JBC Ideal method for primary cell transfection

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M705608200 on September 26, 2007

J. Biol. Chem., Vol. 282, Issue 48, 34929-34937, November 30, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
282/48/34929    most recent
M705608200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Google Scholar
Right arrow Articles by Saito, Y.
Right arrow Articles by Fukai, F.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Saito, Y.
Right arrow Articles by Fukai, F.

A Peptide Derived from Tenascin-C Induces β1 Integrin Activation through Syndecan-4*Formula

Yohei Saito{ddagger}, Hisae Imazeki{ddagger}, Shogo Miura{ddagger}, Tomohisa Yoshimura{ddagger}, Hiroaki Okutsu{ddagger}, Yosei Harada{ddagger}, Toshiyuki Ohwaki{ddagger}, Osamu Nagao{ddagger}, Sadahiro Kamiya{ddagger}, Ryo Hayashi§, Hiroaki Kodama§, Hiroshi Handa, Toshimichi Yoshida||, and Fumio Fukai{ddagger}1

From the {ddagger}Department of Molecular Patho-Physiology, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba 278-8510, Japan, the §Department of Biochemistry, Faculty of Science and Engineering, Saga University, Saga 849-0922, Japan, the Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan, and the ||Department of Pathology and Matrix Biology, Mie University Graduate School of Medicine, Mie 514-8507, Japan

Received for publication, July 9, 2007 , and in revised form, September 21, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Tenascin-C (TN-C) is unique for its cell adhesion modulatory function. We have shown that TNIIIA2, a synthetic 22-mer peptide derived from TN-C, stimulated β1 integrin-mediated cell adhesion of nonadherent and adherent cell types, by inducing activation of β1 integrin. The active site of TNIIIA2 appeared cryptic in the TN-C molecule but was exposed by MMP-2 processing of TN-C. The following results suggest that cell surface heparan sulfate (HS) proteoglycan (HSPG), including syndecan-4, participated in TNIIIA2-induced β1 integrin activation: 1) TNIIIA2 bound to cell surface HSPG via its HS chains, as examined by photoaffinity labeling; 2) heparitinase I treatment of cells abrogated β1 integrin activation induced by TNIIIA2; 3) syndecan-4 was isolated by affinity chromatography using TNIIIA2-immobilized beads; 4) small interfering RNA-based down-regulation of syndecan-4 expression reduced TNIIIA2-induced β1 integrin activation, and consequent cell adhesion to fibronectin; 5) overexpression of syndecan-4 core protein enhanced TNIIIA2-induced activation of β1 integrin. However, treatments that targeted the cytoplasmic region of syndecan-4, including ectopic expression of its mutant truncated with the cytoplasmic domains and treatment with protein kinase C{alpha} inhibitor Gö6976, did not influence the TNIIIA2 activity. These results suggest that a TNIIIA2-related matricryptic site of the TN-C molecule, exposed by MMP-2 processing, may have bound to syndecan-4 via its HS chains and then induced conformational change in β1 integrin necessary for its functional activation. A lateral interaction of β1 integrin with the extracellular region of the syndecan-4 molecule may be involved in this conformation change.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Tenascin (TN)-C2 is one of the most intriguing extracellular matrix (ECM) proteins (1–3). TN-C is expressed predominantly during embryogenesis, wound healing, and neoplastic processes, in which alternative mRNA splicing within the fibronectin (FN) type III-like (FN-III) repeats can generate different TN-C isoforms (4). Multifunctional properties have been identified for TN-C, including effects on cell adhesion, migration, proliferation, survival, and differentiation. The effects of TN-C on cell adhesion are particularly complex; the TN-C substrate supports attachment of some cell types, but is nonadhesive or even repulsive for other cell types (5–7). Based on these antipodal effects on cell adhesion, TN-C is multifunctional and is therefore classified as an adhesion modulatory ECM protein, a so-called "matricellular" protein (8). Various TN-C molecule domains, especially FN-III repeats, including the alternative splicing domains, have been implicated in its function as a matricellular protein. However, their contributions to the adhesion modulatory effects of TN-C are not completely understood.

