Diacylglycerol kinase α mediates HGF-induced Rac activation and membrane ruffling by regulating atypical PKC and RhoGDI

Diacylglycerol kinases (DGKs) convert diacylglycerol (DAG) into phosphatidic acid (PA), acting as molecular switches between DAG- and PA-mediated signaling. We previously showed that Src-dependent activation and plasma membrane recruitment of DGKα are required for growth-factor-induced cell migration and ruffling, through the control of Rac small-GTPase activation and plasma membrane localization. Herein we unveil a signaling pathway through which DGKα coordinates the localization of Rac. We show that upon hepatocyte growth-factor stimulation, DGKα, by producing PA, provides a key signal to recruit atypical PKCζ/ι (aPKCζ/ι) in complex with RhoGDI and Rac at ruffling sites of colony-growing epithelial cells. Then, DGKα-dependent activation of aPKCζ/ι mediates the release of Rac from the inhibitory complex with RhoGDI, allowing its activation and leading to formation of membrane ruffles, which constitute essential requirements for cell migration. These findings highlight DGKα as the central element of a lipid signaling pathway linking tyrosine kinase growth-factor receptors to regulation of aPKCs and RhoGDI, and providing a positional signal regulating Rac association to the plasma membrane.     

Role of monokine induced by interferon-γ in liver injury induced by hepatitis B virus in mice

The chemokine monokine induced by interferon-γ (Mig) is involved in the recruitment of inflammatory cells and liver injury during hepatitis B virus (HBV) infection. HBV protein X contributes to Mig expression in vitro by activation of nuclear factor (NF)-κB; however, the molecular mechanisms by which HBV induces Mig expression in vivo are unknown. In this paper, we established a mouse model for HBV study by tail vein injection of HBV genome-containing adenovirus vectors. Host immune response to the secreted hepatitis B surface antigen and e antigen was detected and serum alanine aminotransferase (ALT) was elevated at different time points. We also demonstrated that peripheral and intrahepatic Mig expression was increased after Ad-HBV infection. This was followed by inflammatory cell migration and formation of inflammatory foci in the liver. In addition, NF-κB p65 subunit translocated from the cytoplasm to the nucleus, and phosphoinositide 3-kinase/Akt, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) were to some extent phosphorylated after HBV injection. Following tail vein injection of Mig siRNA/in vivo-jetPEI-Gal complex, Mig expression was partially suppressed, inflammatory cell migration was inhibited, serum level of ALT were reduced. In conclusion, through NF-κB activation, HBV induced Mig expression in vivo, which recruited peripheral inflammatory cells to the liver and resulted in liver damage. Phosphorylation of phosphoinositide 3-kinase/Akt, ERK and JNK but not p38 might involved in the molecular mechanisms underlying HBV induced Mig expression in vivo.     

α5β1-Integrin controls ebolavirus entry by regulating endosomal cathepsins

Integrins are involved in the binding and internalization of both enveloped and nonenveloped viruses. By using 3 distinct cell systems—CHO cells lacking expression of α₅β₁-integrin, HeLa cells treated with siRNA to α₅-integrin, and mouse β₁-integrin knockout fibroblasts, we show that α₅β₁-integrin is required for efficient infection by pseudovirions bearing the ebolavirus glycoprotein (GP). These integrins are necessary for viral entry but not for binding or internalization. Given the need for endosomal cathepsins B and L (CatB and CatL) to prime GPs for fusion, we investigated the status of CatB and CatL in integrin-positive and integrin-negative cell lines. α₅β₁-Integrin-deficient cells lacked the double-chain (DC) forms of CatB and CatL, and this correlated with decreased CatL activity in integrin-negative CHO cells. These data indicate that α₅β₁-integrin-negative cells may be refractory to infection by GP pseudovirions because they lack the necessary priming machinery (the double-chain forms of CatB and CatL). In support of this model, we show that GP pseudovirions that have been preprimed in vitro to generate the 19-kDa form of GP overcome the requirement for α₅β₁-integrin for infection. These results provide further support for the requirement for endosomal cathepsins for ebolavirus infection, identify the DC forms of these cathepsins as previously unrecognized factors that contribute to cell tropism of this virus, and reveal a previously undescribed role for integrins during viral entry as regulators of endosomal cathepsins, which are required to prime the entry proteins of ebolavirus and other pathogenic viruses.     

