All ligands of the epidermal growth factor receptor (EGFR), which has important roles in development and disease, are released from the membrane by proteases. In several instances, ectodomain release is critical for activation of EGFR ligands, highlighting the importance of identifying EGFR ligand sheddases. Here, we uncovered the sheddases for six EGFR ligands using mouse embryonic cells lacking candidate-releasing enzymes (a disintegrin and metalloprotease [ADAM] 9, 10, 12, 15, 17, and 19). ADAM10 emerged as the main sheddase of EGF and betacellulin, and ADAM17 as the major convertase of epiregulin, transforming growth factor α, amphiregulin, and heparin-binding EGF-like growth factor in these cells. Analysis of adam9/12/15/17− /− knockout mice corroborated the essential role of adam17− /− in activating the EGFR in vivo. This comprehensive evaluation of EGFR ligand shedding in a defined experimental system demonstrates that ADAMs have critical roles in releasing all EGFR ligands tested here. Identification of EGFR ligand sheddases is a crucial step toward understanding the mechanism underlying ectodomain release, and has implications for designing novel inhibitors of EGFR-dependent tumors.

Background: As important players in cell-to-cell communication, exosomes (exo) are believed to play a similar role in promoting fracture healing. This study investigated whether exosomes derived from bone marrow mesenchymal stem cells (BMMSC-Exos) could improve fracture healing of nonunion. Methods: BMMSC-Exos were isolated and transplanted into the fracture site in a rat model of femoral nonunion (Exo group) every week. Moreover, equal volumes of phosphate-buffered saline (PBS) and exosome-depleted conditioned medium (CM-Exo) were injected into the femoral fracture sites of the rats in the control and CM-Exo groups. Bone healing processes were recorded and evaluated by radiographic methods on weeks 8, 14 and 20 after surgery. Osteogenesis and angiogenesis at the fracture sites were evaluated by radiographic and histological methods on postoperative week 20. The expression levels of osteogenesis-or angiogenesis-related genes were evaluated in vitro by western blotting and immunohistochemistry. The ability to internalize exosomes was assessed using the PKH26 assay. Altered proliferation and migration of human umbilical vein endothelial cells (HUVECs) and mouse embryo osteoblast precursor cells (MC3TE-E1s) treated with BMMSC-Exos were determined by utilizing EdU incorporation, immunofluorescence staining, and scratch wound assay. The angiogenesis ability of HUVECs was evaluated through tube formation assays. Finally, to explore the effect of exosomes in osteogenesis via the BMP-2/ Smad1/RUNX2 signalling pathway, the BMP-2 inhibitors noggin and LDN193189 were utilized, and their subsequent effects were observed. Results: BMMSC-Exos were observed to be spherical with a diameter of approximately 122 nm. CD9, CD63 and CD81 were expressed. Transplantation of BMMSC-Exos obviously enhanced osteogenesis, angiogenesis and bone healing processes in a rat model of femoral nonunion. BMMSC-Exos were taken up by HUVECs and MC3T3-E1 in vitro, and their proliferation and migration were also improved. Finally, experiments with BMP2 inhibitors confirmed that the BMP-2/Smad1/RUNX2 signalling pathway played an important role in the pro-osteogenesis induced by BMMSC-Exos and enhanced fracture healing of nonunion.

Periostin is a fasciclin-containing adhesive glycoprotein that facilitates the migration and differentiation of cells that have undergone epithelial-mesenchymal transformation during embryogenesis and in pathological conditions. Despite the importance of post-transformational differentiation as a general developmental mechanism, little is known how periostin's embryonic expression is regulated. To help resolve this deficiency, a 3.9-kb periostin proximal promoter was isolated and shown to drive tissue-specific expression in the neural crest-derived Schwann cell lineage and in a subpopulation of periostin-expressing cells in the cardiac outflow tract endocardial cushions. In order to identify the enhancer and associated DNA binding factor(s) responsible, in vitro promoter dissection was undertaken in a Schwannoma line. Ultimately a 304-bp(peri) enhancer was identified and shown to be capable of recapitulating 3.9 kb(peri-lacZ)in vivo spatiotemporal patterns. Further mutational and EMSA analysis helped identify a minimal 37-bp region that is bound by the YY1 transcription factor. The 37-bp enhancer was subsequently shown to be essential for in vivo 3.9 kb(peri-lacZ) promoter activity. Taken together, these studies identify an evolutionary-conserved YY1-binding 37-bp region within a 304-bp periostin core enhancer that is capable of regulating simultaneous novel tissue-specific periostin expression in the cardiac outflow-tract cushion mesenchyme and Schwann cell lineages.

Severe spinal cord injury (SCI) is caused by external mechanical injury, resulting in unrecoverable neurological injury. Recent studies have shown that exosomes derived from bone marrow mesenchymal stem cells (BMSCs-Exos) might be valuable paracrine molecules in the treatment of SCI. In this study, we designed SCI models in vivo and in vitro and then investigated the possible mechanism of successful repair by BMSCs-Exos. In vivo, we established one Sham group and two SCI model groups. The Basso, Beattie, Bresnahan (BBB) scores showed that BMSCs-Exos could effectively promote the recovery of spinal cord function. The results of the Nissl staining, immunohistochemistry, and TUNEL/NeuN/DAPI double staining showed that BMSCs-Exos inhibited neuronal apoptosis. Western blot analysis showed that the protein expression level of Bcl-2 was significantly increased in the BMSCs-Exos group compared with the PBS group, while the protein expression levels of Bax, cleaved caspase-3, and cleaved caspase-9 were significantly decreased. The results of western bolt and qRT-PCR demonstrated that BMSCs-Exos could activate the Wnt/β-catenin signaling pathway effectively. In vitro, we found that inhibition of the Wnt/β-catenin signaling pathway could promote neuronal apoptosis following lipopolysaccharide (LPS) induction. These results demonstrated that BMSCs-Exos may be a promising therapeutic for SCI by activating the Wnt/β-catenin signaling pathway.