Insight into the biology of Macrophage Migration Inhibitory Factor （MIF） revealed by the cloning of its cell surface receptor

The recent cloning of MIF receptor fills an important gap in our understanding of the molecular biology and immunology of MIF. The MIF receptor, like MIF, does not fall into any established family of protein mediators, providing both new challenges and opportunities for the structural and functional analysis of MIF signal transduction.     

Cell electrophoresis — a method for cell separation and research into cell surface properties

In this paper, we discuss the application of various methods of cell electrophoresis in research into cell surface properties (analytical methods), and the separation of uniform cell subpopulations from cell mixtures (preparative methods). The emphasis is on the prospects of the development of simplified and versatile methodologies, i.e. microcapillary cell electrophoresis and horizontal cell electrophoresis under near-isopycnic conditions. New perspectives are considered on the use of analytical and preparative cell electrophoresis in research on cell differentiation, neoplastic transformation, cell-cell interactions and the biology of stem cells. Paper authored by participants of the international conference: XXXIV Winter School of the Faculty of Biochemistry, Biophysics and Biotechnology of Jagiellonian University, Zakopane, March 7–11, 2007, “The Cell and Its Environment”. Publication cost was covered by the organisers of this meeting.     

Indoleamine 2, 3-dioxygenase （IDO） is essential for dendritic cell activation and chemotactic responsiveness to chemokines

Indoleamine 2, 3-dioxygenase (IDO) is a rate-limiting enzyme for the tryptophan catabolism. In human and murine cells, IDO inhibits antigen-specific T cell proliferation in vitro and suppresses T cell responses to fetal alloantigens during murine pregnancy. In mice, IDO expression is an inducible feature of specific subsets of dendritic cells (DCs),and is important for T cell regulatory properties. However, the effect of IDO and tryptophan deprivation on DC functions remains unknown. We report here that when tryptophan utilization was prevented by a pharmacological inhibitor of IDO, 1-methyl tryptophan (1MT), DC activation induced by pathogenic stimulus lipopolysaccharide (LPS) or inflammatory cytokine TNF-α was inhibited both phenotypically and functionally. Such an effect was less remarkable when DC was stimulated by a physiological stimulus, CD40 ligand. Tryptophan deprivation during DC activation also regulated the expression of CCR5 and CXCR4, as well as DC responsiveness to chemokines. These results suggest that tryptophan usage in the microenvironment is essential for DC maturation, and may also play a role in the regulation of DC migratory behaviors.     

LPS—induced activation of phospholipase A2 phospholipase C and protein kinase C of murine macrophage—like cell lines （J774 and P388D1）

A murine macrophage-like cell line J774,acquired,in response to LPS,an ability to kill tumor necrosis factor(TNF)-insensitive target P815 mastocytoma cells whereas another cell line,P388D1 did not ,LPS triggered signaling mechanisms between the two cell lines were compared with an aim to inquire about the possible nature of the above-mentioned difference,The results whowed that two cell lines respond to LPS-treatment by parallel activation of both phospholipases C and A2 (PLC and PLA2) to approximately the same extent.The maximum response of toth enzymes of J774 cells was noted within 10 min the treatment whereas that of P388D1 cells required more than 20 min,The other properties of LPS-responsive enzymes studied were similar between two cell lines,including Activation of PLC and PLA2 and PKC in macrophages by LPS. Ca2 augmentation of enzyme activation,participation of guanine nucleotide binding(G) proteins in the initial activation preocesses,and inhibition of enzyme activation by the prior treatment of cells with choleraor pertussis toxinsetc.Moreover,LPS-triggered activation of PLC and PLA2 was found to be followed by the increase of PKC activities in both cell lines.Inspite of these similarities.J774 cells possessed both basic and acidicforms of PKC activities,while P 388 D1 cells owned only PKC of basic form,Nevertheless,the question why J774 cells but not P388D1 cells,can acquire the tumoricidal activity,aganist P815,cells following LPStreatment rematins to be answered.     

Directional migration of leukocytes：their pathological roles in inflammation and strategies for development of anti－inflammatory therapies

Directional migration of leukocytes is indispensable to innate immunity for host defense. However, recruitment of leukocytes to a site of tissue injury also constitutes a leading cause for inflammatory responses. Mechanistically, it involves a cascade of cellular events precisely regulated by temporal and spatial presentation of a repertoire of molecules in the migrating leukocytes and their surroundings (microenvironments). Here I will summarize the emerging evidence that has shed lights on the underlying molecular mechanism for directional migration of leukocytes, which has guided the therapeutical development for innovative anti-inflammatory medicines.     

