Calcium (Ca2+) signaling is the first messenger signal exhibited by osteocytes. The present study aimed to better understand the link between Ca2+ concentration, and the levels of bone mineralization regulator proteins [phosphate-regulating neutral endopeptidase on chromosome X (PHEX), matrix extracellular phosphoglycoprotein (MEPE) and dentin matrix protein 1 (DMP1)] and the levels of oxidative stress in osteocytes. The viability of MLO-Y4 cells was determined using the live/dead assay following treatment with various Ca2+ concentrations (1.8, 6, 12, 18, 24 and 50 mM) for different durations (15 and 60 min, and 24 h). Superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and NADPH oxidase (NOX) enzymes were analyzed using a colorimetric method. Apoptosis was detected by caspase-3 analysis. Furthermore, the protein expression levels of PHEX, MEPE and DMP1 were analyzed using immunoblotting, and oxidative stress was examined using the total antioxidant and total oxidant status (TOS) assay. Notably, after 15 min, there were more live cells than dead cells; however, after 60 min, the number of dead cells was increased following treatment with 24 and 50 mM Ca2+. After 24 h, there were more dead cells than live cells following treatment with 50 mM Ca2+. After 24 h of Ca2+ treatment, the highest protein expression levels of PHEX, MEPE and DMP1 were measured in cells treated with 24 mM Ca2+. In addition, as Ca2+ concentration increased, the TOS and the oxidative stress index values were also increased. In conclusion, these results suggested that 24 mM Ca2+ may trigger bone mineralization proteins, such as PHEX, MEPE and DMP1, and could be considered an applicable dosage for the treatment of bone damage in the future.
Osteoblasts and osteoclasts are the major cells of bone that function in bone formation and bone resorption, respectively. Osteocytes are embedded in the mineralized bone matrix and responsible for the regeneration of adult bone cells (
These proteins are secreted by osteocytes and have an essential role in bone modeling and remodeling (
Previous studies have shown that these proteins also have a function in the extracellular matrix as a transcription factor (
There are limited number of studies investigating the Ca2+ induced expression levels of bone mineralization proteins in osteocytes. Our study was designed to test the hypothesis, if such biological changes occur as a result of changes in Ca2+ concentration, there would be changes in the expression levels of PHEX, MEPE and DMP1 which play an important role in bone formation while osteocytes induced by different Ca2+ concentrations. On the other hand, we have shown the apoptosis by evaluating caspase-3 levels and oxidative stress-related enzymes CAT, SOD, GSH, NOX and TAS/TOS levels of MLO-Y4 cell in the presence of different concentrations of Ca2+.
MLO-Y4 cells were purchased from the Kerafast Company. Cells were grown to confluence in type I rat tail collagen-coated cell culture dishes at 37°C in a humidified atmosphere containing 5% CO2 air in 5% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., 16000044) supplemented α-MEM medium (Gibco; Thermo Fisher Scientific, Inc., 12571063), 5% calf serum (HyClone, SH30401) and 1% penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc., 15140122).
The cell viability of MLO-Y4 cell lines was tested using a live/dead assay (Sigma-Aldrich; Merck KGaA, CBA415) at various Ca2+ (Sigma-Aldrich; Merck KGaA, 10043-52-4) concentrations (1.8, 6, 12, 18, 24 and 50 mM). After MLO-Y4 osteocytes were seeded 5×103 cells/cm2 in 48 collagen-coated well plates and incubated for 72 h. The cells were treated with Ca2+ at 1.8, 6, 12, 18, 24 and 50 mM concentrations for 15, 60 min and 24 h. After the treatment of MLO-Y4 cells with different Ca2+ concentrations for 15 min, 60 min and 24 h, all media were removed and live/dead assay was performed at the end of the time period. The pictures were captured from the same region of interest for each group. At the same time, the percentages of live and dead cells were calculated.
