共查询到20条相似文献,搜索用时 15 毫秒
1.
Christiane Kummer Brian G. Petrich David M. Rose Mark H. Ginsberg 《The Journal of biological chemistry》2010,285(13):9462-9469
Extracellular antagonists of α4 integrin are an effective therapy for several autoimmune and inflammatory diseases; however, these agents that directly block ligand binding may exhibit mechanism-based toxicities. Inhibition of α4 integrin signaling by mutations of α4 that block paxillin binding inhibits inflammation while limiting mechanism-based toxicities. Here, we test a pharmacological approach by identifying small molecules that inhibit the α4 integrin-paxillin interaction. By screening a large (∼40,000-compound) chemical library, we identified a noncytotoxic inhibitor of this interaction that impaired integrin α4-mediated but not αLβ2-mediated Jurkat T cell migration. The identified compound had no effect on α4-mediated migration in cells bearing the α4(Y991A) mutation that disrupts the α4-paxillin interaction, establishing the specificity of its action. Administration of this compound to mice led to impaired recruitment of mononuclear leukocytes to a site of inflammation in vivo, whereas an isomer that does not inhibit the α4-paxillin interaction had no effect on α4-mediated cell migration, cell spreading, or recruitment of leukocytes to an inflammatory site. Thus, a small molecule inhibitor that interferes with α4 integrin signaling reduces α4-mediated T cell migration in vivo, thus providing proof of principle for inhibition of α4 integrin signaling as a target for the pharmacological reduction of inflammation. 相似文献
2.
Sundaravadivel Balasubramanian Lakeya Quinones Harinath Kasiganesan Yuhua Zhang Dorea L. Pleasant Kamala P. Sundararaj Michael R. Zile Amy D. Bradshaw Dhandapani Kuppuswamy 《PloS one》2012,7(9)
The adhesion receptor β3 integrin regulates diverse cellular functions in various tissues. As β3 integrin has been implicated in extracellular matrix (ECM) remodeling, we sought to explore the role of β3 integrin in cardiac fibrosis by using wild type (WT) and β3 integrin null (β3−/−) mice for in vivo pressure overload (PO) and in vitro primary cardiac fibroblast phenotypic studies. Compared to WT mice, β3−/− mice upon pressure overload hypertrophy for 4 wk by transverse aortic constriction (TAC) showed a substantially reduced accumulation of interstitial fibronectin and collagen. Moreover, pressure overloaded LV from β3−/− mice exhibited reduced levels of both fibroblast proliferation and fibroblast-specific protein-1 (FSP1) expression in early time points of PO. To test if the observed impairment of ECM accumulation in β3−/− mice was due to compromised cardiac fibroblast function, we analyzed primary cardiac fibroblasts from WT and β3−/− mice for adhesion to ECM proteins, cell spreading, proliferation, and migration in response to platelet derived growth factor-BB (PDGF, a growth factor known to promote fibrosis) stimulation. Our results showed that β3−/− cardiac fibroblasts exhibited a significant reduction in cell-matrix adhesion, cell spreading, proliferation and migration. In addition, the activation of PDGF receptor associated tyrosine kinase and non-receptor tyrosine kinase Pyk2, upon PDGF stimulation were impaired in β3−/− cells. Adenoviral expression of a dominant negative form of Pyk2 (Y402F) resulted in reduced accumulation of fibronectin. These results indicate that β3 integrin-mediated Pyk2 signaling in cardiac fibroblasts plays a critical role in PO-induced cardiac fibrosis. 相似文献
3.
Background
Osteosarcoma is characterized by a high malignant and metastatic potential. CCL5 (previously called RANTES) was originally recognized as a product of activated T cells, and plays a crucial role in the migration and metastasis of human cancer cells. It has been reported that the effect of CCL5 is mediated via CCR receptors. However, the effect of CCL5 on migration activity and integrin expression in human osteosarcoma cells is mostly unknown.Methodology/Principal Findings
Here we found that CCL5 increased the migration and expression of αvβ3 integrin in human osteosarcoma cells. Stimulation of cells with CCL5 increased CCR5 but not CCR1 and CCR3 expression. CCR5 mAb, inhibitor, and siRNA reduced the CCL5-enhanced the migration and integrin up-regulation of osteosarcoma cells. Activations of MEK, ERK, and NF-κB pathways after CCL5 treatment were demonstrated, and CCL5-induced expression of integrin and migration activity was inhibited by the specific inhibitor and mutant of MEK, ERK, and NF-κB cascades. In addition, over-expression of CCL5 shRNA inhibited the migratory ability and integrin expression in osteosarcoma cells.Conclusions/Significance
CCL5 and CCR5 interaction acts through MEK, ERK, which in turn activates NF-κB, resulting in the activations of αvβ3 integrin and contributing the migration of human osteosarcoma cells. 相似文献4.
5.
