MiR-107 suppresses cell proliferation and tube formation of Ewing sarcoma cells partly by targeting HIF-1β

MicroRNAs serve a crucial role in the regulation of malignant biological behavior of Ewing’s sarcoma (ES). Abnormal expression of miR-107 has been reported in a cohort of cancers, while its exact function in ES remains unclear. Hence, we explored the expression of miR-107 in ES cells and detected its effects on the malignant phenotype of ES cells. Firstly, we perceived the under-expression of miR-107 in human ES cells contrast with the human mesenchymal stem cells. Over-expression of miR-107 restrained cell proliferation and tube formation, arrested cell cycle progression, and facilitated cell apoptosis in SK-ES-1 and RD-ES cell lines. Furthermore, hypoxia inducible factor-1β (HIF-1β) was assumed as a target gene of miR-107. We confirmed the target role of HIF-1β in ES cells. Finally, restoring the expression of HIF-1β could partly abolish miR-107-mediated tumor suppression in ES cells. In conclusion, our results advised that miR-107 suppressed the malignant biological ability of ES cells through targeting HIF-1β.     

Notch inhibition promotes fetal liver stem/progenitor cells differentiation into hepatocytes via the inhibition of HNF-1β

In a previous study, the Notch pathway inhibited with N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (also called DAPT) was shown to promote the differentiation of fetal liver stem/progenitor cells (FLSPCs) into hepatocytes and to impair cholangiocyte differentiation. The precise mechanism for this, however, was not elucidated. Two mechanisms are possible: Notch inhibition might directly up-regulate hepatocyte differentiation via HGF (hepatocyte growth factor) and HNF (hepatocyte nuclear factor)-4α or might impair cholangiocyte differentiation thereby indirectly rendering hepatocyte differentiation as the dominant state. In this study, HGF and HNF expression was detected after the Notch pathway was inhibited. Although our initial investigation indicated that the inhibition of Notch induced hepatocyte differentiation with an efficiency similar to the induction via HGF, the results of this study demonstrate that Notch inhibition does not induce significant up-regulation of HGF or HNF-4α in FLSPCs. This suggests that Notch inhibition induces hepatocyte differentiation without the influence of HGF or HNF-4α. Moreover, significant down-regulation of HNF-1β was observed, presumably dependent on an impairment of cholangiocyte differentiation. To confirm this presumption, HNF-1β was blocked in FLSPCs and was followed by hepatocyte differentiation. The expression of markers of mature cholangiocyte was impaired and hepatocyte markers were elevated significantly. The data thus demonstrate that the inhibition of cholangiocyte differentiation spontaneously induces hepatocyte differentiation and further suggest that hepatocyte differentiation from FLSPCs occurs at the expense of the impairment of cholangiocyte differentiation, probably being enhanced partially via HNF-1β down-regulation or Notch inhibition.     

Caveolin-1 inhibits oligodendroglial differentiation of neural stem/progenitor cells through modulating β-catenin expression

In the present study, we aim to elucidate the role of caveolin-1 (Cav-1) in modulating oligodendroglial differentiation of neural progenitor cells (NPCs) in vivo and in vitro. For in vivo experiments, we investigated oligodendroglial differentiation by detecting the expressions of 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) and β-catenin in the brains of wild type mice and Cav-1 knockout mice. Cav-1 knockout mice revealed more oligodendroglial differentiation, but lower levels of β-catenin expression than wild type mice. For in vitro experiments, we observed the potential roles of Cav-1 in modulating β-catenin expression and oligodendroglial differentiation in isolated cultured NPCs by manipulating Cav-1 expression with Cav-1 scaffolding domain peptide and Cav-1 RNA silencing approach. In the differentiating NPCs, Cav-1 scaffolding domain peptide markedly inhibited oligodendroglial formation, but up-regulated the expression of β-catenin. In contrast, the knockdown of Cav-1 promoted oligodendroglial differentiation of NPCs, but down-regulated the expression of β-catenin. Taken together, these results directly prove that caveolin-1 can inhibit oligodendroglial differentiation of NPCs through modulating β-catenin expression.     