To detect cellular oxidative stress, the activity of SOD (YLA0115RA), CAT (YLA0123RA), GSH (YLA0121RA) and NOX (YLA1501RA) enzymes were analyzed by using colorimetric diagnostic kits (Shanghai YL Biotech, China). MLO-Y4 cells (1×106 cells) were seeded in collagen-coated 6 well plates and incubated for 72 h. After the incubation period, the cells were treated with the different concentrations of Ca2+ (1.8, 6, 12, 18, 24 and 50 mM) containing media for 15 and 60 min. High concentrations of calcium led to elevated cell death in 24 h and it was difficult to measure the oxidant-antioxidant enzymes SOD, CAT, GSH, NOX levels in dead cells. So we determined the enzyme levels in 15 and 60th min. At the end of each time point, cells were washed by PBS three times and lysed with the ultrasonic waves to collect. Samples were centrifugated at 2×103 g for 5 min, the supernatants were collected and added to ELISA plate wells with HRP-conjugate reagent and incubated at 37°C for 60 min. After the incubation, the ELISA plate well was washed five times with the wash buffer. Then, chromogen solution was added to each well and avoided the light for 15 min at 37°C. After that, stop solution was added to each well. OD value of each well was measured at 450 nm and the activities were calculated according to the standard curve.
To determine antioxidant status and oxidant status according to the different concentrations of Ca2+ in MLO-Y4 cells, Rel Assay Diagnostic Total Antioxidant Status (TAS) and Total Oxidant Status (TOS) kits (Rel Assay Diagnostic) were utilized. Erel's method was used for the calculations (
Cell lysates were prepared in ice-cold RIPA buffer (Cell Signaling Technology, Inc.) after MLO-Y4 cell lines were treated with six different Ca2+ concentrations for 24 h. Cell lysates were spun to remove cellular debris by centrifuge at 12×103 g for 10 min at +4°C. Equal amounts of proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% polyacrylamide gels. The gels were transferred on PVDF membrane by using a wet transfer system overnight and were labeled to perform immunoblot analysis with PHEX (Thermo Fisher Scientific, Inc., bs-12313R), MEPE (bioss, bs-8689R), DMP1 (biorbyt, orb255063), GAPDH (Cell Signaling Technology, Inc.) and Horseradish peroxidase-labeled antirabbit secondary antibodies (Cell Signaling Technology, Inc.). Proteins were visualized by using Image Studio Lite Ver 4.0 in LI-COR. Protein-content determination was measured by the method described by Bradford.
The activity of the caspase-3 was measured using a colorimetric assay (Elabscience). Centrifugation at 1×103 g for 10 min was used to collect cells that had been exposed to six different concentrations of Ca2+ for 15 and 60 min. Lysis buffer was used to lyse the pelleted cells. Then, the cell lysates were incubated on ice for 10 min and were centrifuged at 14×103 g for 1 min. Following the centrifugation, supernatants were transferred to new tubes. For measuring caspase-3 enzyme activity, 100 µl of the samples were added to the wells, after the incubation period 100 µl Biotinylated Detection Antibody working solution, 100 µl HRP conjugate working solution, 90 µl Substrate Reagent and 50 µl Stop Solution applied to each well respectively and incubated for 2 h at 37°C in CO2 incubator. Absorbances of the samples were read under 450 nm via the ELISA reader (Biotek).
Data are expressed as mean ± standard deviation of three independent determinations. The non-parametric approach was preferred and the Kruskal Wallis analysis of variance was used for comparison between groups. When a significant difference was detected as a result of the Kruskal Wallis test, the post hoc Dunn's test was used. In the Kruskal Wallis analysis of variance results, P<0.05 was accepted as significant. All analyses were performed with SPSS 25.0 (IBM SPSS Statistics 25 software (IBM Corp.) package program.
Since Ca2+ has a critical role in bone mineralization, we have designed to investigate the effect of Ca2+ concentration on cell viability in an experiment that consists of six different Ca2+ concentrations and three-time points. After MLO-Y4 cells were treated with 1.8, 6, 12, 18, 24 and 50 mM Ca2+ for 15 min, cell viability was determined by live/death assay. After Ca2+ treatment at the 15th min, there were much more live MLO-Y4 cells than dead MLO-Y4 cells in all Ca2+ concentrations (
Ca2+ has important role between the free radical and enzymatic antioxidant capacity in bone metabolism. Therefore, we aimed to show how Ca2+ stimulation regulates the CAT, SOD, GSH and NOX levels. After MLO-Y4 cells were treated with Ca2+ (1.8, 6, 12, 18, 24 and 50 mM) for 15 and 60 min, CAT, SOD, GSH and NOX levels were determined by ELISA assay.