Daniel Dongwei Wu Fuqiang Zhang Feng Hao Jerold Chun Xuemin Xu Mei-Zhen Cui 《The Journal of biological chemistry》2014,289(9):5774-5783
Lysophosphatidic acid (LPA), a potent bioactive lipid found in atherosclerotic lesions, markedly induces smooth muscle cell (SMC) migration, which is an important process in atherogenesis. Therefore, understanding the mechanism of LPA-induced SMC migration is important. Several microarray databases suggest that the matricellular protein Cyr61 is highly induced by LPA. We hypothesized that Cyr61 mediates LPA-induced cell migration. Our data show that LPA induced temporal and spatial expression of Cyr61, which promptly accumulated in the cellular Golgi apparatus and then translocated to the extracellular matrix. Cyr61 antibody blockade and siRNA inhibition both diminished LPA-induced SMC migration, indicating a novel regulatory role of Cyr61. SMCs derived from LPA receptor 1 (LPA1) knock-out mice lack the ability of Cyr61 induction and cell migration, supporting the concept that LPA1 is required for Cyr61 expression and migration. By contrast, PPARγ was not found to be involved in LPA-mediated effects. Furthermore, focal adhesion kinase (FAK), a nonreceptor tyrosine kinase important for regulating cell migration, was activated by LPA at a late time frame coinciding with Cyr61 accumulation. Interestingly, knockdown of Cyr61 blocked LPA-induced FAK activation, indicating that an LPA-Cyr61-FAK axis leads to SMC migration. Our results further demonstrate that plasma membrane integrins α6β1 and ανβ3 transduced the LPA-Cyr61 signal toward FAK activation and migration. Taken together, these data reveal that de novo Cyr61 in the extracellular matrix bridges LPA and integrin pathways, which in turn, activate FAK, leading to cell migration. The current study provides new insights into mechanisms underlying cell migration-related disorders, including atherosclerosis, restenosis, and cancers. 相似文献
6.
Majumder M Tutunea-Fatan E Xin X Rodriguez-Torres M Torres-Garcia J Wiebe R Timoshenko AV Bhattacharjee RN Chambers AF Lala PK 《PloS one》2012,7(4):e35094
Introduction and Objectives
Lymphatic metastasis is a common occurrence in human breast cancer, mechanisms remaining poorly understood. MDA-MB-468LN (468LN), a variant of the MDA-MB-468GFP (468GFP) human breast cancer cell line, produces extensive lymphatic metastasis in nude mice. 468LN cells differentially express α9β1 integrin, a receptor for lymphangiogenic factors VEGF-C/-D. We explored whether (1) differential production of VEGF-C/-D by 468LN cells provides an autocrine stimulus for cellular motility by interacting with α9β1 and a paracrine stimulus for lymphangiogenesis in vitro as measured with capillary-like tube formation by human lymphatic endothelial cells (HMVEC-dLy); (2) differential expression of α9 also promotes cellular motility/invasiveness by interacting with macrophage derived factors; (3) stable knock-down of VEGF-D or α9 in 468LN cells abrogates lymphangiogenesis and lymphatic metastasis in vivo in nude mice.Results
A comparison of expression of cyclo-oxygenase (COX)-2 (a VEGF-C/-D inducer), VEGF-C/-D and their receptors revealed little COX-2 expression by either cells. However, 468LN cells showed differential VEGF-D and α9β1 expression, VEGF-D secretion, proliferative, migratory/invasive capacities, latter functions being stimulated further with VEGF-D. The requirement of α9β1 for native and VEGF-D-stimulated proliferation, migration and Erk activation was demonstrated by treating with α9β1 blocking antibody or knock-down of α9. An autocrine role of VEGF-D in migration was shown by its impairment by silencing VEGF-D and restoration with VEGF-D. 468LN cells and their soluble products stimulated tube formation, migration/invasiveness of HMVEC-dLy cell in a VEGF-D dependent manner as indicated by the loss of stimulation by silencing VEGF-D in 468LN cells. Furthermore, 468LN cells showed α9-dependent stimulation of migration/invasiveness by macrophage products. Finally, capacity for intra-tumoral lymphangiogenesis and lymphatic metastasis in nude mice was completely abrogated by stable knock-down of either VEGF-D or α9 in 468LN cells.Conclusion
Differential capacity for VEGF-D production and α9β1 integrin expression by 468LN cells jointly contributed to their lymphatic metastatic phenotype. 相似文献7.
Tomoya Isaji Yuya Sato Tomohiko Fukuda Jianguo Gu 《The Journal of biological chemistry》2009,284(18):12207-12216
N-Glycosylation of integrin α5β1 plays a crucial role
in cell spreading, cell migration, ligand binding, and dimer formation, but
the detailed mechanisms by which N-glycosylation mediates these
functions remain unclear. In a previous study, we showed that three potential
N-glycosylation sites (α5S3–5) on the β-propeller of
the α5 subunit are essential to the functional expression of the
subunit. In particular, site 5 (α5S5) is the most important for its
expression on the cell surface. In this study, the function of the
N-glycans on the integrin β1 subunit was investigated using
sequential site-directed mutagenesis to remove the combined putative
N-glycosylation sites. Removal of the N-glycosylation sites
on the I-like domain of the β1 subunit (i.e. the Δ4-6
mutant) decreased both the level of expression and heterodimeric formation,
resulting in inhibition of cell spreading. Interestingly, cell spreading was
observed only when the β1 subunit possessed these three
N-glycosylation sites (i.e. the S4-6 mutant). Furthermore,
the S4-6 mutant could form heterodimers with either α5S3-5 or α5S5
mutant of the α5 subunit. Taken together, the results of the present
study reveal for the first time that N-glycosylation of the I-like
domain of the β1 subunit is essential to both the heterodimer formation
and biological function of the subunit. Moreover, because the
α5S3-5/β1S4-6 mutant represents the minimal
N-glycosylation required for functional expression of the β1
subunit, it might also be useful for the study of molecular structures.Integrin is a heterodimeric glycoprotein that consists of both an α
and a β subunit (1). The
interaction between integrin and the extracellular matrix is essential to both
physiologic and pathologic events, such as cell migration, development, cell
viability, immune homeostasis, and tumorigenesis
(2,
3). Among the integrin
superfamily, β1 integrin can combine with 12 distinct α subunits
(α1–11, αv) to form heterodimers, thereby acquiring a wide
variety of ligand specificity
(1,
4). Integrins are thought to be
regulated by inside-out signaling mechanisms that provoke conformational
changes, which modulate the affinity of integrin for the ligand
(5). However, an increasing
body of evidence suggests that cell-surface carbohydrates mediate a variety of
interactions between integrin and its extracellular environment, thereby
affecting integrin activity and possibly tumor metastasis as well
(6–8).Guo et al. (9)
reported that an increase in β1–6-GlcNAc sugar chains on the
integrin β1 subunit stimulated cell migration. In addition, elevated
sialylation of the β1 subunit, because of Ras-induced STGal-I transferase
activity, also induced cell migration
(10,
11). Conversely, cell
migration and spreading were reduced by the addition of a bisecting GlcNAc,
which is a product of N-acetylglucosaminyltransferase III
(GnT-III),2 to the
α5β1 and α3β1 integrins
(12,
13). Alterations of
N-glycans on integrins might also regulate their cis interactions
with membrane-associated proteins, including the epidermal growth factor
receptor, the galectin family, and the tetraspanin family of proteins
(14–19).In addition to the positive and negative regulatory effects of
N-glycan, several research groups have reported that
N-glycans must be present on integrin α5β1 for the
αβ heterodimer formation and proper integrin-matrix interactions.
