Studies on the Pinctada fucata BMP-2 Gene : Structural Similarity and Functional Conservation of Its Osteogenic Potential within the Animal Kingdom

Bonemorphogenetic protein (BMP)-2 plays an important role inmorphogenesis in both vertebrates and invertebrates. BMP-2 is one of the most powerful bioactive substances known to induce the osteogenic differentiation of mesenchymal cells. We examined the structural and functional conservation of Pinctada fucata BMP-2 in inducing osteogenesis in the murine mesenchymal stem cells, C3H10T1/2. Exposure of C3H10T1/2 cells to the recombinant mature fragment of Pinctada fucata BMP-2 resulted in osteoblastic differentiation.The sequence, SVPKPCCVPTELSSL, within theC-terminal portion ofPinctada fucataBMP-2, is homologous to the knuckle epitope of human BMP-2.This synthetic polypeptide was able to induce differentiation of C3H10T1/2 along the osteoblastic lineage, as confirmed by an increase in alkaline phosphatase activity, and the accumulation of calcium, as determined by von Kossa staining. Furthermore, using immunohistochemical staining, we observed an increased expression of collagen type I, osteopontin, and osteocalcin, which are known markers of osteogenesis. These results show that BMP-2 is conserved, not only in terms of its homology at the amino acid sequence, but also in terms of driving the formation of hard tissues in vertebrates and invertebrates.


Introduction
The transforming growth factor-beta (TGF-) supergene family plays various roles in the regulation of cell growth, cell specialization, and morphogenesis [1,2].More than 40 genes have been identified in the mammalian TGF- family [3], including the bone morphogenetic proteins (BMPs).BMPs are well-known regulators of bone and cartilage formation in vertebrates.
The BMPs and their homologs are also involved in the regulation of morphogenesis in the shells of mollusks [4][5][6][7].Hard tissue in invertebrates is equivalent to bone of vertebrates in terms of the organization of accumulated minerals.Although it is to be expected that BMP-2 or -4 participates in the formation of hard tissues such as the shells of mollusks, direct proof has not been reported.Previously, to examine the role of BMP-2 in morphogenesis and hard tissues formation of Pinctada fucata, we cloned the Pinctada fucata BMP-2 (PfBMP-2) gene using DNA obtained by PCR-based genome amplification as the probe [8].The PfBMP-2 gene was expressed strongly in the inner part of the mantle tissue, corresponding to the nacreous aragonite shell layer.This suggested that BMP-2 has a key role in the nacreous layer formation.Recently, indirect proof that PfBMP-2 participates in the hard tissue formation of P. fucata was reported [9].In that report, it was observed that there is a delay in the speed of regeneration in the inner shell surface following the inhibition of calcineurin by an intramuscular injection of cyclosporine A. The morphological abnormality of the crystal surface was also observed, mainly in aragonite regions, as well as a decrease in BMP-2 expression, suggesting an important role for BMP-2 in the nacreous layer formation [9].This result also indicated that PfBMP-2 functions via a calcineurin/NFAT4 pathway.
Although the mechanism of BMP-2 gene induction in invertebrates is not well understood, a large number of vertebrate studies have attempted to outline the process.Runx2/ Cbfa-1 binds to the 5  flanking region and activates the transcription of human BMP-2 [10].The relationship between calcineurin and BMP-2 expression has been reported in vertebrates [11].In that report, the authors showed that BMP-2 transcription was suppressed by cyclosporine but stimulated by ionomycin treatment and was stimulated in cells transduced with an adenovirus carrying the activated form of NFAT4.These results suggested that NFAT4 induces BMP-2 transcription via calcineurin during chondrogenesis.
Concerning a common factor in a bone and mollusk shell formation, some reports suggest that a BMP-2-like diffusible osteoinductive factor exists in the nacreous layer of the pearl oyster although the substances have not yet been identified [12][13][14][15].Although not conclusive, these results imply a common signal transduction pathway between vertebrate and invertebrate hard tissue formations [16].One candidate is BMP-2 or a fragment of the protein.If this reported diffusible factor is BMP-2 or a fragment, BMP-2 could represent a universal common factor, not only in the morphogenesis, but also in hard tissue formation in vertebrates and invertebrates animal.If true, it is necessary to prove its existence in the nacreous layer and to measure its potential to induce osteogenic differentiation.It would also be necessary to show that BMP-2 is functionally conserved across species.Among bone morphogenetic proteins and their homologs in invertebrates, the only factors that have been proven to be compatible with vertebrate are Drosophila Dpp and A60 [17].
In this paper, we examined the osteoinductive potential of Pf BMP-2 on the differentiation of mesenchymal cells C3H10T1/2 in vitro.Using histological analyses, our findings show that a purified recombinant 10-kDa mature fragment of Pf BMP-2 can induce osteogenic differentiation in C3H10T1/2 cells.Furthermore, we identified the functional domain of Pf BMP-2.The recombinant Pf BMP-2 or the synthetic polypeptide of the functional domain has the potential to be applied to regenerative medicine to a defective human bone.

