This study was approved by the Ethics Committee of Wonkwang University following the guidelines for the experimental use of animals. Twelve newborn ICR mice (1 day of age) were purchased from Damul Science (Jungeub, Korea). The newborn mice were anesthetized and sacrificed with 70% ethanol. The calvaria of the newborn mice were dissected, and the bones were digested 5 times with 0.1% collagenase (Wako, Osaka, Japan) and 0.2% dispase (Roche, Basel, Switzerland). The cells isolated in the last 3 digestions were combined and cultured in α-minimum essential medium (α-MEM) containing 10% FBS, 100 U/ml penicillin and 100 µg/m1 streptomycin (Gibco-BRL, Grand Island, NY, USA). The primary osteoblasts were plated at a density of 1×10⁵ cells/6-well plates in the presence of 50 µg/ml ascorbic acid and 10 mM β-glycerol phosphate (Sigma-Aldrich, St. Louis, MO, USA), and the culture medium was replaced every 3 days.

Total RNA was extracted from the cultured mouse calvaria cells every three days, using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. One microgram of total RNA was reverse transcribed using M-MLV reverse transcriptase (Promega, Madison, WI, USA) and random primers (Invitrogen), according to the manufacturer's instructions. For RT-PCR analysis, Ex Taq polymerase (Takara, Shiga, Japan) was used with primers specific to the LOX family genes and collagen, type I, alpha 2 (COL1A2). The primer sequences used for RT-PCR are available upon request. The thermal cycling parameters were as follows: 25–30 cycles at 94°C for 30 sec, 55–60°C for 30 sec, and 72°C for 30 sec, with a pre-denaturation cycle at 94°C for 4 min, and a final extension at 72°C for 7 min. The amplified PCR products were analyzed by electrophoresis on 2% agarose gels. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. All RT-PCR analyses were performed in the linear range of amplification.

Total cDNA from the cultured mouse calvaria cells was prepared every 3 days as described above. For qPCR, a total volume of 10 µl, which contained 5 µl of SYBR select master mix (Applied Biosystems, Foster City, CA, USA), 1 µl of the total cDNA, and 10 pmoles of primers, was amplified using the StepOnePlus instrument (Thermo Scientific, Waltham, MA, USA). The thermal parameters involved an initial step at 95°C for 10 min, followed by 40 cycles of 15 sec at 95°C, and then 60 sec at 60°C. The primer sequences used for qPCR are presented in Table I. The analyses were performed in triplicate for 3 independent experiments. Relative expression levels were obtained using the ΔΔCt method, as previously described (17). GAPDH was used as an internal control, and the fold changes were calculated using the values of day 0 as a calibrator. Data from 3 independent experiments are presented as the means ± SD.

This study was approved by the Ethics Committee of Wonkwang University following the guidelines for the experimental use of animals. Two rabbits (15 weeks of age; Damul Science) were used for the generation of a polyclonal antibody to LOX. After obtaining the polyclonal antibodies, the animals were anesthetized by an intramuscular injection of xylazine (10 mg/kg) and ketamine (50 mg/kg). After confirming that the hearts had stopped, the animals were sacrificed according to the guidelines. A recombinant form of the human LOX protein was expressed and purified as previously reported (13), and a polyclonal antibody to LOX was generated by immunizing rabbits with the LOX protein. Rabbits were injected intramuscularly with 300–400 µg of the purified protein in a buffer containing 6 M urea, 250 mM imidazol and 10 mM K₂HPO₄ on days 0, 14 and 21. After the final injection, the rabbits were bled on days 7 and 14, and the antibodies were then tested using an enzyme-linked immunosorbent assay (ELISA). Antibodies were purified using a Protein A Agarose kit (KPL, Gaithersburg, MD, USA) according to the manufacturer's instructions. Culture medium from the mouse calvaria cell cultures was collected and concentrated 10-fold using an Amicon 10 kDa cut-off filter (EMD Millipore, Billerica, MA, USA). Following concentration, samples of equivalent protein concentrations were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% polyacrylamide gel and electrophoretically transferred onto a PVDF membrane (EMD Millipore). The membrane was blocked with 5% skim milk in Tris-buffered saline with Tween-20 (TBST) for 2 h at room temperature, and was then incubated with a 1:1,000 dilution of the anti-LOX polyclonal antibody in TBST overnight at 4°C. The membrane was washed with TBST and incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin secondary antibody (#G21234; Invitrogen) for 2 h at room temperature. The membrane was developed using the ECL Prime Western Blot Detection kit (GE Healthcare UK Ltd., Buckinghamshire, UK), according to the manufacturer's instructions. For quantitative analysis, data from 3 independent experiments were analyzed using Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA) and presented as the means ± SD.

