Obesity is associated with increased production of inflammatory mediators in adipose tissue, which contributes to chronic inflammation and insulin resistance. Midkine (MK) is a heparin-binding growth factor with potent proinflammatory activities. We aimed to test whether MK is associated with obesity and has a role in insulin resistance. It was found that MK was expressed in adipocytes and regulated by inflammatory modulators (TNF-α and rosiglitazone). In addition, a significant increase in MK levels was observed in adipose tissue of obese ob/ob mice as well as in serum of overweight/obese subjects when compared with their respective controls. In vitro studies further revealed that MK impaired insulin signaling in 3T3-L1 adipocytes, as indicated by reduced phosphorylation of Akt and IRS-1 and decreased translocation of glucose transporter 4 (GLUT4) to the plasma membrane in response to insulin stimulation. Moreover, MK activated the STAT3-suppressor of cytokine signaling 3 (SOCS3) pathway in adipocytes. Thus, MK is a novel adipocyte-secreted factor associated with obesity and inhibition of insulin signaling in adipocytes. It may provide a potential link between obesity and insulin resistance.
In this paper, we propose the analytical solution of nano-rod antennas utilizing a cylindrical harmonics expansion. By treating the metallic nano-rods as plasmonic cavities, we derive closed-form expressions for both the internal and the radiated fields, as well as the resonant condition and the radiation efficiency. With our theoretical model, we show that besides the plasmonic resonances, efficient radiation takes advantage of (a) rendering a large value of the rods' radius and (b) a central-fed profile, through which the radiation efficiency can reach up to 70% and even higher in a wide frequency band. Our theoretical expressions and conclusions are general and pave the way for engineering and further optimization of optical antenna systems and their radiation patterns.
This study investigates the role of glycogenolysis in stimulated release of ATP as a transmitter from astrocytes. Within the last 20 years our understanding of brain glycogenolysis has changed from it being a relatively uninteresting process to being a driving force for essential brain functions like production of transmitter glutamate and homoeostasis of potassium ions (K+) after their release from excited neurons. Simultaneously, the importance of astrocytic handling of adenosine, its phosphorylation to ATP and release of some astrocytic ATP, located in vesicles, as an important transmitter has also become to be realized. Among the procedures stimulating Ca2+-dependent release of vesicular ATP are exposure to such transmitters as glutamate and adenosine, which raise intra-astrocytic Ca2+ concentration, or increase of extracellular K+ to a depolarizing level that opens astrocytic L-channels for Ca2+ and thereby also increase intra-astrocytic Ca2+ concentration, a prerequisite for glycogenolysis. The present study has confirmed and quantitated stimulated ATP release from well differentiated astrocyte cultures by glutamate, adenosine or elevated extracellular K+ concentrations, measured by a luciferin/luciferase reaction. It has also shown that this release is virtually abolished by an inhibitor of glycogenolysis as well as by inhibitors of transmitter-mediated signaling or of L-channel opening by elevated K+ concentrations.
Stimulation of Ca2+-dependent astrocytic ATP release by glutamate, adenosine or high K+ concentration is abolished by inhibition of glycogenolysis, probably due to signaling inhibition. This is consistent with the K+-mediated pathway, but glycogenolysis-sensitive steps in the transmitter pathways are unknown.
adenosine; astrocyte; ATP release; glutamate; glycogen; potassium; DAB, 1,4-dideoxy-1,4-imino-D-arabinitol; dBcAMP, dibutyryl cAMP; DMEM, Dulbecco’s Minimum Essential Medium; ERK, extracellular-signal-regulated kinase; GABA, γ-aminobutyric acid; MEK, [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase)] kinase; MPEP, 2-methyl-6-(phenylethynyl)-pyridine; RLU, relative light unit(s)
Nitric oxide derived from iNOS in activated T cells negatively regulates Th17 cell differentiation.