Consistent with this hypothesis, in the presence of the glycosylation
inhibitor, tunicamycin, normal integrin-substrate binding and transport to the
cell surface are inhibited
(20). Moreover, treatment of
purified integrin with N-glycosidase F blocked both the inherent
association of the subunits and the interaction between integrin and
fibronectin (FN) (21). These
results suggest that N-glycosylation is essential to the functional
expression of α5β1. However, because integrin α5β1
contains 26 potential N-linked glycosylation sites, 14 in the α
subunit and 12 in the β subunit, identification of the sites that are
essential to its biological functions is key to understanding the molecular
mechanisms by which N-glycans alter integrin function. Recently, our
group determined that N-glycosylation of the β-propeller domain
on the α5 subunit is essential to both heterodimerization and biological
functions of the subunit. Furthermore, we determined that sites 3–5 are
the most important sites for α5 subunit-mediated cell spreading and
migration on FN (22). The
purpose of this study was to clarify the roles of N-glycosylation of
the β1 subunit. Therefore, we performed combined substitutions in the
putative N-glycosylation sites by replacement of asparagine residues
with glutamine residues. We subsequently introduced these mutated genes into
β1-deficient epithelial cells (GE11). The results of these mutation
experiments revealed that the N-glycosylation sites on the I-like
domain of the β1 subunit, sites number 4–6 (S4-6), are essential to
both heterodimer formation and biological functions, such as cell
spreading. 相似文献
8.
Valeria Benedusi Francesca Martorana Liliana Brambilla Adriana Maggi Daniela Rossi 《The Journal of biological chemistry》2012,287(43):35899-35911
9.
Aurelia Raducanu Ernst B. Hunziker Inga Drosse Attila Asz��di 《The Journal of biological chemistry》2009,284(35):23780-23792
The lack of β1 integrins on chondrocytes leads to severe chondrodysplasia associated with high mortality rate around birth. To assess the impact of β1 integrin-mediated cell-matrix interactions on the function of adult knee joints, we conditionally deleted the β1 integrin gene in early limb mesenchyme using the Prx1-cre transgene. Mutant mice developed short limbed dwarfism and had joint defects due to β1 integrin deficiency in articular regions. The articular cartilage (AC) was structurally disorganized, accompanied by accelerated terminal differentiation, altered shape, and disrupted actin cytoskeleton of the chondrocytes. Defects in chondrocyte proliferation, cytokinesis, and survival resulted in hypocellularity. However, no significant differences in cartilage erosion, in the expression of matrix-degrading proteases, or in the exposure of aggrecan and collagen II cleavage neoepitopes were observed between control and mutant AC. We found no evidence for disturbed activation of MAPKs (ERK1/2, p38, and JNK) in vivo. Furthermore, fibronectin fragment-stimulated ERK activation and MMP-13 expression were indistinguishable in control and mutant femoral head explants. The mutant synovium was hyperplastic and frequently underwent chondrogenic differentiation. β1-null synoviocytes showed increased proliferation and phospho-focal adhesion kinase expression. Taken together, deletion of β1 integrins in the limb bud results in multiple abnormalities of the knee joints; however, it does not accelerate AC destruction, perturb cartilage metabolism, or influence intracellular MAPK signaling pathways.Chondrocytes of the articular cartilage (AC)2 secrete a unique set of extracellular matrix (ECM) molecules that assemble into interactive associates composed of collagens, proteoglycans (PGs), and non-collagenous glycoproteins (1). The fibrillar collagen meshwork supplies cartilage with its tensile strength, whereas the hydrated glycosaminoglycan (GAG) chains of PGs (mainly aggrecan) generate an osmotic swelling pressure that resists compressive forces. In diarthrodial joints, the molecular composition and the physical properties of the cartilage are principal determinants for the shock-absorbing function of articular surfaces upon mechanical loading. During the development of osteoarthritis (OA), an imbalance between anabolic and catabolic processes increases the proteolysis of PGs and collagens (2, 3), which eventually leads to the mechanical weakening of the AC and culminates in its progressive destruction. Physiological and pathological remodeling of the AC ECM is primarily attributed to the activities of matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin-like repeat (ADAMTS) proteases (4, 5) and is controlled by the communication between the cells and their environment.An increasing amount of evidence suggests that interactions between chondrocytes and the ECM through the integrin family of heterodimeric (αβ) transmembrane receptors play a central role in cartilage function (6). Integrins connect the pericellular matrix to cytoskeletal and intracellular signaling complexes and modulate various cellular functions, including survival, proliferation, differentiation, and matrix assembly and metabolism (7, 8). Chondrocytes express several integrin receptors for cartilage matrix ligands, such as α1β1, α2β1, and α10β1 (for collagen II); α5β1, αvβ3, and αvβ5 (for