Mesenchymal stem cell (MSC)–derived exosome plays a central role in the cell-free therapeutics involving MSCs and the contents can be customized under disease-associated microenvironments. However, optimal MSC-preconditioning to enhance its therapeutic potential is largely unknown. Here, we show that preconditioning of gingival tissue-derived MSCs (GMSCs) with tumor necrosis factor-alpha (TNF- α ) is ideal for the treatment of periodontitis. TNF- α stimulation not only increased the amount of exosome secreted from GMSCs, but also enhanced the exosomal expression of CD73, thereby inducing anti-inflammatory M2 macrophage polarization. The effect of GMSC-derived exosomes on inflammatory bone loss were examined by ligature-induced periodontitis model in mice. Local injection of GMSC-derived exosomes significantly reduced periodontal bone resorption and the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, and these effects were further enhanced by preconditioning of GMSCs with TNF- α . Thus, GMSC-derived exosomes also exhibited anti-osteoclastogenic activity. Receptor activator of NF- κ B ligand (RANKL) expression was regulated by Wnt5a in periodontal ligament cells (PDLCs), and exosomal miR-1260b was found to target Wnt5a-mediated RANKL pathway and inhibit its osteoclastogenic activity. These results indicate that significant ability of the TNF- α -preconditioned GMSC-derived exosomes to regulate inflammation and osteoclastogenesis paves the way for establishment of a therapeutic approach for periodontitis.

Various mitogenic stimuli such as epidermal growth factor (EGF), fibroblast growth factor (FGF), and phorbol 12,13-dibutyrate (PDBu) activate the Ras-Raf-MEK-ERK pathway, but the regulatory mechanism of this pathway remains to be investigated. Here we found that in 293 cells, mammalian Sprouty2 and Sprouty4 were rapidly induced by EGF, FGF, and PDBu in an ERK pathway-dependent manner. Forced expression of Sprouty2 and Sprouty4 inhibited FGF-induced ERK activation but did not affect EGF- or PDBu-induced ERK activation. To examine whether endogenous Sproutys were also selective inhibitors, we generated a dominant negative form of Sprouty2 (Y55A) and Sprouty4 (Y53A) in which conserved tyrosine residues were mutated. These mutants reverted the suppressive effect of both Sprouty2 and Sprouty4 but not that of RasGAP or SPRED (Sprouty-related EVH1 domain-containing protein), another Sprouty-related Ras suppressor. Expression of dominant negative Sprouty2 and Sprouty4 enhanced and prolonged FGF- but not EGF-induced ERK activation in 293 cells. In PC12 cells, endogenous Sprouty4 was also induced by FGF. Overexpression of wild-type Sprouty4 blocked FGF-induced differentiation, whereas Y53A-Sprouty4 enhanced it. These observations suggest that endogenous Sprouty2 and Sprouty4 are physiological negative feedback regulators of growth factor-mediated ERK pathway and that there are Sprouty-sensitive and -insensitive ERK activation pathways. Finding a dominant negative form of Sproutys will facilitate the study of the molecular mechanism and physiological function of Sproutys.

The signalling cascade including Raf, mitogen-activated protein kinase (MAPK) kinase and extracellular-signal-regulated kinase (ERK) is important in many facets of cellular regulation. Raf is activated through both Ras-dependent and Ras-independent mechanisms, but the regulatory mechanisms of Raf activation remain unclear. Two families of membrane-bound molecules, Sprouty and Sprouty-related EVH1-domain-containing protein (Spred) have been identified and characterized as negative regulators of growth-factor-induced ERK activation. But the molecular functions of mammalian Sproutys have not been clarified. Here we show that mammalian Sprouty4 suppresses vascular epithelial growth factor (VEGF)-induced, Ras-independent activation of Raf1 but does not affect epidermal growth factor (EGF)-induced, Ras-dependent activation of Raf1. Sprouty4 binds to Raf1 through its carboxy-terminal cysteine-rich domain, and this binding is necessary for the inhibitory activity of Sprouty4. In addition, Sprouty4 mutants of the amino-terminal region containing the conserved tyrosine residue, which is necessary for suppressing fibroblast growth factor signalling, still inhibit the VEGF-induced ERK pathway. Our results show that receptor tyrosine kinases use distinct pathways for Raf and ERK activation and that Sprouty4 differentially regulates these pathways.

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