Interactions of cells with the ECM are largely mediated by members of the integrin superfamily of adhesive receptors. The most unique feature of integrins is their ability to alter ligand binding and signaling activities. Because integrin-mediated cell-ECM interactions play key roles in maintaining normal cellular functions, affinity changes in integrins are critical for the anchorage-dependent cellular processes, such as growth, survival, migration, and differentiation. Integrin activation is considered to be regulated by "inside-out" signals from the cell interior, that is, those triggered by extracellular stimuli. However, the molecular pathways leading to inside-out activation of integrins are still poorly understood.

There have been many studies indicating that ECM proteins such as FNs, laminins, and collagens have biologically active cryptic sites. These matricryptic sites are exposed through biological processes such as proteolytic cleavage and conformational change in response to multimerization of ECM proteins, binding to other molecules, or cell-mediated mechanical forces (9, 10). Davis et al. (11) coined the term "matricryptins" to describe biologically active proteolytic fragments of ECM proteins. Matricryptic sites and matricryptins of ECM proteins have been implicated in a variety of events governed by cell-ECM interactions.

We have previously found that FN has both cell adhesion sites and a matricryptic site opposing cell adhesion. The 22-mer matricryptin, FNIII14, derived from the 14th FN-III repeat, strongly suppresses β1 integrin-mediated cell adhesion to FN, whose activity depends on its C-terminal amino acid sequence, YTIYVIAL (12). This cryptic antiadhesive site is exposed by either FN degradation with MMP-2, or FN interaction with heparin (13). As a negative modulator of cell-ECM interaction, FNIII14 influences physiological cellular processes such as survival (14) and differentiation (15), as well as pathological events such as tumor metastasis (16).

There are several sequences similar to the YTIYVIAL sequence of FN in other ECM proteins including TN-C. Two analogous sequences, YTITIRGV and YTIYLNGD, are present in the FN-III repeat A2 of the alternative splicing region and the C terminus fibrinogen-globe, respectively, of the human TN-C molecule (supplemental Fig. S1). These analogous sequences may be involved in TN-C cell adhesion modulatory activity. We have investigated the effects of synthetic TN-C peptides containing these analogous sequences on cell adhesion to FN. Surprisingly, TNIIIA2, a 22-mer TN-C peptide containing YTITIRGV, stimulated cell adhesion to FN by inducing conformational and functional activation of β1 integrin. The active site of TNIIIA2 appears cryptic but was exposed by MMP-2 processing. Some of our results suggest that cell surface heparan sulfate (HS) proteoglycans (HSPG), including syndecan-4, participated in β1 integrin activation in response to TNIIIA2, without requiring its cytoplasmic domains. Thus, a TNIIIA2-related matricryptic site of TN-C molecule, exposed by MMP-2 processing, may induce a lateral interaction of β1 integrin with the syndecan-4 ectodomain, resulting in conformational change in β1 integrin necessary for its functional activation. The cell adhesion-modulatory activity of TN-C, at least in part, may be due to the TNIIIA2-related matricryptic site/matricryptin.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture, Transfection, and Treatment with Enzymes or Methyl-β-cyclodextrin—SV40-transformed human embryonic lung fibroblasts WI38VA13 and normal mouse fibroblasts NIH3T3 were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 10% CS, respectively. Human erythroleukemia cells K562 and human Burkitt's lymphoma cells Ramos were cultured in RPMI1640 containing 10% fetal calf serum. Human umbilical vein endothelial cells (HUVEC) (Clontecs, Sanko Junyaku Co.) were grown and treated as described earlier (16). WI38VA13 cells were treated with 0.1 unit/ml of heparitinase I and 0.5 unit/ml of chondroitinase ABC (Seikagaku Kogyo) at 37 °C for 30 min. Plasmid DNAs for syndecan-4 (S4, S4{Delta}R) were kindly provided by Dr. John R. Couchman (Imperial College London). At 24 h following transfection using Transfast (Promega), NIH3T3 cells were diluted and grown in medium containing 1 mg/ml G418 (Calbiochem) to select clones stably expressing syndecan-4. Overexpression of syndecan-4 by the clones was confirmed by immunoblot analysis using anti syndecan-4 mAb (N-19).

