Substance P Activates the Wnt Signal Transduction Pathway and Enhances the Differentiation of Mouse Preosteoblastic MC3T3-E1 Cells

Recent experiments have explored the impact of Wnt/β-catenin signaling and Substance P (SP) on the regulation of osteogenesis. However, the molecular regulatory mechanisms of SP on the formation of osteoblasts is still unknown. In this study, we investigated the impact of SP on the differentiation of MC3T3-E1 cells. The osteogenic effect of SP was observed at different SP concentrations (ranging from 10⁻¹⁰ to 10⁻⁸ M). To unravel the underlying mechanism, the MC3T3-E1 cells were treated with SP after the pretreatment by neurokinin-1 (NK1) antagonists and Dickkopf-1 (DKK1) and gene expression levels of Wnt/β-catenin signaling pathway components, as well as osteoblast differentiation markers (collagen type I, alkaline phosphatase, osteocalcin, and Runx2), were measured using quantitative polymerase chain reaction (PCR). Furthermore, protein levels of Wnt/β-catenin signaling pathway were detected using Western blotting and the effects of SP, NK1 antagonist, and DKK1 on β-catenin activation were investigated by immunofluorescence staining. Our data indicated that SP (10⁻⁹ to 10⁻⁸ M) significantly up-regulated the expressions of osteoblastic genes. SP (10⁻⁸ M) also elevated the mRNA level of c-myc, cyclin D1, and lymphocyte enhancer factor-1 (Lef1), as well as c-myc and β-catenin protein levels, but decreased the expression of Tcf7 mRNA. Moreover, SP (10⁻⁸ M) promoted the transfer of β-catenin into nucleus. The effects of SP treatment were inhibited by the NK1 antagonist and DKK1. These findings suggest that SP may enhance differentiation of MC3T3-E1 cells via regulation of the Wnt/β-catenin signaling pathway.     

Different Cell Responses to Hinokitiol Treatment Result in Senescence or Apoptosis in Human Osteosarcoma Cell Lines

Hinokitiol is a tropolone-related compound isolated from the heartwood of cupressaceous plants. It is known to exhibit various biological functions including antibacterial, antifungal, and antioxidant activities. In the study, we investigated the antitumor activities of hinokitiol against human osteosarcoma cells. The results revealed that hinokitiol treatment inhibited cell viability of human osteosarcoma U-2 OS and MG-63 cells in the MTT assay. Further study revealed that hinokitiol exposure caused cell cycle arrest at the S phase and a DNA damage response with the induction of γ-H2AX foci in both osteosarcoma cell lines. In U-2 OS cells with wild-type tumor suppressor p53, we found that hinokitiol exposure induced p53 expression and cellular senescence, and knockdown of p53 suppressed the senescence. However, in MG-63 cells with mutated p53, a high percentage of cells underwent apoptosis with cleaved-PARP expression and Annexin V staining after hinokitiol treatment. In addition, up-regulated autophagy was observed both in hinokitiol-exposed U-2 OS and MG-63 cells. As the autophagy was suppressed through the autophagy inhibitor chloroquine, hinokitiol-induced senescence in U-2 OS cells was significantly enhanced accompanying more abundant p53 expression. In MG-63 cells, co-treatment of chloroquine increased hinokitiol-induced apoptosis and decreased cell viability of the treated cells. Our data revealed that hinokitiol treatment could result in different cell responses, senescence or apoptosis in osteosarcoma cell lines, and suppression of autophagy could promote these effects. We hypothesize that the analysis of p53 status and co-administration of autophagy inhibitors might provide more precise and efficacious therapies in hinokitiol-related trials for treating osteosarcoma.     

