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Introduction

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 (1). Osteocytes continue to increase with age and bone size. Because of the limited access to the osteocytes in the bone matrix, knowledge of its functions remained unclear. Due to their particular morphology, they have multiple functions and communication features (2). Kato et al developed MLO-Y4 cells, which are remarkably close to primary osteocytes (3) and this cell line, which has high expression of osteocalcin, connexin 43 but low alkaline phosphatase, was obtained from a transgenic mouse. Ca2+ and phosphate metabolism is regulated by the osteocyte lacuno-canalicular, which contains multiple proteins such as Phosphate regulating neutral endopeptidase on chromosome X (PHEX), Matrix extracellular phosphoglycoprotein (MEPE), sclerostin, and Dentin matrix protein 1 (DMP1) that can affect systemic mineral homeostasis (4). Osteocytes also play a role in the regulation of calcium and phosphate metabolism which contains multiple proteins such as phosphate regulating neutral endopeptidase on chromosome X (PHEX), matrix extracellular phosphoglycoprotein (MEPE), sclerostin, and dentin matrix protein 1 (DMP1) that can affect systemic mineral homeostasis.

These proteins are secreted by osteocytes and have an essential role in bone modeling and remodeling (5). DMP1 is highly expressed in osteocytes and has a function in bone mineralization. MEPE is a bone mineralization regulator that modifies mineralization within the osteocyte microenvironment in response to mechanical loading. Additionally, MEPE and DMP1 belong to the same SIBLING family (6). Another osteocyte specific protein is PHEX which is related to biomineralization and phosphate homeostasis and sclerostin inhibits osteoblastic bone formation and is expressed and released in osteocytes and other terminally differentiated cell types embedded inside mineralized matrices (7).

Previous studies have shown that these proteins also have a function in the extracellular matrix as a transcription factor (8,9). Ling et al showed that bone mineralization was decreased in DMP1 null mice (10). MEPE protein expression was shown in human bone osteocytes and bone marrow by Rowe et al (11). Over-expression of MEPE protein in bone causes a mineralization defect in a murine mice model (12). Lu et al reported that MEPE protein was produced by both osteoblasts and osteocytes during skeletogenesis as early two days of the post-natal period (13). It is therefore clear that PHEX, MEPE and DMP1 are secreted by osteocytes. Ca2+ is an important intracellular secondary messenger and it is stored in the skeletal system in a form that is linked to carbonated apatite crystals. Ca2+, on the other hand, is found in soluble form in the extracellular domain (14). In osteoblast cells, increased extracellular Ca2+ has been shown to trigger intracellular Ca2+ signaling (15). In osteoprogenitor cells, increased Ca2+ concentration has also been shown to boost proliferation, differentiation, protein matrix production, and mineralized nodule formation in a dose-dependent manner (16). Ca2+ and phosphate in the cell culture medium are often analyzed and imaged, especially to quantify mineral deposition rates (17). The measurement of Ca/P ratios used for the identification of the mineralization in the medium can be compared to the ratio in tissue. Similarly, just demonstrating that the culture's Ca2+ or phosphate concentration increases over time is insufficient because numerous anionic matrix molecules can bind Ca2+, and the culture's development typically entails changes in matrix protein phosphorylation (18). The cells must produce a collagen matrix on which the mineral will deposit, and that matrix must contain proteins and peptides that support mineralization (19). The addition of serum used in cell culture (which increases Ca2+ concentration) provides both mineralization inhibitors and beneficial growth factors (20,21).

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+.

Materials and methods

Cell culture

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).

Live/dead cells assay

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.

CAT, SOD, GSH and NOX measurements

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.

Determination of the total antioxidant, oxidant status and oxidative stress index

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 (22). Oxidative Stress Index (OSI) was measured as: OSI=[(TOS, µmol H2O2 equivalent/l)/(TAS, mmol Trolox equivalent/l) ×10].

Immunoblotting for PHEX, MEPE, DMP1 and GAPDH

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.

Caspase-3 analysis

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).

Statistical analysis

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.

