Zinc modulation of osterix in MC3T3-E1 cells

Reagents

Chemicals were obtained from Sigma (St. Louis, MO, USA). Cell culture reagents α-MEM, penicillin & streptomycin (PN/ST) and fetal bovine serum (FBS) were obtained from Gibco Laboratories (Grand Island, NY, USA). Primers for osterix and housekeeping gene (glyceraldehyde 3-phosphate dehydrogenase, GAPDH) were obtained from Invitrogen (Grand Island, NY, USA). Antibody for osterix was obtained from Abcam. Antibody for GAPDH as loading control was obtained from Cell Signaling (Danvers, MA, USA). Horseradish peroxidase-labeled donkey anti-rabbit immunoglobulin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Cell culture and zinc-treatment

Mouse calvariae origin osteoblastic MC3T3-E1 cells (ATCC, CRL-2593) were seeded at a density of 1 × 10⁵ and maintained in regular osteoblast growth medium (α-MEM with 10% FBS, 1 mM sodium pyruvate and 1% PN/ST) in an under a humidified atmosphere of 5% CO₂ at a 37°C. After reaching 80-90% confluence, cells were treated with zinc (as 0, 0.5, 1, 5, 15 µM ZnCl₂) and cultured for 1 day and 3 days. N,N,N′,N′-tetrakis(2-pyridyl-methyl)-ethylenediamine (TPEN, 5 µM) as an intracellular zinc chelator was added at the same time of zinc treatment to deplete cellular zinc level which was adjusted by the addition of ZnCl₂. Normal osteogenic medium (OSM, growth media plus 10 mM β-glycerophosphate + 50 µg/mL ascorbic acid) was used as a normal control for 1 day and 3 days of culture.

Cell proliferation by MTT assay

Osteoblast viability was evaluated by MTT assay. Briefly, MC3T3-E1 cells were plated onto 96-well plates at a density of 1 × 10⁴ cells/well and maintained in growth media for 24 hours. After reaching 80–85% confluence, cells were treated with zinc for 1 day and 3 days. Then, the cell was incubated with 10 µL of MTT solution (5 mg/mL) for an additional 3 hours. The resulting formazan crystals were dissolved in filtered dimethyl sulfoxide. The formation of formazan was measured by reading absorbance at a wavelength of 570 nm using Micro Elisa reader (Asys Hitech Expert 96; Asys Co., Eugendorf, Austria).

Cellular and media alkaline phosphatase (ALP) activity assay

Cellular (synthesized) and medium (secreted) ALP activities were measured based on colorimetric enzymatic activity [17]. MC3T3-E1 cells were treated with 0, 0.5, 1, 5, 15 µM zinc and OSM as a control for 1 day and 3 days. Cells were lysed of 0.02% Triton-X before sonicating for 30s twice on ice. These sonicated lysates were centrifuged at 12,000 × g for 15 minutes. The supernatant (cytoplasmic fraction) was collected and kept at -70°C until analysis. ALP activity in cellular cytosolic supernatant or medium was measured using para-nitrophenyl-phosphate (PNPP) as a substrate. Its conversion to para-nitrophenol (PNP) as the product was measured at 405 nm. Cellular ALP activity was normalized to protein concentration measured by the Bradford method. The enzyme activity was expressed as nmol PNPP/min/mg of protein (cellular) or nmol PNPP/min/mL (medium). A minimum of four dishes was assayed and experiments were repeated 2–3 times.

Real-time reverse transcription polymerase chain reaction (RT-PCR) for osterix mRNA expression

Real-time RT-PCR was performed for the quantification of osterix transcripts. Briefly, total RNA was prepared using Trizol reagent (Invitrogen) and cDNA was synthesized using a reverse transcription kit (Bioneer, Daejeon, Korea). Synthesized cDNAs were stored at −20°C for future analysis. Real-time RT-PCR was performed using the first-strand cDNA as the template in 20 µL SYBR Green PCR Master Mix (Takara Bio Inc., Shiga, Japan) using a fluorometric thermal cycler (Applied Biosystems 7500; Applied Biosystems, Foster City, CA, USA). Sequences of forward and reverse primers are listed in Table 1. PCR amplification condition was: 2 minutes at 50°C, 95°C initial activation step (10 minutes) and then 40 cycles of 95°C for denaturation (30 seconds), 60°C for annealing (45 seconds) and 70°C for extension (33 seconds). The progress of the PCR amplification was monitored in real-time by fluorescent measurement during each amplification cycle. The level of osterix mRNAs was normalized against the endogenous reference and expressed relative to the calibrator using the 2^−∆∆Ct method. All reactions were performed in duplicate. Nuclease free water and 96-well optical plates were used for PCR amplification with appropriate caps.