Endothelial FGFR1 deficiency induces AcS-DKP-resistant EndMT by regulating TGFβ signal pathway

正Objective To investigate the role of fibroblast growth factor receptor (FGFR) 1 in endothelial to-mesenchymal transition (EndMT) and epithelial-to-mesenchymal transition (EMT),and to find out a new strategy to study the vascular endothelial function of diabetic renal fibrosis.Methods Culture media from FRS2 knockdown HMVECs was transferred to HK-2 cells.Western blot and immunofluorescence staining were used to measure EMT markers and key moleculars of transforming growth factor (TGFβ).Re-     

Exogenous IGF-1 promotes hair growth by stimulating cell proliferation and down regulating TGF-β1 in C57BL/6 mice in vivo

ObjectiveInsulin-like growth factor 1 (IGF-1) increases the growth of cultured hair follicles and plays a role in regulating hair migration during the development of hair follicles in transgenic mice. However, the exogenous effect of IGF-1 on hair growth in wild-type mice has not been reported. In the present study, we examined whether IGF-1 was an important regulator of hair follicle growth in wide-type mice in vivo.DesignC57BL/6 mice were injected with different concentrations of IGF-1 on dorsal skin. The treated tissues were analyzed by immunoassay methods for TGF-β1 and BrdU.ResultsLocal injection of IGF-1 increased hair follicle number and prolonged the growing phase during the transition from anagen to telogen. Meanwhile, immunology analyses revealed that IGF-1 also stimulated the proliferation of follicle cells in anagen of the matrix and down regulated TGF-β1 expression in hair follicles.ConclusionsThese observations suggest that IGF-1 is an effective stimulator of hair follicle development in wide-type mice in vivo and may be a promising drug candidate for baldness therapy.     

Investigation of T-cell receptor-γ gene rearrangement in gastrointestinal lymphomas by PCR-SSCP analysis

AIM: To analyze the characterization of T-cell receptor-γ (TCR-γ) gene rearrangement in the gastrointestinal lymphomas and evaluate the value of PCR-SSCP analysis in gastrointestinal lymphomas investigation. METHODS: TCR-γ gene rearrangement segments of gastrointestinal lymphomas were cloned and sequenced. Single clone plasmid and mixed clone plsamids were subsequently submitted to PCR-SSCP analysis to investigate the relationship between the number of amplified clones and band patterns of the amplified products. The PCR products of TCR-γ gene rearrangement of 40 gastrointestinal lymphomas were electrophoresed on agarose gels and the positive cases on agarose gels were studied by SSCP analysis. RESULTS: The sequencing showed that TCR-γ gene rearrangement of the gastrointestinal lyrnphomas included functional gene and pseudogene with extensive variety in the junctional regions. In SSCP analysis, the number of the single-stranded bands was about two times of the number of amplified clones, and double-stranded band became broad with the increased number of the amplified clones. Thirteen of the 25 B-cell gastrointestinal lymphornas and 14 of the 15 gastrointestinal T-cell lymphomas were positive detected on agarose gel electrophoresis. Of the positive cases detected by SSCP analysis, 3 B-cell lyrnphomas and 13 T-cell lyrnphomas showed positive bands. The other cases showed only smears. The rearranged pattern included 13 monoallelic gene rearrangements and 3 biallelic or oligoclonal gene rearrangements. CONCLUSION: The pattern of TCR-γ gene rearrangement in gastrointestinal lymphomas are similar to that of the nodular lymphomas. PCR-SSCP analysis for TCR-γ gene rearrangement can be applied both for adjuvant diagnosis of gastrointestinal lymphomas and analysis of the gene rearrangement pattern. The ratio of TCR-γ gene rearrangements occurred in T-cell gastrointestinal lymphomas is significantly higher than that in B-cell gastrointestinal lymphomas. The gene rearrangement pattern involves monoallelic and biallelic (or oligoclonal) gene rearrangement.     