Preparation of the Recombinant Mature Fragment of
PfBMP-2.Expression and purification of the recombinant mature fragment of Pf BMP-2 in E. coli were performed, as described in previously [19].

Synthetic Peptides Production.
Synthetic peptides, PGS-VPKPCCVPTELSSLSLL (corresponding to the amino acid sequence of Pf BMP-2 that showed the highest homology with the knuckle epitope of human BMP-2) and KIPKASSVPTEL-SAISTLYL (corresponding to amino acids 73-92 of human BMP-2 and acting as a positive control for this study) were purchased from Operon Biotechnologies, Inc. (Tokyo, Japan) and were dissolved in pure water to a concentration of approximately 20 mg/mL.2.4.Cell Culture.C3H10T1/2 mesenchymal stem cells (clone 8) were purchased from Human Science Research Resources Bank (Osaka, Japan).C3H10T1/2 cells were plated into a 40 mm culture dish (Nunc, Roskilde, Denmark) or a 48-well cell culture plate (Iwaki Glass, Tokyo, Japan) at a density of 1.4 × 10 4 /dish and 1.2 × 10 3 /well, respectively.C3H10T1/2 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Gibco BRL, Gaithersburg, MD) supplemented with 10% FBS (Gibco BRL) and 100 g/mL streptomycin (Gibco-BRL) at 37 ∘ C in a 5% CO 2 humidified incubator.For experiments, cells were grown in the presence of Pf BMP-2 or the synthetic peptides at the indicated concentrations and time periods as described for each figure, with regular changes of media and new compound supplementations at each media change.

Alkaline Phosphatase (ALP) Activity Assay.
To assay the enzymatic activity of ALP, the culture medium was removed and cell layers were washed with phosphate buffered saline (PBS, pH 7.4).Cells were lysed in 200 L of lysis buffer (0.1 M NaHCO 3 -Na 2 CO 3 , 2 mM MgCl 2 , 0.1% Triton X-100) and the lysate transferred to a microtube for centrifugation at 15,000 rpm for 5 min at 4 ∘ C. 100 L of supernatant was transferred to a new microtube.The reaction was started by adding p-nitrophenyl phosphate to the tube at a final concentration of 8 mM.The reaction was incubated for 30 min at 37 ∘ C and then stopped by the addition of 100 L of 1 M NaOH.The absorbance was measured at 405 nm.ALP activity was expressed as micromoles of p-nitrophenol (p-NP) produced per minute per milligram of protein.The protein content was determined using the Pierce BCA Protein Assay Kit (Pierce Chemical Company, Rockford, IL, USA).

Alkaline Phosphatase Staining.
For ALP staining, the culture medium was removed, and the C3H10T1/2 cells were washed twice with PBS before fixing with 4% paraformaldehyde in PBS for 1 min at room temperature.The cell layers were then rinsed with 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.05% tween 20.Staining was performed using an Alkaline Phosphatase Detection Kit (Chemicon, Temecula, CA, USA).