fibronectin); and α6β1 (for laminin) (6, 9). We have previously demonstrated that β1fl/fl-Col2a1cre+ mice, in which the floxed β1 integrin gene (β1fl/fl) was deleted using the chondrocyte-specific Col2a1cre transgene, display severe chondrodysplasia and a high mortality rate at birth (10). Homozygous mutant mice exhibit multiple growth plate abnormalities during endochondral bone formation, characterized by defects in chondrocyte adhesion, shape, proliferation, cytokinesis, and actin organization. In addition, the cartilage matrix shows a sparse, distorted collagen network. Similar, but milder abnormalities were found in mice lacking the collagen-binding integrin α10β1 or integrin-linked kinase in cartilage (11, 12).Although these works have identified β1 integrins as essential regulators of growth plate development, the role of integrins in joint morphogenesis, adult joint function, and pathology is incompletely understood. In the embryonic mouse limb culture system, administration of β1 and α5 blocking antibodies or RGD peptides induced ectopic joint formation between proliferating and hypertrophic chondrocytes of the growth plate, suggesting that α5β1 integrin controls the decision between cartilage differentiation and joint formation during development (13). In adult joints, increased immunostaining of β1 integrin was reported in osteoarthritic monkey cartilage compared with normal cartilage (14) and in human OA samples at minimally damaged locations compared with areas with more severe lesions (15). In another study, the neoexpression of α2, α4, and β2 integrins was observed in osteoarthritic human femoral head cartilage (16). In vitro experiments have suggested that signaling through the fibronectin (FN) receptor α5β1 integrin is pivotal to prevent cell death of normal and osteoarthritic human articular chondrocytes (17). FN fragments (FN-fs) present in synovial fluid and cartilage of OA patients have been implicated in cartilage breakdown (18–21). Human AC chondrocytes treated with the central, 110–120-kDa cell-binding FN-f but not with intact FN were shown to increase MMP-13 synthesis through the stimulation of α5β1 integrin and the subsequent activation of the proline-rich tyrosine kinase-2 and mitogen-activated protein kinases (MAPKs) ERK-1/2, JNK, and p38 (22, 23). Similarly, an adhesion-blocking antibody against α2β1 integrin induced the phosphorylation of MAPKs in human AC chondrocytes (22). Treatment of cultured rabbit synovial fibroblasts with central FN-fs or activating antibodies against α5β1 integrin elevated MMP-1 and MMP-3 expression (24). Although these experiments suggest that blocking integrin signaling through α2β1/α5β1 in response to degradation fragments may attenuate OA, mice lacking α1β1 integrin are prone to osteoarthritis (25). Knee joints of α1-null mice display precocious PG loss, cartilage erosion associated with increased MMP-2 and MMP-3 expression, and synovial hyperplasia.To further explore the role of β1 integrins in joint biology, here we report the deletion of the floxed β1 integrin gene in embryonic limb bud mesenchymal cells using the Prx1cre transgene (26). β1fl/fl-Prx1cre+ mice were born alive with short limbs due to the lack of β1 integrin heterodimers on chondrocytes. We found that β1 integrin deficiency in knee joints leads to multiple abnormalities of the AC and the synovium, but it is not associated with accelerated AC destruction, perturbed AC metabolism, and MAPK signaling. Our data suggest that β1 integrins are required for the proper structural organization of the AC by anchoring chondrocytes to the ECM, but signaling through β1 integrins is less important for normal cartilage homeostasis. 相似文献
10.
Natalia Ninkina Owen M. Peters Natalie Connor-Robson Olga Lytkina Essam Sharfeddin Vladimir L. Buchman 《The Journal of biological chemistry》2012,287(53):44471-44477
In neuronal synapses, neurotransmitter-loaded vesicles fuse with presynaptic plasma membrane in a complex sequence of tightly regulated events. The assembly of specialized SNARE complexes plays a pivotal role in this process. The function of the chaperone cysteine string protein α (CSPα) is important for synaptic SNARE complex formation, and mice lacking this protein develop severe synaptic dysfunction and neurodegeneration that lead to their death within 3 months after birth. Another presynaptic protein, α-synuclein, also potentiates SNARE complex formation, and its overexpression rescues the phenotype of CSPα null mutant mice, although these two proteins use different mechanisms to achieve this effect. α-Synuclein is a member of a family of three related proteins whose structural similarity suggests functional redundancy. Here, we assessed whether γ-synuclein shares the ability of α-synuclein to bind synaptic vesicles and ameliorate neurodegeneration caused by CSPα deficiency in vivo. Although the N-terminal lipid-binding domains of the two synucleins showed similar affinity for purified synaptic vesicles, the C-terminal domain of γ-synuclein was not able to interact with synaptobrevin-2/VAMP2. Consequently, overexpression of γ-synuclein did not have any noticeable effect on the phenotype of CSPα null mutant mice. Our data suggest that the functions of α- and γ-synucleins in presynaptic terminals are not fully redundant. 相似文献
11.