The total amine oxidase activity of the mouse calvaria cells was assessed using a peroxidase-coupled fluorometric assay, as previously described (18). Following concentration using an Amicon 10 kDa cut-off filter (EMD Millipore), the cell medium was pre-incubated, with or without 1 mM BAPN, for 1 h at 37°C in the presence of 1.2 M urea and 50 mM sodium borate, pH 8.2. The cell medium was further incubated with 20 nM calf skin collagen type I (Sigma-Aldrich) for 2 h at 37°C. Fluorescence was measured using the SpectraMax M3 microplate reader (Molecular Devices, Sunnyvale, CA, USA) with excitation and emission set at 500 and 650 nm, respectively. Data from 3 independent experiments are presented as the means ± SD.

Primary osteoblasts were plated at a density of 2×10⁴ cells/48 wells in the presence of 50 µg/ml ascorbic acid and 10 mM β-glycerol phosphate (Sigma-Aldrich). BAPN (0, 1 or 2 mM) was added to the culture medium, and the medium was replaced every 3 days. For Alizarin redARS staining, cultured cells were fixed in 3.7% formalin and stained for 10 min with 2% ARS (Sigma-Aldrich), pH 4.2. After repeated washing with distilled water, the bound ARS was dissolved in 10% cetylpyridinium chloride monohydrate, pH 7.0 (Sigma-Aldrich). The absorbance was measured at 545 nm using a microplate reader, and data from 3 independent experiments are presented as the means ± SD.

Statistical analyses were performed by one-way ANOVA, followed by a multiple-comparison Tukey's test, using SPSS 12.0 software. P-values <0.05 were considered to indicate statistically significant differences.

To investigate the temporal expression of the LOX family genes during osteoblast differentiation, we induced the differentiation of primary cultured mouse calvaria cells in the presence of ascorbic acid and β-glycerol phosphate, as previously described (19). qPCR analysis of several well-known marker genes associated with osteoblast differentiation, such as the genes encoding alkaline phosphatase (ALP), osteo-pontin (OPN), bone sialoprotein (BSP), osteocalcin (OCN) and runt-related transcription factor 2 (RUNX2), confirmed that osteoblast differentiation occurred in the primary mouse calvaria cells that we used (data not shown). For expression analysis of the LOX family genes at the mRNA level, total RNA was isolated every 3 days, and RT-PCR analysis was performed using primers derived from the non-conserved 3′-UTR regions of the LOX family genes. Throughout the differentiation period of the primary calvaria cells, only LOX was predominantly expressed, whereas the expression of the other LOX family genes was barely detectable (Fig. 1A). For quantitative analysis, qPCR was performed on the LOX family genes. The expression of LOX increased until day 9 of differentiation and then gradually diminished during the later stages of differentiation (Fig. 1B). The expression of the other LOX family genes was undetectable throughout the differentiation period. COL1A2, which encodes the α2-chain of mouse collagen type I, produced an expression pattern similar to that of LOX; its expression increased until day 9, and then gradually diminished thereafter (Fig. 1).

We assessed the total amine oxidase activity of the differentiating primary calvaria cells using the media of cultured cells, in the presence and absence of BAPN, which is a well-known specific inhibitor of LOX-derived amine oxidase activity. Total amine oxidase activity was evaluated using collagen type I as a substrate. We noted that total amine oxidase activity increased until day 9 of differentiation, and then gradually decreased thereafter (Fig. 2). Moreover, total amine oxidase activity was inhibited by treatment with 1 mM BAPN compared to the background level for all samples tested (Fig. 2). The temporal pattern of total amine oxidase activity during osteoblast differentiation closely resembled the mRNA expression pattern observed for LOX.

To evaluate LOX expression at the protein level, we performed western blot analyses of the culture media collected throughout the osteoblast differentiation period. The culture medium was collected every 3 days, concentrated 10-fold, and then subjected to western blot analysis with an antibody specific to LOX. Analogous to the total amine oxidase activity, the highest expression level of LOX protein was detected on day 9 of differentiation, and then decreased thereafter, reaching the basal level (Fig. 3). These results suggest that the total amine oxidase activity of the differentiating mouse calvaria cells was derived from LOX protein, which was expressed and secreted into the culture medium.

To investigate the effects of LOX on the osteoblast differentiation of primary mouse calvaria cells, we inhibited the amine oxidase activity of LOX by including BAPN in the culture medium. We then assessed the effects of BAPN on mineral nodule formation, as well as its effects on the expression of osteoblast marker genes, namely ALP, OPN, BSP, OCN and RUNX2. We noted that BAPN substantially reduced the expression of all the marker genes tested in a dose-dependent manner (Fig. 4). Moreover, ARS staining suggested that the inhibitory effect of BAPN on mineral nodule formation was not evident during the early stages of differentiation (days 0–12), but became distinct on day 15, and even clearer during the later stages of osteoblast differentiation (Fig. 5).