Von Kossa Silver Staining.
For von Kossa staining, the culture medium was removed and the C3H10T1/2 cells were washed twice with PBS and fixed with 10% neutral formalin solution for 30 min.The monolayers were then rinsed extensively with deionized water.The cells were treated with 5% AgNO 3 solution under ultraviolet light for 15 min.Plates were then rinsed with deionized water and the cells were fixed with 5% sodium thiosulfate for 3-5 min.
2.8.Immunohistochemistry. C3H10T1/2 cells were fixed with 10% neutral buffered formalin and washed with PBS.The fixed cells were then treated with 0.5% Nonidet P-40 for 10 min at room temperature for permeabilization and blocked with 10% serum for 30 min at 20 ∘ C. The cells were then incubated with antibodies against collagen type I, osteocalcin, and osteopontin (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 1 : 1000 dilution for 1 h.The cells were then washed and incubated with an appropriate biotinylated secondary antibody (1/4000) for 30 min followed by streptavidin-conjugated horseradish peroxidase for 20 min at room temperature.Proteins were counterstained with 3,30diaminobenzidine for visualization under the microscope.For the negative control, we used cells cultured for the same period of time in the absence of the synthetic peptides.

Structural Conservation of PfBMP-2.
To examine the structural homology between Pinctada fucata, Drosophilidae family members, and vertebrate BMP-2, we performed a Dot matrix analysis [18] of the conserved amino acid sequences.As shown in Figure 1, Pf BMP-2 has a high overall amino acid sequence homology with that of hBMP-2 and Xenopus BMP-2 (Figures 1(a) and 1(d)), as well as that of the Drosophilidae homolog Dpp and Chordata Amphioxus BMP2/4 (Figures 1(c) and 1(e)).Human BMP-2 also has high homology with that of the Drosophilidae homolog Dpp and Chordata Amphioxus BMP2/4 (Figures 1(f) and 1(g)).The degree of homology is remarkable, even in the mature C-terminal portion of Pf BMP-2 and the Drosophilidae homolog 60A (Figure 1(b)).

Effect of the PfBMP-2 Mature Domain on the Differentiation of C3H10T1/2 Cells.
To examine the osteogenic differentiative ability of Pf BMP-2, we used the mouse C3H10T1/2 cell line, a murine embryonic mesenchymal cell line [20].Previous reports showed that a synthetic peptide corresponding to residues 68-87 (NSVNSKIPKACCVPTELSAI) of the knuckle epitope of human BMP-2 could induce ectopic bone formation in vivo [21,22], suggesting that this sequence is the functional domain of the mature human BMP-2.Another study identified that a peptide corresponding to amino acids 73-92 (KIPKASSVPTELSAISTLYL) of human BMP-2, which substituted Cys78, Cys79, and Met89 with Ser (S), Ser (S), and Thr (T), respectively, induced a higher alkaline phosphatase (ALP) activity than the original 68-87 peptide [22].This 73-92 peptide competes with rhBMP-2 (recombinant human BMP-2) and can significantly inhibit the binding of rhBMP-2 to both receptors type IA and type II; this suggests that the substituted knuckle epitope has receptor binding capability [22].Therefore, we used this substituted synthetic polypeptide as a positive control for the induction of osteogenesis.
We examined the functional conservation of the osteoinductive potential of Pf BMP-2.Osteogenic differentiation in vitro was first confirmed using ALP activity.Figures 2(a 3 shows the specific activity of ALP at various intervals after the addition of either control medium, recombinant Pf BMP-2, or the hBMP-2 positive control polypeptide, respectively.ALP activity in C3H10T1/2 cells treated with the recombinant Pf BMP-2 was significantly greater than that of the untreated cells (versus ⧫-⧫) and was twice that of the synthetic polypeptide of the knuckle epitope ( -versus ◼-◼) at about 7 days.
ALP and von Kossa staining methods are commonly used to indicate the presence of osteoblasts.Therefore, we examined the effects of BMP-2 on the osteogenic differentiation of C3H10T1/2 cells using these histochemical techniques.As shown in Figure 4(a), ALP-positive cells were observed at 6 days in C3H10T1/2 cells treated with the recombinant Pf BMP-2 (1) and the hBMP-2 positive control (2), whereas there was negligible staining of the control C3H10T1/2 cells (3).We also examined the mineralized nodule formation in the cultures using von Kossa staining.As shown in Figure 4(b), mineralized nodule formation was observed at 6 days following the addition of either the recombinant Pf BMP-2 (1) or hBMP-2 positive control polypeptide (2), as compared with the control cells (3).This mineralized matrix indicates the differentiation of C3H10T1/2 stem cells into osteoblasts.Light microscopy showed that the undifferentiated C3H10T1/2 stem cells had a spindle-shaped fibroblastic phenotype (Figures 4(a To determine the functional domain of Pf BMP-2, a pairwise sequence alignment between Pf BMP-2 and human BMP-2 was performed.As shown in Figure 5, the alignment depicts a high homology between these two mature proteins.The Pinctada fucata sequence contains almost the same residues of human BMP-2 (68-87) (NSVNSKIPKACCVPT-ELSAI).Therefore, we synthesized an additional polypeptide (PGSVPKPCCVPTELSSLSLL) containing the amino acid sequence of Pf BMP-2 that showed the highest homology with the knuckle epitope of human BMP-2 and examined its osteogenic activity (herein referred to as Pf BMP-2 synthetic polypeptide).Figure 6 shows the specific ALP activity observed at various intervals after the addition of the Pf BMP-2 synthetic polypeptide.Both the Pf BMP-2 and hBMP-2 positive control synthetic polypeptides specifically increased ALP activity in C3H10T1/2 cells as compared with the untreated cells (◼-◼ and - versus ⧫-⧫).These increases were approximately three times higher at 21 days in the treated cells over the control cells.We then used the ALP and von Kossa histological analyses as before.As shown in Figure 7(a), ALP-positive cells were observed at 21 days in C3H10T1/2 cells treated with the Pf BMP-2 and hBMP-2 positive control synthetic polypeptides.In contrast, the staining for control C3H10T1/2 cells was negative.Furthermore, mineralized nodule formation was observed at 21 days for both synthetic polypeptides but not in the control cells (Figure 7(b)).