Yuya Sato Tomoya Isaji Michiko Tajiri Shumi Yoshida-Yamamoto Tsuyoshi Yoshinaka Toshiaki Somehara Tomohiko Fukuda Yoshinao Wada Jianguo Gu 《The Journal of biological chemistry》2009,284(18):11873-11881
Recently we reported that N-glycans on the β-propeller domain
of the integrin α5 subunit (S-3,4,5) are essential for α5β1
heterodimerization, expression, and cell adhesion. Herein to further
investigate which N-glycosylation site is the most important for the
biological function and regulation, we characterized the S-3,4,5 mutants in
detail. We found that site-4 is a key site that can be specifically modified
by N-acetylglucosaminyltransferase III (GnT-III). The introduction of
bisecting GlcNAc into the S-3,4,5 mutant catalyzed by GnT-III decreased cell
adhesion and migration on fibronectin, whereas overexpression of
N-acetylglucosaminyltransferase V (GnT-V) promoted cell migration.
The phenomenon is similar to previous observations that the functions of the
wild-type α5 subunit were positively and negatively regulated by GnT-V
and GnT-III, respectively, suggesting that the α5 subunit could be
duplicated by the S-3,4,5 mutant. Interestingly GnT-III specifically modified
the S-4,5 mutant but not the S-3,5 mutant. This result was confirmed by
erythroagglutinating phytohemagglutinin lectin blot analysis. The reduction in
cell adhesion was consistently observed in the S-4,5 mutant but not in the
S-3,5 mutant cells. Furthermore mutation of site-4 alone resulted in a
substantial decrease in erythroagglutinating phytohemagglutinin lectin
staining and suppression of cell spread induced by GnT-III compared with that
of either the site-3 single mutant or wild-type α5. These results, taken
together, strongly suggest that N-glycosylation of site-4 on the
α5 subunit is the most important site for its biological functions. To
our knowledge, this is the first demonstration that site-specific modification
of N-glycans by a glycosyltransferase results in functional
regulation.Glycosylation is a crucial post-translational modification of most secreted
and cell surface proteins (1).
Glycosylation is involved in a variety of physiological and pathological
events, including cell growth, migration, differentiation, and tumor invasion.
It is well known that glycans play important roles in cell-cell communication,
intracellular signal transduction, protein folding, and stability
(2,
3).Integrins comprise a family of receptors that are important for cell
adhesion. The major function of integrins is to connect cells to the
extracellular matrix, activate intracellular signaling pathways, and regulate
cytoskeletal formation (4).
Integrin α5β1 is well known as a fibronectin
(FN)3 receptor. The
interaction between integrin α5 and FN is essential for cell migration,
cell survival, and development
(5–8).
In addition, integrins are N-glycan carrier proteins. For example,
α5β1 integrin contains 14 and 12 putative N-glycosylation
sites on the α5 and β1 subunits, respectively. Several studies
suggest that N-glycosylation is essential for functional integrin
α5β1. When human fibroblasts were cultured in the presence of
1-deoxymannojirimycin, which prevents N-linked oligosaccharide
processing, immature α5β1 integrin appeared on the cell surface,
and FN-dependent adhesion was greatly reduced
(9). Treatment of purified
integrin α5β1 with N-glycosidase F, which cleaves between
the innermost N-acetylglucosamine (GlcNAc) and asparagine
N-glycan residues of N-linked glycoproteins, prevented the
inherent association between subunits and blocked α5β1 binding to
FN (10).A growing body of evidence indicates that the presence of the appropriate
oligosaccharide can modulate integrin activation.
N-Acetylglucosaminyltransferase III (GnT-III) catalyzes the addition
of GlcNAc to mannose that is β1,4-linked to an underlying
N-acetylglucosamine, producing what is known as a
“bisecting” GlcNAc linkage as shown in
Fig. 1B. GnT-III is
generally regarded as a key glycosyltransferase in N-glycan
biosynthetic pathways and contributes to inhibition of metastasis. The
introduction of a bisecting GlcNAc catalyzed by GnT-III suppresses additional
processing and elongation of N-glycans. These reactions, which are
catalyzed in vitro by other glycosyltransferases, such as
N-acetylglucosaminyltransferase V (GnT-V), which catalyzes the
formation of β1,6 GlcNAc branching structures
(Fig. 1B) and plays
important roles in tumor metastasis, do not proceed because the enzymes cannot
utilize the bisected N-glycans as a substrate. Introduction of the
bisecting GlcNAc to integrin α5 by overexpression of GnT-III resulted in
decreased in ligand binding and down-regulation of cell adhesion and migration
(11–13).
Contrary to the functions of GnT-III, overexpression of GnT-V promoted
integrin α5β1-mediated cell migration on FN
(14). These observations
clearly demonstrate that the alteration of N-glycan structure
affected the biological functions of integrin α5β1. Similarly
characterization of the carbohydrate moieties in integrin α3β1 from
non-metastatic and metastatic human melanoma cell lines showed that expression
of β1,6 GlcNAc branched structures was higher in metastatic cells
compared with non-metastatic cells, confirming the notion that the β1,6
GlcNAc branched structure confers invasive and metastatic properties to cancer
cells. In fact, Partridge et al.
