p47^phox-Nox2-dependent ROS Signaling Inhibits Early Bone Development in Mice but Protects against Skeletal Aging^*

Animals

Time-impregnated female C57BL/6J-Ncf1M1J/J (p47^phox−/−) mice and their wild type controls were obtained from The Jackson Laboratory (Bar Harbor, ME). Dams and litters were housed in polycarbonate cages in an Association of Laboratory Animal Care-approved animal facility in an environmentally controlled room at 22 °C with a 12-h light/dark cycle and fed standard rodent chow ad libitum throughout experimental period including pregnancy and lactation. Male wild type control and p47^phox−/− pups (n = 10 per group) were fed chow ad libitum as previously described (18), for 6 weeks and 2 years. We have recorded body weights on a weekly basis and food intake daily for 7 days after 1, 4, and 12 weeks on the diet. After the mice were sacrificed, serum, legs, and vertebrae were collected and stored at −80 °C until use.

Bone Analyses

Peripheral quantitative computerized tomography (pQCT) was performed on formalin-fixed left tibia for bone mineral density (BMD) measurement using a method established in our laboratory (19). A STRATEC XCT 960 M unit (XCT Research SA, Norland Medical Systems, Fort Atkins, WI) specifically configured for small bone specimens was utilized. Software version 5.4 was used with thresholds of 570 mg/cm³ to distinguish cortical bone and 214 mg/cm³ to distinguish trabecular from cortical and subcortical bone. Tibial BMD and bone mineral content were calculated. The position for pQCT scanning was defined at a distance from proximal tibia 1 mm below growth plate corresponding to 7% of the total length of the tibia. The distance between each scanning was 1 mm, and total of five scans (five slices) were carried out. The data are expressed as the means of three contiguous slices with the greatest trabecular bone density.

Sequential dehydration of right rear tibial was carried out using different concentrations of alcohol. Proximal tibial bone samples were embedded, cut, and Masson and TRAP (tartrate-resistant acid phosphatase) stained by standard histology special procedures (20, 21). For histomorphometric analysis, sections were read in a blinded fashion. Parameters of cancellous bones in the proximal tibia were measured with a digitizing morphometric system, which consists of an epifluorescent microscope (model BH-2; Olympus), a color video camera, and a digitizing pad (Numonics 2206) coupled to a computer (Sony) and a morphometry program from OsteoMetrics, Inc. Total bone area, total bone surface, osteoblast surface, osteoclast surface, and eroded surface were obtained by manual tracing. Vertebrae (L4) histology and immunostaining including above unstained tibia sections using TNFα antibody (Cell Signaling) were carried out using standard protocol from VectaStain ABC kit (Vector Laboratory, Burlingame, CA).

Microcomputed tomography measurements of trabecular of the tibial bone after the above pQCT process were evaluated by using Scanco microcomputed tomography scanner (μCT-40; Scanco Medical AG, Bassersdorf, Switzerland) at 6-μm isotropic voxel size with x-ray source power of 55 kV and 145 μA and integration time of 300 ms. The gray scale images were processed using a low pass Gaussian filter (σ = 0.8, support = 1) to remove noise, and a fixed threshold of 220 was used to extract the mineralized bone from the soft tissue and marrow phase. Cancellous bone was separated from the cortical regions by semiautomatically drawn contours. A total of 120 slices starting from about 1 mm distal to growth plate, constituting 0.70-mm length, was evaluated for trabecular bone structure by using software provided by Scanco, as described in detail previously (22).

Three-point bending of the left femur was performed at room temperature using a miniature bending apparatus with the posterior femoral surface lying on lower supports (7 mm apart) and the left support immediately proximal to the distal condyles. Load was applied to the anterior femoral surface by an actuator midway between the two supports moving at a constant rate of 3 mm/min to produce a physiological in vivo strain rate of 1% for the average mouse femur. The mechanical properties including ultimate strength/stress and stiffness were recorded with a digital caliper.