RORγt is necessary for the generation of TH17 cells but the molecular mechanisms for the regulation of TH17 cells are still not fully understood. We show that activation of CD4+ T cells results in the expression of inducible nitric oxide synthase (iNOS). iNOS-deficient mice displayed enhanced TH17 cell differentiation but without major effects on either TH1 or TH2 cell lineages, whereas endothelial NOS (eNOS) or neuronal NOS (nNOS) mutant mice showed comparable TH17 cell differentiation compared with wild-type control mice. The addition of N6-(1-iminoethyl)-l-lysine dihydrochloride (L-NIL), the iNOS inhibitor, significantly enhanced TH17 cell differentiation, and S-nitroso-N-acetylpenicillamine (SNAP), the NO donor, dose-dependently reduced the percentage of IL-17–producing CD4+ T cells. NO mediates nitration of tyrosine residues in RORγt, leading to the suppression of RORγt-induced IL-17 promoter activation, indicating that NO regulates IL-17 expression at the transcriptional level. Finally, studies of an experimental model of colitis showed that iNOS deficiency results in more severe inflammation with an enhanced TH17 phenotype. These results suggest that NO derived from iNOS in activated T cells plays a negative role in the regulation of TH17 cell differentiation and highlight the importance of intrinsic programs for the control of TH17 immune responses.
Atherosclerotic plaque vulnerability is associated with cerebrovascular events in patients with carotid atherosclerosis. The aim of this study was to investigate the expression of inflammatory factors in carotid artherosclerotic plaques in order to explore its clinical significance in patients with carotid stenosis. Forty three patients with carotid stenosis were divided into symptomatic group (n=24) and asymptomatic group (n=19) based on clinical manifestation. All patients were treated with selective standard carotid endarterectomy (CEA); the carotid atherosclerotic plaques were removed surgically and studied pathologically to investigate the expression of nuclear factor-kappa κ (NF-κB), CD68 and CD105. The plaques were grouped into stable and unstable plaques based on thickness of the fibrous cap and the area of lipid-rich core in the plaques. The proportion of unstable plaques were significantly higher in symptomatic group than in asymptomatic group (70.8% vs. 63.2%, P=0.026). Results of immunohistochemisty staining showed that the expression of NF-κB, CD68 and CD105 in unstable plaques was higher than stable plaques (P<0.001). The association of the higher expression of these factors with instability of carotid plaque needs to be clarified in future study.
Carotid stenosis; nuclear factor-kappa κ (NF-κB); CD68; CD105; vulnerable plaque
Obesity is associated with a state of chronic low-grade inflammation, which contributes to insulin resistance and type 2 diabetes. However, the molecular mechanisms that link obesity to inflammation are not fully understood. Follistatin-like 1 (FSTL1) is a novel proinflammatory cytokine that is expressed in adipose tissue and secreted by preadipocytes/adipocytes. We aimed to test whether FSTL1 could have a role in obesity-induced inflammation and insulin resistance. It was found that FSTL1 expression was markedly decreased during differentiation of 3T3-L1 preadipocytes but reinduced by TNF-α. Furthermore, a significant increase in FSTL1 levels was observed in adipose tissue of obese ob/ob mice, as well as in serum of overweight/obese subjects. Mechanistic studies revealed that FSTL1 induced inflammatory responses in both 3T3-L1 adipocytes and RAW264.7 macrophages. The expression of proinflammatory mediators including IL-6, TNF-α, and MCP-1 was upregulated by recombinant FSTL1 in a dose-dependent manner, paralleled with activation of the IKKβ-NFκB and JNK signaling pathways in the two cell lines. Moreover, FSTL1 impaired insulin signaling in 3T3-L1 adipocytes, as revealed by attenuated phosphorylation of both Akt and IRS-1 in response to insulin stimulation. Together, our results suggest that FSTL1 is a potential mediator of inflammation and insulin resistance in obesity.
The La-related protein 1 (LARP1) has been found to be a RNA binding protein and was related to spermatogenesis, embryogenesis and cell-cycle progression. The aim of this study was to investigate the prognostic value of LARP1 in hepatocellular carcinoma (HCC).
LARP1 expression was examined in 15 HCC cell lines and 272 clinical specimens using real-time PCR, immunohistochemistry (IHC) and western blot analysis (WB). LARP1 expression was also studied in 6 paired HCC lesions and the adjacent non-cancerous tissue samples. Statistical analyses were applied to derive association between LARP1 expression scores and clinical characters as well as patient survival.
mRNA and protein levels of LARP1 were higher in HCC cell lines and HCC lesions than in normal liver epithelial cells and the paired adjacent noncancerous tissues. LARP1 expression was correlated to survival time, vital status, tumor size and Child-Pugh score. Overall survival analysis showed HCC patients with high LARP1 expression level had lower survival rate (P < 0.01). Importantly, this correlation remained significant in patients with early-stage HCC or with normal serum AFP level.