Peptides—The GRGDSP was purchased from IWAKI. Peptides derived from human FN (FNIII14: TEATITGLEPGTEYTIYVIAL), TN-C (TNIIIA2), their mutants (supplemental Fig. S1 and supplemental Table S1), and CS-1 (LHPGEILDVPST) were synthesized using the solid phase strategy combined with the Boc and Fmoc chemistry, in which a Cys was added to the C terminus of each peptide to increase their activity by dimerization and to facilitate coupling to SG-beads. For a photoaffinity cross-linking of a putative cell surface receptor of TNIIIA2, a photosensitive derivative of TNIIIA2, RSTDLPGLKAAT-p-benzoyl-phenylalanine (Bpa)-YTITIRGVK (biotin) C (biotinylated TNIIIA2-Bpa), which has been shown to retain the TNIIIA2 activity (17), was synthesized using standard solid state methodology (18). These synthetic peptides were purified by reversed-phase HPLC and characterized by mass spectrometry.

Antibodies—Function-blocking mAbs against integrin subunits {alpha}4 (P1H4), {alpha}5 (P1D6), and β2 (WT.3) were purchased from CHEMICON, while that against human integrin subunit β1 (DE9) was from UPSTATE and that against mouse integrin subunit β1 (Ha2/5) was from BD Biosciences. mAbs recognizing the active conformation of integrin subunit β1, i.e. AG89 (MBL), HUTS-4 (CHEMICON), and 9EG7 (BD Biosciences), were purchased as noted. Antibodies reactive with the heparan sulfate (HS) chain (10E4) and an anti-{Delta}HS mAb (3G10) recognizing a neoepitope generated by heparitinase digestion of HSPG were purchased from Seikagaku Kogyo. Anti-syndecan-4 (N-19, 5G9), anti-syndecan-1(C-20), and anti-β1 integrin (M106) antibodies were from Santa Cruz Biotechnology. Anti-VCAM-1 mAb was from BD Biosciences.

Confocal Microscopy—Cells were seeded on FN-coated cover glasses, incubated with or without TNIIIA2 for 1 h, fixed with 4% paraformaldehyde and permeabilized with 0.03% Triton X-100. For immunostaining of β1 integrins, the cells were not permeabilized. The cells were stained with anti-vinculin Ab (Simga) and rhodamine-phalloidin (Invitrogen), or an anti-integrin β1 mAb (AG89 and HUTS-4), and mounted on slides. Confocal microscope images were obtained with an inverted microscope (Fluoview FV1000, Olympus) fitted with a x60 UPlanSApo oil-immersion objective (NA 1.35) and Fluoview software.

Cell Adhesion Assay—Cell adhesion assay was performed as described previously (19). Ramos cell adhesion to HUVEC was performed as reported previously (16).

Flow Cytometric Analysis of β1 Integrin Activation—Activation of β1 integrins on the cells was evaluated by flow cytometric analysis (FACS Calibur and FACS Aria: Becton Dickinson; Cytoron: Ortho Diagnostics) using three different mAbs, i.e. AG89, HUTS-4 and 9EG7, recognizing the active β1 integrin conformation-specific epitope, as described previously (13).

Recombinant TN-C Protein and Its Processing with Proteinases—A recombinant protein containing the FN-III repeats A1–4 of human TN-C (rA1–4) was prepared as described previously (20). Briefly, cDNA encoding the region was generated by PCR using human fetal brain Marathon-Ready cDNA (BD Biosciences Clontech) as the template and primers consisting of 5'-AGTGGATCCACTGAACAAGCCCCTGAGC-3' and 5'-CCCAAGCTTGGGCAGTTCGTTCAGCACCAGAGA-3'. rA1–4 was processed in a dialysis bag (Sanko) at room temperature as follows. rA1-4 mixed with trypsin (1:200, w/w) was dialyzed first against PBS(-) for 6–24 h, next against PBS(-) containing 1 mM diisopropyl fluorophosphate and then against the serum-free medium. rA1–4 protein mixed with MMP-2 (1:100, w/w) and 2 mM p-aminophenylmercuric acetate was dialyzed first against the buffer containing 2 mM CaCl2 for 6–24 h, next against the buffer containing MMP-2 inhibitor GM6001 (10 µM) and then against the serum-free medium. As a control, rA1–4 protein was treated under the same conditions without MMP-2.

