Kielin/chordin‐like protein enhances induction of osteoblast differentiation by Bone Morphogenetic Protein‐2

Bone morphogenetic proteins (BMPs) play a key role in embryonic differentiation for osteoblast and bone formation. Kielin/chordin‐like protein (Kcp) is known to enhance the effects of BMP signaling. Here, we present ALP activity, gene expression, and calcification data demonstrating that Kcp affects the differentiation of C2C12 myoblasts into osteoblasts. We report that the presence of Kcp enhances the ability of BMP‐2 to induce the differentiation of C2C12 myoblasts into osteoblasts. Additionally, BMP‐2‐mediated stimulation of phosphorylated Smad1/5 was apparently enhanced in the presence of Kcp. The present findings may contribute to progression toward the clinical use of BMPs for treatment of bone fracture, osteoarthritis, and other similar conditions.

Kielin/chordin-like protein (Kcp), a large secreted protein that contains 18 cysteine-rich domains, has become widely known for its regulatory effects on bone morphogenic proteins [7]. It is generally expressed in the kidneys and brain during all stages of the embryonic developmental process, with abundant amounts localized in renal tubules of the kidney cortex, though not within the nephrogenic zone [8]. Kcp has both suppressive and enhancing effects on some cytokines, such as enhancement of BMPs, and suppression of other cytokines such as TGF-b or activin [7]. Although the mechanism of the effects of Kcp has not been completely revealed, it is regarded as a direct action of binding to these cytokines, due to its paracrine function [8].
The present study was conducted to investigate the sensitivity and effects of Kcp as an enhancer of BMP for induction of osteoblast differentiation utilizing C2C12 mouse cells in vitro. The results show that Kcp enhances the BMP effect by increasing activation of reactions of Smad protein with BMP type-II receptors by phosphorylation.

Cell cultures
C2C12 myoblasts derived from mouse samples were used in the experiments. For cell proliferation, 15% fetal bovine serum (FBS) containing DMEM medium was used, while 2.5% FBS containing DMEM was utilized for cell differentiation.

Determination of alkaline phosphatase activity
In order to analyze differentiation of C2C12 myoblasts into osteoblasts, alkaline phosphatase (ALP) activity was determined as a marker. Myoblasts in a 96-well dish (1 9 10 4 cells per well) were inoculated with 15% fetal bovine serum (FBS) containing DMEM medium 1 day before treatment with reagents for cell proliferation. BMP-2 (300 ngÁmL À1 ) and Kcp (400 ngÁmL À1 ) with 2.5% FBS containing DMEM were added; then, incubation was performed at 37°C for 72 h. Following differentiation, 1% NP-40 containing physiological saline solution was added and then subjected to destruction using an ultrasonic destructor. To determine ALP activity within the cells, an ALP activity reagent containing disodium-P nitrophenyl phosphate hydroxylate was added and incubation performed for 15 min at 37°C, and then, absorbance at 405 nm was measured.
Nickel pull-down assay of ALP activity with Kcp full-length plasmid C2C12 myoblasts in 6-well dishes (1 9 10 5 cells per well) containing DMEM with 2.5% FBS were inoculated with Kcp full-length (Kcp FL) plasmid, constructed by Vector-Builder Japan and termed pPB[Exp]-Puro-EF1A > lgj-sig/ mKcp[NM-00102998.4]/Myc/6xHis (VB220208-1023cxt), and Lipofectamine 3000 (Thermo Fisher Scientific); then, incubation was performed at 37°C for 72 h. Using 2 mL of supernatant from a dish with cell lysate containing histidine (His) tagged with full-length Kcp, a nickel pull-down assay was conducted. Ni-NTA agarose beads were prepared with a high affinity for His-tagged proteins and used with His-tagged full-length Kcp to bind to the nickel-charged resin, after which the resin was washed thoroughly to remove any unbound or nonspecifically bound proteins. Finally, the His-tagged protein was eluted from the resin using a buffer containing a high concentration of imidazole, which competes with the His residues for binding to nickel ions. A sample containing 0.5 mL of the eluted Kcp FL was utilized as a reagent for determining ALP activity performed with partial length Kcp. Myoblasts in a 96-well dish (1 9 10 4 cells per well) were inoculated with 15% fetal bovine serum (FBS) containing DMEM medium 1 day before treatment with reagents for cell proliferation. Finally, DMEM containing 2.5% FBS with BMP-2 and 10% of the Kcp FL from the nickel pull-down assay were added, and incubation was performed at 37°C for 72 h.