LARP1 protein may represent a promising biomarker for predicting the prognosis of HCC, including in early-stage and AFP-normal patients.
LARP1; Hepatocellular carcinoma; AFP; Prognosis
The aim of this study was to analyze the changes in serum protein levels in the progression of hepatitis B using isobaric tags for relative and absolute quantitation (iTRAQ) analysis, in addition to comparing the serum protein levels of patients with chronic hepatitis B (CHB), patients with hepatitis B virus-induced acute-on-chronic liver failure (HBV-induced ACLF) and normal individuals. Protein analysis was performed on 15 serum samples using iTRAQ. The study population included healthy controls (n=5), patients with CHB (n=5) and patients with HBV-induced ACLF (n=5). Western blotting was used to verify the results in an additional nine serum samples from healthy controls, patients with CHB and patients with HBV-induced ACLF (n=3, respectively). Using iTRAQ analysis, 16 different serum proteins with ≥1.5-fold differences in expression levels were identified in the patients with CHB and ACLF compared with the healthy controls. Five of those proteins, C-reactive protein precursor, hemoglobin β chain variant Hb S-Wake, apolipoprotein J precursor, platelet factor 4 precursor and vitronectin, which demonstrated the greatest differences in their expression levels and the most significant correlation with liver diseases, were subsequently verified using western blotting. The western blotting results were consistent with the results from the iTRAQ. Two of the five proteins are not classified by biological process, and the biological functions of all the proteins in HBV-induced ACLF remain unclear. This preliminary study demonstrated that a correlation between the expression of various serum proteins and the different pathogenetic conditions induced by HBV may exist. The analysis of a larger number of samples is required to identify potential protein biomarkers that may be involved in the pathogenesis and progression of hepatitis B.
serum; proteomics; isobaric tags for relative and absolute quantitation; hepatitis B
Interactions between stem cells and extracellular matrix (ECM) are requisite for inducing lineage-specific differentiation and maintaining biological functions of mesenchymal stem cells by providing a composite set of chemical and structural signals. Here we investigated if cell-deposited ECM mimicked in vivo liver's stem cell microenvironment and facilitated hepatogenic maturation. Decellularization process preserved the fibrillar microstructure and a mix of matrix proteins in cell-deposited ECM, such as type I collagen, type III collagen, fibronectin, and laminin that were identical to those found in native liver. Compared with the cells on tissue culture polystyrene (TCPS), bone marrow mesenchymal stem cells (BM-MSCs) cultured on cell-deposited ECM showed a spindle-like shape, a robust proliferative capacity, and a suppressed level of intracellular reactive oxygen species, accompanied with upregulation of two superoxide dismutases. Hepatocyte-like cells differentiated from BM-MSCs on ECM were determined with a more intensive staining of glycogen storage, an elevated level of urea biosynthesis, and higher expressions of hepatocyte-specific genes in contrast to those on TCPS. These results demonstrate that cell-deposited ECM can be an effective method to facilitate hepatic maturation of BM-MSCs and promote stem-cell-based liver regenerative medicine.
Cytoscape is a free software package for visualizing, modeling and analyzing molecular and genetic interaction networks. This protocol explains how to use Cytoscape to analyze the results of mRNA expression profiling, and other functional genomics and proteomics experiments, in the context of an interaction network obtained for genes of interest. Five major steps are described: (i) obtaining a gene or protein network, (ii) displaying the network using layout algorithms, (iii) integrating with gene expression and other functional attributes, (iv) identifying putative complexes and functional modules and (v) identifying enriched Gene Ontology annotations in the network. These steps provide a broad sample of the types of analyses performed by Cytoscape.