Photoaffinity Labeling—WI38VA13 cells (3 x 105) spread on a 6-well plate were incubated with biotinylated TNIIIA2-Bpa (5 µg/ml) at 37 °C for 1 h in the presence or absence of heparin (20 µg/ml) and then irradiated with UV (two 15-watt lamps, 365 nm) for 30 min on ice at a distance of 10 cm. Cells were dissolved with 1 ml of lysis buffer (10 mM Tris-HCl buffer (pH 7.4) containing 0.15 M NaCl, 1% Nonidet P-40 and 2 mM phenylmethylsulfonyl fluoride). Aliquots of the lysates were subjected to immunoblot analysis using avidin-conjugated peroxidase. Another aliquot of the lysate was mixed with avidin-conjugated beads to precipitate the biotinylated TNIIIA2-Bpa-linked molecules. After washing the beads extensively with the lysis buffer, the bound materials were eluted with Laemmli's buffer and then subjected to immunoblot analysis using anti-HS and anti-β1 (DE9) mAbs.

Affinity Chromatography—WI38VA13 cells (1 x 109) dissolved with 10 mM Tris-HCl buffer, pH 7.4 containing 0.15 M NaCl, 4 M urea, and 2 mM phenylmethylsulfonyl fluoride was incubated at 4 °C for 16 h with TNIIIA2-immobilized SG beads, which have been successfully used as an affinity resin showing minimal nonspecific interactions (21). After washing the beads with the lysis buffer containing 4 M urea, the bound materials were eluted with 1 M NaCl. To digest GAG chains, the purified sample was dialyzed (10 mM Tris-HCl, pH 7.0, 20 mM sodium acetate, 0.1% CHAPS) and then treated with heparitinase I (0.1 milliunits/ml) and chondroitinase ABC (1 unit/ml) at room temperature for 18 h, and then subjected to immunoblot analysis using anti-{Delta}HS (3G10), anti-syndecan-4 (D-16 and N-19), and anti-syndecan-1 (C-20) mAbs.

Syndecan-4 Knockdown by RNAi—siRNAs of syndecan-4 (340CACCGAACCCAAGAAACTA358), (714GGGTGAGGTCAACCTAATA732) (GenBankTM/EMBL/DDBJ accession no. D13292) and the negative control siRNA were obtained from RNAi, Japan. The siRNAs were transfected into cells by using Lipofectamine 2000 (Invitrogen), and the cells were used for experiments at 72 h after transfection. RNAi-mediated knockdown of syndecan-4 expression was verified by flow cytometry and immunoblot analysis using anti-syndecan-4 mAb (5G9).


Figure 1
View larger version (92K):
[in this window]
[in a new window]

