miR-19b enhances osteogenic differentiation of mesenchymal stem cells and promotes fracture healing through the WWP1/Smurf2-mediated KLF5/β-catenin signaling pathway

Bone marrow mesenchymal stem cell (BMSC)-derived exosomes have been found to enhance fracture healing. In addition, microRNAs contributing to the healing of various bone fractures have attracted widespread attention in recent years, but knowledge of the mechanisms by which they act is still very limited. In this study, we clarified the function of altered microRNA-19b (miR-19b) expression in BMSCs in fracture healing. We modulated miR-19b expression via mimics/inhibitors in BMSCs and via agomirs in mice to explore the effects of these changes on osteogenic factors, bone cell mineralization and the healing status of modeled fractures. Through gain- and loss-of function assays, the binding affinity between miR-19b and WWP1/Smurf2 was identified and characterized to explain the underlying mechanism involving the KLF5/β-catenin signaling pathway. miR-19b promoted the differentiation of human BMSCs into osteoblasts by targeting WWP1 and Smurf2. Overexpression of WWP1 or Smurf2 degraded the target protein KLF5 in BMSCs through ubiquitination to inhibit fracture healing. KLF5 knockdown delayed fracture healing by modulating the Wnt/β-catenin signaling pathway. Furthermore, miR-19b enhanced fracture healing via the KLF5/β-catenin signaling pathway by targeting WWP1 or Smurf2. Moreover, miR-19b was found to be enriched in BMSC-derived exosomes, and treatment with exosomes promoted fracture healing in vivo. Collectively, these results indicate that mesenchymal stem cell-derived exosomal miR-19b represses the expression of WWP1 or Smurf2 and elevates KLF5 expression through the Wnt/β-catenin signaling pathway, thereby facilitating fracture healing.Subject terms: Diseases, Biotechnology     

Anti-Hair Loss Effect of Adenosine Is Exerted by cAMP Mediated Wnt/β-Catenin Pathway Stimulation via Modulation of Gsk3β Activity in Cultured Human Dermal Papilla Cells

In the present study, we investigated the molecular mechanisms of adenosine for its hair growth promoting effect. Adenosine stimulated the Wnt/β-catenin pathway by modulating the activity of Gsk3β in cultured human dermal papilla cells. It also activated adenosine receptor signaling, increasing intracellular cAMP level, and subsequently stimulating the cAMP mediated cellular energy metabolism. The phosphorylation of CREB, mTOR, and GSK3β was increased. Furthermore, the expression of β-catenin target genes such as Axin2, Lef1, and growth factors (bFGF, FGF7, IGF-1) was also enhanced. The inhibitor study data conducted in Wnt reporter cells and in cultured human dermal papilla cells demonstrated that adenosine stimulates Wnt/β-catenin signaling through the activation of the adenosine receptor and Gsk3β plays a critical role in transmitting the signals from the adenosine receptor to β-catenin, possibly via the Gαs/cAMP/PKA/mTOR signaling cascade.     

Euphorbiasteroid Abrogates EGFR and Wnt/β-Catenin Signaling in Non-Small-Cell Lung Cancer Cells to Impart Anticancer Activity

EGFR and Wnt/β-catenin signaling pathways play a prominent role in tumor progression in various human cancers including non-small-cell lung carcinoma (NSCLC). Transactivation and crosstalk between the EGFR and Wnt/β-catenin pathways may contribute to the aggressiveness of cancers. Targeting these oncogenic pathways with small molecules is an attractive approach to counteract various types of cancers. In this study, we demonstrate the effect of euphorbiasteroid (EPBS) on the EGFR and Wnt/β-catenin pathways in NSCLC cells. EPBS induced preferential cytotoxicity toward A549 (wildtype EGFR-expressing) cells over PC-9 (mutant EGFR-expressing) cells. EPBS suppressed the expression of EGFR, Wnt3a, β-catenin, and FZD-1, and the reduction in β-catenin levels was found to be mediated through the activation of GSK-3β. EPBS reduced the phosphorylation of GSK-3β^S9 with a parallel increase in β-TrCP and phosphorylation of GSK-3β^Y216. Lithium chloride treatment increased the phosphorylation of GSK-3β^S9 and nuclear localization of β-catenin, whereas EPBS reverted these effects. Forced expression or depletion of EGFR in NSCLC cells increased or decreased the levels of Wnt3a, β-catenin, and FZD-1, respectively. Overall, EPBS abrogates EGFR and Wnt/β-catenin pathways to impart its anticancer activity in NSCLC cells.     