Serum Bone Turnover Markers

The serum bone formation marker alkaline phophatase (ALP) and the serum bone resorption marker C-terminal telopeptides of type I collagen (CTX-1) RatLaps were measured by Rat-MID^TM ALP ELISA and RatLaps^TM ELISA, respectively, from Nordic Biosciences Diagnostic (Herlev, Denmark). Serum total osteocalcin levels were measured by an ELISA-based kit from TAKARA (TAKARA Bio Inc.). ELISA for mouse mTRAP5b levels was performed using enzyme immunoassay kits from TSZ ELISA (TSZ Scientific).

Real Time RT-PCR Analysis

Mouse femur bone RNA and osteoblastic cell RNA were extracted using TRI reagent (MRC Inc., Cincinnati, OH) according to the manufacturer's recommendation followed by DNase digestion and column cleanup using Qiagen mini columns. Briefly, for RNA isolation from bone tissue, at the time of sacrifice, the right femur was taken, and bone marrow cells were flushed with Eagle's minimum essential medium + Hanks' salts after cleaning the surrounding connective tissue. Femoral bones were then frozen in liquid nitrogen and smashed. Femoral bone was placed in 1000 μl of TRI reagent and homogenized using a Polytron aggregate (Kinematica, Luzern, Switzerland). 100 μl of 1-bromo-3-chloropropane was added, and the mixture was centrifuged for 15 min at speed of 16,000 rpm, 4 °C. 450 μl of supernatant was taken, and an equal volume of isopropanol was added and centrifuged for an additional 15 min (16,000 rpm, 4 °C). After washing the RNA pellet with 75% ethanol, isolated RNA was resuspended in RNase-free water. For RNA isolation from cultured cells, treated cells from 6-well plates were washed twice with PBS, and 1000 μl of TRI reagent was added into each well. Cells were scraped into a 1.5-ml Eppendorf tube. RNA preparation was identical to that of isolation of RNA from bone tissue. Reverse transcription was carried out using an iScript cDNA synthesis kit from Bio-Rad. Real time RT-PCR was carried out using SYBR Green and an ABI 7500 fast sequence detection system (Applied Biosystems, Foster City, CA) (23) (Table 1).

Western Blotting and Antibody Array

Femoral bone tissue proteins were extracted using a cell lysate buffer as described previously (24). Collagen 1, Runx2, RANKL, TNFα, and MMP9 protein expression in bone tissue was assessed by standard Western immunoblotting using antibodies recognizing these proteins (Santa Cruz Biotechnology, Inc.) followed by incubation with secondary antibodies conjugated with horseradish peroxidase (Santa Cruz). β-Actin protein in bone tissue was analyzed by immunoblotting, using mouse monoclonal antibody recognizing β-actin (Sigma) followed by incubation with a secondary anti-mouse antibody conjugated with horseradish peroxidase (Santa Cruz). Immunoblots were visualized using SuperSignal West Pico chemiluminescent (Pierce). Quantitation of the intensity of the bands in the autoradiograms was performed using a VersaDoc^TM imaging system (Bio-Rad). Mouse inflammation antibody arrays were carried out using proteins isolated either from vertebrae or cultured cells according to the procedure provided by manufacture in detail (RayBiotech, Inc., catalog no. AAM-INF-1-8).

Cell Culture, DNA Constructs, and Transient Transfection

Neonatal wild type and p47^phox knock-out mouse calvarial cells were isolated from 3-day-old pups using sequential collagenase digestion using a method described previously (25). Cells were cultured in α-minimum essential medium (Invitrogen) supplemented with 10% FBS (Hyclone, Logan, UT), penicillin (100 units/ml), streptomycin (100 μg/ml), and glutamine (4 mm). To generate replicative senescent calvarial cells, we cultured 2 × 10⁴ cells/well in a 6-well plate, when cells became ∼80% confluent after 5–6 days they were passaged. This procedure was repeated up to 20 times for a total of 20 passages. Transfections of Nox4 shRNA (Sigma-Aldrich; NM_015760/trcn0000076586/pLKO.1) into calvarial cells were performed using protocols provided by the manufacturer. Empty vectors of pLKO.1 entry was used as control for Nox4 shRNA. ALP staining for assessing cell differentiation was performed based on method published previously (20).