MicroRNA-331-3p Suppresses Cervical Cancer Cell Proliferation and E6/E7 Expression by Targeting NRP2

Aberrant expression of microRNAs (miRNAs) is involved in the development and progression of various types of cancers. In this study, we investigated the role of miR-331-3p in cell proliferation and the expression of keratinocyte differentiation markers of uterine cervical cancer cells. Moreover, we evaluated whether neuropilin 2 (NRP2) are putative target molecules that regulate the human papillomavirus (HPV) related oncoproteins E6 and E7. Cell proliferation in the human cervical cancer cell lines SKG-II, HCS-2, and HeLa was assessed using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. Cellular apoptosis was measured using the TdT-mediated dUTP nick end labeling (TUNEL) and Annexin V assays. Quantitative RT-PCR was used to measure the messenger RNA (mRNA) expression of the NRP2, E6, E7, p63, and involucrin (IVL) genes. A functional assay for cell growth was performed using cell cycle analyses. Overexpression of miR-331-3p inhibited cell proliferation, and induced G2/M phase arrest and apoptosis in SKG-II, HCS-2 and HeLa cells. The luciferase reporter assay of the NRP2 3′-untranslated region revealed the direct regulation of NRP2 by miR-331-3p. Gene expression analyses using quantitative RT-PCR in SKG-II, HCS-2, and HeLa cells overexpressing miR-331-3p or suppressing NRP2 revealed down-regulation of E6, E7, and p63 mRNA and up-regulation of IVL mRNA. Moreover, miR-331-3p overexpression was suppressed NRP2 expression in protein level. We showed that miR-331-3p and NRP2 were key effectors of cell proliferation by regulating the cell cycle, apoptosis. NRP-2 also regulates the expression of E6/E7 and keratinocyte differentiation markers. Our findings suggest that miR-331-3p has an important role in regulating cervical cancer cell proliferation, and that miR-331-3p may contribute to keratinocyte differentiation through NRP2 suppression. miR-331-3p and NRP2 may contribute to anti-cancer effects.     

Structural Characterization of the Interaction of Hypoxia Inducible Factor-1 with Its Hypoxia Responsive Element at the −964G > A Variation Site of the HLA-G Promoter Region

Human Antigen Leukocyte-G (HLA-G) gene encodes an immune checkpoint molecule that has restricted tissue expression in physiological conditions; however, the gene may be induced in hypoxic conditions by the interaction with the hypoxia inducible factor-1 (HIF1). Hypoxia regulatory elements (HRE) located at the HLA-G promoter region and at exon 2 are the major HIF1 target sites. Since the G allele of the −964G > A transversion induces higher HLA-G expression when compared to the A allele in hypoxic conditions, here we analyzed HIF1-HRE complex interaction at the pair-atom level considering both −964G > A polymorphism alleles. Mouse HIF2 dimer crystal (Protein Data Bank ID: 4ZPK) was used as template to perform homology modelling of human HIF1 quaternary structure using MODELLER v9.14. Two 3D DNA structures were built from 5′GCRTG’3 HRE sequence containing the −964G/A alleles using x3DNA. Protein-DNA docking was performed using the HADDOCK v2.4 server, and non-covalent bonds were computed by DNAproDB server. Molecular dynamic simulation was carried out per 200 ns, using Gromacs v.2019. HIF1 binding in the HRE containing −964G allele results in more hydrogen bonds and van der Waals contact formation than HRE with −964A allele. Protein-DNA complex trajectory analysis revealed that HIF1-HRE-964G complex is more stable. In conclusion, HIF1 binds in a more stable and specific manner at the HRE with G allele.     

Enhanced Anti-Atherosclerotic Efficacy of pH-Responsively Releasable Ganglioside GM3 Delivered by Reconstituted High-Density Lipoprotein

Recently, the atheroprotective role of endogenous GM3 and an atherogenesis-inhibiting effect of exogenous GM3 suggested a possibility of exogenous GM3 being recruited as an anti-atherosclerotic drug. This study seeks to endow exogenous GM3 with atherosclerotic targetability via reconstituted high-density lipoprotein (rHDL), an atherosclerotic targeting drug nanocarrier. Unloaded rHDL, rHDL loaded with exogenous GM3 at a low concentration (GM3_L-rHDL), and rHDL carrying GM3 at a relatively high concentration (GM3_H-rHDL) were prepared and characterized. The inhibitory effect of GM3-rHDL on lipid deposition in macrophages was confirmed, and GM3-rHDL did not affect the survival of red blood cells. In vivo experiments using ApoE^−/− mice fed a high fat diet further confirmed the anti-atherosclerotic efficacy of exogenous GM3 and demonstrated that GM3 packed in HDL nanoparticles (GM3-rHDL) has an enhanced anti-atherosclerotic efficacy and a reduced effective dose of GM3. Then, the macrophage- and atherosclerotic plaque-targeting abilities of GM3-rHD, most likely via the interaction of ApoA-I on GM3-rHDL with its receptors (e.g., SR-B1) on cells, were certified via a microsphere-based method and an aortic fragment-based method, respectively. Moreover, we found that solution acidification enhanced GM3 release from GM3-rHDL nanoparticles, implying the pH-responsive GM3 release when GM3-rHDL enters the acidic atherosclerotic plaques from the neutral blood. The rHDL-mediated atherosclerotic targetability and pH-responsive GM3 release of GM3-rHDL enhanced the anti-atherosclerotic efficacy of exogenous GM3. The development of the GM3-rHDL nanoparticle may help with the application of exogenous GM3 as a clinical drug. Moreover, the data imply that the GM3-rHDL nanoparticle has the potential of being recruited as a drug nanocarrier with atherosclerotic targetability and enhanced anti-atherosclerotic efficacy.     