Trichosanthin inhibits T cell activation by interfering with the recruitment of ZAP—70 to CD3 chain

Plant protein Trichosanthin(Tk) has been shown in our previous experiments to suppress antigenic response of T cells.Here we explored its inhibitory mechanisms on the proliferation of human Jurkat leukemia T cell triggered by anti-CD3 McAb,By examination of tyrosine phosphorylation of cell lysate,we were able to show that Tk could interfere with the PTK-related activity in the TCR/CD3-initiated signal transduction in addition to blocking the phosphorylation of PKC.As shown in our experiment the expression intensity of ZAP-70,a kind of protein tyrosine kinase,was not changed but its phosphorylation could be inhibited.When physical link between CD3 ζ chain and ZAP-70 was further examined by using coimmunoprecipitation after pluse-treatment of the cell line with Tk,the anti-CD3 McAb-induced recruitment of ZAP-70 to CD3 ζ chain was observed to be blocked in some extent.This may account for,at least in part,how Trichosanthin was able to inhibit the TCR-triggered T cell proliferation.     

P48—Triggered transmembrane signaling transduction of human monocytes：modilization of calcium ion and activation of protein kinase C（PKC）

P48 is a cytokine which induces monocyte differentiation and the induction of cytotoxic activity.In this study,the signal transduction events involved in the stimulation of monocytes with the membrane form of P48 (mP48) were investigated.Monocyte stimulation with mP48 was found to involve the mobilization of intracellular calcium(Ca^2 ) and the activation and translocation of PKC from the cytosol to the membrane.Membane P48 induced a rapid rise of intracellular Ca^2 in a dose dependent maner.Similarly,the stimulation of monocytes with P48 was found to involve the activation and translocation of PKC.The translocation of PKC was rapid(within 0-5min) yet transient with PKC activity returning to control levels by 8 min.The functional role of protein kineses in P48 induced TNF secretion was studied using various kinese inhibitors.The PKC inhibitors,H-7 and sphingosine,were found to inhibit P48 induced TNF secretion with 50% inhibition at 5μM.HA1004,which inhibts cyclic nucleotide-dependent kinase(PKA,Ki 1.2μM),did not inhibit TNF secretion.H-8(PKA inhibitor) was found to be an effective inhibitor of TNF secretion only at high concentrations(30μM).The Calmodulin-dependent kinase inhibitor,W7(Ki 12μM) was found to be effective at concentration above 5μM.These findings suggest that P48-triggered TNF secretion involves transmembrane Ca^2 signaling and the subsequent activation of at least two protein kineses,PKC and CaMK.     

BST2 promotes cell proliferation,migration and induces NF-κB activation in gastric cancer

Objectives

To investigate the functional roles of bone marrow stromal cell antigen 2 (BST2) in gastric cancer (GC) cells and its implications in the development of GC patients.

Results

BST2 was frequently overexpressed in GC tissues compared with the adjacent non-tumorous tissues, and high BST2 expression was correlated with tumor stage and lymphatic metastasis. Furthermore, in vitro experiments demonstrated that knockdown of BST2 by siRNA inhibited cell proliferation, induced apoptosis and repressed cell motility in GC cells. In addition, the pro-tumor function of BST2 in GC was mediated partly through the NF-κB signaling.

Conclusion

BST2 possesses the oncogenic potential in GC by regulating the proliferation, apoptosis, and migratory ability of GC cells, thereby BST2 could be a potential therapeutic target for the treatment of GC.

     

Targeting of alpha(v) integrins interferes with FAK activation and smooth muscle cell migration and invasion

Aberrant migration of smooth muscle cells (SMCs) is a key feature of restenosis. Since extracellular matrix proteins and their receptors of the integrin family play a critical role in this process, it is instrumental to understand their contribution to cell migration and invasive motility of SMC on the molecular level. Therefore, we investigated the role of alpha(v)-containing integrins expressed by primary human coronary artery smooth muscle cells (hCASMCs) in vitronectin (VN)-initiated signaling events and cell migration. In hCASMC plated on VN, alpha(v)-containing integrins were localized at focal adhesion sites. Haptotactic stimulation through VN led to a dose-dependent increase in cell migration and concomitantly to enhanced tyrosine phosphorylation of focal adhesion kinase. Both events were completely blocked by a specific inhibitor of integrin alpha(v). Additionally, the integrin alpha(v) inhibitor abolished PDGF-BB-stimulated chemotactic migration. Confocal microscopy confirmed the increased tyrosine phosphorylation at VN-initiated focal contact sites in hCASMC, that was abolished upon alpha(v) inhibition. In vitro invasion of hCASMC was severely compromised in the presence of the integrin alpha(v) inhibitor paralleled by decreased levels of secreted matrix metalloprotease 2 (MMP-2). Together, integrin alpha(v) inhibition abrogates tyrosine phosphorylation at focal adhesion sites and diminishes MMP-2 secretion leading to reduced migration and invasion of hCASMCs.     