SOD and CAT increased and reached the maximum levels in 12 mM Ca2+ in 15 min (
Previous studies have shown that ROS and/or antioxidant systems are related to the pathogenesis of bone loss. In our study; TAS and TOS levels were measured at the end of the 24 h of different concentrations of Ca2+ application, and OSI was calculated (
PHEX, MEPE and DMP1 play an important role in bone remodeling. Therefore, to assess the expression levels of PHEX, MEPE and DMP1 in MLO-Y4 cells, we performed Western Blot analysis. Immunoblot analysis revealed that Ca2+ upregulates expressions of PHEX, MEPE and DMP1 in a dose dependent manner. The highest expression levels of PHEX, MEPE and DMP1 were seen at 24 mM Ca2+ treatment. We also observed a depletion/feedback in the expression of PHEX, MEPE and DMP1 at 50 mM Ca2+ concentration. The expression of PHEX, a pro-mineralization gene, was notably increased at the 24 mM Ca2+ but was suppressed at 50 mM Ca2+; the highest tested concentration of Ca2+. Increased exposure to Ca2+ appeared to alter the interaction of other osteoblast mineralization-associated genes. Our data show that optimum Ca2+ concentration induces bone mineralization while high Ca2+ concentration cause depletion of PHEX, MEPE and DMP1 expression (
Caspase-3 levels were studied in MLYO-4 cells grown in complete media supplemented with different concentrations of Ca2+ for 15 and 60 min. Caspase-3 levels were significantly elevated by the 12, 24 and 50 mM Ca2+ when compared to 1.8 mM Ca2+ treated control cells in both timelines. The rate of rising was most visible in 12 mM compared to other groups in 15 and 60 min. Incubation of MLO-Y4 cells with 1.8, 12, 24 and 50 mM of Ca2+ for 60 min led to increased caspase-3 levels compared to the 15 min treatment for each (
Calcium plays an important role in many basic biological processes like the muscle system, neural system, enzyme activity, hormone metabolism, and membrane permeability. Moreover, Ca2+ is an important structural component of the skeleton. Additionally, control of Ca2+ ion in the extracellular fluid is vital for many metabolic activities and some endocrine control systems have developed to maintain constant Ca2+ concentration (
Our results indicate that Ca2+ concentration significantly inhibits cell viability in a dose and time-dependent manner (
Cellular oxidative stress disrupts bone remodeling and leads to an imbalance between osteoclast and osteoblast activity. Previous studies have shown that ROS and/or antioxidant systems are related to the pathogenesis of bone loss (
In the present study, antioxidant-oxidant (CAT, SOD, GSH and NOX) enzyme levels were measured after 15 and 60 min of Ca2+ administration in MLO-Y4 cells (
Some important clinical problems arise in deficiency or inactivation of PHEX and MEPE and it is important to understand the metabolism of these proteins. The inactivation of PHEX or/and DMP1 has been shown to result in an increased fibroblast growth factor 23 (FGF23) (
Increased levels of both PHEX and MEPE in the treatment of 24 mM Ca2+ are important for osteocytes metabolism and they might be used in bone treatment in the future. PHEX, MEPE, and DMP1 have also been demonstrated to be controlled during loading and unloading settings in previous investigations (
In a summary, our results suggest that high Ca2+ concentrations can perturb osteocyte cell hemostasis which leads to tissue damage and apoptosis of the bone and result in metabolic bone diseases such as osteoporosis. 24 mM Ca2+ concentration can trigger bone mineralization markers such as PHEX, MEPE and DMP1 can be a convenient dosage for the treatment of bone damage.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
BOD and OA designed all experimental procedures of the study, and BOD conducted cell culture and the live/dead cells assay. ACD and ERK conducted CAT, SOD, GSH and NOX measurements, determination of TAS and TOS, immunoblotting for PHEX, MEPE, DMP1 and GAPDH, and caspase-3 analysis. ACD and ERK confirm the authenticity of all the raw data. JC calculated the percentage of live and dead cells. All authors read and approved the final manuscript.
Not applicable.
Not applicable.
All authors declare that they have no competing interests.