The acquisition of metastasis potential is a critical point for malignant tumors. Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) is a potential tumor suppress gene and frequently down-regulated in malignant tumors. It has been implicated that overexpression of MDA-7 led to proliferation inhibition in many types of human tumor. Invasion is an important process which is potential to promote tumor metastasis. However, the role and potential molecular mechanism of mda-7/IL-24 to inhibit the invasion of human melanoma cancer is not fully clear. In this report, we identified a solid role for mda-7/IL-24 in invasion inhibition of human melanoma cancer LiBr cells, including decreasing of adhesion and invasion in vitro, blocking cell cycle, down-regulating the expression of ICAM-1, MMP-2/9, CDK1, the phosphorylation of ERK and Akt, NF-κB and AP-1 transcription activity. Meanwhile, there was an increased expression of PTEN in mda-7/IL-24 over-expression LiBr cells. Our results demonstrated that mda-7/IL-24 is a potential invasion suppress gene, which inhibits the invasion of LiBr cells by the down-regulation of ICAM-1, MMP-2/9, PTEN, and CDK1 expression. The molecular pathways involved were the MAPK/ERK, PI3K-Akt, NF-κB, and AP-1. These findings suggest that mda-7/IL-24 may be used as a possible therapeutic strategy for human melanoma cancer.
Melanoma; mda-7; Invasion
Cytoscape is open-source software for integration, visualization and analysis of biological networks. It can be extended through Cytoscape plugins, enabling a broad community of scientists to contribute useful features. This growth has occurred organically through the independent efforts of diverse authors, yielding a powerful but heterogeneous set of tools. We present a travel guide to the world of plugins, covering the 152 publicly available plugins for Cytoscape 2.5–2.8. We also describe ongoing efforts to distribute, organize and maintain the quality of the collection.
Brain excitation increases neuronal Na+ concentration by 2 major mechanisms: (i) Na+ influx caused by glutamatergic synaptic activity; and (ii) action-potential-mediated depolarization by Na+ influx followed by repolarizating K+ efflux, increasing extracellular K+ concentration. This review deals mainly with the latter and it concludes that clearance of extracellular K+ is initially mainly effectuated by Na+,K+-ATPase-mediated K+ uptake into astrocytes, at K+ concentrations above ~10 mM aided by uptake of Na+,K+ and 2 Cl− by the cotransporter NKCC1. Since operation of the astrocytic Na+,K+-ATPase requires K+-dependent glycogenolysis for stimulation of the intracellular ATPase site, it ceases after normalization of extracellular K+ concentration. This allows K+ release via the inward rectifying K+ channel Kir4.1, perhaps after trans-astrocytic connexin- and/or pannexin-mediated K+ transfer, which would be a key candidate for determination by synchronization-based computational analysis and may have signaling effects. Spatially dispersed K+ release would have little effect on extracellular K+ concentration and allow K+ accumulation by the less powerful neuronal Na+,K+-ATPase, which is not stimulated by increases in extracellular K+. Since the Na+,K+-ATPase exchanges 3 Na+ with 2 K+, it creates extracellular hypertonicity and cell shrinkage. Hypertonicity stimulates NKCC1, which, aided by β-adrenergic stimulation of the Na+,K+-ATPase, causes regulatory volume increase, furosemide-inhibited undershoot in [K+]e and perhaps facilitation of the termination of slow neuronal hyperpolarization (sAHP), with behavioral consequences. The ion transport processes involved minimize ionic disequilibria caused by the asymmetric Na+,K+-ATPase fluxes.
action potential; astrocyte; computational analysis; Kir channel; Na+,K+-ATPase; neuron; brain potassium homeostasis; slow neuronal hyperpolarization
Traditionally, astrocytic mRNA and protein expression are studied by in situ hybridization (ISH) and immunohistochemically. This led to the concept that astrocytes lack aralar, a component of the malate-aspartate-shuttle. At least similar aralar mRNA and protein expression in astrocytes and neurons isolated by fluorescence-assisted cell sorting (FACS) reversed this opinion. Demonstration of expression of other astrocytic genes may also be erroneous. Literature data based on morphological methods were therefore compared with mRNA expression in cells obtained by recently developed methods for determination of cell-specific gene expression. All Na,K-ATPase-α subunits were demonstrated by immunohistochemistry (IHC), but there are problems with the cotransporter NKCC1. Glutamate and GABA transporter gene expression was well determined immunohistochemically. The same applies to expression of many genes of glucose metabolism, whereas a single study based on findings in bacterial artificial chromosome (BAC) transgenic animals showed very low astrocytic expression of hexokinase. Gene expression of the equilibrative nucleoside transporters ENT1 and ENT2 was recognized by ISH, but ENT3 was not. The same applies to the concentrative transporters CNT2 and CNT3. All were clearly expressed in FACS-isolated cells, followed by biochemical analysis. ENT3 was enriched in astrocytes. Expression of many nucleoside transporter genes were shown by microarray analysis, whereas other important genes were not. Results in cultured astrocytes resembled those obtained by FACS. These findings call for reappraisal of cellular nucleoside transporter expression. FACS cell yield is small. Further development of cell separation methods to render methods more easily available and less animal and cost consuming and parallel studies of astrocytic mRNA and protein expression by ISH/IHC and other methods are necessary, but new methods also need to be thoroughly checked.