5-Bromo-3,4-dihydroxybenzaldehyde Promotes Hair Growth through Activation of Wnt/β-Catenin and Autophagy Pathways and Inhibition of TGF-β Pathways in Dermal Papilla Cells

Various studies addressing the increasing problem of hair loss, using natural products with few side effects, have been conducted. 5-bromo-3,4-dihydroxybenzaldehyde (BDB) exhibited anti-inflammatory effects in mouse models of atopic dermatitis and inhibited UVB-induced oxidative stress in keratinocytes. Here, we investigated its stimulating effect and the underlying mechanism of action on hair growth using rat vibrissa follicles and dermal papilla cells (DPCs), required for the regulation of hair cycle and length. BDB increased the length of hair fibers in rat vibrissa follicles and the proliferation of DPCs, along with causing changes in the levels of cell cycle-related proteins. We investigated whether BDB could trigger anagen-activating signaling pathways, such as the Wnt/β-catenin pathway and autophagy in DPCs. BDB induces activation of the Wnt/β-catenin pathway through the phosphorylation of GSG3β and β-catenin. BDB increased the levels of autophagic vacuoles and autophagy regulatory proteins Atg7, Atg5, Atg16L, and LC3B. We also investigated whether BDB inhibits the TGF-β pathway, which promotes transition to the catagen phase. BDB inhibited the phosphorylation of Smad2 induced by TGF-β1. Thus, BDB can promote hair growth by modulating anagen signaling by activating Wnt/β-catenin and autophagy pathways and inhibiting the TGF-β pathway in DPCs.     

Exploring In Vitro Biological Cellular Responses of Pegylated β-Cyclodextrins

βCDPEG5 and βCDPEG2 are two derivatives comprising seven PEG linear chains of 5 and 2 kDa, respectively, conjugated to βCD. As βCDPEGs display different physicochemical properties than their precursors, they could also trigger distinct cellular responses. To investigate the biological behavior of βCDPEGs in comparison to their parent compounds, we performed broad toxicological assays on RAW 264.7 macrophages, MC3T3-E1 osteoblasts, and MDCK cells. By analyzing ROS and NO₂⁻ overproduction in macrophages, we found that βCDPEGs induced a moderate stress response without affecting cell viability. Although MC3T3-E1 osteoblasts were more sensitive than MDCK cells to βCDPEGs and the parent compounds, a similar pattern was observed: the effect of βCDPEG5 on cell viability and cell cycle progression was larger than that of βCDPEG2; PEG2 affected cell viability and cell cycle more than βCDPEG2; cell post-treatment recovery was favorable in all cases, and the compounds had similar behaviors regarding ROS generation. The effect on MDCK cell migration followed a similar pattern. In contrast, for osteoblasts, the interference of βCDPEG5 with cell migration was smaller than that of βCDPEG2; likewise, the effect of PEG2 was shorter than its conjugate. Overall, the covalent conjugation of βCD and PEGs, particularly to yield βCDPEG2, improved the biocompatibility profile, evidencing that a favorable biological response can be tuned through a thoughtful combination of materials. Moreover, this is the first time that an in vitro evaluation of βCD and PEG has been presented for MC3T3-E1 and MDCK cells, thus providing valuable knowledge for designing biocompatible nanomaterials constructed from βCD and PEGs.     

Emodin prevents intima thickness via Wnt4/Dvl-1/β-catenin signaling pathway mediated by miR-126 in balloon-injured carotid artery rats

Neointimal proliferation after vascular injury is a key mechanism of restenosis, a major cause of percutaneous transluminal angioplasty failure and artery bypass occlusion. Emodin, an anthraquinone with multiple physiological activities, has been reported to inhibit proliferation of vascular smooth muscle cells (VSMCs) that might cause intimal arterial thickening. Thus, in this study, we established a rat model of balloon-injured carotid artery and investigated the therapeutic effect of emodin and its underlying mechanism. Intimal thickness was analyzed by hematoxylin and eosin staining. Expression of Wnt4, dvl-1, β-catenin and collagen was determined by immunohistochemistry and/or western blotting. The proliferation of VSMC was evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and electron microscopy. MicroRNA levels were quantified by real-time quantitative PCR. Emodin relieved injury-induced artery intimal thickness. Results of western blots and immunohistochemistry showed that emodin suppressed expression of signaling molecules Wnt4/Dvl-1/β-catenin as well as collagen protein in the injured artery. In addition, emodin enhanced expression of an artery injury-related microRNA, miR-126. In vitro, MTT assay showed that emodin suppressed angiotensin II (AngII)-induced proliferation of VSMCs. Emodin reversed AngII-induced activation of Wnt4/Dvl-1/β-catenin signaling by increasing expression of miR-126 that was strongly supported by transfection of mimic or inhibitor for miR-126. Emodin prevents intimal thickening via Wnt4/Dvl-1/β-catenin signaling pathway mediated by miR-126 in balloon-injured carotid artery of rats.     