Live/dead cells assay at 15 min. Live/dead assay of MLO-Y4 cells incubated with different concentrations of Ca2+ (1.8, 6, 12, 18, 24 and 50 mM) for 15 min. All live cells (green) on the left side and all dead cells (red) on the right side were shown with arrows.
Live/dead cells assay at 60 min. Live/dead assay of MLO-Y4 cells incubated with different concentrations of Ca2+ (1.8, 6, 12, 18, 24 and 50 mM) for 60 min. All live cells (green) on the left side and all dead cells (red) on the right side were shown with arrows.
Live/dead cells assay at 24 h. Live/dead assay of MLO-Y4 cells incubated with different concentrations of Ca2+ (1.8, 6, 12, 18, 24 and 50 mM) for 24 h. All live cells (green) on the left side and all dead cells (red) on the right side were shown with arrows.
Percentages of live and dead cells. Percentages of live and dead cell incubated with different concentrations of Ca2+ (1.8, 6, 12, 18, 24 and 50 mM) for 15 min, 60 min and 24 h.
CAT, SOD, GSH and NOX levels at 15 min. The role of the Ca2+ on oxidant/antioxidant balance in MLO-Y4 at the incubation of 15 min. (A) CAT, (B) SOD, (C) GSH and (D) NOX levels were measured in MLO-Y4 cells treated with Ca2+ (1.8, 6, 12, 18, 24 and 50 mM) for 15 min. *P<0.05 vs. 1.8 mM Ca2+ group. CAT, catalase; SOD, superoxide dismutase; GSH, glutathione; NOX, NADPH oxidase.
CAT, SOD, GSH and NOX levels at 60 min. The role of the Ca2+ on oxidant/antioxidant balance in MLO-Y4 at the incubation of 60 min. (A) CAT, (B) SOD, (C) GSH and (D) NOX levels were measured in MLO-Y4 cells treated with Ca2+ (1.8, 6, 12, 18, 24 and 50 mM) for 60 min. *P<0.05 vs. 1.8 mM Ca2+ group. CAT, catalase; SOD, superoxide dismutase; GSH, glutathione; NOX, NADPH oxidase.
PHEX, DMP1 and MEPE expression levels. The Ca2+ promotes the PHEX, DMP1/MEPE axis. Western blotting analysis of PHEX, MEPE, DMP1 and GAPDH levels in MLO-Y4 cells treated with different concentrations of Ca2+. Each Ca2+ concentration was analyzed densitometrically according to its GAPDH (n=2), (_1 is sample one, _2 is sample two of each Ca2+ concentration). Two sibling gels were prepared, run and transferred in the same electrophoresis equipment. 24_2, 50_1 and 50_2 samples were loaded to the sibling gel. PHEX, phosphate-regulating neutral endopeptidase on chromosome X; DMP1, dentin matrix protein 1; MEPE, matrix extracellular phosphoglycoprotein.
Caspase-3 levels at 15 and 60 min. Evaluation of caspase-3 levels of MLO-Y4. Caspase-3 levels were measured, and comparison over time (15, 60 min) was made between four groups (1.8, 12, 24 and 50 mM) significant differences between the groups vs. 1.8 mM Ca2+ concentration group were indicated as *P<0.05.
TAS, TOS and OSI were measured in MLO-Y4 cells after 24 h of Ca2+ application.
Experimental group | TAS (mmol/l) | TOS (µmol/l) | OSI (arbitrary unit) |
---|---|---|---|
1.8 mM Ca | 0.366 | 4.580 | 1.250 |
6 mM Ca | 0.180 | 4.580 | 2.544 |
12 mM Ca | 0.252 | 17.328 |
6.877 |
18 mM Ca | 0.383 | 18.244 |
4.758 |
24 mM Ca | 0.149 |
21.374 |
14.376 |
50 mM Ca | 0.390 | 27.786 |
7.130 |
TAS and TOS were measured in MLO-Y4 cells after 24-h calcium application. OSI was measured as: OSI=[(TOS, µmol H2O2 equivalent/l)/(TAS, mmol Trolox equivalent/l) ×10].
P<0.05 compared to control (1.8 mM calcium) group. TAS, total antioxidant status; TOS, total oxidant status; OSI, oxidative stress index.