The β3‐integrin endothelial adhesome regulates microtubule‐dependent cell migration

Integrin β3 is seen as a key anti‐angiogenic target for cancer treatment due to its expression on neovasculature, but the role it plays in the process is complex; whether it is pro‐ or anti‐angiogenic depends on the context in which it is expressed. To understand precisely β3's role in regulating integrin adhesion complexes in endothelial cells, we characterised, by mass spectrometry, the β3‐dependent adhesome. We show that depletion of β3‐integrin in this cell type leads to changes in microtubule behaviour that control cell migration. β3‐integrin regulates microtubule stability in endothelial cells through Rcc2/Anxa2‐driven control of active Rac1 localisation. Our findings reveal that angiogenic processes, both in vitro and in vivo, are more sensitive to microtubule targeting agents when β3‐integrin levels are reduced.     

NELL‐1 promotes cell adhesion and differentiation via integrinβ1

NELL‐1 (Nel‐like molecule‐1) is a secreted osteogenic growth factor first identified in human craniosynostosis (CS) patients. NELL‐1 protein has been observed to promote bone and cartilage differentiation and to suppress adipogenesis in both in vitro and in vivo models. Despite these findings, the cell surface receptors of NELL‐1 have remained unknown. In this study, we observed for the first time that NELL‐1 promotes cell adherence in multiple cell lines, including ST2, C3H10T1/2, M2‐10B4, ATDC5, and MC3T3 cells. Additionally, we found that NELL‐1 binds to extracellular Integrinβ1 and induces cell focal adhesion. By utilizing siRNA methods, we determined that NELL‐1 cell surface binding and enhanced cell attachment were dependent on Integrinβ1 expression. Finally, we observed that pre‐coating of culture dishes or PLGA (polylactic‐co‐glycolic acid) scaffold with NELL‐1 resulted in a significant increase in both cell attachment and osteogenic differentiation. Our results identify for the first time a cell surface target of NELL‐1, Integrinβ1, and elucidate new functions of NELL‐1 in promoting cell adherence and osteogenic differentiation. J. Cell. Biochem. 113: 3620–3628, 2012. © 2012 Wiley Periodicals, Inc.     

Mitochondrial PKC‐ε deficiency promotes I/R‐mediated myocardial injury via GSK3β‐dependent mitochondrial permeability transition pore opening

Mitochondrial fission is critically involved in cardiomyocyte apoptosis, which has been considered as one of the leading causes of ischaemia/reperfusion (I/R)‐induced myocardial injury. In our previous works, we demonstrate that aldehyde dehydrogenase‐2 (ALDH2) deficiency aggravates cardiomyocyte apoptosis and cardiac dysfunction. The aim of this study was to elucidate whether ALDH2 deficiency promotes mitochondrial injury and cardiomyocyte death in response to I/R stress and the underlying mechanism. I/R injury was induced by aortic cross‐clamping for 45 min. followed by unclamping for 24 hrs in ALDH2 knockout (ALDH2^?/?) and wild‐type (WT) mice. Then myocardial infarct size, cell apoptosis and cardiac function were examined. The protein kinase C (PKC) isoform expressions and their mitochondrial translocation, the activity of dynamin‐related protein 1 (Drp1), caspase9 and caspase3 were determined by Western blot. The effects of N‐acetylcysteine (NAC) or PKC‐δ shRNA treatment on glycogen synthase kinase‐3β (GSK‐3β) activity and mitochondrial permeability transition pore (mPTP) opening were also detected. The results showed that ALDH2^?/? mice exhibited increased myocardial infarct size and cardiomyocyte apoptosis, enhanced levels of cleaved caspase9, caspase3 and phosphorylated Drp1. Mitochondrial PKC‐ε translocation was lower in ALDH2^?/? mice than in WT mice, and PKC‐δ was the opposite. Further data showed that mitochondrial PKC isoform ratio was regulated by cellular reactive oxygen species (ROS) level, which could be reversed by NAC pre‐treatment under I/R injury. In addition, PKC‐ε inhibition caused activation of caspase9, caspase3 and Drp1Ser⁶¹⁶ in response to I/R stress. Importantly, expression of phosphorylated GSK‐3β (inactive form) was lower in ALDH2^?/? mice than in WT mice, and both were increased by NAC pre‐treatment. I/R‐induced mitochondrial translocation of GSK‐3β was inhibited by PKC‐δ shRNA or NAC pre‐treatment. In addition, mitochondrial membrane potential (?Ψ_m) was reduced in ALDH2^?/? mice after I/R, which was partly reversed by the GSK‐3β inhibitor (SB216763) or PKC‐δ shRNA. Collectively, our data provide the evidence that abnormal PKC‐ε/PKC‐δ ratio promotes the activation of Drp1 signalling, caspase cascades and GSK‐3β‐dependent mPTP opening, which results in mitochondrial injury‐triggered cardiomyocyte apoptosis and myocardial dysfuction in ALDH2^?/? mice following I/R stress.     