Betacellulin inhibits osteogenic differentiation and stimulates proliferation through HIF-1α

Cellular signaling via epidermal growth factor (EGF) and EGF-like ligands can determine cell fate and behavior. Osteoblasts, which are responsible for forming and mineralizing osteoid, express EGF receptors and alter rates of proliferation and differentiation in response to EGF receptor activation. Transgenic mice over-expressing the EGF-like ligand betacellulin (BTC) exhibit increased cortical bone deposition; however, because the transgene is ubiquitously expressed in these mice, the identity of cells affected by BTC and responsible for increased cortical bone thickness remains unknown. We have therefore examined the influence of BTC upon mesenchymal stem cell (MSC) and pre-osteoblast differentiation and proliferation. BTC decreases the expression of osteogenic markers in both MSCs and pre-osteoblasts; interestingly, increases in proliferation require hypoxia-inducible factor-alpha (HIF-α), as an HIF antagonist prevents BTC-driven proliferation. Both MSCs and pre-osteoblasts express EGF receptors ErbB1, ErbB2, and ErbB3, with no change in expression under osteogenic differentiation. These are the first data that demonstrate an influence of BTC upon MSCs and the first to implicate HIF-α in BTC-mediated proliferation.     

Adrenomedullin promotes human endothelial cell proliferation via HIF-1α

Adrenomedullin (ADM) and hypoxia-inducible factor-1α (HIF-1α) are important pro-proliferation genes in response to hypoxic stress. Although it was reported that ADM is a target gene for HIF-1, recent studies also showed that ADM regulates HIF-1 expression and its activity; however, the mechanism of action remains unknown. Two stable human endothelial cell lines with HIF-1α knockdown by hy926-siHIF-1α or HMEC-siHIF-1α were established. mRNA and protein expression of ADM and HIF-1α in EA.hy926 and HMEC1 cells were examined under hypoxic stress. Upon ADM treatment, cell proliferation was investigated and the expression profiles of HIF-1α and its target genes (VEGF, PFKP, PGK1, and AK1) were examined. Furthermore, the proline hydroxylase (PHD) mRNA level and its activity were investigated. We observed that mRNA and protein expression of ADM in hypoxia are earlier events than HIF-1α in EA.hy926 and HMEC1 cells. ADM-promoted cell proliferation of endothelial cells, which was HIF-1α dependent. We also found that ADM up-regulated the mRNA and protein expressions of HIF-1α- and HIF-1-targeted genes, and ADM up-regulated the protein expressions of HIF-1α through down-regulation of PHD mRNA expression and PHD activity.     

20-Hydroxyecdysone inhibits inflammation via SIRT6-mediated NF-κB signaling in endothelial cells

20-Hydroxyecdysone (20E) is known to have numerous pharmacological activities and can be used to treat diabetes and cardiovascular diseases. However, the protective effects of 20E against endothelial dysfunction and its targets remain unclear. In the present study, we revealed that 20E treatment could modulate the release of the endothelium-derived vasomotor factors NO, PGI₂ and ET-1 and suppress the expression of ACE in TNF-α-induced 3D-cultured HUVECs. In addition, 20E suppressed the expression of CD40 and promoted the expression of SIRT6 in TNF-α-induced 3D-cultured HUVECs. The cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS) and molecular docking results demonstrated that 20E binding increased SIRT6 stability, indicating that 20E directly bound to SIRT6 in HUVECs. Further investigation of the underlying mechanism showed that 20E could upregulate SIRT6 levels and that SIRT6 knockdown abolished the regulatory effect of 20E on CD40 in TNF-α-induced HUVECs, while SIRT6 overexpression further improved the effect of 20E. Moreover, we found that 20E could reduce the acetylation of NF-κB p65 (K310) through SIRT6, but the catalytic inactive mutant SIRT6 (H133Y) did not promote the deacetylation of NF-κB p65, suggesting that the inhibitory effect of 20E on NF-κB p65 was dependent on SIRT6 deacetylase activity. Additionally, our results indicated that 20E inhibited NF-κB via SIRT6, and the expression of CD40 was increased in HUVECs treated with SIRT6 siRNA and NF-κB inhibitor. In conclusion, the present study demonstrates that 20E exerts its effect through SIRT6-mediated deacetylation of NF-κB p65 (K310) to inhibit CD40 expression in ECs, and 20E may have therapeutic potential for the treatment of cardiovascular diseases.     