S1P Lyase Regulates Intestinal Stem Cell Quiescence via Ki-67 and FOXO3

Background: Reduction of the Sphingosine-1-phosphate (S1P) degrading enzyme S1P lyase 1 (SGPL1) initiates colorectal cancer progression with parallel loss of colon function in mice. We aimed to investigate the effect of SGPL1 knockout on the stem cell niche in these mice. Methods: We performed immunohistochemical and multi-fluorescence imaging on tissue sections of wildtype and SGPL1 knockout colons under disease conditions. Furthermore, we generated SGPL1 knockout DLD-1 cells (SGPL1^−/−M.Ex1) using CRISPR/Cas9 and characterized cell cycle and AKT signaling pathway via Western blot, immunofluorescence, and FACS analysis. Results: SGPL1 knockout mice were absent of anti-Ki-67 staining in the stem cell niche under disease conditions. This was accompanied by an increase of the negative cell cycle regulator FOXO3 and attenuation of CDK2 activity. SGPL1^−/−M.Ex1 cells show a similar FOXO3 increase but no arrest of proliferation, although we found a suppression of the PDK1/AKT signaling pathway, a prolonged G1-phase, and reduced stem cell markers. Conclusions: While already established colon cancer cells find escape mechanisms from cell cycle arrest, in vivo SGPL1 knockout in the colon stem cell niche during progression of colorectal cancer can contribute to cell cycle quiescence. Thus, we propose a new function of the S1P lyase 1 in stemness.     

Melatonin-Mediated Colonic Microbiota Metabolite Butyrate Prevents Acute Sleep Deprivation-Induced Colitis in Mice

Radical cure colitis is a severe public health threat worldwide. Our previous studies have confirmed that melatonin can effectively improve gut microbiota disorder and mucosal injury caused by sleep deprivation (SD). The present study further explored the mechanism whereby exogenous melatonin prevented SD-induced colitis. 16S rRNA high-throughput sequencing and metabolomics analysis were used to explore the correlation between SD-induced colitis and intestinal microbiota and metabolite composition in mice. Fecal microbiota transplantation (FMT) and melatonin or butyrate supplementation tests verified the core role of gut microbiota in melatonin-alleviating SD-induced colitis. Further, in vitro tests studied the modulatory mechanism of metabolite butyrate. The results demonstrated that SD leads to reductions in plasma melatonin levels and colonic Card9 expression and consequent occurrence of colitis and gut microbiota disorder, especially the downregulation of Faecalibacterium and butyrate levels. The FMT from SD-mice to normal mice could restore SD-like colitis, while butyrate supplementation to SD-mice inhibited the occurrence of colitis, but with no change in the plasma melatonin level in both treatments. However, melatonin supplementation reversed all inductions in SD-mice. In intestinal epithelial cells, the inflammatory ameliorative effect of butyrate was blocked with pretreatments of HDAC3 agonist and HIF-1α antagonist but was mimicked by GSK-3β and p-P65 antagonists. Therefore, the administration of MLT may be a better therapy for SD-induced colitis relative to butyrate. A feasible mechanism would involve that melatonin up-regulated the Faecalibacterium population and production of its metabolite butyrate and MCT1 expression and inhibited HDAC3 in the colon, which would allow p-GSK-3β/β-catenin/HIF-1α activation and NF-κB/NLRP3 suppression to up-regulate Card9 expression and suppress inflammation response.