astrocyte culture; BAC transgenic animals; fluorescence-assisted cell sorting; GFAP; immunohistochemistry; in situ hybridization
The involvement of glycogenolysis, occurring in astrocytes but not in neurons, in learning is undisputed (Duran et al., 2013). According to one school of thought the role of astrocytes for learning is restricted to supply of substrate for neuronal oxidative metabolism. The present “perspective” suggests a more comprehensive and complex role, made possible by lack of glycogen degradation, unless specifically induced by either (1) activation of astrocytic receptors, perhaps especially β-adrenergic or (2) even small increases in extracellular K+ concentration above its normal resting level. It discusses (1) the known importance of glycogenolysis for glutamate formation, requiring pyruvate carboxylation; (2) the established role of K+-stimulated glycogenolysis for K+ uptake in cultured astrocytes, which probably indicates that astrocytes are an integral part of cellular K+ homeostasis in the brain in vivo; and (3) the plausible role of transmitter-induced glycogenolysis, stimulating Na+,K+-ATPase/NKCC1 activity and thereby contributing both to the post-excitatory undershoot in extracellular K+ concentration and the memory-enhancing effect of transmitter-mediated reduction of slow neuronal afterhyperpolarization (sAHP).
astrocyte; glutamate; glycogen; learning; slow neuronal afterhyperpolarization
Survivin, a member of the family of inhibitor of apoptosis proteins, functions as a key regulator of mitosis and programmed cell death. YM155, a novel molecular targeted agent, suppresses survivin, which is overexpressed in many tumor types. The aim of this study was to determine the antitumor activity of YM155 in SK-NEP-1 cells.
SK-NEP-1 cell growth in vitro and in vivo was assessed by MTT and nude mice experiments. Annexin V/propidium iodide staining followed by flow cytometric analysis was used to detect apoptosis in cell culture. Then gene expression profile of tumor cells treated with YM155 was analyzed with real-time PCR arrays. We then analyzed the expression data with MEV (Multi Experiment View) cluster software. Datasets representing genes with altered expression profile derived from cluster analyses were imported into the Ingenuity Pathway Analysis tool.
YM155 treatment resulted in inhibition of cell proliferation of SK-NEP-1cells in a dose-dependent manner. Annexin V assay, cell cycle, and activation of caspase-3 demonstrates that YM155 induced apoptosis in SK-NEP-1 cells. YM155 significantly inhibited growth of SK-NEP-1 xenografts (YM155 5 mg/kg: 1.45 ± 0.77 cm3; YM155 10 mg/kg: 0.95 ± 0.55 cm3) compared to DMSO group (DMSO: 3.70 ± 2.4 cm3) or PBS group cells (PBS: 3.78 ± 2.20 cm3, ANOVA P < 0.01). YM155 treatment decreased weight of tumors (YM155 5 mg/kg: 1.05 ± 0.24 g; YM155 10 mg/kg: 0.72 ± 0.17 g) compared to DMSO group (DMSO: 2.06 ± 0.38 g) or PBS group cells (PBS: 2.36 ± 0.43 g, ANOVA P < 0.01). Real-time PCR array analysis showed between Test group and control group there are 32 genes significantly up-regulated and 54 genes were significantly down-regulated after YM155 treatment. Ingenuity pathway analysis (IPA) showed cell death was the highest rated network with 65 focus molecules and the significance score of 44. The IPA analysis also groups the differentially expressed genes into biological mechanisms that are related to cell death, cellular function maintenance, cell morphology, carbohydrate metabolism and cellular growth and proliferation. Death receptor signaling (3.87E-19), TNFR1 signaling, induction of apoptosis by HIV1, apoptosis signaling and molecular mechanisms of cancer came out to be the top four most significant pathways. IPA analysis also showed top molecules up-regulated were BBC3, BIRC3, BIRC8, BNIP1, CASP7, CASP9, CD5, CDKN1A, CEBPG and COL4A3, top molecules down-regulated were ZNF443, UTP11L, TP73, TNFSF10, TNFRSF1B, TNFRSF25, TIAF1, STK17A, SST and SPP1, upstream regulator were NR3C1, TP53, dexamethasone , TNF and Akt.