  		FIGURE 1.
Stimulation of cell adhesion to FN by TNIIIA2. A, WI38VA13 cells were allowed to adhere on FN for 1 h in the absence (Control) or presence of Tnfbg and TNIIIA2 or its control TNIIIA2scr at 25 µg/ml. The adhered cells were stained with crystal violet. Similar results were obtained in four independent experiments. Scale bars, 100 µm. B, WI38VA13 cells adhered to FN in the absence (upper panels) or presence (lower panels) of TNIIIA2 (25 µg/ml) were fixed, permeabilized, and then incubated with anti-vinculin (panels a and d) or rhodamine-phalloidin (panels b and e). Photographs c and f are merged images. Data shown are representative of four individual experiments. Scale bars, 20 µm.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Stimulation of Cell Adhesion to FN by a TN-C-derived Peptide—We first examined whether synthetic TN-C peptides containing analogous sequences, YTITIRGV and YTIYLNGD in their C terminus, termed TNIIIA2 and TNfbg, respectively (supplemental Fig. S1), exerted any effect on cell adhesion to FN. WI38VA13 cells were seeded onto FN-coated plates, with or without one of the synthetic peptides. In the absence of these peptides, the cells began attaching to the FN substrate about 30 min after seeding, while many cells had not spread, even after 60 min (Fig. 1A). Focal contact did not appear in these attached cells (Fig. 1B). In the presence of TNIIIA2, most cells had attached completely within 60 min, and many had already spread on the FN (Fig. 1A). There was a concomitant appearance of focal contact and actin stress fiber reorganization (Fig. 1B). A control peptide, TNIIIA2scr, in which the YTITIRGV sequence is shuffled (RITYITGV), and TNfbg were inactive (Figs. 1A and 2A). TNIIIA2-stimulated cell adhesion in a dose-dependent manner, and this was inhibited by function-blocking mAbs directed to integrin subunits {alpha}4, {alpha}5, and β1, but not β2 (Fig. 2, A and B).

We then examined the effect of TNIIIA2 on adhesion of nonadherent hematopoietic progenitor cell lines, K562 (which exclusively expresses {alpha}5β1 as a β1 class integrin) and Ramos (which only expresses {alpha}4β1 of the β1 integrins), whose integrins are known to be in their inactive states. K562 cells attached specifically to FN only in the presence of Mn2+, an integrin activator (Fig. 2C). TNIIIA2 was capable of inducing K562 cell attachment to FN without Mn2+, and this was blocked by mAbs anti-{alpha}5 and β1, but not {alpha}4 (Fig. 2D). TNIIIA2 also induced Ramos cell attachment to FN without Mn2+, and this was blocked by CS-1 and an anti-{alpha}4 mAb, but not by anti-{alpha}5 mAb (Fig. 2E). Ramos cells attached to HUVEC via {alpha}4β1-VCAM-1 interaction, for which Ramos cells and HUVEC had to be pretreated with Mn2+ and TNF-{alpha}, respectively (Fig. 2F). TNIIIA2 induced Ramos cell attachment onto HUVEC without Mn2+, in an {alpha}4β1-VCAM-1 interaction-specific manner (Fig. 2G). Thus, the effects of TNIIIA2 on nonadherent cell adhesion lead us to speculate that this peptide may induce activation of β1 integrin.


Figure 2
View larger version (20K):
[in this window]
[in a new window]

  		FIGURE 2.
Effects of TNIIIA2 on integrin-mediated cell adhesion. WI38VA13 (A and B), K562 (C and D), and Ramos (E–G) cells suspended in serum-free medium (1 x 104/ml) in the presence of TNIIIA2 or TNIIIA2scr (50 µg/ml) were seeded with or without Mn2+ (1 mM), anti-integrin mAb (20 µg/ml), or CS-1 peptide (200 µg/ml) into a 96-well plates coated with the FN (0.5 µg/ml in A and B;5 µg/ml in C–E). F and G, Ramos cells were seeded onto a HUVEC monolayer which was prepared on a 48-well plate and treated as indicated. HUVEC were pretreated with or without TNF-{alpha} (10 ng/ml) for 24 h (16). In all experiments, cells were allowed to adhere for 1 h, and the number of cells either attached (white bars) or spread (gray bars) was counted as described previously (12). Each point represents the mean ± S.E. of triplicate determinations. One of three individual experiments is shown.

 
TNIIIA2 Induces Activation of β1 Integrin—The status of β1 integrin activation can be evaluated by its accessibility to anti-β1 integrin mAbs, such as AG89 (22) and HUTS-4 (23), both of which recognize the active conformation-specific epitope of β1 subunit, regardless of the {alpha} subunit. We performed flow cytometric analysis with those mAbs to investigate whether TNIIIA2 was able to influence the conformational status of β1 integrin (Fig. 3, A and B).