ALP activity staining
After culturing with reagents for BMP-2 and Kcp, cells were treated with 10% formalin for 20 min at room temperature, then an acetone/ethanol solution was added, and incubation continued for 1 min at room temperature. For ALP activity staining, cells were treated with an ALP staining solution containing Naphthol AS-mX and Fast blue BB salt and then semi-dissolved with N-N diethyl formalin for 15 min at 37°C. Purple staining in the cells was considered to indicate the portion possessing ALP activity.

Alizarin red staining
To determine the amounts that showed differentiation into osteoblasts, the degree of osteoblast calcification was determined using Alizarin red staining and its absorbance. In the same manner noted above, C2C12 myoblasts in a 96-well dish (1 9 10 4 cells per well) were inoculated with 15% FBS containing DMEM medium 1 day before treatment with reagents for cell proliferation. BMP-2 (100 ngÁmL À1 ) and Kcp (500 ngÁmL À1 ) were added with 2.5% FBS containing DMEM, and then, incubation was performed at 37°C for 72 h. Culturing was then continued for 11 more days with differentiation medium containing ascorbic acid (50 lLÁmL À1 ), b-glycerophosphate (10 mM), and dexamethasone (10 nM), which was changed every 3 days. For staining, cells were treated with 95% ethanol for 20 min at room temperature; then, a calcification staining mixture containing 1% Alizarin red S and ammonium solutions was added for 5 min at room temperature. Parts with dark reddish staining were considered to indicate osteoblast calcification. Consequently, after drying the stained cells, 10% cetylpyridinium chloride hydrate was applied to dissolve them and absorbance at 570 nm was measured.

ALP and osteocalcin gene expressions
Quantitative evaluation of osteoblast mRNA expression in C2C12 cells in the presence or absence of BMP-2 and Kcp was performed. C2C12 myoblasts in a 6-well dish (1 9 10 5 cells per well) were inoculated with BMP-2 (100 ngÁmL À1 ) or Kcp (500 ngÁmL À1 ) with 2.5% FBS containing DMEM and then incubated at 37°C for 72 h. Expression of marker genes, such as ALP and osteocalcin, was evaluated for determining osteoblast differentiation. Total RNA was extracted from C2C12 cells using TRIzol reagent (Life Technologies, Carlsbad, CA, USA). After inducing a reverse transcription reaction with 59 RT Master mix (catalog number FSQ-201; TOYOBO, Kita-ku, Osaka, Japan), gene expressions were quantified using realtime PCR with THUNDERBIRDÒ qPCR Mix (catalog number QPS-101, TOYOBO) for ALP and with glyceraldehyde 3-phosphate (GAPDH) from a TaqMan TM Gene Expression Assay Kit (Thermo Fisher Scientific) for osteocalcin, and then, the levels of expression were normalized based on the value for GAPDH.
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Results and Discussion
ALP activity measurement, and quantitative analysis of ALP and osteocalcin gene expression for differentiation into osteoblasts Results showing promotion of differentiation of C2C12 myoblasts into osteoblasts with BMP-2 and Kcp are presented in Fig. 1. For this analysis, increased ALP activity was considered to indicate increased differentiation into osteoblasts. With stable concentrations of BMP-2 and Kcp, the activity of ALP was apparently increased in the presence of BMP-2, while addition of Kcp enhanced that increase induced by BMP-2 ( Fig. 1A(i)). ALP activity staining was also performed to visualize the increased activity, with purple staining indicating osteoblasts. Without reagents or in the presence of Kcp alone, no color change was observed, whereas under the condition with both BMP-2 and Kcp, several of the cells changed to purple (Fig. 1A(ii)). Additionally, quantitative mRNA expression of ALP and osteocalcin genes were analyzed by normalizing their values with the value of GAPDH. The amounts of ALP and osteocalcin genes were obviously increased in the presence of BMP-2 with Kcp as compared to BMP-2 alone (Fig. 1B) Kcp FL also has an enhanced effect to block induction of BMP-2 into osteoblasts (Fig. S1).

