Materials and reagents
Fetal bovine serum (FBS), FITC‐conjugated phalloidin and DAPI stain, IRDye fluorescent-labeled secondary antibodies, and alpha modification of Eagle minimal essential medium (α‐MEM) were procured from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). The TRAP staining kit and cell counting kit‐8 (CCK-8) were procured from Sigma‐Aldrich (St. Louis, MO, USA). ELISA kits against CTX‐1, TRACP‐5b, BGP, and PINP were procured from Cusabio Biotech (Wuhan, China). RANKL and M-CSF were bought from R&D Systems (Minneapolis, MN, USA). The specific primary antibodies, including PDK1 (rabbit, cat. no.: 5662S), p-AKT (phosphor T308; rabbit, cat. no.: 13038S), AKT (rabbit, cat. no.: 4685S), p-IκBα (rabbit, cat. no.: 2859S), IκBα (rabbit, cat. no.: 4812S), p-P65 (phosphor Ser536; rabbit, cat. no.: 3033S), P65 (rabbit, cat. no.: 8242S), RANK (rabbit, cat. no.: 4845S), and NFATC1 (rabbit, cat. no.: 5861S) were procured from Cell Signaling Technology (Danvers, MA, USA). The specific primary antibody, Ctsk (rabbit, cat. no.: ab187647), was purchased from Abcam (Cambridge, UK).
Generation and identification of PDK1-cKO (RANKCre. PDK1flox/flox) mice
PDK1flox/flox and RANKCre mice were designed by GemPharmatech (Nanjing, China). Cre recombinase expression was regulated via RANK promoter transcriptional control. PDK1flox/flox and RANKCre mice were mated to produce RANKCre. PDK1flox/+ mice. RANKCre. PDK1flox/+ and PDK1flox/flox were mice mated to produce PDK1-cKO (RANKCre. PDK1flox/flox) and wild-type (WT) (PDK1flox/flox) mice (Fig. 1A). Mice were kept in individually ventilated cages (temperature: 22–26°C; humidity: 50–60%; dark/light: 12/12 hours) with free to eat standard feed and freshwater. Mice were numbered with ear tags 2 weeks after birth. Rat tails were cut into 1 mm segments for PCR. DNA was acquired utilizing a TIANamp genomic DNA extraction kit (TIANGEN Biotech, Beijing, China) as per the instructions stipulated by the manufacturer. Primers were provided by Shanghai Sangon Biological Engineering Technology (Shanghai, China) (Table 1). The PCR reaction system was as follows: 25 μL 2× Tag MasterMix, 2 μL primer F, 2 μL primer F, 1 μL template, and 20 μL ddH2O. The PCR reaction conditions were set according to the 2× Tag MasterMix instructions (Shanghai Sangon Biological Engineering Technology, Shanghai, China). The PCR products were used for horizontal agarose gel electrophoresis (Fig. 1B). Mice were weighed weekly and weights were recorded. All of the animal experimentations were undertaken in compliance with the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020). Mice were anesthetized via of 2% sevoflurane in a special container, CO2 was then injected into the container at a rate that replaced 25% of container volume per minute. When it was confirmed that the mice were dead, the CO2 was turned off. The execution dates ranged from November 2019 to May 2021.
Alizarin Red and Alcian Blue staining to visualize mice skeletons
At 8 weeks, WT and PDK1-cKO mice were euthanized, placed on a foam board in a prone position, and photographed utilizing an X-ray imager (Faxitron MX20/DC2; voltage: 5.0 KV; time of exposure: 6.0 s). Mice were observed for skeletal deformities and variation and then dissected after photography. The main viscera were dissociated to observe whether there was any variation. The whole skeleton was carefully dissociated and muscle tissue was removed as much as possible, and then the skeleton was fixed in 95% alcohol for 3 d, which was then digested in acetone for 48 h to remove excess adipose tissue. The skeleton was stained with pre-configured staining solution for 5 d (75% alcohol: 0.3% Alcian Blue: 0.1% Alizarin Red: glacial acetic acid = 1:1:1:17). After staining, the skeleton was transferred to 1% KOH and soaked for 48 hours, and then placed to different solutions for 24 hours (glycerol: 1% KOH = 1:4; glycerol: 1% KOH = 1:1; glycerol: 1% KOH = 4:1). After the muscle tissue was transparent, the staining results were observed.