Results

The effects of Ca2+ concentration cell viability in MLO-Y4 cells

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 (Fig. 1). Based on the above results, we questioned whether there was a correlation between the incubation period of Ca2+ and cell viability. Therefore; we set up two experiments as 60 min and 24 h at the same Ca2+ concentrations. Dead cells were increased at 24 and 50 mM Ca2+ concentrations in 60 min (Fig. 2). In 24 h of Ca2+ treatment, there were much more dead cells than live MLO-Y4 cells on 50 mM Ca2+ treatment (Fig. 3). The percentage of live/dead cells were shown in Fig. 4.

Figure 1. 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.

Figure 2. 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.

Figure 3. 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.

Figure 4. 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.

The Ca2+ increases CAT, SOD, GSH and NOX levels in MLO-Y4 cell lines

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 (Fig. 5A and B). GSH levels in different Ca2+ inductions did not differ significantly (Fig. 5C). In 15 min, the higher Ca2+ levels steadily raised NOX levels (Fig. 5D). 18, 24 and 50 mM Ca2+ concentrations led to the depletion of antioxidant enzymes such as CAT and SOD. The significantly increased NOX activity was observed in 18, 24 and 50 mM Ca2+ concentrations (P<0.05) (Fig. 5D). 24 mM Ca2+ concentration led to a significant increase in CAT levels within 60 min of treatment (Fig. 6A). SOD activity gradually increased by the elevated concentrations of Ca2+ in 60 min. The statistically significant difference in SOD was observed in 24 and 50 mM Ca2+ concentrations compared to that of 1.8 mM Ca2+ treatment in 60 min (Fig. 6B). There was no significant change in GSH activity in different Ca2+ points (Fig. 6C). NOX activity was at the highest level in 24 and 50 mM compared to that of 1.8 mM Ca2+ concentrations in 15 and 60 min (Fig. 6D). Our results clearly show that 12 mM Ca2+ concentration augments CAT and SOD levels, but from the beginning of 12 mM Ca2+ application NOX enzyme levels significantly increased in dose dependent on manner in 15 and 60 min. 24 mM Ca2+ concentration significantly increased SOD and CAT levels in 60 min. GSH levels of MLO-Y4 cells were stable by the induction of different Ca2+ concentrations and different time points. NOX activity gradually increased concentration and time.

Figure 5. 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.

Figure 6. 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.

Ca2+ induces bone mineralization by triggering crosstalk between ROS dependent activation and PHEX, MEPE, DMP1

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 (Table I). While the amount of Ca2+ increased, total oxidant status and OSI were significantly increased at 12, 18, 24 and 50 mM Ca2+ (P<0.05 compared to 1.8 mM Ca2+).

Table I. TAS, TOS and OSI were measured in MLO-Y4 cells after 24 h of Ca2+ application.

Table I.

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	2.544a
12 mM Ca	0.252	17.328a	6.877a
18 mM Ca	0.383	18.244a	4.758a
24 mM Ca	0.149a	21.374a	14.376a
50 mM Ca	0.390	27.786a	7.130a

{ label (or @symbol) needed for fn[@id='tfn1-mmr-26-06-12876'] } 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].

a P<0.05 compared to control (1.8 mM calcium) group. TAS, total antioxidant status; TOS, total oxidant status; OSI, oxidative stress index.

Ca2+ upregulates crosstalk between PHEX, MEPE, DMP1 expression and bone mineralization

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 (Fig. 7).

Figure 7. 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 analysis

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 (Fig. 8). The data obtained from our results show all (12, 24 and 50 mM) Ca2+ concentrations stimulated the caspase-3 activity which is the key factor for the initiation of the apoptotic pathway.

Figure 8. 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.

Discussion

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 (23). Additionally, the breakdown of mesoporous bioactive glass scaffold releases active substances like calcium that have the potential to increase gene expression and cause osteoblast development and they also suggested that electrical stimulation could encourage cellular differentiation by controlling calcium ionic pathways and increasing calcium ion inflow into cells (24,25). Total calcium levels in healthy persons are normally kept within the range of 2.2-2.6 mM, while ionized calcium levels are usually maintained between 1.1-1.3 mM. However, soluble Ca2+ levels in the bone interstitial fluid may be higher, since previous studies demonstrated that the levels of Ca2+ ranged from 8 to 40 mM at the nearest area of the resorbing osteoclasts (26,27). Ca2+concentrations can range from 8 to 40 mM in the microenvironment of the resorbing osteoclast. Osteocytes can also release considerable quantities of Ca2+, as seen during lactation, implying that they are exposed to high extracellular Ca2+ concentrations (27). Elevated serum free calcium concentrations may have an impact on the maximal calcium concentration in the interstitial fluid of the bone that is closely next to osteoclasts. According to Sugimoto et al, bone remodeling depends on the presence of mononuclear cells and a high calcium concentration at the resorptive site (28).

