Dexamethasone stimulates nephronectin expression and mediates mineralization in MDPC-23 cell via Akt/mTOR signaling pathway

Dexamethasone (Dex) is frequently used for induction of cellular mineralization in vitro. However, the underlying mechanism of its influence on mineralization remains incompletely characterized. Nephronectin (Npnt), a novel RGD-containing matrix protein, was reported to promote osteoblast differentiation and mineralization, moreover, its mRNA expression was found to be up-regulated by bone morphogenetic protein 2 (BMP2) and 1α25-dihydroxyvitamin D3 in concentrationand time-dependent manner. The present work seeks to uncover the effect of Dex in the regulation of Npnt mRNA expression and possible signaling pathways that involved in the Dex-mediated mineralization. The results conclude that Dex promotes cellular mineralization in MDPC-23 cell in a dose-dependent manner via Akt/mTOR signaling pathway, simultaneously, it up-regulated Npnt mRNA expression during the differentiation process of MDPC-23 cell. Correspondence to: Jia Tang, Division of Biochemistry, Department of Oral Biology, School of Dentistry, Health Sciences University of Hokkaido, 1757-Kanazawa, Ishikari-Tobetsu, 061-0293, Hokkaido, Japan. E-mail: tangjia@ hoku-iryo-u.ac.jp


Introduction
Dexamethasone (Dex) is a type of synthetic glucocorticoid (GC) analogue, which exhibits intensive GC activity without showing any mineralocorticoid activity. Its GC activity is 30 times as potent as cortisol [1], the natural adrenal steroid secreted by adrenal cortex. Dex and other GCs are widely used in anti-inflammatory (eg. rheumatoid arthritis) and immunosuppressive treatments following organ transplant. However, the dosage should be carefully fine-tuned to avoid unwanted side effects, as long-term use of GCs often results in osteoporosis [2].
In the in vitro experiments, Dex in combination with β-glycerophosphate (β-GP) and ascorbic acid (AA) are frequently used for induction of osteogenic commitment and differentiation [3]. Stem cell (eg. bone marrow mesenchymal stem cell) [4] and certain mineralizing cell lines (eg. MC3T3-E1 cell) [5] are promising candidates for the approaches. Efforts have been ongoing in elucidating the underlying molecular mechanism of Dex-related effects on osteoblast differentiation. Yamanouchi et al. reported a concentration-dependent (10nM, 100nM, 1µM) effect of Dex on alkaline phosphatase activity in human osteoblast cell [6]. Ogata et al. examined osteoblastic cell lines (UMR 106-6 and ROS 17/2.8 cell) after exposure to a single dose of Dex (10nM). The researchers found that there was a significantly sixfold increase of bone sialoprotein (BSP) expression.
Further, they identified a GC response element in BSP gene promoter [7]. Aside from BSP, secretion of alkaline phosphatase (ALP) and osteocalcin (OCN) were augmented by the same dosage of Dex as well in another group's study using human iliac trabecular bone cells [8]. More recently, a detailed characterization of the effects of Dex on long-term culture of mesenchymal stromal cells revealed that, the Dex exerts biphasic effect on cells.
In the early stage, transcription factors including Runx2 and osterix (Osx) are up-regulated to promote osteogenic differentiation, when the cells enter the late stage of culture, protein expression of GC receptors (GR) decreases in a time and dose dependent manner, leading to weakened or even frigid response of Runx2 and Osx to Dex stimulation; moreover, Dex also promotes the expression of an inhibitory protein histone deacetylase 6 (HDAC6) and forms a complex with GR, which further compromises the chance of Dex binding to GR [9]. Nephronectin (Npnt) was identified to stimulate the differentiation and mineralization of MDPC-23 cell when coated to polystyrene [10]. Further, over-expression of Npnt in MC3T3-E1 cell accelerates the mineralization process in vitro [11]. Recently, Kamijo's group reported Npnt can be up-regulated upon exposure to both bone morphogenetic protein 2 (BMP-2) [12], a well-established osteogenic molecule [13], and 1α-25-dihydroxyvitamin D3 [14], an essential calcium homeostasis regulator [15,16]. These clues suggest Npnt is positively involved in the process of cellular differentiation toward hard tissue forming direction. Based on the above-noted evidences in relation to the osteogenic capacity of Dex, it is thus of particular interest to investigate whether Dex plays some role in regulation of Npnt expression. The purpose of current work was therefore to examine the Npnt expression after treatment by Dex and explore possible signaling pathway that is involved in Dex-mediated mineralization in MDPC-23 cell.