PDGFRβ triggered by bFGF promotes the proliferation and migration of endothelial progenitor cells via p-ERK signalling

We have examined the effects of bFGF (basic fibroblast growth factor) on p-ERK (phosphorylated extracellular signal-regulated kinase) through PDGFRβ (platelet-derived growth factor receptor β) in the proliferation and migration of EPCs (endothelial progenitor cells). EPC migration was detected using the Transwell system. The expression of PDGFRβ mRNA and protein, total ERK and p-ERK proteins was respectively assessed by real-time PCR and Western blottings. bFGF promote the proliferation and migration of EPCs, the effects of bFGF being implemented by activating ERK signalling through the expression of PDGFRβ, whereas an anti-bFGF antibody and inhibitor of PDGF (platelet-derived growth factor) receptor kinase (AG1296) could respectively decrease the expression of PDGFRβ mRNA and protein and p-ERK protein. Total ERK protein did not change under the same experimental conditions, and an inhibitor of p-ERK (PD98059) inhibited the proliferation and migration of EPCs. The findings strongly suggest that a PDGFRβ/p-ERK signalling pathway triggered by bFGF plays an important role in the proliferation and migration of EPCs.     

Interactions of primary neuroepithelial progenitor and brain endothelial cells： distinct effect on neural progenitor maintenance and differentiation by soluble factors and direct contact

Neurovascular interactions are crucial for the normal development of the central nervous system. To study suchinteractions in primary cultures, we developed a procedure to simultaneously isolate neural progenitor and endothelialcell fractions from embryonic mouse brains. Depending on the culture conditions endothelial cells were found to favormaintenance of the neuroprogenitor phenotype through the production of soluble factors, or to promote neuronal differ-entiation of neural progenitors through direct contact. These apparently opposing effects could reflect differential cellularinteractions needed for the proper development of the brain.     

HIF-1α protein is upregulated in HIF-2α depleted cells via enhanced translation

The hypoxia-inducible factors HIF-1 and HIF-2 are primarily regulated via stabilization of their respective α-subunits under hypoxic conditions. Previously, compensatory upregulation of one HIF-α-subunit upon depletion of the other α-subunit was described, yet the underlying mechanism remained elusive. Here we provide evidence that enhanced HIF-1α protein expression in HIF-2α knockdown (k/d) cells neither results from elevated HIF-1α mRNA expression, nor from increased HIF-1α protein stability. Instead, we identify enhanced HIF-1α translation as molecular mechanism. Moreover, we found elevated levels of the RNA-binding protein HuR and provide evidence that HuR is critical for the compensatory HIF-1α regulation in HIF-2α k/d cells.     

Oncostatin M inhibits TGF-β1-induced CTGF expression via STAT3 in human proximal tubular cells

Matricellular proteins play a critical role in the development of tubulointerstitial fibrosis and renal disease progression. Connective tissue growth factor (CTGF/CCN2), a CCN family member of matricellular proteins, represents an important mediator during development of glomerular and tubulointerstitial fibrosis in progressive kidney disease. We have recently reported that oncostatin M (OSM) is a potent inhibitor of TGF-β1-induced CTGF expression in human proximal tubular cells (PTC). In the present study we examined the role of TGF-β1- and OSM-induced signaling mechanisms in the regulation of CTGF mRNA expression in human proximal tubular HK-2 cells. Utilizing siRNA-mediated gene silencing we found that TGF-β1-induced expression of CTGF mRNA after 2h of stimulation at least partially depends on SMAD3 but not on SMAD2. In contrast to TGF-β1, OSM seems to exert a time-dependent dual effect on CTGF mRNA expression in these cells. While OSM led to a rapid and transient induction of CTGF mRNA expression between 15min and 1h of stimulation it markedly suppressed basal and TGF-β1-induced CTGF mRNA levels thereafter. Silencing of STAT1 or STAT3 attenuated basal CTGF mRNA levels indicating that both STAT isoforms may be involved in the regulation of basal CTGF mRNA expression. However, knockdown of STAT3 but not STAT1 prevented OSM-mediated suppression of basal and TGF-β1-induced upregulation of CTGF mRNA expression. Together these results suggest that the inhibitory effect of OSM on TGF-β1-induced CTGF mRNA expression is mainly driven by STAT3, thereby providing a signaling mechanism whereby OSM may contribute to tubulointerstitial protection.     