Metformin protects against intestinal barrier dysfunction via AMPKα1‐dependent inhibition of JNK signalling activation

Disruption of the intestinal epithelial barrier, that involves the activation of C‐Jun N‐terminal kinase (JNK), contributes to initiate and accelerate inflammation in inflammatory bowel disease. Metformin has unexpected beneficial effects other than glucose‐lowering effects. Here, we provided evidence that metformin can protect against intestinal barrier dysfunction in colitis. We showed that metformin alleviated dextran sodium sulphate (DSS)‐induced decreases in transepithelial electrical resistance, FITC‐dextran hyperpermeability, loss of the tight junction (TJ) proteins occludin and ZO‐1 and bacterial translocation in Caco‐2 cell monolayers or in colitis mice models. Metformin also improved TJ proteins expression in ulcerative colitis patients with type 2 diabetes mellitus. We found that metformin ameliorated the induction of colitis and reduced the levels of pro‐inflammatory cytokines IL‐6, TNF‐a and IL‐1β. In addition, metformin suppressed DSS‐induced JNK activation, an effect dependent on AMP‐activated protein kinase α1 (AMPKα1) activation. Consistent with this finding, metformin could not maintain the barrier function of AMPKα1‐silenced cell monolayers after DSS administration. These findings highlight metformin protects against intestinal barrier dysfunction. The potential mechanism may involve in the inhibition of JNK activation via an AMPKα1‐dependent signalling pathway.     

Laminar shear stress elicit distinct endothelial cell e‐selectin expression pattern via TNFα and IL‐1β activation

The ability to discriminate cell adhesion molecule expression between healthy and inflamed endothelium is critical for therapeutic intervention in many diseases. This study explores the effect of laminar flow on TNFα‐induced E‐selectin surface expression levels in human umbilical vein endothelial cells (HUVECs) relative to IL‐1β‐induced expression via flow chamber assays. HUVECs grown in static culture were either directly (naïve) activated with cytokine in the presence of laminar shear or pre‐exposed to 12 h of laminar shear (shear‐conditioned) prior to simultaneous shear and cytokine activation. Naïve cells activated with cytokine in static served as control. Depending on the cell shear history, fluid shear is found to differently affect TNFα‐induced relative to IL‐1β‐induced HUVEC expression of E‐selectin. Specifically, E‐selectin surface expression by naïve HUVECs is enhanced in the 8–12 h activation time range with simultaneous exposure to shear and TNFα (shear‐TNFα) relative to TNFα static control whereas enhanced E‐selectin expression is observed in the 4–24 h range for shear‐IL‐1β treatment relative to IL‐1β static control. While exposure of HUVECs to shear preconditioning mutes shear‐TNFα‐induced E‐selectin expression, it enhances or down‐regulates shear‐IL‐1β‐induced expression dependent on the activation period. Under dual‐cytokine‐shear conditions, IL‐1β signaling dominates. Overall, a better understanding of E‐selectin expression pattern by human ECs relative to the combined interaction of cytokines, shear profile and history can help elucidate many disease pathologies. Biotechnol. Bioeng. 2013; 110: 999–1003. © 2012 Wiley Periodicals, Inc.     

Kappa opioids promote the proliferation of astrocytes via Gβγ and β‐arrestin 2‐dependent MAPK‐mediated pathways

GTP binding regulatory protein (G protein)‐coupled receptors can activate MAPK pathways via G protein‐dependent and ‐independent mechanisms. However, the physiological outcomes correlated with the cellular signaling events are not as well characterized. In this study, we examine the involvement of G protein and β‐arrestin 2 pathways in kappa opioid receptor‐induced, extracellular signal‐regulated kinase 1/2 (ERK1/2)‐mediated proliferation of both immortalized and primary astrocyte cultures. As different agonists induce different cellular signaling pathways, we tested the prototypic kappa agonist, U69593 as well as the structurally distinct, non‐nitrogenous agonist, C(2)‐methoxymethyl salvinorin B (MOM‐Sal‐B). In immortalized astrocytes, U69593, activated ERK1/2 by a rapid (min) initial stimulation that was sustained over 2 h and increased proliferation. Sequestration of activated Gβγ subunits attenuated U69593 stimulation of ERK1/2 and suppressed proliferation in these cells. Furthermore, small interfering RNA silencing of β‐arrestin 2 diminished sustained ERK activation induced by U69593. In contrast, MOM‐Sal‐B induced only the early phase of ERK1/2 phosphorylation and did not affect proliferation of immortalized astrocytes. In primary astrocytes, U69593 produced the same effects as seen in immortalized astrocytes. MOM‐Sal‐B elicited sustained ERK1/2 activation which was correlated with increased primary astrocyte proliferation. Proliferative actions of both agonists were abolished by either inhibition of ERK1/2, Gβγ subunits or β‐arrestin 2, suggesting that both G protein‐dependent and ‐independent ERK pathways are required for this outcome.     