Flow Cytometric Measurement of ROS

The cell-permeable dye 2,7-dichlorodihydrofluorescein diacetate (2,7-DCF-DA) (Sigma-Aldrich) becomes fluorescent upon reaction with ROS. 2,7-DCF-DA was dissolved in dimethyl sulfoxide and stored as 50 mm stock. Bone marrow cells or calvarial cells were loaded with 10 μm 2,7-DCF-DA for 30 min and then washed three times with PBS before they were harvested. Washed cells were resuspended in 500 μl of PBS and kept on ice until flow cytometric analysis was started. ROS measurement was immediately carried out by flow cytometry using FACSort (Becton-Dickinson, Rutherford, NJ) with a 488-nm excitation beam. The signals were obtained using a 530-nm band pass filter for DCF. Each determination was based on the mean fluorescence intensity of 5000 cells. Basal and phorbol 12-myristate 13-acetate-stimulated hydrogen peroxide production was measured in neonatal calvarial cells using the Amplex Red hydrogen peroxide/peroxidase assay (Invitrogen, Molecular Probes) as per the manufacturer's instructions and our previous description (26).

Cell Proliferation Assay

Nonradioactive cell proliferation assay was performed following the protocol provided by manufacturer (Promega Corporation, part no. TB169). According to the manufacturer's instructions, this assay measures absorbance at 490 nm, and there is a linear correlation (the correlation coefficient of the line is 0.997) between cell number and absorbance at 490 nm; absorbance was converted to cell number.

Senescence-associated β-Galactosidase (SABG) Staining and Activity Assay

SABG activity assay was performed by β-galactosidase enzyme assay kit (Promega) measured the absorbance at 420 nm according to the manufacturer's instructions. Cell β-galactosidase staining was also performed according to a method published previously (23). Senescent cells were identified as blue-stained cells by standard light microscopy.

Statistical Analyses

The data are expressed as means ± S.E. t test or one-way or two-way analysis of variance followed by Student-Newman-Keuls post hoc analysis was used to compare the treatment groups. Values were considered statistically significant at p < 0.05.

Increased Bone Formation, Mass, and Strength in Young p47^phox−/− Mice, Is Reversed in Old p47^phox−/− Mice Compared with Their Age-matched Wild Type Controls