(15) reported that
GnT-V-modified N-glycans containing
poly-N-acetyllactosamine, the preferred ligand for galectin-3, on
surface receptors oppose their constitutive endocytosis, promoting
intracellular signaling and consequently cell migration and tumor
metastasis.Open in a separate windowFIGURE 1.Potential N-glycosylation sites on the α5 subunit and its
modification by GnT-III and GnT-V. A, schematic diagram of
potential N-glycosylation sites on the α5 subunit. Putative
N-glycosylation sites are indicated by triangles, and point
mutations are indicated by crosses (N84Q, N182Q, N297Q, N307Q, N316Q,
N524Q, N530Q, N593Q, N609Q, N675Q, N712Q, N724Q, N773Q, and N868Q).
B, illustration of the reaction catalyzed by GnT-III and GnT-V.
Square, GlcNAc; circle, mannose. TM, transmembrane
domain.In addition, sialylation on the non-reducing terminus of N-glycans
of α5β1 integrin plays an important role in cell adhesion. Colon
adenocarcinomas express elevated levels of α2,6 sialylation and
increased activity of ST6GalI sialyltransferase. Elevated ST6GalI positively
correlated with metastasis and poor survival. Therefore, ST6GalI-mediated
hypersialylation likely plays a role in colorectal tumor invasion
(16,
17). In fact, oncogenic
ras up-regulated ST6GalI and, in turn, increased sialylation of
β1 integrin adhesion receptors in colon epithelial cells
(18). However, this is not
always the case. The expression of hyposialylated integrin α5β1 was
induced by phorbol esterstimulated differentiation in myeloid cells in which
the expression of the ST6GalI was down-regulated by the treatment, increasing
FN binding (19). A similar
phenomenon was also observed in hematopoietic or other epithelial cells. In
these cells, the increased sialylation of the β1 integrin subunit was
correlated with reduced adhesiveness and metastatic potential
(20–22).
In contrast, the enzymatic removal of α2,8-linked oligosialic acids from
the α5 integrin subunit inhibited cell adhesion to FN
(23). Collectively these
findings suggest that the interaction of integrin α5β1 with FN is
dependent on its N-glycosylation and the processing status of
N-glycans.Because integrin α5β1 contains multipotential
N-glycosylation sites, it is important to determine the sites that
are crucial for its biological function and regulation. Recently we found that
N-glycans on the β-propeller domain (sites 3, 4, and 5) of the
integrin α5 subunit are essential for α5β1
heterodimerization, cell surface expression, and biological function
(24). In this study, to
further investigate the underlying molecular mechanism of GnT-III-regulated
biological functions, we characterized the N-glycans on the α5
subunit in detail using genetic and biochemical approaches and found that
site-4 is a key site that can be specifically modified by GnT-III. 相似文献
12.
Background
Matrix metalloproteinase-2 (MMP-2) is a key regulator in the migration of tumor cells. αvβ3 integrin has been reported to play a critical role in cell adhesion and regulate the migration of tumor cells by promoting MMP-2 activation. However, little is known about the effects of MMP-2 on αvβ3 integrin activity and αvβ3 integrin-mediated adhesion and migration of tumor cells.Methodology/Principal Findings
Human melanoma cells were seeded using an agarose drop model and/or subjected to in vitro analysis using immunofluorescence, adhesion, migration and invasion assays to investigate the relationship between active MMP-2 and αvβ3 integrin during the adhesion and migration of the tumor cells. We found that MMP-2 was localized at the leading edge of spreading cells before αvβ3 integrin. αvβ3 integrin-mediated adhesion and migration of the tumor cells were inhibited by a MMP-2 inhibitor. MMP-2 cleaved fibronectin into small fragments, which promoted the adhesion and migration of the tumor cells.Conclusion/Significance
MMP-2 cleaves fibronectin into small fragments to enhance the adhesion and migration of human melanoma cells mediated by αvβ3 integrin. These results indicate that MMP-2 may guide the direction of the tumor cell migration. 相似文献13.
S. Fabio Falsone Andreas J. Kungl Angelika Rek Roberto Cappai Klaus Zangger 《The Journal of biological chemistry》2009,284(45):31190-31199
α-Synuclein is an intrinsically unstructured protein that binds to membranes, forms fibrils, and is involved in neurodegeneration. We used a reconstituted in vitro system to show that the molecular chaperone Hsp90 influenced α-synuclein vesicle binding and amyloid fibril formation, two processes that are tightly coupled to α-synuclein folding. Binding of Hsp90 to monomeric α-synuclein occurred in the low micromolar range, involving regions of α-synuclein that are critical for vesicle binding and amyloidogenesis. As a consequence, both processes were affected. In the absence of ATP, the accumulation of non-amyloid α-synuclein oligomers prevailed over fibril formation, whereas ATP favored fibril growth. This suggests that Hsp90 modulates the assembly of α-synuclein in an ATP-dependent manner. We propose that Hsp90 affects these folding processes by restricting conformational fluctuations of α-synuclein. 相似文献
14.
15.