Protein kinase B (PKB or Akt)/mechanistic target of rapamycin (mTOR) signaling pathway inhibition
MDPC-23 cells were seeded into 24-well plate (For mineralization assay) or 12-well plate (For gene expression analysis) (seeding density: 1×10 4 /mL), maintained in CM for four or five days. Afterwards, the cells were exposed to Akt inhibitor (600 nM, 6 µM, 60 µM, dissolved in DMSO) (14870, Cayman) or mTOR inhibitor (1 µM, 10 µM, 100 µM, dissolved in DMSO) (KU0063794, Wako) in DMEM (FBS free) for two hours; in control and Dex groups cells were treated by the DMSO vehicle only. Following inhibition, the media containing inhibitors or DMSO vehicle were aspirated and changed according to the purpose of experiment. For the mineralization test, Dex was added in conjunction with β-GP and AA in DMEM (5%FBS). For the gene expression experiment, Dex alone was added. As the vehicle for Dex, PBS was added into the control group.

Cell viability assay
MDPC-23 cells were seeded into 96-well plate (tissue culture treated polystyrene, 3860-096, Iwaki) at the density of 1×10 3 /well in CM. At day three, cells were treated with various concentrations of Akt and mTOR inhibitors dissolved in DMEM in the absence of FBS for two hours in incubator. Subsequently, inhibitors containing media were aspirated and replaced with CM in the presence of Dex (100 nM) or PBS vehicle (Control). Cell Counting Kit-8 (CCK-8) reagent (10 µL/ well) (347-07621, Dojindo, Japan) was added the next day (day four) and the cells were incubated for another 1h and 45min, absorbance was read at the wavelength of 450nm.

Alizarin red staining
Cells were maintained in CM until confluence, afterwards, various concentrations of Dex (0.01, 0.1, 1, 10, 100 nM in PBS) were added to cells in the presence or absence of AA or β-GP (10 mM, Wako). Staining was conducted on day eight. Detailed description of staining and CPC extraction is given out elsewhere [10].

Statistical analysis
Results were presented as means ± SD, and the difference was assessed by post hoc Tukey HSD test. Statistical differences were determined based on the p value lower than 0.05.

