The PDZ-GEF Gef26 regulates synapse development and function via FasII and Rap1 at the Drosophila neuromuscular junction

Guanine nucleotide exchange factors (GEFs) are essential for small G proteins to activate their downstream signaling pathways, which are involved in morphogenesis, cell adhesion, and migration. Mutants of Gef26, a PDZ-GEF (PDZ domain-containing guanine nucleotide exchange factor) in Drosophila, exhibit strong defects in wings, eyes, and the reproductive and nervous systems. However, the precise roles of Gef26 in development remain unclear. In the present study, we analyzed the role of Gef26 in synaptic development and function. We identified significant decreases in bouton number and branch length at larval neuromuscular junctions (NMJs) in Gef26 mutants, and these defects were fully rescued by restoring Gef26 expression, indicating that Gef26 plays an important role in NMJ morphogenesis. In addition to the observed defects in NMJ morphology, electrophysiological analyses revealed functional defects at NMJs, and locomotor deficiency appeared in Gef26 mutant larvae. Furthermore, Gef26 regulated NMJ morphogenesis by regulating the level of synaptic Fasciclin II (FasII), a well-studied cell adhesion molecule that functions in NMJ development and remodeling. Finally, our data demonstrate that Gef26-specific small G protein Rap1 worked downstream of Gef26 to regulate the level of FasII at NMJs, possibly through a βPS integrin-mediated signaling pathway. Taken together, our findings define a novel role of Gef26 in regulating NMJ development and function.     

Aggregation of human dental pulp cells into 3D spheroids enhances their migration ability after reseeding

Multicellular three-dimensional (3D) spheroids allow intimate cell–cell communication and cell–extracellular matrix interaction. Thus, 3D cell spheroids better mimic microenvironment in vivo than two-dimensional (2D) monolayer cultures. The purpose of this study was to evaluate the behaviors of human dental pulp cells (DPCs) cultured on chitosan and polyvinyl alcohol (PVA) membranes. The protein expression of hypoxia-inducible factor 1-α (HIF-1α) and vascular endothelial growth factor (VEGF), and the migration ability of the DPCs from 2D versus 3D environments were investigated. The results showed that both chitosan and PVA membranes support DPCs aggregation to form multicellular spheroids. In comparison to 2D cultures on tissue culture polystyrene, DPC spheroids exhibited higher protein expression of HIF-1α and VEGF. The treatment with YC-1 (inhibitor to HIF-1α) blocked the upregulation of VEGF, indicating a downstream event to HIF-1α expression. When DPC spheroids were collected and subjected to the transwell assay, the cells growing outward from 3D spheroids showed greater migration ability than those from 2D cultures. Moreover, DPCs aggregation and spheroid formation on chitosan membrane were abolished by Y-27632 (inhibitor to Rho-associated kinases), whereas the inhibitory effect did not exist on PVA membrane. This suggests that the mechanism regulating DPCs aggregation and spheroid formation on chitosan membrane is involved with the Rho-associated kinase signaling pathway. In summary, the multicellular spheroid structure was beneficial to the protein expression of HIF-1α and VEGF in DPCs and enhanced the migration ability of the cells climbing from spheroids. This study showed a new perspective in exploring novel strategies for DPC-based research and application.     

Mutual regulation between IGF-1R and IGFBP-3 in human corneal epithelial cells

The insulin-like growth factor type 1 receptor (IGF-1R) is part of the receptor tyrosine kinase superfamily. The activation of IGF-1R regulates several key signaling pathways responsible for maintaining cellular homeostasis, including survival, growth, and proliferation. In addition to mediating signal transduction at the plasma membrane, in serum-based models, IGF-1R undergoes SUMOylation by SUMO 1 and translocates to the nucleus in response to IGF-1. In corneal epithelial cells grown in serum-free culture, however, IGF-1R has been shown to accumulate in the nucleus independent of IGF-1. In this study, we report that the insulin-like growth factor binding protein-3 (IGFBP-3) mediates nuclear translocation of IGF-1R in response to growth factor withdrawal. This occurs via SUMOylation by SUMO 2/3. Further, IGF-1R and IGFBP-3 undergo reciprocal regulation independent of PI3k/Akt signaling. Thus, under healthy growth conditions, IGFBP-3 functions as a gatekeeper to arrest the cell cycle in G0/G1, but does not alter mitochondrial respiration in cultured cells. When stressed, IGFBP-3 functions as a caretaker to maintain levels of IGF-1R in the nucleus. These results demonstrate mutual regulation between IGF-1R and IGFBP-3 to maintain cell survival under stress. This is the first study to show a direct relationship between IGF-1R and IGFBP-3 in the maintenance of corneal epithelial homeostasis.     

