Stem Cell

Glial maturation factor-β deficiency prevents oestrogen deficiency-induced bone loss by remodelling the actin network to suppress adipogenesis of bone marrow mesenchymal stem cells

Time Stamp: November 13, 2024 7:00 PM
Source Node: 735544

Preparation of PMOP patient-derived bone samples

This study was approved by the Ethics Committee of Minhang Hospital of Fudan University. A total of 19 patients who underwent spine-related surgeries at Shanghai Minhang Hospital of Fudan University were enrolled in this study. All patients signed written informed consent. The inclusion criteria are listed in Table S1. The vertebral bone samples were collected as described previously [19].

Generation of GMFB knockout rats

In this study, female Sprague‒Dawley rats (6–12 weeks old) were purchased from Slack Laboratory Animal Company, Shanghai. GMFB knockout rats were generated via the CRISPR‒Cas9 system. PCR analyses of genomic DNA were used to identify the genotype of the transgenic rats. All the studies performed were approved by the Institutional Animal Care and Use Committee of Fudan University.

Preparation of the OVX rat model

In this study, 8-week-old SD female GMFB knockout and wild-type rats were selected for ovariectomy. There was no significant difference in the initial body weight of the rats. A sham surgery or an ovariectomy was performed on the rats after anaesthesia via pentobarbital sodium (body weight of 50 mg/kg, i.p.). The ovariectomy was performed according to a previous study [20], and the rats were then randomly divided into 4 groups, each with eight mice: I, sham-operated wild-type rats; II, sham-operated GMFB KO rats; III, OVX wild-type rats; IV, OVX GMFB KO rats.

BMD measurement

Eight weeks after OVX, the BMD of the femur was measured via dual-energy X-ray absorptiometry (DXA; GE Healthcare, Madison, WI, USA).

Human BMD was measured using a DXA fan-beam bone densitometer (Hologic QDR 4500 A; Hologic) at the lumbar spine, total hip, and femoral neck 1–2 days before surgery. The X-ray absorptiometer was calibrated, and reference values were obtained according to a previous study [21]. The regions of severe scoliosis, fracture, and sites of operation were excluded from the BMD measurements.

Micro-CT analysis

The harvested bone tissues were fixed in 4% polyoxymethylene for 2 days and then scanned and analysed using micro-computed tomography (micro-CT; Bruker Corp., Billerica, MA, USA). The scanner was set at a spatial resolution of 8 μm per pixel, a voltage of 55 kV, and a current of 114 mA. The proximal tibia was used to determine the ratio of the trabecular bone volume/tissue volume (BV/TV), the trabecular thickness (Tb.Th), the trabecular number (Tb.N), and the trabecular separation (Tb.Sp). The region of interest (ROI) of the cortical bone was 30% of the length of the middle tibia, and the bone volume (BV/TV) and cortical thickness (Ct. Th) were calculated.

Histomorphometric assay and toluidine blue staining

Histomorphometric analysis was performed as previously described [22]. The rats were injected with calcein (15 mg/kg) 8 days and 2 days before euthanasia. Tibias were soaked in 70% ethanol and embedded in methyl methacrylate. The samples were sectioned into 5 μm sections for dynamic histomorphometry. The mineral apposition rate (MAR) and bone formation rate (BFR/BS) were calculated for each sample. In addition, the bone sections were processed for toluidine blue staining. The sample area selected for bone analysis was a 1 mm² area within the metaphyseal secondary spongiosa, originating 1 mm below the growth plate.

Immunohistochemical and immunofluorescence

Immunohistochemical staining was performed via a standard protocol as previously described [23]. The bone sections were digested with 0.05% trypsin at 37 °C for 15 min for antigen retrieval and subsequently incubated with primary antibodies against osteocalcin (Abcam, ab83976) and GMFB (Proteintech, 10690-1-AP) at 4 °C overnight. Then, immunoactivity was detected via an HRP-streptavidin detection system (DAKO, Carpinteria, CA, USA).

For immunostaining, the sections were incubated with primary antibodies against GMFB (Proteintech, 10690-1-AP), CD105 (R&D Systems, AF6440), and NFATc2 (Cell Signaling Technology, 4389S). Nuclei were counterstained with DAPI (Sigma, D9542). The sections were then observed under a fluorescence microscope (Olympus, Japan).

Primary BMSC isolation and culture

We flushed the bone marrow of the tibias and femurs to isolate primary rat BMSCs. The cells were cultured in the following growth medium (GM): low-glucose Dulbecco’s modified Eagle’s medium (LG-DMEM) (Gibco, 11885084) supplemented with 10% foetal bovine serum (FBS) (Gibco, A5670701), 2 mM glutamine (Sigma, 1294808), 100 mg/mL streptomycin (Sigma, P4458), and 100 U/mL penicillin (Sigma, P4458). The medium was changed twice a week.