Inhibition effect of N-acetyl-seryl-aspartyllysyl-proline on myofibroblast differentiation by regulating acetylated tubulin α in silicotic rat model

<正>Objective To explore the inhibition effect and mechanism of N-acetyl-seryl-aspartyl-lysyl-proline(AcSDKP)on myofibroblast differentiation via regulating acetylated tubulinα(Ac-Tubα)in vivo and in vitro.Methods Silicotic model was made by SiO_2 douched and divided into 6 groups as follows:control(4w,8w)group,silicotic model(4w,8w)group and post-or pre-treatment     

MiR-361-5p promotes oxygen–glucose deprivation/re-oxygenation induced neuronal injury by negatively regulating SQSTM1 in vitro

Metabolic Brain Disease - It has been reported that microRNAs (miRNAs) play essential roles in cerebral ischemia and reperfusion (I/R) injury. This study aimed to explore the role of miR-361-5p in...     

Mangiferin exerts neuroprotective effects against focal cerebral ischemia in mice by regulating NF-κB signaling pathway

Purpose: Mangiferin is a natural free radical scavenging antioxidant that induces excitation of the central nervous system. However, the mechanism of neuroprotective effect of mangiferin on focal cerebral ischemia has not been fully investigated. The aim of this study was to investigate the protective effect of mangiferin on focal cerebral ischemia in mice.

Methods: Middle cerebral artery occlusion (MCAO) was performed to investigate the effect of mangiferin on focal cerebral ischemia. Mice were randomly divided into 5 groups: sham, MCAO, MCAO?+?5 mg/kg mangiferin, MCAO?+?20 mg/kg mangiferin and MCAO?+?5 mg/kg nimodipine. Neurobehavioral scores, brain edema, brain injury scores, relative infarct size and expression of some inflammatory factors in the brain were evaluated. NF-κB pathway was detected by Western blotting and immunofluorescence.

Results: The results showed that mangiferin effectively attenuated MCAO-induced brain injury, including improvement of neurological impairment, reduction of brain edema, and reduction of infarct size. Compared with the MCAO group, mangiferin significantly inhibited MCAO-induced neuroinflammation, which can be proved by reduced expression levels of TNF-α, IL-1β, iNOS and COX-2. In addition, we found that phosphorylation of IκBα was inhibited and the expression of NF-κB p65 in the nucleus was reduced after the addition of mangiferin.

Conclusion: Our study suggested that mangiferin exerts neuroprotective effects on focal cerebral ischemia in mice by regulating the NF-κB signaling pathway. Mangiferin may be an effective treatment for cerebral ischemia and other neurological disorders.

     

Innate pro–B-cell progenitors protect against type 1 diabetes by regulating autoimmune effector T cells