To determine p47^phox−/− mouse bone phenotypes at both age of 6 weeks and 2 years, we first analyzed tibial bone by pQCT (peripheral quantitative computed tomography scan). At 6 weeks of age, BMD (with exception of trabecular BMD) and bone mineral content were both significantly higher in p47^phox−/− mice compared with those from wild type animals (Fig. 1A). To our surprise, at 2 years of age, all pQCT parameters measured in the p47^phox−/− mice were significantly lower relative to their wild type counterparts (Fig. 1A). To determine whether the observed lower bone quantity, i.e. bone mass, is associated with poor bone quality, three-point bone bending tests were performed to evaluate bone strength in femurs. As expected, and consistent with our pQCT analysis, both stiffness and peak load were significantly higher in 6-week-old p47^phox−/− mice and lower in 2-year-old p47^phox−/− mice compared with their respective wild type controls (Fig. 1B). The observed differences in bone quantity and quality between p47^phox−/− and wild type mice at 6 weeks and 2 years were reflected by the bone metabolite levels in the serum. Levels of bone formation markers osteocalcin and ALP were significantly higher in serum of 6 weeks p47^phox−/− mice, whereas they were lower in 2-year-old p47^phox−/− mice compared with their respective age-matched wild type controls (Fig. 1C). On the other hand, bone resorption marker CTX-1, reflecting osteoclast activity, was only significantly different between p47^phox−/− and wild type mice at 6 weeks (Fig. 1C). TRAP5b, believed to be a marker of osteoclast number, was significantly different between p47^phox−/− and wild type mice at age 2 years, but not at 6 weeks (Fig. 1C). Bone histomorphometric analytical data further supported these results on bone mass and bone metabolic markers. In 6-week-old mice, with the exception of osteoblast surfaces, increased osteoblast indices including osteoblast number and bone volume were found in p47^phox−/− mice compared with their respective controls (Fig. 2A). Decreased osteoclast surface accompanied by erosion surface was also observed in p47^phox−/− mice compared with their control at 6 weeks of age (Fig. 2A). In contrast to 6-week-old p47^phox−/− mice, 2-year-old p47^phox−/− mice had significantly decreased bone volume and osteoblast number and increased osteoclast number but no differences in osteoclast surface and erosion surface compared with their wild type controls (Fig. 2B). Despite changes in osteoclast indices in both young and aged p47^phox−/− mice, we could not quantify obvious differences between control and p47^phox−/− on bone sections using TRAPase staining (data not shown). These data suggest that osteoblast bone formation was largely affected in both rapidly growing and aged p47^phox−/− mice, although there were some osteoclast bone resorption changes. Moreover, microcomputed tomography analysis confirmed the increased bone volume in 6-week-old p47^phox−/− mice and decreased bone volume in 2-year-old p47^phox−/− mice compared with their respective wild type controls (Fig. 2C). The changes of an index of bone strength, polar moment of inertia (J; mm4) (Fig. 2C), was especially consistent with the femur bending test in p47^phox−/− mice at 6 weeks and 2 years compared with their controls.

Reduced ROS Generation and p47^phox-Nox2 Signaling Is Associated with Increased Osteoblast Differentiation in Early Life but Enhanced Inflammation in Aged p47^phox−/− Mice

To explore mechanistic underpinnings of observed bone mass and quality changes with age in p47^phox−/− mice compared with their wild type controls, we isolated primary bone marrow cells and neonatal osteoblastic calvarial cells. Flow cytometric analysis for total ROS production in bone marrow cells using DFC fluorescence revealed that ROS levels in mice increased with age (p < 0.001, between wild type), but that ROS production in p47^phox−/− mice was significantly lower at 6 weeks and 2 years than their corresponding wild type controls (p < 0.001) (Fig. 3A). Interestingly, according to this measurement, ROS levels in 2-year-old p47^phox−/− mice were similar to those from 6-week-old wild type mice. Next, isolated neonatal calvarial cells were utilized to determine whether p47^phox-Nox2-dependent ROS production and Nox2 signaling were abolished in osteoblastic cells from p47^phox−/− mice. Calvarial cells from neonatal p47^phox−/− mice had lower basal production of hydrogen peroxide resulting from the action of superoxide dismutase on Nox-generated superoxide, as measured using Amplex red, compared with cells isolated from wild type mice (Fig. 3B) (p < 0.05). In addition, hydrogen peroxide production in response to treatment with the Nox2 activator phorbol 12-myristate 13-acetate was significantly lower in osteoblastic calvarial cells from p47^phox−/− mice compared with cells from wild type controls (Fig. 3C). These data indicate that p47^phox-Nox2-dependent ROS generating signaling in osteoblastic cells from p47^phox−/− mice is indeed impaired. To determine osteoblastic cell differentiation potential of calvarial cells in the absence of functioning Nox2 signaling from p47^phox−/− mice, we cultured these cells in the presence of osteoblastogenic medium for 12 days. Evaluated by ALP staining, we found that cells from p47^phox−/− mice have increased differentiation potential compared with cells isolated from wild type neonates (Fig. 3D) (p < 0.05). Expression of Nox4, another major isoform of Nox in nonphagocytic osteoblasts, was increased in cells from p47^phox−/− mice (Fig. 3E); however, knocking down Nox4 gene expression using Nox4 shRNA (Fig. 3E) was associated with decreased ALP gene expression compared with cells from p47^phox−/− mice with control shRNA (Fig. 3F). Interestingly, ALP gene expression was also significantly lower in cells from wild type control animals with Nox4 shRNA compared with without Nox4 shRNA (Fig. 3F). These data suggest that p47^phox-Nox2-dependent ROS suppresses osteoblast differentiation, but Nox4 is at least required for increased osteoblast differentiation in p47^phox−/− mice.