The present study demonstrates that YM155 treatment resulted in apoptosis and inhibition of cell proliferation of SK-NEP-1cells. YM155 had significant role and little side effect in the treatment of SK-NEP-1 xenograft tumors. Real-time PCR array analysis firstly showed expression profile of genes dyes-regulated after YM155 treatment. IPA analysis also represents new molecule mechanism of YM155 treatment, such as NR3C1 and dexamethasone may be new target of YM155. And our results may provide new clues of molecular mechanism of apoptosis induced by YM155.
YM155; SK-NEP-1; Survivin; Apoptosis; Real-time PCR array
High serum levels of lipopolysaccharide (LPS) with LPS-MD-2/TLR4 complex activated NF-kb and cytokine cause hepatic necrosis in animal models. We investigated the dynamic changes of LPS levels in patients with acute on chronic hepatitis B liver failure (ACHBLF).
We enrolled ACHBLF patients for a 12-week study. Patients’ LPS levels were measured along with 10 healthy controls. Patients on supportive care and recovered without intervention(s) were analyzed. Patients’ LPS levels during the disease progression phase, peak phase, and remission phase were compared with healthy controls.
Among 30 patients enrolled, 25 who received interventions or expired during the study period were excluded from the analysis, five patients on supportive care who completed the study were analyzed. Significant abnormal distributions of LPS levels were observed in patients in different phases (0.0168±0.0101 in progression phase; 0.0960±0.0680 in peak phase; 0.0249±0.0365 in remission phase; and 0.0201±0.0146 in controls; respectively, p<0.05). The highest level of LPS was in the peak phase and significantly elevated when compared to controls (0.0201±0.0146 vs. 0.0960±0.0680, p = 0.007). There were no statistically significant differences in LPS levels between healthy controls and subjects in the progression phase or remission phase. Dynamic changes of LPS were correlated with MELD-Na in the progression phase (p = 0.01, R = 0.876) and in the peak phase (p = 0.000, R = −1.00).
Significant abnormal distributions of LPS levels were observed in ACHBLF with the highest level in the peak phase. The dynamic changes of LPS were correlated with disease severity and suggested LPS causing secondary hepatic injury.
Elevated extracellular free fatty acids (FFAs) can induce pancreatic beta cell apoptosis, thereby contributing to the pathogenesis of type 2 diabetes mellitus (T2D). Mitochondrial dysfunction has been implicated in FFA-induced beta cell apoptosis. However, molecular mechanisms linking mitochondrial dysfunction and FFA-induced beta cell apoptosis are not clear. Dynamin-related protein 1 (DRP-1) is a mitochondrial fission modulator. In this study, we investigated its role in FFA-induced INS-1 beta cell apoptosis. DRP-1 protein was promptly induced in INS-1 cells and rat islets after stimulation by FFAs, and this DRP-1 upregulation was accompanied by increased INS-1 cell apoptosis. Induction of DRP-1 expression significantly promoted FFA-induced apoptosis in DRP-1 WT (DRP-1 wild type) inducible INS-1-derived cell line, but not in DRP-1K38A (a dominant negative mutant of DRP-1) inducible INS-1-derived cell line. To validate these in vitro results, we transplanted DRP-1 WT or DRP-1 K38A cells into renal capsules of streptozotocin (STZ)-treated diabetic mice to study the apoptosis in xenografts. Consistent with the in vitro results, the over-expression of DRP-1 led to aggravated INS-1-derived cell apoptosis triggered by FFAs. In contrast, dominant-negative suppression of DRP-1 function as represented by DRP-1 K38A significantly prevented FFA-induced apoptosis in xenografts. It was further demonstrated that mitochondrial membrane potential decreased, while cytochrome c release, caspase-3 activation, and generation of reactive oxygen species (ROS) were enhanced by the induction of DRP-1WT, but prevented by DRP-1 K38A in INS-1-derived cells under FFA stimulation. These results indicated that DRP-1 mediates FFA-induced INS-1-derived cell apoptosis, suggesting that suppression of DRP-1 is a potentially useful therapeutic strategy for protecting against beta cell loss that leads to type 2 diabetes.