Murine model of prostate cancer-induced osteolysis
Anesthetization of eight‐week-old PDK1-cKO and WT mice (n = 6) was done by inhalation of sevoflurane and preoperative IP injection of penicillin (200 U/g) to avoid infections. The needle was injected into the proximal tibia head of the right lower extremity with a microsyringe, then rotated 2–3 mm. A 10 μL cell suspension containing 5×105 RM-1 was slowly injected, then the syringe was removed, the site was disinfected with iodophor, and mice were placed back into their cages after waking up. After 2 weeks, the tibia of the right lower limb was fixed in 4% paraformaldehyde for micro-CT detection.
Detection of bone conversion markers
Eight‐week-old mice venous blood was harvested and centrifugated for 20 minutes at 1600 rpm. Bone formation markers (PINP, BGP) and bone resorption markers (TRAC-5b, CTX-I) were detected using an ELISA kit (Wuhan Huamei Biological Engineering Co., Ltd., Wuhan, China) as per the instructions stipulated by the manufacturer.
Micro-CT scanning and 3D reconstruction of the tibia
Two weeks after the murine model of prostate cancer-induced osteolysis was established, the right tibia was taken for scanning and 3D reconstruction was conducted using a micro‐CT scanner (SkyScan1072) (voltage: 70 KV; current: 200 μA; resolution: 10 μm). The osteolysis area was randomly selected for quantitative analysis. The main analysis indexes were structure model index (SMI), connectivity density (Conn‐Dens, 1 mm3), trabecular separation (Tb. Sp, μm), trabecular thickness (Tb. Th, μm), trabecular number (Tb. N, 1 mm), and bone volume to tissue volume (BV/TV, %).
TRAP and HE staining of the tibial bone
After micro-CT scanning, the tibial bones were decalcified in 10 percent ethylenediaminetetraacetic acid (EDTA) for a fortnight at a temperature of 4°C ensued by continuous dehydration in 40 percent, 75 percent, and 95 percent ethanol for 60 minutes, followed by two dehydrations in 100 percent ethanol with each for 30 min. Tissue specimens were cleared in xylene for 15 minutes before being infiltrated with paraffin for 3 hours. The sequential segments were split into 5 μm slices, followed by staining with HE and TRAP, and subsequently visualized utilizing an inverted light microscope (Nikon Eclipse TS100, Tokyo, Japan).
Bone marrow-derived macrophage cell extraction and osteoclast differentiation
Eight-week-old PDK1-cKO and WT mice were euthanized. The intact femur and tibia were isolated after disinfection in 75% alcohol for 5 min. The ends of the femur and tibia were cut off, the α-MEM was drained with a 1 mL syringe, and the bone marrow cavity was rinsed 3 times. The collected rinse solution was sieved with a 200 μm sterile filter, then the supernatant was discarded and subjected to suspension in α-MEM comprising 10 percent FBS and M-CSF (25 ng/mL). The medium was transferred to a T-75 culture flask and subjected to culturing at a temperature of 37°C with a CO2 concentration of 5%. The α-MEM comprising 10 percent FBS and M-CSF (25 ng/mL) was replaced at a frequency of once every two days. After 4 days, several bone marrow-derived macrophage cells (BMMs) were observed, which were digested and suspended in α-MEM with 10 percent FBS and M-CSF (25 ng/mL). BMMs were kept in 96-well plates (8000/well), then subsequently placed in a cell incubator for culturing. After 24 h, the α-MEM comprising 10 percent FBS, RANKL (100 ng/mL), and M-CSF (25 ng/mL) was used to replace the medium. Roughly 6 d after RANKL induction, multinucleated osteoclasts were observed under a microscope. After discarding the medium, rinse operation was down two times using PBS, and then 4% paraformaldehyde was added for fixation at room temperature (RT) for 30 min. The fixative was subsequently discarded and washing routine was conducted twice with PBS, and TRAP dye was added at RT for 1 h. Then, the TRAP dye was discarded, followed by two-time rinse using PBS and air seasoning. Finally, pictures were taken and the number of osteoblasts was calculated.
BMMs proliferation/viability assay
BMMs of PDK1-cKO and WT mice were plated in 96-well plates (6000/well) and subjected to culturing in α-MEM comprising M-CSF (25 ng/mL). Then, after 48hours, each well was added 10 μL CCK-8, followed by incubation at a temperature of 37°C for 2 hours, and values recording of OD at 450 nm utilizing a microplate reader. The proliferation activity of PDK1-cKO and WT mice was statistically analyzed according to the OD value.