The above observed results may explain bone phenotype and metabolism in p47^phox−/−mice during early development (age 6 weeks) compared with their wild type controls. However, it is still not clear why ROS production is reduced but both bone quantity and quality are reversed in p47^phox−/− mice at age 2 years compared with their wild type control. Although p47^phox and Nox2 themselves are different genes, deletion of p47^phox or Nox2 in mice are animal models for CGD, and they all produce less ROS. Age-dependent increases in inflammation, such as rheumatoid arthritis, have been reported in Nox2-deficient mice (6). We therefore next explored bone inflammation in p47^phox−/− mice to determine whether this was associated with their lower bone mass compared with wild type controls at age 2 years. Proteins were isolated from vertebrae, and Western blots showed a decrease in RANKL expression with no obvious differences in the other two inflammatory markers, TNFα and MMP9, between 6-week-old p47^phox−/− and wild type animals (Fig. 4A). Col-1 (type 1 collagen) and Runx2 expression were higher in bone from p47^phox−/− mice compared with wild type controls at age 6 weeks (Fig. 4A). In 2-year-old mice, however, RANKL, TNFα, and MMP9 were all expressed at significantly higher levels in bone from p47^phox−/− mice compared with those from wild type controls (Fig. 4A). On the other hand, Runx2 expression was lower in bone from p47^phox−/− mice (Fig. 4A). Although specific cell types were not defined, the increased TNFα expression in bone from 2-year-old p47^phox−/− mice was further confirmed using antibody immunostaining of decalcified vertebrae bone sections, and TNFα was intensively stained red (Fig. 4B). In accordance with changes of protein levels, mRNA expression of these pro-inflammatory cytokines were also greater in bone of p47^phox−/− mice at 2 years compared with their age-matched controls (Fig. 4C) (p < 0.05). However, there were no differences in mRNA expression of these pro-inflammatory cytokines between wild type and p47^phox−/− mice at age 6 weeks (Fig. 4C). Gene expression of osteoblast differentiation and activity markers revealed significantly higher mRNA expression of ALP, osteocalcin, and Runx2 in bone of p47^phox−/− mice at 6 weeks compared with their wild type controls (Fig. 4C). Among these markers, only osteocalcin was found to be significantly decreased in bone from p47^phox−/− mice at 2 years compared with their controls (Fig. 4C). Proteins isolated from vertebrae were further analyzed using inflammation antibody array (Fig. 4D). Although the signature of inflammation was slightly different between p47^phox−/− and control mice at 2 weeks of age, significant amounts of inflammatory factors were found increased in their expression from p47^phox−/− compared with their control mice at 2 years of age.