Effects of BMP-2 on differentiation into osteoblasts with different concentrations of Kcp
ALP activity was measured after altering the concentrations of BMP-2 and Kcp. Analysis of the increased rate of ALP activity with a stable concentration of BMP-2 (100 ngÁmL À1 ) and various concentrations of Kcp (0-1,000 ngÁmL À1 ) noted statistically significant values with a Kcp concentration of 100 ngÁmL À1 ( Fig. 2A,B), indicating that an extremely low concentration of Kcp has no effect on ALP activity. Moreover, according to the results shown by a graph, ALP activity did not increase in a dose-dependent manner as seen with Kcp. Furthermore, using a stable concentration of Kcp (500 ngÁmL À1 ), a suitable concentration of BMP-2 for inducing a significant increase in ALP was examined, with the results indicating that > 100 ngÁmL À1 of BMP-2 had an adequate effect to increase the activity of ALP (Fig. 2C,D).

Enhancement of BMP-2-induced calcification in osteoblasts by Kcp
To examine calcification of osteoblasts, use of Alizarin red staining was effective to determine differentiation into osteoblasts. First, C2C12 cells were incubated with BMP-2 and Kcp for 3 days, and then, the conditions were altered to differentiation medium lacking BMP-2 or Kcp, which was changed every 3 days. On Day 14, there was an obvious increase in number of cells stained with Alizarin red when incubated in the presence of both BMP-2 and Kcp, with similar results noted when in the presence of BMP-2 alone (Fig. 3A,  B). In contrast, nearly no calcification was observed in the control cells or when incubated with only Kcp. the Smad protein complex that is transferred inside of the nucleus to regulate transcription DNA [9]. To examine the level of activity and means of increasing the effect of BMP-2 by Kcp, western blotting analysis was performed to detect the amount of activated Smad proteins in each sample. Accumulation of phosphorylated Smad1/5 by BMP-2 stimulation was apparently enhanced in its presence (Fig. 4A,B), suggesting that Kcp acts to promote the phosphorylation of Smad proteins within cells via receptor activation (Fig. 4C).

Enhancing effect of Kcp on BMP-2 and its effectiveness
Previous studies have shown the existence of antagonists that bind to BMP and inhibit its function. These are mainly classified into four types, that is, Chordin, Noggin, DAN family, and Folistatin, and indicated to inhibit the function of BMP with a negative feedback system [10]. In contrast to those antagonists, a few reports have noted factors that enhance the effects of BMP. As mentioned above, Kcp, which possesses abundant cysteine residues, has been detected in an embryonic renal mesenchyme cDNA library [8], and reported to contain 18 cysteine-rich coding repeats and a von Willebrand factor D domain [11]. Generally, induction of bone formation by mammalian cells requires a large amount of BMP. Based on the present findings showing the enhancing effect of Kcp on BMP-2, it is speculated that this BMP agonist diminishes the amount of BMPs available for regeneration and formation of bone. Heparin, composed of sulfated polysaccharides and regarded as another BMP agonist, similarly enhances BMPs to inhibit their transformation into myogenic cells, and induction of osteoblast and bone formation [12]. A previous study found that 5 lgÁmL À1 of heparin had an effect on differentiation of C2C12 cells into osteoblasts with 100 ngÁmL À1 of BMP-2 [12]. In the present experiments, in comparison with the effect of heparin, 100 ngÁmL À1 of Kcp produced an adequate effect for 100 ngÁmL À1 of BMP-2, with a significant difference ( Fig. 2A,B). Furthermore, use of 500 ngÁmL À1 of Kcp provided nearly the maximum effect with 100 ngÁmL À1 of BMP-2 on C2C12 cells for osteoblast differentiation (Fig. 2C,D). Regarding the effectiveness of the enhancing effect on BMP-2, it is suggested that Kcp is superior to heparin for osteoblastic differentiation. It has been shown that Kcp can bind directly to BMP-7 and promote binding to BMPR-1A, leading to positive regulation of BMP signaling [8]. That study also found that Kcp binds directly to BMP-7 and enhances BMP-7-mediated phosphorylation of Smad1/ 5/8, a downstream signaling event of BMP signaling. Based on the present findings, it is considered that the same occurs with respect to the interaction of BMP-2 and Kcp. Additional studies will be needed to fully elucidate the molecular mechanisms underlying this interaction.
In conclusion, the present results indicate that as compared to its presence alone, a low amount of BMP-2 with Kcp has a definite effect on enhancing ALP activity to induce differentiation of C2C12 cells into osteoblasts.

Supporting information
Additional supporting information may be found online in the Supporting Information section at the end of the article.