Adipogenic differentiation assay

To induce adipogenic differentiation, the following BMSCs were cultured in adipogenic medium (AM): LG-DMEM containing 10% foetal bovine serum (FBS) (Gibco, 11885084), 2 mM glutamine (Sigma, 1294808), 100 mg/mL streptomycin (Sigma, P4458), 100 U/mL penicillin (Sigma, P4458), 0.5 mM isobutylmethylxanthine (IBMX) (Sigma, I5879), 1 μM Dex (Sigma, D4902), 10 μM insulin (Sigma, 91077 C), and 200 μM indomethacin (Sigma, I7378) for 7 days. Lipid formation was detected by Oil Red O (Sigma, O0625) staining.

Phalloidin staining of F-actin

To visualize F-actin in cells, cultured BMSCs were fixed and stained with phalloidin (200 nM, 40734ES80; Yeasen, Shanghai, China). The cell nucleus was then stained with DAPI. The mean F-actin intensity in each group was measured using ImageJ software (USA).

Calcium imaging

WT and KO BMSCs were incubated with GM or AM for 30 min and then loaded with Fluo-4 AM (5 μM, Beyotime, S1060) for approximately 30 min. Calcium imaging was performed with a fluorescence microscope system (Olympus, Japan). Changes in the intracellular Ca²⁺ concentration were measured from fluorescence images of Fluo-4 AM (excitation at 488 nm, emission at 520 ± 20 nm).

Western blotting analysis

The cells were lysed using RIPA buffer supplemented with proteinase and phosphatase inhibitors. Total lysates were separated by sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked and then incubated with primary antibodies at 4 °C overnight. The primary antibodies used were as follows: GMFB (10690-1-AP, 1:1000, Proteintech), β-actin (20536-1-AP, 1:2000, Proteintech), ERK (4695T, 1:1000, Cell Signaling Technology), phospho-ERK (4370T, 1:1000, Cell Signaling Technology), JNK (9252T, 1:1,000, Cell Signaling Technology), phospho-JNK (4668T, 1:1000, Cell Signaling Technology), P38 (8690T, 1:1000, Cell Signaling Technology), phospho-P38 (4511T, 1:1000, Cell Signaling Technology), β-catenin (8480T, 1:1000, Cell Signaling Technology), NFATc2 (4389S, 1:1000, Cell Signaling Technology), and Lamin-B1 (13435T, 1:1000, Cell Signaling Technology). The membrane was washed and visualized with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (DAKO, Carpinteria, CA, USA). The corresponding bands were detected using an enhanced chemiluminescent (ECL) detection kit (36208ES60, Yeasen).

Quantitative reverse transcription polymerase chain reaction

Total RNA was isolated from samples using TRIzol (10296010; Invitrogen, Carlsbad, CA, USA) and reverse transcribed into cDNA using PrimeScript RT polymerase (RR036A; TaKaRa, Kusatsu, Shiga, Japan). Real-time reverse transcriptase RT‒PCR was performed using a GeneAmp PCR System 9600 (PerkinElmer, Waltham, MA, USA). The primer sequences are listed in Table S2 and were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). Amplification reactions were set up in 25 μl reaction volumes containing SYBR Green Premix (FP205, Tiangen, Beijing, China), primers and cDNA.

RNA sequencing

Total RNA was isolated from adipogenic differentiated BMSCs from WT and KO rats via TRIzol reagent. RNA library construction, sequencing and analysis were performed by Shanghai Oebiotech Co. Ltd. (Shanghai, China). GO and KEGG analyses were subsequently performed to determine the biological functions of these genes.

Virus packaging and injection

The recombinant adeno-associated serotype 9 virus for gene delivery of the shRNA of the GMFB system (rAAV9-shRNA-Cherry, 5′-GCUUCAUUGUGUAUAGUUAUA-3′, 5′-UAACUAUACACAAUGAAGCGA-3′) was generated by Shanghai Taitool Bioscience Co., Ltd. (Shanghai, China). Clones were confirmed by DNA sequencing prior to use, and virus titres were determined by dot blot. An AAV viral titre of 5 × 10¹² vector genomes/ml was used in the study. rAAV9-Cherry was used as a control. The rats received 20 μl of either rAAV9-shRNA-Cherry or rAAV9-Cherry via periosteal injection into the medullary cavity of the femur twice per month for 2 months.

Statistical analysis

The data were statistically analysed using Prism 8 software, and the results are presented as the means ± standard deviations (SDs). Statistical analyses were conducted via one-way analysis of variance (ANOVA) or Student’s t-test. Correlation analysis was performed using Pearson product‒moment correlation. The enumeration data were compared via the chi-square test. p < 0.05 was considered statistically significant.