Table 1
Primer sequences for osterix used for quantitative RT-PCR

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Protein extraction and Western blot analysis

Cells were lysed with radioimmunoprecipitation assay (RIPA) buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM ethylene glycol tetraacetic acid, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerol, 1 mM Na₃VO₄, 1 µg/mL leupeptin and protease inhibitor mixture. Cell lysates were centrifuged at 12,000 × g for 15 minutes at 4°C. Supernatants were collected and stored at −70°C. Protein quantification was performed with Bradford protein assay reagent (Bio-Rad, Tokyo, Japan) [18].

For Western blot analysis, proteins (50 µg) were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane. The membranes were blocked for non-specific binding with 5% skim milk in phosphate-buffered saline containing 0.1% Tween-20 (PBS-T) for 3 hours at room temperature and then incubated with specific primary antibodies (target proteins) at 4°C overnight. After three washes with PBS-T, the membranes were incubated with secondary antibodies conjugated to horse radish peroxidase (sc-2004; Santa Cruz Biotechnology). Signals were visualized with an enhanced chemiluminescence kit (Super Pico Detection Reagent; Pierce, County, WA, USA) and quantified using ChemiDoc Gel Quantification System (Bio-Rad).

Nuclear protein extraction

After treatment, the cells were washed with PBS and cell pellets were resuspended in Buffer A (1 M HEPES (pH 7.9), 1 M KCl, 1 mM MgCl₂, 0.1 mM ethylenediaminetetraacetic acid [EDTA] plus protease inhibitor cocktail [Cell Signaling, Danvers, MA, USA], 0.1 M PMSF, 0.1 M dithiothreitol [DTT]). Samples were incubated with 10% Nonidet P-40, and then centrifuged at 14,000 g for 1 minute at 4°C. Nuclei were pelleted by centrifugation, rinsed briefly with Buffer A and resuspended with Buffer C (1 M HEPES [pH 7.9], 1 M KCl, 4 M NaCl, 0.1 mM EDTA plus protease inhibitor cocktail [Cell signaling], 0.1 M PMSF, 0.1 M DTT), and incubated on ice for 20 min. After centrifugation at 14,000 g for 5 min at 4°C, and the supernatants were retained for transcription factor osterix Western blotting.

Statistical analysis

Values were analyzed with SPSS program (IBM Corp., Armonk, NY, USA). One-way analysis of variance was used to test the effects of different zinc levels within the time point. Once significance was detected, Turkey's honestly significant difference test was used for comparing differences between groups as post hoc analysis. A Significant difference was considered at p < 0.05.

Effect of zinc on osteoblastic MC3T3-E1 cell morphology

Osteoblastic MC3T3-E1 cells were morphologically shown to be less viable at low cellular zinc levels (0–1 µM zinc) on both day 1 and 3. Although cells were still attached to dishes at lower zinc levels, viable cells were less than those at higher zinc levels (5–15 µM). As culture days were extended, cells on day 3 were much less viable at lower zinc levels (0–0.5 µM) than those on day 1 (Fig. 1).

Fig. 1
Morphological characteristics of osteoblastic MC3T3-E1 cells treated with various concentrations of Zn for day 1 and day 3. Photographs were taken with a phase-contrast microscope at 100× magnification.
OSM, normal osteogenic differentiation media.

