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Chondrocyte intracellular calcium, cytoskeletal organization, and gene expression responses to dynamic osmotic loading 总被引:6,自引:0,他引:6
While chondrocytes in articular cartilage experience dynamic stimuli from joint loading activities, few studies have examined the effects of dynamic osmotic loading on their signaling and biosynthetic activities. We hypothesize that dynamic osmotic loading modulates chondrocyte signaling and gene expression differently than static osmotic loading. With the use of a novel microfluidic device developed in our laboratory, dynamic hypotonic loading (200 mosM) was applied up to 0.1 Hz and chondrocyte calcium signaling, cytoskeleton organization, and gene expression responses were examined. Chondrocytes exhibited decreasing volume and calcium responses with increasing loading frequency. Phalloidin staining showed osmotic loading-induced changes to the actin cytoskeleton in chondrocytes. Real-time PCR analysis revealed a stimulatory effect of dynamic osmotic loading compared with static osmotic loading. These studies illustrate the utility of the microfluidic device in cell signaling investigations, and their potential role in helping to elucidate mechanisms that mediate chondrocyte mechanotransduction to dynamic stimuli. cartilage; calcium signaling; actin cytoskeleton; aggrecan 相似文献
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Cell-type specific adhesive interactions of skeletal myoblasts with thrombospondin-1. 总被引:9,自引:1,他引:9 下载免费PDF全文
Thrombospondin-1 (TSP-1) is an extracellular matrix glycoprotein that may play important roles in the morphogenesis and repair of skeletal muscle. To begin to explore the role of thrombospondin-1 in this tissue, we have examined the interactions of three rodent skeletal muscle cell lines, C2C12, G8, and H9c2, with platelet TSP-1. The cells secrete thrombospondin and incorporate it into the cell layer in a distribution distinct from that of fibronectin. Myoblasts attach and spread on fibronectin- or thrombospondin-coated substrates with similar time and concentration dependencies. Whereas cells adherent on fibronectin organize actin stress fibers, cells adherent on TSP-1 display prominent membrane ruffles and lamellae that contain radial actin microspikes. Attachment to thrombospondin-1 or the 140-kDa tryptic fragment is mediated by interactions with the type 1 repeats and the carboxy-terminal globular domain. Attachment is not inhibited by heparin, GRGDSP peptide, or VTCG peptide but is inhibited by chondroitin sulphate A. Integrins of the beta 1 or alpha V subgroups do not appear to be involved in myoblast attachment to TSP-1; instead, this process depends in part on cell surface chondroitin sulphate proteoglycans. Whereas the central 70-kDa chymotryptic fragment of TSP-1 does not support myoblast attachment, the carboxy-terminal domain of TSP-1 expressed as a fusion protein in the bacterial expression vector, pGEX, supported myoblast attachment to 30% the level of intact TSP-1. Thrombospondin-4 (TSP-4) is also present in skeletal muscle and a fusion protein containing the carboxy-terminal domain of TSP-4 also supported myoblast adhesion, although this protein was less active on a molar basis than the TSP-1 fusion protein. Thus, the carboxyterminal domain of TSP-1 appears to contain a primary attachment site for myoblasts, and this activity is present in a second member of the thrombospondin family. 相似文献
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Geisler SB Robinson D Hauringa M Raeker MO Borisov AB Westfall MV Russell MW 《Genomics》2007,89(4):521-531
Cytoskeletal adaptor proteins serve vital functions in linking the internal cytoskeleton of cells to the cell membrane, particularly at sites of cell-cell and cell-matrix interactions. The importance of these adaptors to the structural integrity of the cell is evident from the number of clinical disease states attributable to defects in these networks. In the heart, defects in the cytoskeletal support system that surrounds and supports the myofibril result in dilated cardiomyopathy and congestive heart failure. In this study, we report the cloning and characterization of a novel cytoskeletal adaptor, obscurin-like 1 (OBSL1), which is closely related to obscurin, a giant structural protein required for sarcomere assembly. Multiple isoforms arise from alternative splicing, ranging in predicted molecular mass from 130 to 230 kDa. OBSL1 is located on human chromosome 2q35 within 100 kb of SPEG, another gene related to obscurin. It is expressed in a broad range of tissues and localizes to the intercalated discs, to the perinuclear region, and overlying the Z lines and M bands of adult rat cardiac myocytes. Further characterization of this novel cytoskeletal linker will have important implications for understanding the physical interactions that stabilize and support cell-matrix, cell-cell, and intracellular cytoskeletal connections. 相似文献