Induction of Early and Late Osteogenic Markers in
C3H10T1/2 Cells.The timing of the expression of each marker gene has been studied in detail in the differentiation process of stem cells.According to those reports, type I collagen is an early marker, alkaline phosphatase is an early/middle marker, and osteocalcin and osteopontin are late markers of bone formation [23,24].In addition, the expression of osteopontin was somewhat earlier than that of osteocalcin in experiments using calvarial osteoblasts [23].
To further confirm the differentiation of C3H10T1/2 cells, the expression of early and late osteogenic marker genes International Journal of Zoology  The bold underline indicates the synthetic polypeptides for examining the osteogenic activity of Pf BMP-2.: denotes identical residues in two BMP-2 orthologs; • denotes a moderately conserved residue.canonical pathway, Runx2 transcription is induced by transcription factors, such as Smad4 and Smad1/5.Runx2 associated with Smad1/5 induces the transcription of target genes involved in bone formation [25,26].In the SMADindependent noncanonical pathways, BMP signaling induces the expression of Dlx5.After that, phosphorylated Dlx5 induces the expression of Runx2 [27,28].Finally, these two pathways converge on Runx2 expression.Runx2 induces the expression of osteogenic marker genes, such as collagen type I, BSP, osteocalcin, and osteopontin [29].However, it remains unclear whether these canonical and noncanonical pathways also mediate the activity of Pf BMP-2 in the nacreous or the prismatic layer formation.Many genes participate in the nacreous or prismatic layer formation, such as the nacrein [30], N16/Pearlin [31,32], N19 [33], Pif [34], and prismalin 14 [35].While it has been reported that c-jun activates nacrein transcription via the AP-1 site located in the 5  flanking region [36], there are very few other reports in this field.Thus, studying the regulation of genes involved in hard tissue formation of mollusk shells is an important future subject.
Previous reports showed that the mature region of recombinant human BMP-2 [37] and the recombinant Drosophila Dpp and A60 homolog can induce bone formation [17] without posttranslational modifications such as glycosylation, phosphorylation, acetylation, sumoylation, or other types of modification.However, with the exception of BMP-3, BMPs are glycosylated on a conserved N-glycosylation sequence (NXS/T) in the mature protein domain [38][39][40], which is important for its function.Indeed, the biological activity of BMP-2 and BMP-7 was reduced following chemical deglycosylation [41], and N-glycosylation at the Asn 73 residue in the wrist epitope of BMP-6 is essential for its recognition by the type I receptor [42].Pf BMP-2 retains this NXS/T sequence.We performed a prediction of the N-glycosylation site of Pf BMP-2 and found two potential N-glycosylation sites at Asn 37 or Asn 97 or both (data not shown).We believe that Nglycosylation at these site(s) may contribute to the function of Pf BMP-2, and this will also be the focus of future studies.