Podosome actin belt formation assay
BMMs were put in 96-well plates (8×103/well) to induce mature osteoclasts (same induction process as described above). When mature osteoclasts were observed, cells were gently rinsed twice using 1× PBS, fixed using percent paraformaldehyde at RT for 10 minutes, and rinsed thrice using 1× PBS; then cells were permeated using 0.1 percent Triton X-100 in PBS for 5 min at RT, blocked using percent BSA in PBS for 30 minutes, rinsed 2 times with 0.2 percent BSA, diluted with rhodamine-conjugated phalloidin in 0.2 percent BSA (1:100), incubated for 1 hour at RT, rinsed 4 times using 0.2 percent BSA and 4 times using 1× PBS, and stained using DAPI for 5 minutes. Lastly, photos were taken under a fluorescence microscope (Life Technologies, Carlsbad, CA, USA). The data were evaluated utilizing ImageJ (NIH, Bethesda, Maryland, USA).
Hydroxyapatite resorption assay
BMMs were placed in 6-well plates (2×104/well) and induced with α-MEM comprising 10 percent FBS, RANKL (100 ng/mL), and M-CSF (25 ng/mL). When the round-like preosteoclasts were observed under a microscope, they were digested with 0.25% trypsin and placed in a 96-well hydroxyapatite-coated bone absorption plate (2000/well). After 3 d of culturing, the hydroxyapatite coating was absorbed into a transparent area with irregular shape and the culture medium was sucked out. Wells were washed with 1× PBS 3 times and washed with 5% sodium hypochlorite solution for 10 minutes to eliminate the remaining adherent cells. Finally, hydroxyapatite-coated bone absorption plates were washed using 1× PBS thrice, visualized using an inverted microscope, and photographed after air drying. The absorption area was measured using ImageJ (NIH, Bethesda, Maryland, USA).
Quantitative real-time PCR (qPCR)
The total RNA from PDK1-cKO and WT osteoclasts was obtained utilizing a TaKaRa MiniBEST universal RNA extraction kit (Takara Bio Inc, Kyodo, Japan) as per the protocol stipulated by the manufacturer. The reverse transcription of RNA into cDNA was performed. The acquired cDNA was utilized as a template for qPCR, which was carried out on an ABI Prism 7500 system (Thermo Fisher Scientific, Waltham, MA). The PCR cycling setting was as illustrated below: 95°C for 30 seconds; 40 cycles at 95°C for 5 seconds; and 60°C for 34 seconds. Table 1 lists the primers utilized in this research. The relative gene expression was evaluated utilizing the 2-ΔΔCt method.
Western blot analysis and protein extraction
To investigate the impacts of conditional knockout of the PDK1 gene on the early activation of RANKL signaling pathways, BMMs were treated with serum-free starvation for 3 hours and subsequently triggered with RANKL (100 ng/mL) for 5, 10, 20, 30, or 60 min. To evaluate the effects of conditional knockout of PDK1 on the late RANKL activated signal cascade, BMMs were triggered with RANKL (100 ng/mL) for 0, 2, 4, or 6 d; WT mice were used as the control. Total cellular proteins (TCPs) were obtained utilizing a TCP extraction kit (Sigma-Aldrich, St. Louis, MO, USA) as per the protocols stipulated by the manufacturer. TCPs were isolated by 10 percent SDS-PAGE gel and loaded onto nitrocellulose membranes (Thermo Fisher Scientific, Shanghai, China). Blocking of the membranes was done with 5 percent skim milk in 1× TBST for 1 hour at RT, followed by incubation using primary antibodies (for the dilution ratio, refer to the reagent instructions) for 15 h at 4°C, and three-time rinse with 1× TBST. Afterward, membranes were subjected to incubation with IRDye fluorescent-labeled secondary antibodies at RT for 1 h. The corresponding protein bands were imaged using an LI-COR Odyssey SA Infrared Imaging Scanner. Densitometric analyses were measured using ImageJ (NIH, Bethesda, Maryland, USA).
All of the data are presented as the mean ± standard deviation (SD). All the experimentions conducted in this study were replicated thrice unless otherwise noted. Statistical differences were determined using SPSS v22.0 (SPSS Inc., Chicago, IL, USA). To compare the two groups, an unpaired Student's t-test was employed. The significance threshold was P < 0.05.