housekeeping gene MRPS2. Data analyzed by student t-test. (B) Measurement of bone remodeling markers in the serum of 6-month-old female mice [WT n = 8; Osx1-Cre n = 10; Scarb1^fl/fl n = 13; Osx1-Cre;Scarb1^fl/fl n = 12], P1NP, N-terminal propeptide; TRAP, Tartrate-resistant acid phosphatase. (C) Gene expression of alkaline phosphatase, osteocalcin and TRAP in femoral cortical bone of 6-month-old female mice [WT n = 6–9; Osx1-Cre n = 10; Scarb1^fl/fl n = 10–13; Osx1-Cre;Scarb1^fl/fl n = 12]. Transcripts were normalized to MRPS2. Data analyzed by ANOVA, p-values >0.5. (D) Histomorphometric measurements of the trabecular vertebral bone surface (L2). [Osx1-Cre n = 5; Osx1-Cre;Scarb1^fl/fl n = 5]. Data analyzed by Student t-test. N.Ob, osteoblast number. B.Pm, bone perimeter. Ob.S, osteoblast surface. BS, bone surface. N.Oc, osteoclast number. Oc.S, osteoclast surface. Data are shown as mean and standard deviation.

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Statistical analyses were performed using GraphPad Prism (versions 7.0.4 and 8.0.1). Group mean values were compared by Student’s two-tailed t-test, ANOVA or ANOVA repeated measures 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 3 and 4 was performed using R (version 3.6).

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 [29]. For in vitro experiments, the number of replicates was sufficient to provide confidence in the measurements.

Study approval

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 CO₂ 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.

Results

Deletion of Scarb1 increases osteoblast differentiation and protects agains the anti-proliferative effects of OxLDL

Calvaria-derived osteoblasts obtained from Scarb1 KO mice and 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 suggested that Scarb1 could be an important mediator of the effects of OxLDL in osteoblasts by affecting osteoblast proliferation, in addition to apoptosis and differentiation, as previously reported [15].

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 receptor in cells targeted by the Osx1-Cre transgene. To do this, we crossed mice harboring a Scarb1 allele, in which exons 2 to 8 were flanked by loxP sites (Scarb1^fl/fl mice), with transgenic mice expressing Cre recombinase under the control of Osx1 regulatory elements (Osx1-Cre mice) [23]. The phenotype of the experimental mice Osx1-Cre;Scarb1^fl/fl mice was compared with 3 groups of littermate controls, WT, Osx1-Cre, and Scarb1^fl/fl mice. We first quantified the deletion of the Scarb1 gene in cortical bone and found that the levels of Scarb1 floxed exons were 63.4% and 53.1% lower in the tibia of 6-month-old female and 7-month-old male Scarb1^fl/fl;Osx1-Cre mice, respectively, as compared with WT littermate controls, confirming deletion in bone (Fig 2A and 2B). This level of deletion is comparable to the one otained in other experiments where we used Osx1-Cre mice to delete other genes in the osteoblast lineage [24, 30–32]. There was no change of Scarb1 levels in the spleen, confirming the specificity of the deletion (Fig 2A and 2B).

The Osx1-Cre transgene alone causes mild growth plate/cranial defects during early development. However, most of these effects normalize with age [33, 34]. All mice gained weight throughout the observational period and did not present any phenotypic differences. There was no difference in weight, fat mass, and lean mass at 3 and 6 months of age in female mice (Fig 3A–3C). In males, at 3 months of age, there was no difference in weight between the 4 genotypes (p>0.5) (Fig 3D). However, at 7 months, Scarb1^fl/fl male mice weighed 17.9% more than Osx1-Cre (p = 0.0003) and 20.1% more than Osx1-Cre;Scarb1^fl/fl mice (p = 0.0013) respectively. There was no difference in weight between all the other groups (i.e. WT vs Scarb1^fl/fl, WT vs Osx1-Cre, WT vs Osx1-Cre;Scarb1^fl/fl, Osx1-Cre mice vs Osx1-Cre;Scarb1^fl/fl). These results indicate that, in males, the Scarb1^fl/fl group had a higher rate of weight gain compared to Osx1-Cre and Osx1-Cre;Scarb1^fl/fl mice (details provided in the Figure legend). However, as in female mice, there was no difference in the percentage of fat mass and lean mass in male mice (Fig 3E and 3F).

Deletion of Scarb1 using Osx1-Cre does not affect bone mass

Bone mineral density by DXA was measured at 3 and 6 months of age in female mice and at 3 and 7 months of age in male mice. As shown in Fig 4, there was no difference in BMD among the Osx1-Cre;Scarb1^fl/fl male or female mice and the other three groups of littermate controls at any time point.

We also performed micro-CT analysis in 6-month-old females and 7-month-old males. In female mice, there was no difference in the trabecular bone in either the vertebra or the femur in the four groups (Fig 5A and 5B).

In males, there was an increase in trabecular bone of the vertebra in the Osx1-Cre mice and Osx1-Cre;Scarb1^fl/fl mice, however these two groups were not different, indicating that the increase in trabecular bone was due to the Osx1-Cre transgene and not to the deletion of Scarb1 (Fig 6A). In the trabecular bone of the femur, there was no change in bone volume/total volume (Fig 6B). Analysis of the microarchitecture at this site revealed that Osx1-Cre mice and Osx1-Cre;Scarb1^fl/fl mice had increased trabecular number and decreased trabecular separation, compared to WT and Scarb1^fl/fl mice. This increase in trabecular number was previously reported in Osx1-Cre mice [33]. However, as in the case of the cancellous bone in the vertebra, this microarchitectural change was related to the presence of Osx1-Cre and not to the deletion of Scarb1.