Bone marrow-derived mesenchymal stem cells (BMSCs) can reduce liver fibrosis. Apart from the paracrine mechanism by which the antifibrotic effects of BMSCs inhibit activated hepatic stellate cells (HSCs), the effects of direct interplay and juxtacrine signaling between the two cell types are poorly understood. The purpose of this study was to explore the underlying mechanisms by which BMSCs modulate the function of activated HSCs.
We used BMSCs directly and indirectly co-culture system with HSCs to evaluate the anti-fibrosis effect of BMSCs. Cell proliferation and activation were examined in the presence of BMSCs and HGF. c-met was knockdown in HSCs to evaluate the effect of HGF secreted by BMSCs. The TLR4 and Myeloid differentiation primary response gene 88(MyD88) mRNA levels and the NF-kB pathway activation were determined by real-time PCR and western blotting analyses. The effect of BMSCs on HSCs activation was investigated in vitro in either MyD88 silencing or overexpression in HSCs. Liver fibrosis in rats fed CCl4 with and without BMSCs supplementation was compared. Histopathological examinations and serum biochemical tests were compared between the two groups.
BMSCs remarkably inhibited the proliferation and activation of HSCs by interfering with LPS-TLR4 pathway through a cell–cell contact mode that was partially mediated by HGF secretion. The NF-kB pathway is involved in HSCs activation inhibition by BMSCs. MyD88 over expression reduced the BMSC inhibition of NF-kB luciferase activation. BMSCs protected liver fibrosis in vivo.
BMSCs modulate HSCs in vitro via TLR4/MyD88/NF-kB signaling pathway through cell–cell contact and secreting HGF. BMSCs have therapeutic effects on cirrhosis rats. Our results provide new insights into the treatment of hepatic fibrosis with BMSCs.
To take full advantage of high-throughput genetic and physical interaction mapping projects, the raw interactions must first be assembled into models of cell structure and function. PanGIA (for physical and genetic interaction alignment) is a plug-in for the bioinformatics platform Cytoscape, designed to integrate physical and genetic interactions into hierarchical module maps. PanGIA identifies ‘modules’ as sets of proteins whose physical and genetic interaction data matches that of known protein complexes. Higher-order functional cooperativity and redundancy is identified by enrichment for genetic interactions across modules. This protocol begins with importing interaction networks into Cytoscape, followed by filtering and basic network visualization. Next, PanGIA is used to infer a set of modules and their functional inter-relationships. This module map is visualized in a number of intuitive ways, and modules are tested for functional enrichment and overlap with known complexes. The full protocol can be completed between 10 and 30 min, depending on the size of the data set being analyzed.
Background and aim
CD24 expression is associated with human colorectal cancer (CRC). Our previous data indicated that CD24 promoted the proliferation and invasion of colorectal cancer cells through the activation of ERK1/2. Since Src family kinases are frequently deregulated in CRC and closely related to the MAPK signaling pathway, we investigated the impact of Lyn, an important member of SFKs, on CD24-induced ERK1/2 activation in CRC.
Methods and Results
The interaction of CD24 and Lyn was identified by co-immunoprecipitation (Co-IP) and ectopic expression of CD24-induced Lyn activation. Inhibition of Lyn activation by phosphatase PP2 in SW480CD24cells abrogated CD24-induced invasion. The results of the Co-IP and immunofluorescence assay revealed that overexpression of CD24 enhanced the interaction of Lyn and ERK1/2 and induced the nuclear translocation of Lyn. However, inhibition of Lyn activity attenuated CD24-induced ERK1/2 activation, and depletion of CD24 disrupted Lyn-ERK1/2 interaction. Immunohistochemistry analysis for 202 cases of CRC showed that the expression of both CD24 and Lyn was positively correlated with tumor grade, stage, lymph node and distant metastasis. Patients with lower expression of CD24 or Lyn had a higher survival rate. The Cox multivariate analysis showed that CD24 expression, but not Lyn expression, was an independent prognostic factor of CRC.
Our results suggest that Lyn is involved in CD24-induced ERK1/2 activation in CRC. The expression of CD24 is associated with activation of Lyn and ERK1/2, which might be a novel mechanism related to CD24-mediated regulation of CRC development.
CD24; Lyn; ERK1/2; Colorectal cancer