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The cell biology of thrombospondin-1. 总被引:41,自引:0,他引:41
Thrombospondin-1 (TSP-1) is a matricellular protein that regulates cellular phenotype during tissue genesis and repair. It acts as a molecular facilitator by bringing together cytokines, growth factors, matrix components, membrane receptors and extracellular proteases. TSP-1 binds to a wide variety of integrin and non-integrin cell surface receptors. The binding sites for these receptors on TSP-1 are dispersed throughout the molecule, with most domains binding multiple receptors. In some cases, TSP-1 binds to multiple receptors concurrently, and recent data indicate that there is cross-talk between the receptor systems. Thus, TSP-1 may function to direct the clustering of receptors to specialized domains for adhesion and signal transduction. 相似文献
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Cell contact-dependent activation of alpha3beta1 integrin modulates endothelial cell responses to thrombospondin-1 总被引:2,自引:0,他引:2 下载免费PDF全文
Chandrasekaran L He CZ Al-Barazi H Krutzsch HC Iruela-Arispe ML Roberts DD 《Molecular biology of the cell》2000,11(9):2885-2900
Thrombospondin-1 (TSP1) can inhibit angiogenesis by interacting with endothelial cell CD36 or proteoglycan receptors. We have now identified alpha3beta1 integrin as an additional receptor for TSP1 that modulates angiogenesis and the in vitro behavior of endothelial cells. Recognition of TSP1 and an alpha3beta1 integrin-binding peptide from TSP1 by normal endothelial cells is induced after loss of cell-cell contact or ligation of CD98. Although confluent endothelial cells do not spread on a TSP1 substrate, alpha3beta1 integrin mediates efficient spreading on TSP1 substrates of endothelial cells deprived of cell-cell contact or vascular endothelial cadherin signaling. Activation of this integrin is independent of proliferation, but ligation of the alpha3beta1 integrin modulates endothelial cell proliferation. In solution, both intact TSP1 and the alpha3beta1 integrin-binding peptide from TSP1 inhibit proliferation of sparse endothelial cell cultures independent of their CD36 expression. However, TSP1 or the same peptide immobilized on the substratum promotes their proliferation. The TSP1 peptide, when added in solution, specifically inhibits endothelial cell migration and inhibits angiogenesis in the chick chorioallantoic membrane, whereas a fragment of TSP1 containing this sequence stimulates angiogenesis. Therefore, recognition of immobilized TSP1 by alpha3beta1 integrin may stimulate endothelial cell proliferation and angiogenesis. Peptides that inhibit this interaction are a novel class of angiogenesis inhibitors. 相似文献
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Cal S Arguelles JM Fernandez PL López-Otín C 《The Journal of biological chemistry》2001,276(21):17932-17940
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G L Semenza 《Journal of applied physiology》2000,88(4):1474-1480
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Cloning and characterization of angiocidin, a tumor cell binding protein for thrombospondin-1 总被引:1,自引:0,他引:1
Zhou J Rothman VL Sargiannidou I Dimitrov S Qiu C Smith E Sheffield J Sharma M Tuszynski GP 《Journal of cellular biochemistry》2004,92(1):125-146