Faith Hall-Glenn Armen Aivazi Lusi Akopyan Jessica R. Ong Ruth R. Baxter Paul D. Benya Roel Goldschmeding Frans A. van Nieuwenhoven Ernst B. Hunziker Karen M. Lyons 《Journal of cell communication and signaling》2013,7(3):219-230
CCN2 (connective tissue growth factor (CTGF/CCN2)) is a matricellular protein that utilizes integrins to regulate cell proliferation, migration and survival. The loss of CCN2 leads to perinatal lethality resulting from a severe chondrodysplasia. Upon closer inspection of Ccn2 mutant mice, we observed defects in extracellular matrix (ECM) organization and hypothesized that the severe chondrodysplasia caused by loss of CCN2 might be associated with defective chondrocyte survival. Ccn2 mutant growth plate chondrocytes exhibited enlarged endoplasmic reticula (ER), suggesting cellular stress. Immunofluorescence analysis confirmed elevated stress in Ccn2 mutants, with reduced stress observed in Ccn2 overexpressing transgenic mice. In vitro studies revealed that Ccn2 is a stress responsive gene in chondrocytes. The elevated stress observed in Ccn2−/− chondrocytes is direct and mediated in part through integrin α5. The expression of the survival marker NFκB and components of the autophagy pathway were decreased in Ccn2 mutant growth plates, suggesting that CCN2 may be involved in mediating chondrocyte survival. These data demonstrate that absence of a matricellular protein can result in increased cellular stress and highlight a novel protective role for CCN2 in chondrocyte survival. The severe chondrodysplasia caused by the loss of CCN2 may be due to increased chondrocyte stress and defective activation of autophagy pathways, leading to decreased cellular survival. These effects may be mediated through nuclear factor κB (NFκB) as part of a CCN2/integrin/NFκB signaling cascade.
Electronic supplementary material
The online version of this article (doi:10.1007/s12079-013-0201-y) contains supplementary material, which is available to authorized users. 相似文献16.
Brassart-Pasco S Sénéchal K Thevenard J Ramont L Devy J Di Stefano L Dupont-Deshorgue A Brézillon S Feru J Jazeron JF Diebold MD Ricard-Blum S Maquart FX Monboisse JC 《PloS one》2012,7(4):e29587
Background
NC1 domains from α1, α2, α3 and α6(IV) collagen chains were shown to exert anti-tumor or anti-angiogenic activities, whereas the NC1 domain of the α4(IV) chain did not show such activities so far.Methodology/Principal Findings
We demonstrate in the present paper that the NC1 α4(IV) domain exerts a potent anti-tumor activity both in vitro and in an experimental human melanoma model in vivo. The overexpression of NC1 α4(IV) in human UACC-903 melanoma cells strongly inhibited their in vitro proliferative (–38%) and invasive (–52%) properties. MT1-MMP activation was largely decreased and its cellular distribution was modified, resulting in a loss of expression at the migration front associated with a loss of migratory phenotype. In an in vivo xenograft model in athymic nude mice, the subcutaneous injection of NC1 α4(IV)-overexpressing melanoma cells induced significantly smaller tumors (–80% tumor volume) than the Mock cells, due to a strong inhibition of tumor growth. Exogenously added recombinant human NC1 α4(IV) reproduced the inhibitory effects of NC1 α4(IV) overexpression in UACC-903 cells but not in dermal fibroblasts. An anti-αvβ3 integrin blocking antibody inhibited cell adhesion on recombinant human NC1 α4(IV) substratum. The involvement of αvβ3 integrin in mediating NC1 α4(IV) effect was confirmed by surface plasmon resonance (SPR) binding assays showing that recombinant human NC1 α4(IV) binds to αvβ3 integrin (KD = 148±9.54 nM).Conclusion/Significance
Collectively, our results demonstrate that the NC1 α4(IV) domain, named tetrastatin, is a new endogenous anti-tumor matrikine. 相似文献17.
Nicholas J. Anthis Jacob R. Haling Camilla L. Oxley Massimiliano Memo Kate L. Wegener Chinten J. Lim Mark H. Ginsberg Iain D. Campbell 《The Journal of biological chemistry》2009,284(52):36700-36710
Integrins are large membrane-spanning receptors fundamental to cell adhesion and migration. Integrin adhesiveness for the extracellular matrix is activated by the cytoskeletal protein talin via direct binding of its phosphotyrosine-binding-like F3 domain to the cytoplasmic tail of the β integrin subunit. The phosphotyrosine-binding domain of the signaling protein Dok1, on the other hand, has an inactivating effect on integrins, a phenomenon that is modulated by integrin tyrosine phosphorylation. Using full-length tyrosine-phosphorylated 15N-labeled β3, β1A, and β7 integrin tails and an NMR-based protein-protein interaction assay, we show that talin1 binds to the NPXY motif and the membrane-proximal portion of β3, β1A, and β7 tails, and that the affinity of this interaction is decreased by integrin tyrosine phosphorylation. Dok1 only interacts weakly with unphosphorylated tails, but its affinity is greatly increased by integrin tyrosine phosphorylation. The Dok1 interaction remains restricted to the integrin NPXY region, thus phosphorylation inhibits integrin activation by increasing the affinity of β integrin tails for a talin competitor that does not form activating membrane-proximal interactions with the integrin. Key residues governing these specificities were identified by detailed structural analysis, and talin1 was engineered to bind preferentially to phosphorylated integrins by introducing the mutation D372R. As predicted, this mutation affects talin1 localization in live cells in an integrin phosphorylation-specific manner. Together, these results indicate that tyrosine phosphorylation is a common mechanism for regulating integrin activation, despite subtle differences in how these integrins interact with their binding proteins. 相似文献
18.