Alizarin red staining
The alizarin red staining was used to identify the mineralization of MDPC-23 cell. Generally, cells start to mineralize after reaching confluence, the timing of adding Dex was accordingly selected at the day of cell achieving confluence. As Dex has dual roles on osteogenesis, the pharmacological concentration at 1 µM was inhibitory on alkaline phosphatase activity and mineralization of mesenchymal progenitor cells [18]. Therefore, to determine the optimum concentration of Dex in MDPC-23 cell, five different concentrations at the magnitude of nanomolar range were selected in the present experiment. To further testify whether the mineralization of cells could be enhanced synergistically by type I collagen, cells were seeded to non-coated tissue culture polystyrene or fish scale type I collagen (FCOL1)-coated polystyrene in the presence of osteogenic factors including AA, b-GP and Dex.
Type I collagen was an effective osteogenic factor [19]. Therefore, to investigate whether type I collagen could promote the mineralization of cells synergistically with Dex, cells were seeded into cultured plates coated with fish scale type I collagen (FCOL-1) or non-coated plates. The alizarin red staining, a method for evaluating calcific deposition in cells, revealed that, first, no mineralization was observed in groups without β-GP, highlighting β-GP is a pre-requisite for initiating mineralization. Indeed, as the source of phosphate for the formation of hydroxyapatite mineral (Ca10(PO4)6(OH)2) and inducer of osteogenic gene expression [20][21][22], β-GP is of critical importance to in vitro mineralization assay.
Nevertheless, one needs to be careful when interpreting the data, as acellular mineralization occurs when the cells are treated with β-GP at a concentration higher than 2.5 mM, and this could also be detected by alizarin red or von kossa staining [23]. In the current work, no mineralization was observed in cells treated by 10 mM β-GP in the absence of Dex or presence of Dex (0.01-0.1 nM), which indicated that although the concentration of β-GP was much higher than the threshold of acellular mineralization, the cells did not mineralize in groups without or with low concentration of Dex (0.01-0.1 nM). In groups without β-GP, cells did not mineralize even in the presence of the optimum concentration (100 nM) of Dex, indicating β-GP and Dex are mutually indispensable for eliciting early mineralization. Second, the staining intensity was in proportion to the concentration of Dex used, in other words, the mineralization adopts a concentration-dependent increase upon exposure to Dex. Based on the quantification data, it was suggested that a concentration of 100nM exhibited the strongest staining in the three groups ( Figure  1A AA+ β-GP+ non-coated; Figure 1B AA-β-GP+ type I collagencoated; Figure 1A AA-β-GP+ non-coated), although the absolute value of absorbance in AA+ β-GP+ type I collagen-coated Dex (100 nM) was higher than Dex (10 nM), the difference (p>0.05) was not of statistical significance as analyzed by Tukey's test. Interestingly, the quantification result of alizarin red staining ( Figure 1C) indicated a coating of FCOL1 does not exert remarkable synergistic effect with Dex in enhancing mineralization. However, in our previous work, FCOL1 was found to contribute to nearly two-fold enhancement of MDPC-23 cell mineralization at day ten in osteogenic media containing AA and β-GP [19], but without Dex. A possible explanation may be that Dex was such a potent accelerator of mineralization that the potential long term effect of FCOL1 in promoting mineralization was yet to be detected as early as day eight. The present study showing the optimum concentration (100 nM) for Dex correlates well with a previous work demonstrating that 10 -7 /mL of Dex exerted the maximal effect on alkaline phosphatase activity in chick periostea cells [24]. Next, we demonstrated that Dex-induced stimulation of mineralization was accompanied by a concomitant dose-and time-dependent induction of Npnt (Figure 2 A&B), an adhesive matrix protein and strong promoter of osteo/odontogenic differentiation [10,11].

Up-regulation of Npnt stimulated by Dex and the effect of Akt/mTOR inhibition on Npnt expression
Subsequently, since Akt/mTOR signaling pathway is essential in osteo/odontoblast differentiation and bone mass determination [25][26][27]. We used Akt/mTOR inhibitors to elucidate the mechanism by which Dex regulates the Npnt expression in MDPC-23 cells. Figure  2C revealed up-regulation of Npnt was slightly inhibited by the Akt/ mTOR inhibitors, indicating other signaling pathways are involved in the regulation of its expression by Dex. Indeed, the ALK/SMAD4 [12]   and Wnt/β-catenin [28] signaling pathways were noted to play major roles in the up-regulation of Npnt in an osteoblast-like cell. Whether these two signaling pathways identified elsewhere also affect the Dexmediated up-regulation of Npnt in MDPC-23 cell, requires further scrutinization.

The effects of Akt/mTOR inhibition on cell viability and mineralization
In contrast, we noted that the inhibition of Akt and mTOR significantly impaired the viability ( Figure 3), remarkably, 60 µM Akt inhibitor abrogated the viability to nearly zero; mTOR at its highest concentration (100 µM) exhibited but mild attenuation. The mineralization of MDPC-23 cell activated by Dex was impaired in a concentration-dependent manner by Akt/mTOR inhibition ( Figure  4), specifically, a concentration of 6 µM and 60 µM of Akt inhibitor decreased the mineralization by 43% and 74%, respectively.
Similarly, in the groups treated by mTOR inhibitor, the initial concentration of 1 µM did not appear to affect the mineralization, when the concentration was increase to 10µM, the mineralization was abrogated by 32%. Importantly, 100 µM of mTOR inhibitor completely blocked the mineralization while it only moderately hampers the viability of cells.
Taken together, the results verified that Dex stimulates mineralization and simultaneously induces the expression of Npnt mRNA. The blocking of Akt/mTOR signaling pathway significantly attenuates the mineralization of MDPC-23 cell treated by Dex, albeit only slightly down-regulated Npnt expression.