Our results indicate that Ca2+ concentration significantly inhibits cell viability in a dose and time-dependent manner (Fig. 1, Fig. 2, Fig. 3). Changes in Ca2+ concentration have been shown to promote the function and cooperation of the osteocytes and osteoblasts (29). It has also been shown that many biological activities such as muscle contraction and nerve conduction are regulated as a result of these changes in Ca2+ concentration, which occurs outside the cell, especially in the presence of mechanical stress. Osteocytes are derived from the mature osteoblasts that have a more differentiated morphology during the bone formation process and are embedded in the matrix (30). The osteocytes contact with adjacent cells and communicate via dendritic processes in the canaliculi in the mineralized matrix of the bone. Furthermore, osteocytes can communicate with other cells by secreting factors into the bone marrow space or straight into the bloodstream (31,32). Cellular oxidative damage is a basic general mechanism of cell damage and is mainly caused by reactive oxygen species (ROS). While ROS is overproduced, it can cause oxidative stress which is characterized as an imbalance between ROS formation and antioxidant defense systems (33) and NADPH oxidases (NOX) produce ROS in a controlled to specifically regulate signal transduction (34). Oxidative stress can affect age-related diseases, including osteoporosis and significantly affect osteocytes. Increasing concentrations of cytoplasmic Ca2+ causes Ca2+ influx into mitochondria and Ca2+ disrupts metabolism of the cell. Additionally, Ca2+ in the cytoplasm can regulate phosphorylation/dephosphorylation of proteins (35). The lack of an impact of Ca channel inhibitors on expression levels of PHEX, MEPE and DMP1 proteins, SOD, CAT, GSH, NOX and Caspase-3 levels and total antioxidant and oxidant status is a limitation of our work. A further in vivo examination is necessary to determine the effect of high serum calcium concentrations on bone remodeling, which is another limitation of our work.

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 (36). Osteoporosis is a metabolic bone disease characterized by low bone mineral density and a loss in bone mass, making the bone fragile and prone to fracture (37). According to a recent review article by Domazetovic et al, few data have shown how the molecular mechanism changes with apoptosis and oxidative status (38) but, recently, more studies have been aiming to explain the details.