Extracellular ATP promotes breast cancer chemoresistance via HIF-1α signaling

We have previously demonstrated that extracellular adenosine 5''-triphosphate (ATP) promotes breast cancer cell chemoresistance. However, the underlying mechanism remains unclear. Using a cDNA microarray, we demonstrated that extracellular ATP can stimulate hypoxia-inducible factor (HIF) signaling. In this study, we report that hypoxia-inducible factor 1α (HIF-1α) was upregulated after ATP treatment and mediated the ATP-driven chemoresistance process. We aimed to investigate the mechanisms and identify potential clinically relevant targets that are involved. Using mass spectrometry, we found that aldolase A (ALDOA) interacts with HIF-1α and increases HIF-1α expression. We then demonstrated that STAT3-ALDOA mediates ATP-HIF-1α signaling and upregulates the HIF-1 target genes adrenomedullin (ADM) and phosphoinositide-dependent kinase-1 (PDK1). Moreover, we show that PI3K/AKT acts upstream of HIF-1α in ATP signaling and contributes to chemoresistance in breast cancer cells. In addition, HIF-1α-knockdown or treatment with direct HIF inhibitors combined with the ATP hydrolase apyrase in MDA-MB-231 cells induced enhanced drug sensitivity in nude BALB/c mice. We then used in vitro spheroid formation assays to demonstrate the significance of ATP-HIF-1α in mediating chemoresistance. Furthermore, considering that indirect HIF inhibitors are effective in clinical cancer therapy, we treated tumor-bearing BALB/c mice with STAT3 and PI3K/AKT inhibitors and found that the dual-targeting strategy sensitized breast cancer to cisplatin. Finally, using breast cancer tissue microarrays, we found that ATP-HIF-1α signaling is associated with cancer progression, poor prognosis, and resistance to chemotherapy. Taken together, we suggest that HIF-1α signaling is vital in ATP-driven chemoresistance and may serve as a potential target for breast cancer therapies.Subject terms: Breast cancer, Cell signalling     

Mir‐29b promotes human aortic valve interstitial cell calcification via inhibiting TGF‐β3 through activation of wnt3/β‐catenin/Smad3 signaling

Herein, we hypothesized that pro‐osteogenic MicroRNAs (miRs) could play functional roles in the calcification of the aortic valve and aimed to explore the functional role of miR‐29b in the osteoblastic differentiation of human aortic valve interstitial cells (hAVICs) and the underlying molecular mechanism. Osteoblastic differentiation of hAVICs isolated from human calcific aortic valve leaflets obtained intraoperatively was induced with an osteogenic medium. Alizarin red S staining was used to evaluate calcium deposition. The protein levels of osteogenic markers and other proteins were evaluated using western blotting and/or immunofluorescence while qRT‐PCR was applied for miR and mRNA determination. Bioinformatics and luciferase reporter assay were used to identify the possible interaction between miR‐29b and TGF‐β3. Calcium deposition and the number of calcification nodules were pointedly and progressively increased in hAVICs during osteogenic differentiation. The levels of osteogenic and calcification markers were equally increased, thus confirming the mineralization of hAVICs. The expression of miR‐29b was significantly increased during osteoblastic differentiation. Furthermore, the osteoblastic differentiation of hAVICs was significantly inhibited by the miR‐29b inhibition. TGF‐β3 was markedly downregulated while Smad3, Runx2, wnt3, and β‐catenin were significantly upregulated during osteogenic induction at both the mRNA and protein levels. These effects were systematically induced by miR‐29b overexpression while the inhibition of miR‐29b showed the inverse trends. Moreover, TGF‐β3 was a direct target of miR‐29b. Inhibition of miR‐29b hinders valvular calcification through the upregulation of the TGF‐β3 via inhibition of wnt/β‐catenin and RUNX2/Smad3 signaling pathways.     