Thrombospondin-1 (TSP-1) is a matrix protein that has been implicated in mechanisms of tumor progression. Our laboratory previously showed that the CSVTCG (cys-ser-val-thr-cys-gly) sequence of TSP-1 functioned as a tumor cell adhesion domain and CSVTCG peptides as well as an anti-peptide antibody possessed anti-metastatic activity in a murine model of lung metastasis. In a subsequent study, a putative TSP-1 binding protein from lung carcinoma was isolated by CSVTCG-peptide affinity chromatography. In this study, we present the full-length cDNA of this binding protein isolated from a prostate cancer cell (PC3-NI) cDNA library. The purified recombinant protein, termed angiocidin, is a potent inhibitor of tumor growth of Lewis Lung carcinoma in vivo and tumor invasion and angiogenesis in vitro. In addition, the recombinant protein inhibits tumor and endothelial cell proliferation and induces apoptosis. The activity of angiocidin both in vivo and in vitro is partially dependent on its TSP-1 binding activity, since an angiocidin deletion mutant missing a high affinity-binding site for TSP-1 failed to inhibit tumor growth in vivo and was less active in its anti-tumor and anti-angiogenic activities in vitro. These results suggest that the anti-tumor activity of TSP-1 reported in many studies may be mediated in part by binding proteins such as angiocidin. Such proteins may function as tumor-suppressor proteins, which limit the growth of tumors by inhibiting angiogenesis and cell matrix interaction. 相似文献
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Margosio B Rusnati M Bonezzi K Cordes BL Annis DS Urbinati C Giavazzi R Presta M Ribatti D Mosher DF Taraboletti G 《The international journal of biochemistry & cell biology》2008,40(4):700-709
Thrombospondin-1, an antiangiogenic matricellular protein, binds with high affinity to the angiogenic fibroblast growth factor-2, affecting its bioavailability and activity. The present work aimed at further locating the fibroblast growth factor-2 binding site of thrombospondin-1 and investigating its activity, using recombinant thrombospondin-1 proteins. Only recombinant constructs containing the thrombospondin-1 type III repeats bound fibroblast growth factor-2, whereas other domains, including the known anti-angiogenic type I repeats, were inactive. Binding was specific and inhibited by the anti thrombospondin-1 monoclonal antibody B5.2. Surface plasmon resonance analysis on BIAcore revealed a binding affinity (K(d)) of 310nM for the type III repeats and 11nM for intact thrombospondin-1. Since the type III repeats bind calcium, the effect of calcium on thrombospondin-1 binding to fibroblast growth factor-2 was investigated. Binding was modulated by calcium, as thrombospondin-1 or the type III repeats bound to fibroblast growth factor-2 only in calcium concentrations <0.3mM. The type III repeats inhibited binding of fibroblast growth factor-2 to endothelial cells, fibroblast growth factor-2-induced endothelial cell proliferation in vitro and angiogenesis in the chorioallantoic membrane assay in vivo, thus indicating the antiangiogenic activity of the domain. In conclusion, this study demonstrates that the fibroblast growth factor-2 binding site of thrombospondin-1 is located in the type III repeats. The finding that this domain is active in inhibiting angiogenesis indicates that the type III repeats represent a novel antiangiogenic domain of thrombospondin-1. 相似文献
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Leukemic cell intracellular responses to nanosecond electric fields 总被引:13,自引:0,他引:13
Chen N Schoenbach KH Kolb JF James Swanson R Garner AL Yang J Joshi RP Beebe SJ 《Biochemical and biophysical research communications》2004,317(2):421-427
Intense, nanosecond (ns) pulsed electric fields (PEFs) are known to affect the intracellular structures of cells. The probability of preferentially inducing subcellular effects increases with decreasing pulse length while effects on the plasma membrane are diminished. This has been demonstrated by applying electrical pulses of 60 and 10 ns duration with electric field intensities of up to 6.5 MV/m to HL-60 cells. Using confocal microscopy, PEF-induced changes in the integrity of the plasma membrane and nucleus were measured by recording fluorescence changes with propidium iodide (PI) and acridine orange (AO), respectively. Results suggest that high voltage, nsPEFs target the nucleus and modify cellular functions while plasma membrane effects are delayed and become smaller as pulse duration is shortened. Cell viability was not affected by these pulses. In spite of the high pulsed electric fields, thermal effects can be neglected because of the ultrashort pulse duration. The results suggest application of this ultrashort pulse technology to modulate nuclear structure and function for potential therapeutic benefit. 相似文献