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 (Figs. 5 and 6). Additionally, ROS induces the apoptosis of osteoblasts and supports osteoclastogenesis (39). Advanced oxidation products had been shown to inhibit the proliferation and differentiation of rat osteoblastic cells and rat mesenchymal stem cells (40) but osteocytes have been reported as a major source of the cytokine receptor activator of nuclear factor kappa-B ligand (RANKL), its functions as a key factor for osteoclast differentiation and activation (41). Yu et al reported that the treatment with advanced oxidation protein products significantly triggered apoptosis of MLO-Y4 cells and induced the phosphorylation of c-Jun N-terminal kinases (JNK) and p38 mitogen-activated protein kinases. Their findings cumulatively suggest that the advanced oxidation protein products induced activation of JNK/p38 MAPK is reactive oxygen species (ROS)-dependent (42). Superoxide is formed when oxygen acquires an additional electron and oxygen is generally converted by the superoxide dismutase (SOD) which is the first enzymatic structure of the antioxidant defense system (43). Kobayashi et al revealed that mitochondrial superoxide dismutase 2 (Sod2) depletion in osteocytes improved the production of cellular superoxide and Sod2 loss decreased the number of live osteocytes inside the lucano-canalicular networks (44). To estimate oxidative and antioxidative status in cells, TOS and TAS are generally used. While Ca2+ concentration was increased, TOS and the OSI values also increased in our findings (Table I). Glutathione (GSH) is a tripeptide in living organisms and it acts as an antioxidant either directly by interacting with ROS (45). There was no significant difference between GSH levels of different Ca2+ concentrations. CAT is the one of the most important antioxidant enzymes which break down two H2O2 molecules into one molecule of oxygen and two molecules of water (46). Increased concentrations of Ca2+ in 15 min treatments led to elevated CAT activity. Previous studies suggested that concentrations of Ca2+ in the range of 10–15 mM demonstrated a moderate negative effect on cell survival in calvarial cells of the newborn mouse (26). Consistent with these findings, our study demonstrates that 12, 24 and 50 mM Ca2+ concentrations result in elevated caspase-3 levels osteocytes are sensitive to the extracellular concentration of Ca2+, up to around 12 mM in 15 and 60 min of induction (Fig. 7). PHEX, MEPE and DMP1 are essential proteins released from osteocytes and play a significant role in bone mineralization. Topically administered Ca2+ to osteocytes in varying amounts could have a variety of effects. The goal of our study was to reveal the changes in bone mineralization-related protein levels in different Ca2+ concentrations and at different time points. Our results showed that PHEX, MEPE, and DMP1 expression levels correlated to the increased concentrations of Ca2+ (6, 12, 18 mM). The highest increase in PHEX, MEPE and DMP1 expressions was observed at a Ca2+ concentration of 24 mM (Fig. 7). However, the expression levels of the proteins decreased at 50 mM Ca2+ concentration because of the enhanced dead cell ratio.

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) (47), and they play a key role in the progression of hypophosphatemic conditions such as tumor-induced osteomalacia and X-linked hypophosphatemic rickets/osteomalacia. PHEX deficiency results in the overproduction of fibroblast growth factor 23 (FGF23) in osteocytes (48), which leads to hypophosphatemia and impaired vitamin D metabolism (47). Miyagawa et al noted that DMP1 expression in osteocytic cells was increased after the 24 h treatment with 10 mM phosphate (47). Both PHEX and DMP1 are also important proteins on phosphate homeostasis. Because another role of osteocytes is to regulate phosphate (Pi) homeostasis through the release of several molecules such as PHEX, MEPE, DMP1 and FGF23 (49). DMP1 and PHEX inhibit the expression of FGF23, whereas MEPE increases it, although the mechanisms are still unknown (50,51). Both DMP1 and PHEX are excessively expressed in osteocytes and might regulate the maturation of the osteocyte cells via FGF23 regulation of phosphate homeostasis (8). Magne et al cultured M2H4 cells and supplemented the media with 3 mM Pi from day 8 to 20 (20). They have shown that M2H4 cells had more mineralized the dentin extracellular matrix than control conditions (1 mM Pi) and medium supplemented 3 mM Pi accumulate Ca2+ in cell layers (20). In our study, both expressions of PHEX and MEPE increased at 24 mM Ca2+ induced.

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 (52–54). These proteins involved in bone construction are expected to be upregulated in response to anabolic loading, and they are responsible for absorption (55). Gluhak-Heinrich et al (52) revealed that alveolar osteocytes showed high basal levels of MEPE that decreased during the first day of loading compared to that of DMP1. Kulkarni et al (56) have shown that mechanical loading upregulated gene expression of MEPE but not PHEX in MLO-Y4 cells during 1 and 6 h pulsating fluid flow. RANKL plays an important role during the loading/unloading mechanism because unloading increases RANKL which is an essential promoter for osteocytes (57). Osteocytes has dendritic process, which is associated with mechanical stimulation, and this process is regulated by E11 which is the earliest osteocytes protein increased by mechanical loading by osteocytes (58). Moreover, E11, MEPE and DMP1 have higher levels of expression in basal bone than in alveolar bone (59). During the skeleton is exposed to loading, an increase in these proteins secreted from osteocytes can be expected, which is very important in the mineralization of bone tissue. A new strategy for bone treatment can be designed by giving the appropriate Ca2+ concentration to increase the release of these proteins from osteocytes.

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.

Acknowledgements

Not applicable.

Funding

This study was supported by Pamukkale University (grant nos. 2019BSP006 and 2019BSP002).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

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.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

All authors declare that they have no competing interests.

References