Conclusion
BMP-2 induces mesenchymal cell differentiation into osteoblasts in vitro.We examined the functional interchangeability of Pinctada fucata and human BMP-2 synthetic peptides on the osteogenic differentiation of C3H10T1/2 murine mesenchymal stem cell cultures.Exposure of the C3H10T1/2 cells to the recombinant mature fragment of Pf BMP-2 resulted in osteogenic differentiation.The equivalent sequence from the knuckle epitope of human BMP-2 also induced osteogenic differentiation of C3H10T1/2 cells.Osteogenic induction was confirmed by an increase in ALP activity, the accumulation of calcium, and the protein expression of osteogenic markers.These results show that BMP-2 is conserved not only in terms of homology, but also in terms of its function in the formation of hard tissue.
) and 2(b) show the dose-response effects of recombinant Pf BMP-2 and the synthetic polypeptide of the knuckle epitope of human BMP-2 on ALP activity in C3H10T1/2 cells.ALP activity increased with increasing amounts of BMP-2 and reached a plateau at 2.5 g of Pf BMP-2 (Figure2(a)) and 40 g of the synthetic BMP-2 (Figure 2(b)).

Figure 4 :Figure 5 :
Figure 4: Histochemical analysis.C3H10T1/2 cells were plated into 40 mm culture dishes and cultivated for 2 days.Cells were then treated with 250 ng/mL recombinant mature BMP-2 or 1 g/mL synthetic polypeptide of the knuckle epitope of human BMP-2 for 6 days culture before staining.(a) Alkaline phosphatase staining at 6 days.(b) von Kossa staining at 6 days.1: recombinant Pf BMP-2; 2: the synthetic polypeptide of the knuckle epitope of human BMP-2; and 3: control culture.Cells were photographed at 100x magnification.

Figure 6 :
Figure 6: Effects of the Pf BMP-2 synthetic polypeptide and the synthetic polypeptide of the knuckle epitope of human BMP-2 on the endogenous expression of alkaline phosphatase in C3H10T1/2 cells.C3H10T1/2 cells were plated into 48-well dishes for 2 days and then treated with 80 g/well Pf BMP-2 synthetic polypeptide or 80 g/well synthetic polypeptide of the knuckle epitope of human BMP-2.Alkaline phosphatase activity was measured at endpoint, on the days indicated.◼-◼: the knuckle epitope of human BMP-2, -: Pf BMP-2 synthetic polypeptide, and ⧫-⧫: in the absence of protein.

Figure 7 :
Figure 7: Histochemical analysis.C3H10T1/2 cells were plated into 40 mm culture dishes for 2 days.Cells were then treated with 250 ng/mL Pf BMP-2 synthetic polypeptide or 1 g/mL synthetic polypeptide of the knuckle epitope of human BMP-2 for the indicated time periods.(a) Staining for alkaline phosphatase was performed after 21 days.(b) von Kossa staining at 21 days.1: the Pf BMP-2 synthetic polypeptide; 2: the synthetic polypeptide of the knuckle epitope of human BMP-2; 3: control culture.The white arrows indicate the areas of calcification.Cells were photographed at 100x magnification.

3. 4 .Figure 8 :
Figure 8: Immunohistochemical staining for osteogenic markers in C3H10T1/2 cells.C3H10T1/2 cells were plated into 40 mm culture dishes for 2 days and then treated with 250 ng/mL Pf BMP-2 synthetic polypeptide or 1 g/mL synthetic polypeptide of the knuckle epitope of human BMP-2 (hBMP-2) for the indicated time periods.(a) Immunohistochemical staining of collagen type I after 7 days in culture.(b) Immunohistochemical staining of osteopontin after 13 days in culture.(c) Immunohistochemical staining of osteocalcin after 18 days in culture.Cells were photographed at 100x magnification.