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K E Wilson Z Li M Kara K L Gardner D D Roberts 《Journal of immunology (Baltimore, Md. : 1950)》1999,163(7):3621-3628
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Isenberg JS Ridnour LA Dimitry J Frazier WA Wink DA Roberts DD 《The Journal of biological chemistry》2006,281(36):26069-26080
CD36 is necessary for inhibition of some angiogenic responses by the matricellular glycoprotein thrombospondin-1 and is therefore assumed to be the receptor that mediates its anti-angiogenic activities. Although ligation of CD36 by antibodies, recombinant type 1 repeats of thrombospondin-1, or CD36-binding peptides was sufficient to inhibit nitric oxide (NO)-stimulated responses in both endothelial and vascular smooth muscle cells, picomolar concentrations of native thrombospondin-1 similarly inhibited NO signaling in vascular cells from wild-type and CD36-null mice. Ligation of the thrombospondin-1 receptor CD47 by recombinant C-terminal regions of thrombospondin-1, thrombospondin-1 peptides, or CD47 antibodies was also sufficient to inhibit NO-stimulated phenotypic responses and cGMP signaling in vascular cells. Thrombospondin-1 did not inhibit NO signaling in CD47-null vascular cells or NO-stimulated vascular outgrowth from CD47-null muscle explants in three-dimensional cultures. Furthermore, the CD36-binding domain of thrombospondin-1 and anti-angiogenic peptides derived from this domain failed to inhibit NO signaling in CD47-null cells. Therefore, ligation of either CD36 or CD47 is sufficient to inhibit NO-stimulated vascular cell responses and cGMP signaling, but only CD47 is necessary for this activity of thrombospondin-1 at physiological concentrations. 相似文献
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A method is presented for determining the retardation of diffusion of particles inside cells owing to cytoskeletal barriers. The cytoskeletal meshwork is treated as a repeating periodic two-dimensional or three-dimensional lattice composed of elements of given size, shape, and spacing. We derive an analytic expression for the diffusion coefficient relative to that of the cytosol. This expression is evaluated by solving numerically an appropriate boundary-value problem for the Laplace equation. For the two-dimensional case, e.g., diffusion in a membrane, the results are quantitatively similar to those obtained by Saxton (1987. Biophys. J. 52:989-997) using Monte Carlo methods. The three-dimensional results are quantitatively similar to experimental results reported by Luby-Phelps et al. (1987. Proc. Natl. Acad. Sci. USA. 84:4910-4913) for the diffusion of dextran and Ficoll particles in Swiss 3T3 cells. By accounting for geometrical factors, these results allow one to assess the relative contributions of geometrical hindrance and of binding to the cytoskeletal lattice from measurements of intracellular diffusion coefficients of proteins. 相似文献
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The Ena-VASP homology (EVH1) domain is a protein interaction module found in several proteins that are involved in transducing migratory and morphological signals into cytoskeletal reorganization. EVH1 specifically recognizes proline-rich sequences in its binding partners and directs the localization and formation of multicomponent assemblies involved in actin-based motile processes and neural development. The structure of the complex between an EVH1 domain and the target peptide sequence EFPPPPT identifies the interactions responsible for recognition and distinguishes it from other proline-rich binding modules, including SH3 and WW domains. Surprisingly, the EVH1 domain has structural similarity to pleckstrin homology (PH), phosphotyrosine-binding (PTB) and ran-binding (RanBD) domains. 相似文献