Solution-phase synthesis of ICG-001, a β-turn peptidomimetic molecule inhibitor of β-catenin–Tcf-mediated transcription

The solution-phase synthesis of the β-turn peptidomimetic ICG-001, a selective inhibitor of Wnt/β-catenin signalling, which has been found to be important for both initiation and progression of cancers of different tissues and has been exploited as an extremely useful chemogenomic tool, was developed. This route is particularly suitable for the multigram scale preparation of ICG-001.     

Discovery of GSK3β Inhibitors through In Silico Prediction-and-Experiment Cycling Strategy,and Biological Evaluation

Direct inhibitors of glycogen synthase kinase 3β (GSK3β) have been investigated and reported for the past 20 years. In the search for novel scaffold inhibitors, 3000 compounds were selected through structure-based virtual screening (SBVS), and then high-throughput enzyme screening was performed. Among the active hit compounds, pyrazolo [1,5-a]pyrimidin-7-amine derivatives showed strong inhibitory potencies on the GSK3β enzyme and markedly activated Wnt signaling. The result of the molecular dynamics (MD) simulation, enhanced by the upper-wall restraint, was used as an advanced structural query for the SBVS. In this study, strong inhibitors designed to inhibit the GSK3β enzyme were discovered through SBVS. Our study provides structural insights into the binding mode of the inhibitors for further lead optimization.     

The Amyloid Fibril-Forming β-Sheet Regions of Amyloid β and α-Synuclein Preferentially Interact with the Molecular Chaperone 14-3-3ζ

14-3-3 proteins are abundant, intramolecular proteins that play a pivotal role in cellular signal transduction by interacting with phosphorylated ligands. In addition, they are molecular chaperones that prevent protein unfolding and aggregation under cellular stress conditions in a similar manner to the unrelated small heat-shock proteins. In vivo, amyloid β (Aβ) and α-synuclein (α-syn) form amyloid fibrils in Alzheimer’s and Parkinson’s diseases, respectively, a process that is intimately linked to the diseases’ progression. The 14-3-3ζ isoform potently inhibited in vitro fibril formation of the 40-amino acid form of Aβ (Aβ₄₀) but had little effect on α-syn aggregation. Solution-phase NMR spectroscopy of ¹⁵N-labeled Aβ₄₀ and A53T α-syn determined that unlabeled 14-3-3ζ interacted preferentially with hydrophobic regions of Aβ₄₀ (L11-H21 and G29-V40) and α-syn (V3-K10 and V40-K60). In both proteins, these regions adopt β-strands within the core of the amyloid fibrils prepared in vitro as well as those isolated from the inclusions of diseased individuals. The interaction with 14-3-3ζ is transient and occurs at the early stages of the fibrillar aggregation pathway to maintain the native, monomeric, and unfolded structure of Aβ₄₀ and α-syn. The N-terminal regions of α-syn interacting with 14-3-3ζ correspond with those that interact with other molecular chaperones as monitored by in-cell NMR spectroscopy.     

Soy Isoflavones Inhibit Both GPIb-IX Signaling and αIIbβ3 Outside-In Signaling via 14-3-3ζ in Platelet

Soy diet is thought to help prevent cardiovascular diseases in humans. Isoflavone, which is abundant in soybean and other legumes, has been reported to possess antiplatelet activity and potential antithrombotic effect. Our study aims to elucidate the potential target of soy isoflavone in platelet. The anti-thrombosis formation effect of genistein and daidzein was evaluated in ex vivo perfusion chamber model under low (300 s⁻¹) and high (1800 s⁻¹) shear forces. The effect of genistein and daidzein on platelet aggregation and spreading was evaluated with platelets from both wildtype and GPIbα deficient mice. The interaction of these soy isoflavone with 14-3-3ζ was detected by surface plasmon resonance (SPR) and co-immunoprecipitation, and the effect of αIIbβ3-mediated outside-in signaling transduction was evaluated by western blot. We found both genistein and daidzein showed inhibitory effect on thrombosis formation in perfusion chamber, especially under high shear force (1800 s⁻¹). These soy isoflavone interact with 14-3-3ζ and inhibited both GPIb-IX and αIIbβ3-mediated platelet aggregation, integrin-mediated platelet spreading and outside-in signaling transduction. Our findings indicate that 14-3-3ζ is a novel target of genistein and daidzein. 14-3-3ζ, an adaptor protein that regulates both GPIb-IX and αIIbβ3-mediated platelet activation is involved in soy isoflavone mediated platelet inhibition.     