Thrombospondin-4 critically controls transforming growth factor β1 induced hypertrophic scar formation

Transforming growth factor β (TGF-β) is a growth factor presenting important functions during tissue remodeling and hypertrophic scar (HS) formation. However, the underlying molecular mechanisms are largely unknown. In this study, we identified thrombospondin-4 (TSP-4) as a TGF-β1 target that essentially mediates TGF-β1-induced scar formation both in vitro and in vivo. The expression of TSP-4 was compared on both mRNA and protein levels between hypertrophic scar fibroblasts (HSFs) and normal skin fibroblast (NFs) in response to TGF-β1 treatment. Two signaling molecules, Smad3 and p38, were assessed for their importance in regulating TGF-β1-mediated TSP-4 expression. The significance of TSP-4 in controlling TGF-β1-induced proliferation, invasion, migration, and fibrosis in HSFs was analyzed by knocking down endogenous TSP-4 using small hairpin RNA (shRNA) (TSP-4 shRNA). Finally, a skin HS model was established in rats and the scar formation was compared between rats treated with vehicle (saline), TGF-β1, and TGF-β1 + TSP-4 shRNA. The TSP-4 level was significantly higher in HSFs than in NFs and TGF-β1 more potently boosted TSP-4 expression in the former than in the latter. Both Smad3 and p38 essentially mediated TGF-β1-induced TSP-4 expression. TSP-4 shRNA significantly suppressed TGF-β1-stimulated proliferation, invasion, migration, or fibrosis of HSFs in vitro and drastically improved wound healing in vivo. TGF-β1, by activating both Smad3 and p38, induces TSP-4, which in turn not only presents a positive feedback regulation on the activation of Smad3 and p38, but also essentially mediates TGF-β1-induced HS formation. Targeting TSP-4 thus may benefit HS treatment.     

MicroRNA-200c promotes osteogenic differentiation of human bone mesenchymal stem cells through activating the AKT/β-Catenin signaling pathway via downregulating Myd88

During the human bone formation, the event of osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs) is vital, and recent evidence has emphasized the important role of microRNAs (miRNAs) in osteogenic differentiation of hBMSCs. This study aims to examine the potential effects of miR-200c in osteogenic differentiation of hBMSCs and understand their underlying mechanisms. HBMSCs were obtained via human bone marrow. During osteogenic induction and differentiation, cells were transfected with different plasmids with the intention of investigating the roles of miR-200c on osteogenic differentiation, calcium salt deposition, alkaline-phosphatase (ALP) activity, mineralized nodule formation, osteocalcin (OCN) content, and proliferation of osteoblasts. Following transfection, dual luciferase reporter gene assay was conducted so as to explore the correlation between miR-200c and Myd88. Moreover, the AKT/β-Catenin signaling pathway was blocked with an AKT/β-Catenin inhibitor, AKTi, to investigate its involvement. The hBMSCs were successfully isolated from human bone marrow. Myd88 was determined as a target gene of miR-200c. Gain and loss-of-function assays confirmed that overexpression of miR-200c, or silencing of Myd88 promoted osteogenic differentiation, increased calcium salt deposition, ALP activity, mineralized nodule formation, and enhanced the proliferation of osteoblasts following osteogenic differentiation of hBMSCs. Meanwhile, the downregulation of miR-200c has been shown to have the opposite effect. Furthermore, these findings showed that the miR-200c overexpression activated the AKT/β-Catenin signaling pathway by targeting Myd88. To sum up, the miR-200c upregulation induces osteogenic differentiation of hBMSCs by activating the AKT/β-Catenin signaling pathway via the inhibition of Myd88, providing a target for treatment of bone repair.     

Blocking OLFM4/HIF-1α axis alleviates hypoxia-induced invasion,epithelial–mesenchymal transition,and chemotherapy resistance in non-small-cell lung cancer

Hypoxia is a common biological hallmark of solid cancers, which has been proposed to be associated with oncogenesis and chemotherapy resistance. The purpose of the present study was to investigate the role and underlying mechanisms of olfactomedin 4 (OLFM4) in the hypoxia-induced invasion, epithelial–mesenchymal transition (EMT), and chemotherapy resistance of non-small-cell lung cancer (NSCLC). We observed dramatically upregulated expression of OLFM4 in several NSCLC cell lines, and this effect was more pronounced in A549 and H1299 cells. In addition, our data revealed that OLFM4 expression was remarkably increased in both A549 and H1299 cells under hypoxic microenvironment, accompanied by enhanced levels of hypoxia-inducible factor (HIF)-1α protein. The HIF-1α level was elevated in response to hypoxia, resulting in the regulation of OLFM4. Interestingly, OLFM4 was a positive regulator of hypoxia-driven HIF-1α production. Moreover, depletion of OLFM4 modulated multiple EMT-associated proteins, as evidenced by the enhanced E-cadherin levels along with the diminished expression of N-cadherin and vimentin in response to hypoxia, and thus blocked invasion ability of A549 and H1299 cells following exposure to hypoxia. Furthermore, ablation of OLFM4 accelerated the sensitivity of A549 cells to cisplatin under hypoxic conditions, implying that OLFM4 serves as a key regulator in chemotherapeutic resistance under hypoxia. In conclusion, OLFM4/HIF-1α axis might be a potential therapeutic strategy for NSCLC.