Diverse hematopoietic progenitors, including myeloid populations arising in inflammatory and tumoral conditions and multipotent cells, mobilized by hematopoietic growth factors or emerging during parasitic infections, display tolerogenic properties. Innate immune stimuli confer regulatory functions to various mature B-cell subsets but immature B-cell progenitors endowed with suppressive properties per se or after differentiating into more mature regulatory B cells remain to be characterized. Herein we provide evidence for innate pro-B cells (CpG-proBs) that emerged within the bone marrow both in vitro and in vivo upon Toll-like receptor-9 activation and whose adoptive transfer protected nonobese diabetic mice against type 1 diabetes (T1D). These cells responded to IFN-γ released by activated effector T cells (Teffs), by up-regulating their Fas ligand (FasL) expression, which enabled them to kill Teffs through apoptosis. In turn, IFN-γ derived from CpG-proBs enhanced IFN-γ while dramatically reducing IL-21 production by Teffs. In keeping with the crucial pathogenic role played by IL-21 in T1D, adoptively transferred IFN-γ–deficient CpG-proBs did not prevent T1D development. Additionally, CpG-proBs matured in vivo into diverse pancreatic and splenic suppressive FasL^high B-cell subsets. CpG-proBs may become instrumental in cell therapy of autoimmune diseases either on their own or as graft complement in autologous stem cell transplantation.A growing body of evidence attests that immune cells with immunoregulatory functions do not exclusively belong to mature populations of diverse lineage, but also comprise several hematopoietic progenitor subsets. The first subset to be recognized comprised myeloid progenitors that acquired suppressive properties in tumoral and inflammatory environments (1) and played either detrimental or beneficial roles in different pathological situations. We have reported previously that mobilization with hematopoietic growth factors conferred tolerogenic properties to multipotent hematopoietic progenitors at the multipotent progenitor (MPP2) stage of differentiation that enabled them to promote the expansion of regulatory T cells (2, 3), thereby preventing spontaneous autoimmune type 1 diabetes (T1D) in the nonobese diabetic (NOD) mouse model. Moreover, parasitic infections were shown to stimulate via IL-25 the emergence of Th2 cytokine-secreting MPPs (MPP^Th2) that ultimately differentiated into mature cell types with pro-Th2 functions, thus contributing to parasitic clearance (4).Direct interactions between pathogens and hematopoietic stem cells occur through Toll-like receptor (TLR) activation, driving their differentiation along myeloid pathways to enforce anti-infectious defenses (5, 6). TLR agonists also promote hematopoiesis by enhancing the production of the hematopoietic growth factor G-CSF, with whom they synergize to mobilize hematopoietic stem cells from the bone marrow to the periphery (7).TLR-mediated innate-type stimulation by infectious (8) and parasitic (9) agents also plays a major role in promoting the emergence of regulatory B cells (Bregs), along with acquired-type stimulation, such as B-cell receptor (BCR) engagement concomitant or not with CD40 activation (10, 11). Such induced regulatory B-cell functions are believed to be more robust than those expressed by naive and resting B cells, which can nevertheless tolerize naive T cells and induce regulatory T cells (Tregs) (12, 13).Bregs are a heterogeneous lymphocyte subset present among all major B-cell populations (14–17). The rare so-called B10 cells identified by their CD19⁺CD1d^hiCD5⁺ phenotype (18–20), peritoneal CD5⁺ B1a cells (21, 22), large follicular B cells, and activated transitional, marginal zone (MZ) B cells can all acquire regulatory properties. The most immature Breg subset described so far is composed of B220⁺IgM⁺CD21^lowCD93⁺CD23⁺ transitional T2 MZ precursor B (T2 MZP-B) cells, which are continuously produced in adult bone marrow and home to the MZ of the spleen, where they differentiate into IgM^highCD1d^highCD21^highCD23^low MZ B cells (23, 24).The differentiation pathways of the various Breg subsets remain unknown. Only functional precursors, named “B10pro,” which are mature B cells requiring additional BCR-activating antigenic signals to produce immunosuppressive IL-10 but cannot be distinguished from B10 cells by phenotypic criteria, have hitherto been identified (25). It is unknown whether Bregs derive from one or several progenitors or solely from conventional B-cell subsets. Moreover, an immature B-cell progenitor population endowed with suppressive properties per se or after differentiation into more mature Bregs has not been demonstrated as yet.Herein we describe a hematopoietic progenitor population that emerges transiently in vitro and in vivo in the bone marrow of NOD mice, after activation with the TLR-9 agonist CpG and whose adoptive transfer into NOD mice prevents T1D onset. These cells were c-kit^lowSca-1^lowCD127⁺B220⁺CD19⁺IgM⁻CD1d^intCD43⁺, a phenotype consistent with a pro–B-cell stage of differentiation, except for their CD1d expression. The cells differentiated in vivo exclusively into B lymphocytes, at various stages of maturation.Functionally, these TLR-induced hematopoietic progenitors suppressed pathogenic effector T cells (Teffs) by reducing their IL-21 production and by inducing their apoptosis via Fas ligand (FasL). Additionally, the B-cell progeny of CpG-induced proBs continued to express high levels of FasL and to suppress Teffs, and may play a major role in the durable protection against T1D provided by the progenitors in vivo.