Fibronectin-Derived Oligopeptide Stimulates Osteoblast Differentiation Through a Bone Morphogenic Protein 2–Like Signaling Pathway

• Autores: Young Dan Cho, Bong Soo Kim, Chang Seok Lee, Kyung Hwa Kim, Yang-Jo Seol, Yong Moo Lee, In-Chul Rhyu, Young Ku, Hyun Mo Ryoo
• Localización: Journal of periodontology, ISSN 0022-3492, Vol. 88, Nº. 2, 2017, págs. 42-48
• Idioma: inglés
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• Resumen

  • Background: In previous studies by the authors, it was demonstrated that a fibronectin (FN)-derived oligopeptide, termed F20, stimulates osteoblast differentiation in vitro and bone formation in vivo. However, the fundamental molecular mechanism by which F20 stimulates osteogenesis remains unknown. Therefore, in this study the molecular mechanism underlying the effect of F20 in osteoblast differentiation is investigated.

    Methods: The role of F20 in osteoblast differentiation was examined using mouse bone-marrow-derived ST2 cell line. The effect of Smad1/5 was determined following small interfering RNA knockdown. Runt-related transcription factor (Runx) 2, alkaline phosphatase (Alp), and osteocalcin (Oc) mRNA levels were determined by quantitative real-time polymerase chain reaction, and their transcriptional activation was assessed using luciferase reporter assays. Extracellular signal-regulated kinase (ERK) phosphorylation was visualized via immunoblotting.

    Results: Synthetic oligopeptide F20 stimulated expression of bone marker genes Runx2, Alp, and Oc in ST2 cells via Smad and ERK or mitogen-activated protein kinase signaling pathways as did bone morphogenic protein 2 (BMP2). Furthermore, Runx2 acted as a transcription factor during F20-induced osteoblast differentiation.

    Conclusions: Collectively, these results indicate that F20 induces osteoblast differentiation with a pattern similar to that mediated by BMP2 signaling pathway. The authors’ previous data also showed that FN-derived oligopeptide improved wound healing, and it is suggested that F20 might serve as a therapeutic biomolecule to facilitate periodontal tissue regeneration.

    Fibronectin (FN), a glycoprotein of the extracellular matrix (ECM), binds to membrane-spanning receptor proteins termed integrins and plays a critical role in cellular adhesion, proliferation, and differentiation.1,2 Integrins are glycoproteins forming diverse heterodimeric receptors with specific ligand pairs: α1β1, α2β1, α3β1,α4β1, α5β1, α6β1, α8β1, and αvβ3.3 Numerous studies have shown that the cell-binding Arg-Gly-Asp (RGD) motif present in ECM proteins, including FN, vitronectin, and thrombospondin, binds with several integrins that are crucial for cell–ECM interactions.4-6 In particular, α5β1 selectively binds to the RGD motif in FN located in the 10th FN Type III domain in the presence of a synergistic ProHis-Ser-Arg-Asn (PHSRN) sequence in the 9th FN Type III domain;6-8 both the RGD and PHSRN sequences are recognized by integrin α5β1 when the optimal spatial conformation exists between these motifs.9 To better understand the function of the motifs and their intervening sequences, focus has been on analysis of partial FN peptides composed of PHSRN and RGD along with variable amino acids between these motifs indicating optimal conformation.7,10-14 Among these, one that includes the 20 amino acids between PHSRN and RGD was selected, termed “F20,” with the sequence PHSRNSITGTNLTPGYTITVYAVTGRGD, because this peptide showed good osteogenic effect. In particular, the current authors have previously reported the osteogenic potential of F20 toward differentiation of MG637 osteosarcoma and ST214 stromal cells, demonstrating stimulation of alkaline phosphatase (ALP) expression using either a recombinant F20 oligopeptide (0.1 μM)7 purified from an expression vector or a synthetic F20 oligopeptide (1 ng/mL).14 Synthetic F20 showed greater improved effect at low concentration than recombinant F20.

    Bone morphogenic protein 2 (BMP2), a member of the transforming growth factor β superfamily, induces bone and cartilage formation mainly via canonic Smad signaling pathway, which plays a role in the induction of expression of bone marker genes runt-related transcription factor 2 (Runx2), Alp, and osteocalcin (Oc) by BMP2.15,16 In addition to Smads, various signaling pathways are also involved in BMP2-induced osteoblast differentiation; these are collectively called the non-Smad pathway and include extracellular signal-regulated kinase (ERK), p38 mitogen activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), phosphatidylinositol 3-kinase, and protein kinase C and D.17 Runx2 is an essential transcription factor for determination of osteoblast lineage that acts as a master regulator of bone marker gene expression.18 Runx2 gene expression is modulated by diverse extracellular cellular signals including BMP, fibroblast growth factor, and Int/Wingless.19,20 In addition, Runx2 protein stabilization has been shown to be regulated by post-translational modifications including phosphorylation, acetylation, and ubiquitination.21 It has been demonstrated that ERK activation enhances Runx2 transcriptional activity and that BMP2-activated ERK/MAPK stabilizes Runx2 protein by increasing p300 levels and histone acetyl-transferase activity.17 Based on these findings, it was speculated that F20 might also play a role in osteoblast differentiation similar to that shown for BMP2. Therefore, in this study, the molecular mechanism underlying the effect of F20 in osteoblast differentiation was investigated.