Trityl Cation-Catalyzed Hosomi-Sakurai Reaction of Allylsilane with β,γ-Unsaturated α-Ketoester to Form γ,γ-Disubstituted α-Ketoesters

(Ph₃C)[BPh(^F)₄]-catalyzed Hosomi-Sakurai allylation of allylsilanes with β,γ-unsaturated α-ketoesters has been developed to give γ,γ-disubstituted α-ketoesters in high yields with excellent chemoselectivity. Preliminary mechanistic studies suggest that trityl cation dominates the catalysis, while the silyl cation plays a minor role.     

Anti-Cancer Effects of α-Cubebenoate Derived from Schisandra chinensis in CT26 Colon Cancer Cells

α-Cubebenoate derived from Schisandra chinensis has been reported to possess anti-allergic, anti-obesity, and anti-inflammatory effects and to exhibit anti-septic activity, but its anti-cancer effects have not been investigated. To examine the anti-cancer activity of α-cubebenoate, we investigated its effects on the proliferation, apoptosis, and metastasis of CT26 cells. The viabilities of CT26 cells (a murine colorectal carcinoma cell line) and HCT116 cells (a human colon cancer cell line) were remarkably and dose-dependently diminished by α-cubebenoate, whereas the viability of CCD-18Co cells (a normal human fibroblast cell line) were unaffected. Furthermore, α-cubebenoate treatment increased the number of apoptotic CT26 cells as compared with Vehicle-treated cells and increased Bax, Bcl-2, Cas-3, and Cleaved Cas-3 protein levels by activating the MAP kinase signaling pathway. α-Cubebenoate also suppressed CT26 migration by regulating the PI3K/AKT signaling pathway. Furthermore, similar reductions were observed in the expression levels of some migration-related proteins including VEGFA, MMP2, and MMP9. Furthermore, reduced VEGFA expression was found to be accompanied by the phosphorylations of FAK and MLC in the downstream signaling pathway of adhesion protein. The results of the present study provide novel evidence that α-cubebenoate can stimulate apoptosis and inhibit metastasis by regulating the MAPK, PI3K/AKT, and FAK/MLC signaling pathways.     

Selected β2-, β3- and β2,3-Amino Acid Heterocyclic Derivatives and Their Biological Perspective

Heterocyclic moieties, especially five and six-membered rings containing nitrogen, oxygen or sulfur atoms, are broadly distributed in nature. Among them, synthetic and natural alike are pharmacologically active compounds and have always been at the forefront of attention due to their pharmacological properties. Heterocycles can be divided into different groups based on the presence of characteristic structural motifs. The presence of β-amino acid and heterocyclic core in one compound is very interesting; additionally, it very often plays a vital role in their biological activity. Usually, such compounds are not considered to be chemicals containing a β-amino acid motif; however, considering them as this class of compounds may open new routes of their preparation and application as new drug precursors or even drugs. The possibility of their application as nonproteinogenic amino acid residues in peptide or peptide derivatives synthesis to prepare a new class of compounds is also promising. This review highlights the actual state of knowledge about β-amino acid moiety-containing heterocycles presenting antiviral, anti-inflammatory, antibacterial compounds, anaplastic lymphoma kinase (ALK) inhibitors, as well as agonist and antagonists of the receptors.     

α-Linolenic Acid Suppresses Proliferation and Invasion in Osteosarcoma Cells via Inhibiting Fatty Acid Synthase

Fatty acid synthase (FASN) is highly expressed in multiple types of human cancers and is recognized as one of the targets for treating cancer metastasis. α-Linolenic acid is an omega-3 essential fatty acid and it possesses various biological activities. The present study was designed to reveal the effects of α-linolenic acid on osteosarcoma and to reveal whether the mechanism of α-linolenic acid in anticancer activity may be related to FASN inhibition. The cytotoxicity of α-linolenic acid was assessed in osteosarcoma MG63, 143B, and U2OS cells. Cell viability was detected by the MTT assay. The protein expression level was detected by western blotting. Flow cytometry, Annexin V/propidium iodide dual staining, and Hoechst 33258 staining were performed to assess the apoptotic effects. Wound healing assay was applied to detect the inhibitory effect of α-linolenic acid on osteosarcoma cells migration. The results showed that α-linolenic acid downregulated FASN expression. α-Linolenic acid inhibited osteosarcoma cell proliferation and migration in a dose-dependent manner. In addition, α-linolenic acid regulated endoplasmic reticulum transmembrane receptors and signal protein expression in osteosarcoma cells. The findings of the present study suggested that α-linolenic acid suppresses osteosarcoma cell proliferation and metastasis by inhibiting FASN expression, which provides a basis as a potential target for osteosarcoma treatment.