Heide Behrmann Anna Lürick Anne Kuhlee Henning Kleine Balderhaar Cornelia Br?cker Daniel Kümmel Siegfried Engelbrecht-Vandré Ulrich Gohlke Stefan Raunser Udo Heinemann Christian Ungermann 《The Journal of biological chemistry》2014,289(48):33503-33512
Membrane fusion at the vacuole, the lysosome equivalent in yeast, requires the HOPS tethering complex, which is recruited by the Rab7 GTPase Ypt7. HOPS provides a template for the assembly of SNAREs and thus likely confers fusion at a distinct position on vacuoles. Five of the six subunits in HOPS have a similar domain prediction with strong similarity to COPII subunits and nuclear porins. Here, we show that Vps18 indeed has a seven-bladed β-propeller as its N-terminal domain by revealing its structure at 2.14 Å. The Vps18 N-terminal domain can interact with the N-terminal part of Vps11 and also binds to lipids. Although deletion of the Vps18 N-terminal domain does not preclude HOPS assembly, as revealed by negative stain electron microscopy, the complex is instable and cannot support membrane fusion in vitro. We thus conclude that the β-propeller of Vps18 is required for HOPS stability and function and that it can serve as a starting point for further structural analyses of the HOPS tethering complex. 相似文献
19.
Seakwoo Lee Kyle Bowrin Abdel Rahim Hamad Shukti Chakravarti 《The Journal of biological chemistry》2009,284(35):23662-23669
During inflammation, circulating polymorphonuclear neutrophils (PMNs) receive signals to cross the endothelial barrier and migrate through the extracellular matrix (ECM) to reach the injured site. Migration requires complex and poorly understood interactions of chemokines, chemokine receptors, ECM molecules, integrins, and other receptors. Here we show that the ECM protein lumican regulates PMN migration through interactions with specific integrin receptors. Lumican-deficient (Lum−/−) mice manifest connective tissue defects, impaired innate immune response, and poor wound healing with reduced PMN infiltration. Lum−/− PMNs exhibit poor chemotactic migration that is restored with exogenous recombinant lumican and inhibited by anti-lumican antibody, confirming a role for lumican in PMN migration. Treatment of PMNs with antibodies that block β2, β1, and αM integrin subunits inhibits lumican-mediated migration. Furthermore, immunohistochemical and biochemical approaches indicate binding of lumican to β2, αM, and αL integrin subunits. Thus, lumican may regulate PMN migration mediated by MAC-1 (αM/β2) and LFA-1 (αL/β2), the two major PMN surface integrins. We detected lumican on the surface of peritoneal PMNs and not bone marrow or peripheral blood PMNs. This suggests that PMNs must acquire lumican during or after crossing the endothelial barrier as they exit circulation. We also found that peritoneal PMNs do not express lumican, whereas endothelial cells do. Taken together these observations suggest a novel endothelial lumican-mediated paracrine regulation of neutrophils early on in their migration path.Polymorphonuclear neutrophils (PMNs)3 play a major role in the development of inflammatory responses to host injury and infection. Their functions include destruction of invading bacteria and recruitment of macrophages and lymphocytes to the affected site (1). Circulating PMNs sense injury and pathogen signals, cross the vascular endothelium, and migrate to the target tissue; two series of events control this process. The first leads to the slowing down and adherence of circulating PMNs on the vascular endothelium followed by their transendothelial migration or extravasation and activation (2). The second controls the directional migration of PMNs to the injured site through the endothelial basement membrane, a specialized type of ECM, and subsequently the deeper interstitial ECM, along chemokine and cytokine gradients. Leukocyte-to-leukocyte and leukocyte-to-endothelium interactions are important before extravasation. These are mediated by interactions between selectins and their ligands and by β2 (MAC-1 and LFA-1) and β1 (VLA-4–6) integrin interactions with cell adhesion proteins ICAM and PECAM (3). The directional migration of PMNs through the ECM is a complex, multistep process that involves several α and β integrin interactions with ECM proteins. Thus far, a few basement membrane proteins, laminins, entactin, and fibronectin have been identified as specific ligands in regulating migration of PMNs after extravasation (4–6). Additional interstitial ECM proteins and their receptors that modulate PMN migration have yet to be identified. Here we show that the ECM protein lumican is a novel regulator of PMN migration.Lumican is a secreted collagen-binding ECM protein of the corneal, dermal, and tendon stroma, arterial wall, and the intestinal submucosa (7–9). It is a member of the small leucine-rich repeat proteoglycans (10); these were initially investigated in the context of binding collagen and regulating tissue structure and biomechanics (11, 12). A body of literature is beginning to indicate that these proteoglycans interact with cytokines, growth factors, and cell surface receptors to modulate cell adhesion, proliferation, and migration (13–16). Lumican and biglycan, another member of this family of proteoglycans, have been recently shown to regulate host response to pathogen-associated molecular patterns (17, 18). Thus, lumican-deficient (Lum−/−) mice are hyporesponsive to bacterial lipopolysaccharide (LPS) endotoxins, and Lum−/− macrophages in culture produce lower levels of pro-inflammatory cytokines in response to LPS (18). Lumican facilitates innate immune response by binding LPS and CD14, the glycerol phosphatidylinositol-linked cell surface adaptor protein that transfers the LPS signal to toll-like receptor 4 (18). In a corneal injury model neutrophil influx is delayed in the Lum−/− mice (19, 20). Although this may be partly due to impaired innate immune response, it raises the possibility that lumican may have an additional role in neutrophil migration. Here we elucidate a role for lumican in PMN migration. We show that poor chemotactic migration of Lum−/− PMNs can be rescued by exogenous recombinant lumican (rLum) and blocked specifically with antibodies against lumican, β2, β1, and αM integrins. Our results also show that lumican localizes on the surface of extravasated PMNs through its interactions with β2 integrins. The likely source of lumican on neutrophils is the vascular endothelium. 相似文献