GsαR201C and estrogen reveal different subsets of bone marrow adiponectin expressing osteogenic cells

Mice generation

All studies were performed in compliance with relevant Italian laws and Institutional guidelines and all procedures were IACUC approved.

To generate the Rosa26-lsl-GsαR201C vector, the SalI/HindIII cassette was excised from the R201C rat Gsα cDNA, which included a hemagglutinin (HA) epitope as a flag (ATCC 63317, GenBank M12673),32 and inserted it into the pBigT vector (Addgene #21270), which contained a splicing acceptor site of adenovirus (SA), a stop region, including a neo cassette downstream to the PGK promoter and, downstream, a triple SV40 poly-adenylation sequence. The stop cassette was flanked by two loxP sites and followed by the bovine growth hormone poly-adenylation signal (BGHpA). The PacI and AscI restriction sites, placed 5’ to the SA and 3’ to the BGHpA, respectively, were used to subclone the functional sequence into the pRosa26PA plasmid (Addgene #21271), which included the Rosa26 sequences required for targeted recombination into the murine Rosa26 locus. R26-lsl-GsαR201C mice were generated by electroporating the R26-lsl-GsαR201C vector into murine ES CK35 (129/Sv Pas strain) cells. Upon electroporation, mouse ES cells were selected by neo screening, and 216 neo resistant clones were analyzed by multiple PCRs and sequencing. Two positive ES clones were implanted in C57Bl/6 N blastocysts, which, in turn were transferred into surrogate B6CBAF1 female mice. Mouse chimeras were backcrossed with C57Bl/6 N and F1 animals were genotyped by PCR. Heterozygous F1 mice were obtained from both founder clones. Two lines were serially backcrossed and showed regular Mendelian inheritance of the transgenic cassette.

Homozygous (homo) R26-lsl-GsαR201C mice (Rosa26) were crossed with heterozygous (het) Adipoq-Cre mice (#028020, The Jackson Laboratory); the resulting progeny were crossed again with Rosa26 mice to generate Adiponectin-Cre(het);R26-lsl-GsαR201C(homo) (Adq-GsαR201C) mice, which expressed the mutant form of Gsα in adipogenic cells.

To generate Adq-mTmG and Adq-mTmG;GsαR201C lineage reporter mice, we crossed heterozygous Adq-Cre and double heterozygous Adq-GsαR201C mice with homozygous loxP-mT-pA-loxP-mG-pA (mTmG) mice (#007676 The Jackson Laboratory). The triple heterozygous Adq-mTmG;GsαR201C mice harbor one R26 allele with GsαR201C transgene while the other R26 allele contains the mTmG transgene.

All mice were maintained in cabin-type isolators at standard environmental conditions (temperature 22–25 °C, humidity 40%–70%) with 12:12 h dark/light photoperiod. Food and water were provided ad libitum. Mice were genotyped by using the oligonucleotides listed in Table 1.

Table 1 Sequence of primers used for genotyping and qPCRX-ray analysis and micro-CT scanning

Radiographic analyses were performed on femora and tibiae using Faxitron MX-20 Specimen Radiography System (Faxitron X-ray Corp., Wheeling, IL, USA) set at 24–25 kV for 6–8 s with Kodak MIN-R2000 18 × 24 films.

For micro-CT scanning, tibiae were placed within a plastic tube, mounted onto the instrument rotational stage and scanned at 8 μm voxel size using 80 kV, 50 μA X-ray settings and a 1 mm aluminum filter with exposure time of 100 ms per frame, with a Bruker SkyScan 1275 micro-CT (Micro Photonic, Allentown, PA, USA). Three-dimensional reconstruction was performed with Bruker’s NRecon software and visualization occurred using Bruker’s DataViewer and CTscan software programs.

Histology

Mice were euthanized by carbon dioxide inhalation and skeletal segments were dissected and processed for either paraffin embedding, methylmethacrylate (MMA) or gelatin embedding.

For paraffin embedding, samples were fixed with 4% formaldehyde in PBS pH 7.4 for 48 h at 4 °C and decalcified in 10% EDTA for 14–21 days at 4 °C with gentle shaking. Three-micron-thick sections were used for standard histology after staining with Hematoxylin-Eosin (H&E) or with Sirius red to visualize collagen fibers, for Tartrate-Resistant-Acid-Phosphatase (TRAP) histochemistry to highlight cells of the osteoclastic lineage and for histomorphometry.

MMA embedding was performed on undecalcified bone segments. After dissection, bone samples were fixed in 4% formaldehyde for 24 h and dehydrated through a series of increasing ethanol concentrations. Bones were then infiltrated for 3 days with the plastic embedding mixture containing 60 mL of MMA, 35 mL butylmethacrylate, 5 mL methylbenzoate, 1,2 mL polyethylene glycol 400 and 0.8 g of dry benzoyl peroxide. The polymerization mixture was prepared by adding 400 μL of N,N-dimethyl-p-toluidine to the infiltrating solution. Sections of 4–7 μm in thickness were cut from MMA blocks, deplasticized with 2-methoxyethylacetate (all reagents were purchased from Sigma Aldrich, Saint Louis, MO, USA), stained with Von Kossa and counterstained with Van Gieson.