Bone and Osteoblastic Cell Senescence Is Accelerated in Old p47^phox−/− Mice

Progressive accumulation of excess ROS has been suspected to be the leading cause of many tissue inflammation and degenerative diseases and underlies the effects of aging (2). This is somehow paradoxical to our current findings showing that reduced ROS production was associated with increased inflammation in bone from aged p47^phox−/− mice, and this was further associated with decreased bone mass. We therefore asked whether physiological levels of p47^phox-Nox2-dependent ROS signaling protects against the skeletal aging process. SABG activity is the most widely used assay and marker to detect aging or senescent cells both in vivo and in vitro, and the SABG activity in bone was measured in p47^phox−/− and wild type mice at 6 weeks and 2 years. Although there was no significant difference in SABG activity in p47^phox−/− and wild type mice at 6 weeks, SABG activity was significantly higher in p47^phox−/− mice at age 2 years (p < 0.05) (Fig. 5A). It needs to be mentioned that this assay was consistent and generated very small standard deviation (n = 9 was used for in vivo samples), including in ex vivo assay below; error bars are difficult to determine. Each individual sample from p47^phox−/− mice at age 2 years was measured for SABG showed higher than any samples from control mice. In accordance with these results, p16 and p53 gene expression were also significantly higher in bone from 2-year-old p47^phox−/− mice compared with wild type controls (Fig. 5B). There was no difference in expression of these genes between p47^phox−/− and wild type mice at 6 weeks (Fig. 5B). Cell division progression regulator anaphase-promoting complex substrates cyclin A and B were also examined. We found that both cyclin A and B expression in bone were significantly higher in p47^phox−/− mice at 6 weeks but lower in p47^phox−/− mice at 2 years compared with their respective wild type controls (Fig. 5C). This is interesting, because the results implicated that without sufficient p47^phox-Nox2-dependent ROS production, skeletal aging may be accelerated.

Finally, the in vivo results demonstrating accelerated cell senescence were recapitulated using isolated calvarial cell cultures. We tried to establish replicative senescent osteoblastic cells in vitro; we usually need to passage calvarial cell cultures at least up to 20 times as described in detail under “Experimental Procedures” and in our previous study (23). Cells isolated from p47^phox−/− mice showed significantly decreased proliferation and differentiation capacities after only eight passages compared with cells isolated from wild type mice passaged the same number of times (Fig. 6A). Gene expression of ALP, Runx2, p16, and p53 from passage eight calvarial cells from p47^phox−/− mice were further compared with passage 8 cells from wild type controls. Gene expression of ALP and Runx2 gene was significantly lower, whereas that of p16 and p53 genes was significantly higher in passage 8 cells from p47^phox−/− mice compared with those from wild type mice (Fig. 6B). Consistent with cell senescence-associated gene expression, SABG activity was also significantly higher in passage 8 calvarial cells from p47^phox−/− mice compared with their control cells as evaluated by SABG ELISA measurement and SABG blue staining (Fig. 6, C and D). Furthermore, substantial increases in TNFα expression were also found in passage 20 calvarial cells from p47^phox−/− mice compared with wild type control cells (Fig. 6E). As we published previously, to generate replicative senescent primary cells, cells need to be passaged at least 20 times. In current experiment, we only passaged cells from p47^phox−/− mice eight times, and they were quicker to enter senescence program than cells from wild type mice, suggesting that the p47^phox gene exerts roles on anti-osteoblastic cell senescence or skeletal aging. Passage 20 cells from both p47^phox−/− and wild type control mice were further analyzed using inflammation antibody array (Fig. 6F). Expression of large amounts of inflammatory factors was significantly increased in passage 20 cells from p47^phox−/− mice compared with those cells from wild type controls.

Different effects of transient versus sustained inhibition of Nox activity on osteoblastic cell senescence signaling and inflammation from control mice were examined. Fig. 7A shows increased expression of cyclin A and B in passage 2 calvarial cells from p47^phox−/− mice but decreased expression of both cyclin A and B in passage 8 and 20 cells from p47^phox−/− mice compared with cavarial cells from control mice. Calvarial cells from control mice were treated with Nox pan inhibitor diphenyleneiodonium chloride (DPI) for 4, 24, and 72 h with three different doses. We found cell senescence pathway (SABG activity measurement) was significantly lower after 4 h of DPI treatment (50 and 100 nm of DPI) (Fig. 7B). However, SABG activity was increased after 24 and 72 h of 50 nm DPI treatment (Fig. 7B). Similarly, TNFα mRNA expression was decreased after 4 h of 50 nm DPI treatment but significantly increased after 72 h of treatment (Fig. 7C).