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Effect of zinc on osteoblastic MC3T3-E1 cell viability

The effect of zinc on the functional integrity of osteoblasts was assessed by MTT viability assay. MTT assay results revealed that zinc affected cell viability of osteoblastic MC3T3-E1 cells when cells were treated with 0, 0.5, 1, 5, 15 µM ZnCl₂ plus 5 µM TPEN as intercellular zinc chelator during 1 and 3 days (Fig. 2). At day 1, low levels of cellular zinc treatment (0–1 µM) decreased cell viability, compared to normal osteogenic media (OSM), while zinc treatment at higher than 5 µM showed the same viability as shown in normal OSM. However, as culture days extended, cell viability treated with zinc at low levels (0–1 µM) decreased dramatically. At day 3, cells treated with low levels of zinc (0–1 µM) showed very low cell viability, whereas treatment with zinc at high levels (5–15 µM) showed the same viabilities as the control. After treatment with 5 µM TPEN for 1 day and 3 days, 5 µM zinc appeared to be the cut-off zinc level for complete viability which was consistent with the normal osteogenic medium. This cell viability pattern, as based on the measurement of metabolic reduction of MTT by mitochondria dehydrogenases of viable cells, is consistent with the pattern shown in morphological pattern by zinc in Fig. 1.

Fig. 2
Osteoblastic MC3T3-E1 cell proliferation after treatment with various concentrations of Zn for day 1 and day 3. Data are presented as mean ± SEM (n = 10). Different superscript letters mean significantly different by Zn concentration as analyzed one-way analysis of variance, Tukey test (p < 0.05).
SEM, standard error of mean; OSM, normal osteogenic differentiation media.

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Effect of zinc on ALP activity

ALP is a secretory enzyme which is synthesized by osteoblasts and secreted to the extracellular matrix. ALP is an osteoblast marker as well as a zinc-dependent enzyme. Effects of zinc treatment on cellular (synthesized) and medium (secretory) ALP activities of osteoblastic MC3T3-E1 cells are shown in Fig. 3A and B. The activity of cellular and medium ALP by zinc showed the similar patterns which is a zinc concentration-dependent pattern. Medium ALP activity which is the activity of the secretory enzyme showed a more zinc-dependent manner. This might be due to the release of ALP enzyme into the medium.

Fig. 3
Cellular (A) and medium (B) ALP activity in osteoblastic MC3T3-E1 cells treated with various Zn concentration for day 1 and 3. Data are presented as mean ± SEM (n = 3). Different superscript letters mean significantly different by Zn concentration as analyzed one-way analysis of variance, Tukey test (p < 0.05).
ALP, alkaline phosphatase; SEM, standard error of mean; OSM, normal osteogenic differentiation media.

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Zinc modulates osterix gene and protein expression

Osterix gene and protein expressions by zinc for day 1 and 3 are shown in Fig. 4A and B. For osterix gene expression, zinc tended to up-regulate osterix mRNA expression as cellular zinc treatment increased within the range of 0.5–5 µM zinc at day 1 (although without significance at the possibility level of 0.05) (Fig. 4A). Also, 0 µM zinc addition didn't fell into this zinc-dependent manner and 15 µM zinc treatment showed almost the same level of osterix mRNA expression, compared with 5 µM zinc addition. At longer culture period of day 3, down-regulated osterix expression by low zinc treatment (0 and 0.5 µM) was recovered which implies the catching-up mode under cellular low zinc treatment (significance level of p < 0.05).

Fig. 4
Osterix mRNA (A) and protein (B) expression in osteoblastic MC3T3-E1 cells treated with various Zn concentration for day 1 and 3. Data are presented as mean ± SEM (n = 3). Different superscript letters mean significantly different by Zn concentration as analyzed one-way analysis of variance, Tukey test (p < 0.05).
SEM, standard error of mean; OSM, normal osteogenic differentiation media; OSX, osterix.

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Osterix protein expression by zinc showed the pattern of up-regulated as zinc increased at day 1 (Fig. 4B). At day 3 of culture period, the pattern of osterix protein expression showed the catching-up mode which showed the adverse pattern of day 1 and the same pattern of mRNA expression. Both mRNA and protein expression of osterix by zinc implies that low zinc level down-regulated osterix gene and protein expression during short culture period (day 1), however it could be recovered as culture period extended.