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  • The BMP family comprises significant growth factors


    The BMP family comprises significant growth factors for the regeneration of bone and cartilage. Among these, BMP- has been known as an early marker during the proliferation of osteoblast cells, and one of the most potent stimulators of osteoblastic cells differentiation [40]. Studies with COX-2 knockout mice have demonstrated the roles of COX-2 in bone formation [41,42]. In addition to bone resorption, COX-2 may also have a role bone formation [43]. It is known that the effects of BMP-2 on osteoblastic differentiation are enhanced by BMP-2-induced COX-2 expression [44]. As shown in Fig. 4A and Fig. 7A, CCLP gradually triggered BMP 2/4 mRNA and protein overexpression, as compared to the control. An increase in the COX-2 mRNA and protein expression was observed after CCLP treatment, which may accelerate new bone formation. In terms of proliferation of MC3T3-E1 cells, CCLP-treated cells displayed the highest proliferation rate at day 5, which may be attributed to the effect of BMP2/4 and COX-2. In addition, OSC and OPN are closely related to calcium deposition during mineralization. At all tested concentrations, the highest level of ALP activity and expression of OSC mRNA were observed in CCLP-treated cells, which may be attributed mainly to the effect of BMP2/4 and COX-2. To understand the signaling pathway involved in CCLP-induced osteogenesis in MC3T3-E1 cells, we examined the protein expression of ERK, p38, and Smad1/5/8, which are the transcriptional factors involved in the proliferation and differentiation steps that result in the transformation of pre-osteoblasts into osteoblasts. BMP2 protein expression induced p38-, ERK-, and/or Smad1/5/8-dependent pathway [[16], [17], [18],28]. In this study, we found that the bioactive CCLP from C. intestinalis induced p38, ERK, and Smad1/5/8 after 24 h culture. However, Smad1/5/8 activation occurred at 24 h while ERK and p38 occurred at 6 h and 12 h, respectively (Fig. 7C). Previous reports have shown that treatment with latanoprost gene-related peptide induced rapid phosphorylation of ERK1/2 and p38 MAPK signaling pathway in human HaCaT keratinocytes [21]. Calcitonin plays an important role as a paracrine growth factor that upregulates CD44 variant (CD44 v). Calcitonin increases CD44 v expression by binding to its receptor, which activates MAPK kinase and p38 pathways [45,46]. The discovery of the bioactive role of calcitonin obtained from novel natural sources indicated the potential of peptides for pharmaceutical development. Furthermore, the prediction of peptide binding sites was used by the PepSite2 server to predict how likely the candidate peptides bind and interaction with the activin receptor [[47], [48], [49], [50], [51]]. To induce the osteoblast differentiation, these peptides are required to bind to the interaction site of activin receptor and then the enhanced capacity of the peptide activity is related to its ability binding with the residues of the receptors. BMP signaling is mediated through type I and type II BMP receptors. Among receptors, type IIa activin receptor (ActR-IIa) and type IIb activin receptor (ActR-IIb) will bind to BMP ligands. Then, the type I receptor was activated as an effector in the signal transduction by phosphorylating downstream molecules the Smad or/and MAPK pathway [16,50]. This could increase the degree of activity of the receptor and protein. After phosphorylation and activation by receptor kinases, protein is released from the receptor and activate transcriptional responses of specific target genes. Hence, the activin receptor enters in osteoblast differentiation and induces bone formation [50,51]. In this paper, the PepSite2 server is based on the preferred binding of CDGVSTCWLH, ELGNSVHATA, and GGKQNVGFGP residue type in CCLP peptide resulting from a calculation of peptide interactions with activin receptor. These structures of the receptor were scanned by the website server on the protein surface with the spatial position-specific in activin receptor structure and binding sites for the amino acid residues. The six amino acid residues within the sequence of GGKQNVGFGP showed a significant binding relationship with the target activin receptor, indicated that they are likely to interact with the receptor (Table 1). Interestingly, the results indicated that 6 residues of GGKQNVGFGP and 9 residues of ActR-II contribute significantly to the interface (Fig. 8A and B). Note that the core interface residue of ActR-IIb with BMP-2 at several amino acids Trp60, Tyr42 Leu61, Lys56, V55, and Val81. In addition, the functional binding epitope of ActR-IIb for BMP-2 seems to be dominated by Trp60 and to a lesser extent by Tyr42 [51]. Importantly, in comparison with the complex of BMP-2 bound to ActR-II is also similar. These peptides consist of amino acid residues that can exert a highly significant binding on the activin receptor, which may be related to bioactivities of CCLP peptide.