We have reported previously that Osx1-Cre mice exhibit decreased femoral cortical thickness due to decreased periosteal apposition [30, 35]. Analysis of the cortical bone showed a decrease in cortical thickness in both Osx1-Cre and Osx1-Cre;Scarb1^fl/fl female mice (Fig 7A), indicating that the decrease in cortical thickness was due to the effects of the Osx-1-Cre transgene, not to deletion of Scarb1. Femoral length was slightly decreased in Osx1-Cre;Scarb1^fl/fl female mice, compared to WT and Scarb1^fl/fl mice, but there were no differences from Osx1-Cre mice. We also could not detect any change in cortical thickness, total area, medullary area or femoral length in male mice (Fig 7B). Overall these results indicate that deletion of Scarb1 in cells of the osteoblast lineage does not alter bone mass.

Similarly to what observed in mice with global deletion of Scarb1 (Fig 1A), calvaria-derived osteoblasts obtained from Osx1-Cre;Scarb1^fl/fl mice and cultured for 21 days expressed more osteocalcin and alkaline phosphatase compared to calvaria cells extracted from Osx1-Cre mice (Fig 8A). However, consistent with the absence of changes in bone mass measured by BMD or microCT between control and experimental mice, there was no difference in circulating markers of bone remodeling, P1NP and TRAP (Fig 8B), or mRNA expression of osteoblast and osteoclast markers in cortical enriched femoral bone (Fig 8B). Moreover, deletion of Scarb1 in osteoblast progenitors did not affect the number of osteoblasts and osteoclasts in vertebral cancellous bone (Fig 8C). These results confirm that deletion of Scarb1 in cells of the osteoblast lineage, does not affect bone metabolism in vivo.

Discussion

The results presented herein show that deletion of Scarb1 in the entire osteoblast lineage does not affect bone mass in either female or male mice indicating that Scarb1 expression in osteoblasts does not play a major role in skeletal homeostasis.

Previous reports indicated that Scarb1 was implicated in bone metabolism. In vitro studies show that Scarb1 is responsible for selective uptake of cholesteryl esters and estradiol from HDL and LDL [17]. The uptake of these lipoproteins however was similar in osteoblastic cells from WT and Scarb1 KO mice indicating that this process was not the main role of Scarb1 in these cells [17, 21]. Scarb1 binds OxLDL and oxidized phospholipids [1, 12]. 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 [17, 21]. 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 [15]. 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 [36].

In vivo studies also yielded conflicting results. Some studies reported that Scarb1 KO mice have increased trabecular bone at 2 and 4 months of age associated with increased osteoblast surface, mineralized surface, and bone formation rate [17, 20, 21]. In contrast, another study has shown that Scarb1 KO mice have low bone mass and low bone formation compared to WT mice at 16 weeks of age, suggesting that Scarb1 is required for osteoblast differentiation and bone acquisition [37]. 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 scevenger receptor in osteoblasts that is known to bind PC-OxPL, which has deleterious effects on bone homeostasis mainly by affecting osteoblasts [10, 15]. 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 [15] and are protected from the deleterious effect of aging on bone [16]. E06-scFV increases osteoblast number and activity and decreases osteoblast apopostosis, indicating that PC-OxPL affects osteoblasts under physiological conditions [15].

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. It remains possible that OxPLs exert their anti-osteogenic effects indirectly via activation of Scarb1 in other cell types, possibly macrophages that produce anti-osteoblastogenic cytokines such as TNF-α [29]. In addition, PC-OxPL is recognized by other scavenger receptors, such as CD36, and toll-like receptors, such as TLR2, 4 and 6. Therefore, another potential explanation for the lack of a skeletal phenotype in the absence of Scarb1 in osteoblasts progenitors is that other scavenger receptors or toll-like receptors are involved in the deleterious effects of PC-OxPL on bone formation, either directly or indirectly, via other cell types such as macrophages. Thus, identification of the mechanisms by which PC-OxPL affect osteoblasts will require further investigation.

Supporting information

S1 Fig. Schematic representation of the Scrab1 floxed allele.

https://doi.org/10.1371/journal.pone.0265893.s001

(PDF)

S1 Table. TaqMan assays used for quantification of mRNA and genomic DNA by qPCR.

List of the TaqMan primers used in qPCR assays.

https://doi.org/10.1371/journal.pone.0265893.s002

(PPTX)

S1 Dataset.

https://doi.org/10.1371/journal.pone.0265893.s003

(XLSX)

Acknowledgments

We thank Stuart B Berryhill and Qiang Fu for technical assistance.

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

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

Retraction

Figures

Abstract

Introduction

Materials and methods

Animals

Imaging

Histomorphometry

Culture of osteoblastic cells

Genomic DNA isolation and Taqman assay to quantify gene deletion

Quantitative PCR (qPCR)

Bone remodeling markers

Statistics

Study approval

Results

Deletion of Scarb1 increases osteoblast differentiation and protects agains the anti-proliferative effects of OxLDL

Deletion of Scarb1 in Osx1-Cre-targeted cells does not affect body weight and fat mass

Deletion of Scarb1 using Osx1-Cre does not affect bone mass

Discussion

Supporting information

S1 Fig. Schematic representation of the Scrab1 floxed allele.

S1 Table. TaqMan assays used for quantification of mRNA and genomic DNA by qPCR.

S1 Dataset.

Acknowledgments

References