Integrin activation and internalization mediated by extracellular matrix elasticity: A biomechanical model

Cells sense and respond to the elasticity of extracellular matrix (ECM) via integrin-mediated adhesion. As a class of well-documented mechanosenors in cells, integrins switch among inactive, bound, and dissociated states, depending upon the variation of forces acting on them. However, it remains unclear how the ECM elasticity directs and affects the states of integrins and, in turn, their cellular functions. On the basis of our recent experiments, a biomechanical model is proposed to reveal the role of ECM elasticity in the state-switching of integrins. It is demonstrated that a soft ECM can increase the activation level of integrins while a stiff ECM has a tendency to prevent the dissociation and internalization of bound integrins. In addition, it is found that more stable focal adhesions can form on stiffer and thinner ECMs. The theoretical results agree well with relevant experiments and shed light on the ECM elasticity-sensing mechanisms of cells.     

膜型基质金属蛋白酶亚家族和基质金属蛋白酶-2在人恶性造血细胞株中的表达谱及其相关性

为探讨不同恶性血液病中膜型基质金属蛋白酶（memberane-type matrix metalloproteinases, MT-MMPs）和基质金属蛋白酶-2（matrix metalloproteinase-2,MMP-2）的表达差异,用半定量RT-PCR检测了13株不同谱系恶性造血细胞株中MT-MMPs和MMP-2的mRNA表达差异.明胶酶谱法分析各细胞株MMP-2的酶活,并用流式细胞术检测了MT1-MMP蛋白的表达,用SPSS10.0分析了各基因的表达与细胞来源的相关性和各基因相互间表达的相关性.结果提示,各种MT-MMPs和MMP-2在13株细胞中呈现不同的表达谱,其中MT2、MT4、MT1-MMP和MMP-2表达较广泛,而MT3、MT5、MT6-MMP在检测的细胞株中较少表达.统计分析显示,MT-MMPs和MMP-2的表达与细胞的来源无明显的相关性（P>0.1）,而MT1-MMP和MMP-2的表达有相关性（P<0.05）.同时也发现,MT3-MMP和MT5-MMP的表达有相关性（P<0.05）.MT-MMPs在恶性造血细胞中的表达差异,提示它们在不同类型的血液系统肿瘤发展中发挥各自独特的作用.另外,MT1 MMP和MMP-2,MT3-MMP和MT5-MMP之间表达的相关性提示,它们在功能上密切相关.     

Cell adhesion molecules, signal transduction and cell growth

Signals from dynamic cellular interactions between the extracellular matrix and neighboring cells ultimately input into the cellular decision-making process. These interactions form the basis of anchorage-dependent growth. Recent advances have provided the mechanistic details behind the ability of integrins, and other cell adhesion molecules (CAMs), to regulate both early signal transduction events initiated by soluble factors and downstream events more proximally involved in cell cycle progression. These actions appear to depend on the ability of CAMs to initiate the formation of organized structures that permit the efficient flow of information.     

Analysis of directional cell migration on defined FN gradients: role of intracellular signaling molecules

Directional cell migration on extracellular matrix (ECM) plays important roles in embryonic development and adult organisms. To study the mechanisms and signaling pathways involved in the regulation of directional cell migration, we created defined fibronectin (FN) gradients by using microfluidic systems. We found that fibroblasts exhibited haptotaxis towards higher FN concentration on the gradient. Furthermore, the net movements in the direction of FN gradients correlated with the increase in the slope of the gradient although the overall rate of migration was not correlated. Consistent with previous observations on the uniformly coated surface, local higher FN concentration led to reduced migration rate due to increased spreading. Upon transfection of N-WASP or activated Cdc42, but not FAK or Grb7, the cells showed increased directional migration. However, transfection of FAK, but not the other signaling molecules, led to an increase in the persistence of directional cell migration, which is dependent on the slope of the gradient as well as FAK interaction with PI3K. Together, these studies reveal some novel properties of directional cell migration on defined FN gradient and suggested a role for FAK signaling and N-WASP and Cdc42 in the differential regulation of the persistence and rate of directional cell migration.     