Overexpression of osteopontin induces angiogenesis of endothelial progenitor cells via the avβ3/PI3K/AKT/eNOS/NO signaling pathway in glioma cells

Angiogenesis, a hallmark of tumor growth, is regulated by various angiogenic factors. Recent studies have shown that osteopontin (OPN) is a secreted, integrin-binding protein that contributes to glioma progression. However, its effect on the angiogenesis of gliomas is not fully understood. To elucidate the role of OPN in the process of glioma angiogenesis, endothelial progenitor cells (EPCs) were treated with conditioned media of human glioma SHG44 cells overexpressing OPN. Here, we identified that OPN secreted by glioma cells accelerated EPCs angiogenesis in vitro, including proliferation, migration, and tube formation. OPN also induced the activation of AKT and endothelial nitric oxide synthase (eNOS) and increased NO production without affecting the expression of VEGF, VEGFR-1, or VEGFR-2. Moreover, the avβ3 antibody, the PI3-K inhibitor LY294002 and the eNOS inhibitor NMA suppressed the OPN-mediated increase in NO production and angiogenesis in EPCs. Taken together, these results demonstrate that OPN directly stimulates angiogenesis via the avβ3/PI3-K/AKT/eNOS/NO signaling pathway and may play an important role in tumorigenesis by enhancing angiogenesis in gliomas.     

Amyloid-β1–42 oligomer accelerates senescence in adult hippocampal neural stem/progenitor cells via formylpeptide receptor 2

The failure of adult hippocampal neurogenesis is increasingly considered as an important factor in the pathological correlates for memory decline in Alzheimer''s disease (AD). Loss of adult-born neurons and abnormalities of neural stem/progenitor cells (NSPCs) within the dentate gyrus (DG) of adult hippocampus might contribute to this process. In this study, we showed that amyloid-β_1–42 (Aβ₄₂) oligomer triggers senescent phenotype of NSPCs in vitro. Oligomerized Aβ₄₂ induced the production of senescence-associated biomarkers p16 and senescence-associated β-galactosidase (SA-β-gal) in adult mouse hippocampal NSPCs, as well as inhibited cells proliferation and differentiation. In the DG of amyloid precursor protein/presenilin1 (APP/PS1) transgenic mice, the number of senescent NSPCs was significantly increased and senescence-associated protein p16 was upregulated. Formylpeptide receptor 2 (FPR2), one of Aβ₄₂ functional receptors, may be involved in NSPCs senescence. The FPR2 antagonist WRW4 significantly inhibited NSPCs senescence induced by Aβ₄₂. In addition, the activation of p38 mitogen-activated protein kinase (MAPK) in response to the accumulation of reactive oxygen species (ROS) was involved in NSPCs senescence induced by Aβ₄₂. WRW4 inhibited the accumulation of ROS and the activation of p38 MAPK in NSPCs. Our data suggest that Aβ₄₂ accelerates NSPCs senescence via FPR2-dependent activation of its downstream ROS-p38 MAPK signaling, which limits the function of NSPCs and contributes to failure of neurogenesis. This is the first demonstration of NSPCs senescence response to Aβ₄₂.     

Osteocyte-induced angiogenesis via VEGF–MAPK-dependent pathways in endothelial cells

Recently, it has been suggested osteocytes control the activities of bone formation (osteoblasts) and resorption (osteoclast), indicating their important regulatory role in bone remodelling. However, to date, the role of osteocytes in controlling bone vascularisation remains unknown. Our aim was to investigate the interaction between endothelial cells and osteocytes and to explore the possible molecular mechanisms during angiogenesis. To model osteocyte/endothelial cell interactions, we co-cultured osteocyte cell line (MLOY4) with endothelial cell line (HUVECs). Co-cultures were performed in 1:1 mixture of osteocytes and endothelial cells or by using the conditioned media (CM) transfer method. Real-time cell migration of HUVECs was measured with the transwell migration assay and xCELLigence system. Expression levels of angiogenesis-related genes were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The effect of vascular endothelial growth factor (VEGF) and mitogen-activated phosphorylated kinase (MAPK) signaling were monitored by western blotting using relevant antibodies and inhibitors. During the bone formation, it was noted that osteocyte dendritic processes were closely connected to the blood vessels. The CM generated from MLOY4 cells-activated proliferation, migration, tube-like structure formation, and upregulation of angiogenic genes in endothelial cells suggesting that secretory factor(s) from osteocytes could be responsible for angiogenesis. Furthermore, we identified that VEGF secreted from MLOY4-activated VEGFR2–MAPK–ERK-signaling pathways in HUVECs. Inhibiting VEGF and/or MAPK–ERK pathways abrogated osteocyte-mediated angiogenesis in HUVEC cells. Our data suggest an important role of osteocytes in regulating angiogenesis.