LIGHT/IFN‐γ triggers β cells apoptosis via NF‐κB/Bcl2‐dependent mitochondrial pathway

LIGHT recruits and activates naive T cells in the islets at the onset of diabetes. IFN‐γ secreted by activated T lymphocytes is involved in beta cell apoptosis. However, whether LIGHT sensitizes IFNγ‐induced beta cells destruction remains unclear. In this study, we used the murine beta cell line MIN6 and primary islet cells as models for investigating the underlying cellular mechanisms involved in LIGHT/IFNγ – induced pancreatic beta cell destruction. LIGHT and IFN‐γ synergistically reduced MIN6 and primary islet cells viability; decreased cell viability was due to apoptosis, as demonstrated by a significant increase in Annexin V⁺ cell percentage, detected by flow cytometry. In addition to marked increases in cytochrome c release and NF‐κB activation, the combination of LIGHT and IFN‐γ caused an obvious decrease in expression of the anti‐apoptotic proteins Bcl‐2 and Bcl‐xL, but an increase in expression of the pro‐apoptotic proteins Bak and Bax in MIN6 cells. Accordingly, LIGHT deficiency led to a decrease in NF‐κB activation and Bak expression, and peri‐insulitis in non‐obese diabetes mice. Inhibition of NF‐κB activation with the specific NF‐κB inhibitor, PDTC (pyrrolidine dithiocarbamate), reversed Bcl‐xL down‐regulation and Bax up‐regulation, and led to a significant increase in LIGHT‐ and IFN‐γ‐treated cell viability. Moreover, cleaved caspase‐9, ‐3, and PARP (poly (ADP‐ribose) polymerase) were observed after LIGHT and IFN‐γ treatment. Pretreatment with caspase inhibitors remarkably attenuated LIGHT‐ and IFNγ‐induced cell apoptosis. Taken together, our results indicate that LIGHT signalling pathway combined with IFN‐γ induces beta cells apoptosis via an NF‐κB/Bcl2‐dependent mitochondrial pathway.     

Qiliqiangxin attenuates hypoxia‐induced injury in primary rat cardiac microvascular endothelial cells via promoting HIF‐1α‐dependent glycolysis

Protection of cardiac microvascular endothelial cells (CMECs) against hypoxia injury is an important therapeutic strategy for treating ischaemic cardiovascular disease. In this study, we investigated the effects of qiliqiangxin (QL) on primary rat CMECs exposed to hypoxia and the underlying mechanisms. Rat CMECs were successfully isolated and passaged to the second generation. CMECs that were pre‐treated with QL (0.5 mg/mL) and/or HIF‐1α siRNA were cultured in a three‐gas hypoxic incubator chamber (5% CO₂, 1% O₂, 94% N₂) for 12 hours. Firstly, we demonstrated that compared with hypoxia group, QL effectively promoted the proliferation while attenuated the apoptosis, improved mitochondrial function and reduced ROS generation in hypoxic CMECs in a HIF‐1α‐dependent manner. Meanwhile, QL also promoted angiogenesis of CMECs via HIF‐1α/VEGF signalling pathway. Moreover, QL improved glucose utilization and metabolism and increased ATP production by up‐regulating HIF‐1α and a series of glycolysis‐relevant enzymes, including glucose transport 1 (GLUT1), hexokinase 2 (HK2), 6‐phosphofructokinase 1 (PFK1), pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA). Our findings indicate that QL can protect CMECs against hypoxia injury via promoting glycolysis in a HIF‐1α‐dependent manner. Lastly, the results suggested that QL‐dependent enhancement of HIF‐1α protein expression in hypoxic CMECs was associated with the regulation of AMPK/mTOR/HIF‐1α pathway, and we speculated that QL also improved HIF‐1α stabilization through down‐regulating prolyl hydroxylases 3 (PHD3) expression.     

OmpA‐mediated rickettsial adherence to and invasion of human endothelial cells is dependent upon interaction with α2β1 integrin

Rickettsia conorii, a member of the spotted fever group (SFG) of the genus Rickettsia and causative agent of Mediterranean spotted fever, is an obligate intracellular pathogen capable of infecting various mammalian cell types. SFG rickettsiae express two major immunodominant s urface c ell a ntigen (Sca) proteins, OmpB (Sca5) and OmpA (Sca0). While OmpB‐mediated entry has been characterized, the contribution of OmpA has not been well defined. Here we show OmpA expression in Escherichia coli is sufficient to mediate adherence to and invasion of non‐phagocytic human endothelial cells. A recombinant soluble C‐terminal OmpA protein domain (954–1735) with predicted structural homology to the Bordetella pertussis pertactin protein binds mammalian cells and perturbs R. conorii invasion by interacting with several mammalian proteins including β1 integrin. Using functional blocking antibodies, small interfering RNA transfection, and mouse embryonic fibroblast cell lines, we illustrate the contribution of α2β1 integrin as a mammalian ligand involved in R. conorii invasion of primary endothelial cells. We further demonstrate that OmpA‐mediated attachment to mammalian cells is in part dependent on a conserved non‐continuous RGD motif present in a predicted C‐terminal ‘pertactin’ domain in OmpA.Our results demonstrate that multiple adhesin–receptor pairs are sufficient in mediating efficient bacterial invasion of R. conorii.