In vitro assays for adhesion and migration of osteoblastic cells (Saos-2) on titanium surfaces

The first event occurring at the boundary between a metal implant and living tissue is the attachment of cells onto the metal surface of the implant. The attachment characteristics of the metal in this situation are critical in determining its biocompatibility and usefulness as artificial bone and tooth implants. Using the human osteosarcoma cell line Saos-2, we attempted to establish simple and reliable methods for evaluating the attachment of cultured osteoblastic cells onto titanium samples that had been subjected to various surface treatments. Fluorescence actin imaging showed that cells cultured on titanium with hydrofluoric acid etching (HF-Ti) exhibited delayed spreading of their cytoplasm, as compared to cells cultured for the same length of time on nitrided titanium or physically polished titanium. The HF-Ti-cultured cells also exhibited poor assembly of focal contacts, as visualized by vinculin immunofluorescence. Furthermore, in motility assays based on an in vitro wound model, cells cultured on HF-Ti migrated more slowly than cells cultured on other titanium surfaces. These data suggest that Saos-2 cells attach less effectively to the HF-Ti surface. The methods described in this study should be useful for assessing the initial interactions of cultured cells with various materials, including metals.S.G. is the recipient of a grant awarded to foreign students by the government of Japan. This study was supported by the Integrated Center for Science (INCS) at Ehime University.     

Down-regulation of cell surface CXCR6 expression during T cell activation is predominantly mediated by calcineurin

CXCR6, the receptor for the membrane-anchored chemokine, CXCL16, is expressed on a subset of CCR5-bearing memory T cells, and may play a role in recruiting these cells to sites of inflammation. Here, we set out to determine the effect of T cell activation on CXCR6 expression. Highly purified human peripheral blood T cells were cultured for 7-8 days in presence of IL-2 (400 U/ml) to enhance CXCR6 expression. Overnight stimulation with anti-CD3 mAb+anti-CD28 mAb, which resulted in CD69 induction and cytokine (IL-2 and IFN-gamma) production, reduced cell surface expression of CXCR6 by 85% and that of CCR5 by 76%. The Ca(2+) ionophore, ionomycin (125-500 ng/ml), also markedly diminished CXCR6 expression (85%), but without inducing CD69 expression or cytokine production, and reduced CCR5 expression by only 40%. In contrast, the phorbol esters, PdBu or PMA had little effect on CXCR6 expression (23% reduction) but induced CD69 expression and caused a profound down-regulation (92%) of CCR5 expression. Moreover, CCR7, whose expression was low on CXCR6(+) T cells, was little affected by any of these modes of activation. The down-regulation of CXCR6 expression induced by CD3/CD28 activation was blocked by the broad kinase inhibitor, staurosporine, and by the src kinase inhibitor, PP2, but not by the MEK1 inhibitor, U0106. Most interestingly, the calcineurin inhibitor, FK506, consistently inhibited CD3/CD28-induced CXCR6 down-regulation. FK506 also blocked the decrease of CXCR6 expression caused by ionomycin, whereas staurosporine or PP2 had no effect on this decrease. Altogether, these data indicate that CXCR6 expression is down-regulated, independent of CCR5 or CD69 expression and of cytokine induction, by T cell activation signals that involve predominantly the Ca(2+)-dependent calcineurin pathway.     

NG2 proteoglycan increases mesangial cell proliferation and extracellular matrix production

As a membrane-spanning protein, NG2 chondroitin sulfate proteoglycan interacts with molecules on both sides of plasma membrane. The present study explored the role of NG2 in the pathogenesis of diabetic nephropathy. In the normal kidneys, NG2 was observed predominantly in glomerular mesangium, Bowman's capsule and interstitial vessels. Both mRNA and protein expression in kidneys was significantly higher in strepozotocin-induced diabetic rats than that in normal rats. In the cultured rat mesangial cell line HBZY-1, overexpression of NG2 promoted mesangial cell proliferation and extracellular matrix (ECM) production, such as type VI collagen and laminin. Furthermore, target knockdown of NG2 resulted in decreased cell proliferation and ECM formation. The observations suggest that NG2 is up-regulated in diabetic nephropathy. It actively participates in the development and progression of glomerulosclerosis by stimulating proliferation of mesangial cells and deposition of ECM.     

Cell type specificity of a neural cell surface antigen recognized by the monoclonal antibody A2B5

Summary The monoclonal antibody A2B5 reacts with the surface membrane of most neurons in monolayer cultures of cerebellum, retina, spinal cord, and dorsal root ganglion of embryonic and early postnatal C57BL/6J mice maintained in vitro for culture periods of 2 to 10 days. A small percentage of astroglial cells also expresses A2B5 antigen in murine, chicken and rabbit cerebellum, in chicken retina, and in murine spinal cord and dorsal root ganglion. Less mature astroglial cells are stained for A2B5 antigen to a greater extent than the more mature astrocytes. Astrocytes from rat cerebellum and mouse retina were not found to express A2B5 antigen under the present culture conditions. Some of the less mature oligodendrocytes recognized by 04 antibodies express A2B5 antigen, while the more mature 01 antigen and galactocerebroside-positive oligodendrocytes were not found to be A2B5 antigen-positive. Fibroblasts or fibroblast-like cells do not express detectable levels of A2B5 antigen. After fixation of the cells with paraformaldehyde and ethanol, all cell types present in culture are labeled by the A2B5 antibody intracellularly.