Carboxy-Terminal Cementum Protein 1-Derived Peptide 4 (cemp1-p4) Promotes Mineralization through wnt/ß-catenin Signaling in Human Oral Mucosa Stem Cells.

Human cementum protein 1 (CEMP1) is known to induce cementoblast and osteoblast differentiation and alkaline phosphatase (ALP) activity in human periodontal ligament-derived cells in vitro and promotes bone regeneration in vivo. CEMP1's secondary structure analysis shows that it has a random-coiled structure and is considered an Intrinsic Disordered Protein (IDP). CEMP1's short peptide sequences mimic the biological capabilities of CEMP1. However, the role and mechanisms of CEMP1's C-terminal-derived synthetic peptide (CEMP1-p4) in the canonical Wnt/ß-catenin signaling pathway are yet to be described. Here we report that CEMP1-p4 promotes proliferation and differentiation of Human Oral Mucosa Stem Cells (HOMSCs) by activating the Wnt/ß-catenin pathway. CEMP1-p4 stimulation upregulated the expression of ß-catenin and glycogen synthase kinase 3 beta (GSK-3B) and activated the transcription factors TCF1/7 and Lymphoid Enhancer binding Factor 1 (LEF1) at the mRNA and protein levels. We found translocation of ß-catenin to the nucleus in CEMP1-p4-treated cultures. The peptide also penetrates the cell membrane and aggregates around the cell nucleus. Analysis of CEMP1-p4 secondary structure revealed that it has a random-coiled structure. Its biological activities included the induction to nucleate hydroxyapatite crystals. In CEMP1-p4-treated HOMSCs, ALP activity and calcium deposits increased. Expression of Osterix (OSX), Runt-related transcription factor 2 (RUNX2), Integrin binding sialoproptein (IBSP) and osteocalcin (OCN) were upregulated. Altogether, these data show that CEMP1-p4 plays a direct role in the differentiation of HOMSCs to a "mineralizing-like" phenotype by activating the ß-catenin signaling cascade.

Cementum protein 1-derived peptide (CEMP 1-p1) modulates hydroxyapatite crystal formation in vitro.

A cementum protein 1-derived peptide (CEMP1-p1) consisting of 20 amino acids from the CEMP1's N-terminus region: MGTSSTDSQQAGHRRCSTSN, and its role on the mineralization process in a cell-free system, was characterized. CEMP1-p1's physicochemical properties, crystal formation, and hydroxyapatite (HA) nucleation assays were performed. Crystals induced by CEMP1-p1 were analyzed by scanning electron microscopy, Fourier-transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR), X-ray diffraction (XRD), high resolution transmission electron microscopy (HRTEM), and atomic force microscopy. The results indicate that CEMP1-p1 lacks secondary structure, forms nanospheres that organize into three-dimensional structures, possesses affinity to HA, and induces its nucleation. CEMP1-p1 promotes the formation of spherical structures composed by densely packed prism-like crystals, which revealed a Ca/P ratio of 1.56, corresponding to HA. FTIR-ATR showed predominant spectrum peaks that correspond and are characteristic of HA and octacalcium phosphate (OCP). Analysis by XRD indicates that the crystals show planes with a preferential crystalline orientation for HA and for OCP. HRTEM showed interplanar distances that correspond to crystalline planes of HA and OCP. Crystals are composed by superimposed lamellae, which exhibit epitaxial growth, and each layer of the crystals is structured by nanocrystals. This study reveals that CEMP1-p1 regulates HA crystal formation, somehow mimicking the in vivo process of mineralized tissues bioformation.

Synthetic cementum protein 1-derived peptide regulates mineralization in vitro and promotes bone regeneration in vivo.

The use of recombinant proteins has revolutionized the development of biologic pharmaceuticals; however, they are not free of complications. Some have very high molecular weight, some demonstrate in vivo instability, and the high cost of producing them remains a major problem. On the other hand, it has been shown that peptides derived from active domains keep their biologic activity and can trigger events, such as osteogenesis and bone regeneration. Small peptides are advantageous because of their ease of synthesis and handling and their low immunogenic activity. The purpose of this study was to investigate the functions of a synthetic peptide, cementum protein 1-peptide1 (CEMP-1-p1), both in vitro and in vivo. Our results show that CEMP-1-p1 significantly enhanced the proliferation and differentiation of human periodontal ligament cells toward a mineralizing-like phenotype, as evidenced by increasing alkaline phosphatase (ALP)-specific activity and osterix, runt-related transcription factor (RUNX)-2, integrin binding sialoprotein, bone morphogenetic protein-2, osteocalcin, and cementum protein (CEMP)-1 expression at mRNA and protein levels. In vivo assays performed through standardized critical-size calvarial defects in rats treated with CEMP-1-p1 resulted in newly formed bone after 30 and 60 d. These data demonstrate that CEMP-1-p1 is an effective bioactive peptide for bone tissue regeneration. The application of this bioactive peptide may lead to implementing new strategies for the regeneration of bone and other mineralized tissues.-Correa, R., Arenas, J., Montoya, G., Hoz, L., López, S., Salgado, F., Arroyo, R., Salmeron, N., Romo, E., Zeichner-David, M., Arzate, H. Synthetic cementum protein 1-derived peptide regulates mineralization in vitro and promotes bone regeneration in vivo.