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Integrins play a major role in the regulation of cell motility. They physically link the extracellular environment to the cytoskeleton and participate in large protein complexes known as focal adhesions. In this report, it is demonstrated that treatment of tumor cells with the homodimeric disintegrin contortrostatin induces integrin-mediated tyrosine phosphorylation events and causes severe disruptions in the actin cytoskeleton and disassembly of focal adhesion structures without affecting cellular adhesion to a reconstituted basement membrane. Included in this disruption is the tyrosine phosphorylation and altered subcellular localization of FAK. Through use of transfected 293 cells with specific integrin expression profiles and anti-alphavbeta3 mAbs, we demonstrate that these events are mediated exclusively by the alphavbeta3 integrin and are likely the result of contortrostatin-mediated crosslinking of this receptor at the cell surface, since monovalent disintegrins, flavoridin or echistatin do not induce such effects. Further, it is shown that contortrostatin potently inhibits motility in cells expressing the alphavbeta33 integrin. The results of this study describe a novel integrin-mediated mechanism by which cell motility can be inhibited and suggest an alternative approach to therapeutic intervention for cancer invasion and metastasis. 相似文献
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J Scott Isenberg Maria J Calzada Longen Zhou Nenghua Guo Jack Lawler Xue-Qing Wang William A Frazier David D Roberts 《Matrix biology》2005,24(2):110-123
We have reexamined the role of endogenous thrombospondin-1 (TSP1) in growth and motility of vascular smooth muscle cells (SMCs). Based on the ability of aortic-derived SMCs isolated from TSP1 null mice and grown in the absence of exogenous TSP1 to grow at comparable rates and to a slightly higher density than equivalent cells from wild-type mice, TSP1 is not necessary for their growth. Low concentrations of exogenous TSP1 stimulate growth of TSP1 null SMCs, but higher doses of TSP1 or its C-terminal domain are inhibitory. However, SMCs from TSP1 null mice are selectively deficient in chemotactic and proliferative responses to platelet-derived growth factor and in outgrowth in three-dimensional cultures. Recombinant portions of the N- and C-terminal domains of TSP1 stimulate SMC chemotaxis through different integrin receptors. Based on these data, the relative deficiency in SMC outgrowth during an ex vivo angiogenic response of muscle tissue from TSP1 null mice is probably due to restriction of platelet-derived growth factor dependent SMC migration and/or proliferation. 相似文献
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Sandrine Magnetto Gabriella Bruno-Bossio Carole Voland Jean Lecerf Jack Lawler Pierre Delmas Roy Silverstein Philippe Clezardin 《Cell biochemistry and function》1998,16(3):211-221
In this study, we examined the binding of soluble TSP1 (and ox-LDL) to CD36-transfected cells and the mechanisms by which immobilized TSP1 mediated attachment and haptotaxis (cell migration towards a substratum-bound ligand) of these transfected cells. CD36 cDNA transfection of NIH 3T3 cells clearly induced a dramatic increase in binding of both soluble [125I]-TSP1 and [125I]-ox-LDL to the surface of CD36-transfected cells, indicating that there was a gain of function with CD36 transfection in NIH 3T3 cells. Despite this gain of function, mock- and CD36-transfected NIH 3T3 cells attached and migrated to a similar extent on immobilized TSP1. An anti-TSP1 oligoclonal antibody inhibited CD36-transfected cell attachment to TSP1 while function blocking anti-CD36 antibodies, alone or in combination with heparin, did not. A series of fusion proteins encompassing cell-recognition domains of TSP1 was then used to delineate mechanisms by which NIH 3T3 cells adhere to TSP1. Although CD36 binds soluble TSP1 through a CSVTCG sequence located within type 1 repeats,18,19 CD36-transfected NIH 3T3 cells did not attach to immobilized type 1 repeats while they did adhere to the N-terminal, type 3 repeats (in an RGD-dependent manner) and the C-terminal domain of TSP1. Conversely, Bowes melanoma cells attached to type 1 repeats and the N- and C-terminal domains of TSP1. However, CD36 cDNA transfection of Bowes cells did not increase cell attachment to type 1 repeats compared to that observed with mock-transfected Bowes cells. Moreover, a function blocking anti-CSVTCG peptide antibody did not inhibit the attachment of mock- and CD36-transfected Bowes cells to type 1 repeats. It is suggested that CD36/TSP1 interaction does not occur upon cell–matrix adhesion and haptotaxis because TSP1 undergoes conformational changes that do not allow the exposure of the CD36 binding site. © 1998 John Wiley & Sons, Ltd. 相似文献