For gelatin embedding, freshly dissected femora, tibiae and tail vertebrae were fixed in cold 4% formaldehyde solution for 4 h, washed in 1X PBS and decalcified in 0.5 M EDTA at 4 °C. Soft tissues were fixed in 4% formaldehyde for 4 h. Samples were then placed in 20% sucrose and 2% Polyvinylpyrrolidone (PVP) solution in PBS for a further 48 h. Samples were embedded in an 8% porcine gelatin solution containing 20% sucrose and 2% PVP as previously reported.33 Twenty to 50 μm-thick sections were cut, air-dried for 30 min, hydrated with 1X PBS, stained with TO-PRO-3 (#T3605, Thermo Fisher Scientific, Waltham, Massachusetts, USA) for nuclei visualization and imaged with Leica Confocal Microscope (Wetzlar, Germany). For heterotopic transplants, gelatin embedded samples were also used to perform TRAP and Alkaline Phosphatase (ALP) histochemistry.

For measurements of GFP-positive BMSC area (Adq-GFP+ BMSC Ar/Ma.Ar), pictures at 40X magnification were taken with Leica Confocal Microscope, color channel split by ImageJ software and green channel used for quantification of the area of signal that was then normalized on the marrow area. The fraction of GFP-labeled osteocytes (GFP+ osteocytes) was calculated by counting them on bone trabeculae of femora and tibiae.

Histochemistry

TRAP and ALP histochemistry were performed using Sigma Aldrich reagents (Sigma Aldrich). Briefly, for TRAP histochemistry working solution, 50 mg of Naphtol AS-BI phosphate were dissolved in 4 mL N,N-dimethylformamide added to 4 mL acetate buffer and 92 mL of distilled water; then, 150 mg of Tartaric Acid and 30 mg of Fast Garnet were added. The slides were incubated with the working solution at 37 °C. For histochemical detection of ALP on gelatin sections, 30 mg of Naphtol AS Phosphate were dissolved in 0,5 mL N,N-dimethylformamide and added to a 100 mL borate buffer with 100 mg of AS blue BB salt. The solution was added to the slide and incubated for 5–10 min at 37 °C.

Immunohistochemistry

Immunolocalization of OSX, RANKL and ALP was performed using rabbit anti-mouse antibodies (anti-OSX #ab22552, anti-RANKL #ab37415, Abcam, Cambridge, UK; anti-ALP # 11187–1-AP, Proteintech, Rosemont, Illinois, USA) applied at a dilution of 1:200 in PBS + 1% turn BSA, overnight at 4 °C. After repeated washing with PBS, sections were incubated for 30 min with biotin-conjugated swine anti-rabbit IgG (#P0217, Agilent, Santa Clara, CA, USA) 1:200 in PBS + 1% BSA and then exposed for 30 min to peroxidase-conjugated ExtrAvidin (#P0217, Agilent) (1:50 in PBS + 1% BSA). The peroxidase reaction was developed using DAB substrate kit (SK-4100, Vector Laboratories, Burlingame, CA, USA).

Immunolocalization of LEPR, endomucin (EMCN) and alpha-smooth muscle actin (α-SMA) was performed using goat anti-LEPR (#AF497, R&D Systems, Minneapolis, MN, USA), rat anti-EMCN (#ab106100, Abcam) and rabbit anti-α-SMA (#ab5694, Abcam). Twenty-five μm-thick sections from gelatin-embedded samples were rehydrated with PBS and then immunostained overnight. After primary antibody incubation, sections were repeatedly washed with PBS and incubated with appropriate Alexa Fluor 647-conjugated (rabbit anti-goat IgG #A-21446, goat anti-rat IgG #A-21247, goat anti-rabbit IgG #A27040 Thermo Fisher Scientific) secondary antibodies for 1 h at room temperature. Nuclei were counterstained with TO-PRO3.

Histomorphometry

Quantitative bone histomorphometry was conducted on lumbar vertebrae (3rd and 4th) and on distal femora. Experiments were performed in a blinded fashion. Different bone parameters, using standard nomenclature and abbreviations,34 were measured in a region of interest (ROI) in the secondary spongiosa of distal femora, starting 300 μm below the growth plate and for a length of 1 mm, and between the two growth plates in lumbar vertebrae. H&E and Sirius red-stained sections were used to measure trabecular bone volume per tissue volume (BV/TV), osteoblast number per bone surface (N.Ob/BS) and osteoblast surface per bone surface (Ob.S/BS). TRAP-stained sections were used to measure osteoclast number per bone surface (N.Oc/BS) and osteoclast surface per bone surface (Oc.S/BS).

Dynamic bone histomorphometry was performed on lumbar vertebrae dissected from mice that were treated with 30 mg/kg of calcein (Sigma Aldrich), 5 and 2 days before euthanasia. Calcein fluorescent labeling was used to quantify mineralizing surface (MS/BS), mineral apposition rate (MAR) and bone formation rate (BFR/BS).

Bone marrow adiposity was analyzed by manual counting of the number of adipocytes per marrow area (N.Ad/Ma.Ar) and by measuring their area (Ad.Ar/Ma.Ar) in H&E-stained sections of distal femora, according to standard procedures and nomenclature.35,36

Pictures were acquired with an optical microscope (Zeiss Axiophot, Jena, Germany) through a digital camera (Jenoptik ProgrRes C5, Jena, Germany) and all histomorphometric analyses were performed using ImageJ.37

Transplantation assay

BMSCs were isolated from 3-month-old female Adq-mTmG and Adq- mTmG;GsαR201C mice by flushing long bones (femora, tibiae, humeri) and crushing sacral and lumbar vertebrae. Cell suspensions were collected after vigorous pipetting, filtered through a 70 μm nylon mesh cell strainer, and grown at 37 °C 5% CO2 as multiclonal cell strains38 in αMEM Sigma Aldrich) supplemented with 20% Fetal bovine serum (Thermo Fisher Scientific), 1% L-glutamine, 1% penicillin/streptomycin Sigma Aldrich). To isolate BMSCs from the tail vertebrae, the tails were skinned, cleaned of muscle and tendons and incubated in 5 mL of 2 mg·mL−1 Collagenase I solution in HBSS for 15 min and then spun at 200 g (relative centrifugal force) to remove the periosteum. The vertebrae were then minced and incubated in 10 mL of 2 mg·mL−1 Collagenase solution in HBSS for 1 h and spun at 100 g. After digestion, the supernatant was collected, filtered through 70 μm nylon mesh and centrifuged at 1 300 g for 6 min. The resulting pellet was washed with αMEM and cells were grown in the supplemented culture medium reported above.

After 1 week, all cell populations were transplanted. Constructs were made by loading 7 × 106 cells onto 40 mg of ceramic particles (a component of AttraX, NuVasive, San Diego, CA, USA) and transplanted into 2-month-old female CB17.Cg-PrkdscidLystbg-j/Crl (SCID/beige) mice (Charles River, Wilmington, Massachusetts USA) as previously described.39 After 8 weeks, samples were harvested, fixed in 4% formaldehyde in PBS pH 7.4 for 12 h and decalcified in 0.5 mol·L−1 EDTA for 1 week. Samples were then processed for porcine gel embedding as described above. Ten-micron-thick sections were stained with H&E for morphology evaluation and histomorphometric quantification of the different tissue area, and for TRAP and ALP histochemistry. Twenty μm-thick sections were cut and analyzed by confocal microscopy for visualization of GFP and tdTomato fluorescence.

Estradiol treatment

For 17β-estradiol (E2, Sigma Aldrich) treatment, two experimental groups were established: a vehicle group including Rosa26, Adq-GsαR201C and Adq-mTmG;GsαR201C mice (n ≥ 4 for each genotype, 5 months of age) and a E2 group including Rosa26, Adq-GsaR201C mice, Adq-mTmG;GsαR201C mice (n ≥ 4 for each genotype, 5 months of age). E2 was dissolved in ethanol at a concentration of 5 mg·mL−1 and added to 300 mL of drinking water to a final concentration of 4 μg·mL−1. The vehicle group received ethanol in drinking water at a final concentration of 0.1%. Water bottles were changed every week. The dose of E2 ingested was calculated as previously reported.40 After 6 weeks of treatment, mice were sacrificed by CO2 inhalation. Radiographic analyses were performed before the treatment and at sacrifice.

Gene expression analysis by quantitative PCR

Femora and tibiae from 3- and 9-month-old female mice were dissected and snap frozen in liquid nitrogen and kept at −80° until use. Bone samples were homogenized by Mikro-Dismembrator U (Gottingen, Germany) and total RNA was isolated using the TRI Reagent® (Thermo Fisher Scientific) protocol. Reverse transcription was performed by using QuantiTect® Reverse Transcription Kit (Qiagen, Hilden, Germany). cDNA samples were used as templates for quantitative PCR (qPCR) analysis on a 7500 Fast Real-Time PCR System (Applied Biosystem, Waltham, Massachusetts, USA), performed using PowerUP Sybr Green (Thermo Fisher Scientific) and specific primers (Table 1). Gene expression levels of each gene were normalized to GAPDH expression.

Statistical analysis

The comparisons between two groups were performed using the unpaired t-test. Changes in the GFP+ osteocyte fraction and Adq-GFP+ BMSC Area between Adq-mTmG and Adq-mTmG;GsαR201C mice at different ages were analyzed with the two-way ANOVA followed by a Sidak’s multiple comparison test. The comparison of histomorphometrical parameters during estrogen treatment was performed using one-way ANOVA followed by a Tukey’s multiple comparison test. In all experiments a P-value less than 0.05 was considered statistically significant. All graphs and statistical analyses were performed using GraphPad Prism version 8 (GraphPad Software, La Jolla, CA, USA).

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