Unstimulated K562 cells showed only low accessibility to AG89 (Fig. 3A, panel a), while Mn2+ stimulation slightly increased expression of the AG89 epitope (Fig. 3A, panel b). Addition of GRGDSP alone, a ligand of integrin {alpha}5β1, also caused a slight increase in expression of the AG89 epitope (Fig. 3A, panel c), and GRGDSP together with Mn2+ further increased the AG89 epitope expression (Fig. 3A, panel d). TNIIIA2 induced a remarkable increase in expression of the AG89 epitope in a dose-dependent manner even without Mn2+ (Fig. 3, A, panels e and f). Addition of GRGDSP together with TNIIIA2 caused a further but slight increase in expression of the AG89 epitope (Fig. 3A, panel g). TNIIIA2 treatment (at least within 3 h) did not change expression of the β1 integrin subunit under the experimental conditions used here, as confirmed by both flow cytometric and immunoblot analyses (data not shown). Expression of the active β1-specific epitope, in response to TNIIIA2, was similarly confirmed by using either another mAb (HUTS-4) (supplemental Fig. S2A) or another nonadherent cell line, Ramos (supplemental Fig. S2B). Conformational change in the β1 integrin was also investigated using an adherent cell type WI38VA13. The results, characterized by a dramatic conformational change in response to TNIIIA2, were almost the same as those observed with nonadherent cell types, confirmed by using AG89 (Fig. 3B) and HUTS-4 (data not shown). Thus, in both nonadherent and adherent cell types, expression of the AG89 and HUTS-4 epitopes in response to TNIIIA2 correlated well with the effects of this peptide on cell adhesion to FN or HUVEC, suggesting that TNIIIA2 stimulated β1 integrin-mediated cell adhesion by inducing β1 integrin activation.

We next performed immunofluorescence microscopic analysis of β1 integrin, by using the HUTS-4 and AG89 mAbs. Without stimulation, only a low level of β1 integrins expressed the AG89 epitope on K562 cells (Fig. 3C). When the cells were stimulated with TNIIIA2, the dots of punctate staining appeared intensely on the basal surfaces, with a concomitant increase in the number of K562 cells attached to the FN (Fig. 3C). On the other hand, expression of the HUTS-4 epitope was detected peripherally on WI38VA13 cells spreading on the FN (Fig. 3D, left), but became much more remarkable after incubation with TNIIIA2 (Fig. 3D, right). The results show that TNIIIA2 induces a net increase in the quantity of activated β1 integrin on cell surfaces.

Active Site of TNIIIA2 Is Cryptic in the TN-C Molecule—To characterize the active site of TNIIIA2 in the TN-C molecule, we first performed alanine-scanning mutagenesis to identify the amino acid residues in the sequence YTITIRGV, which was essential for the TMIIIA2 activity. Supplemental Table S1 presents the mutant peptides tested and summarizes the results of their abilities to induce β1 integrin activation, as evaluated by flow cytometric analysis using AG89 (data not shown). The TNIIIA2 activity was highly sensitive to single Ala replacement of the two Ile and Val residues at positions 16, 18, and 21, respectively, and the resultant peptides were inactive for β1 integrin activation. The replacement of Arg19 with Ala caused partial reduction in TNIIIA2 activity, while alternative replacement of Arg19 with Glu, resulted in complete loss of the activity, suggesting the necessity of a positive net charge, as well as specific amino acids (two Ile and Val) for β1 integrin activation in this peptide.


Figure 3
View larger version (28K):
[in this window]
[in a new window]

  		FIGURE 3.
TNIIIA2 induces conformational activation of β1 integrins. K562 (A) or WI38VA13 (B) cells suspended in serum-free medium (5 x 104/ml) were incubated with vehicle (panel a), Mn2+ (1 mM)(panel b), GRGDSP (500 µg/ml) (panel c), Mn2+ plus GRGDSP (panel d), TNIIIA2 at 15 µg/ml (panel e) or 30 µg/ml (panel f), or TNIIIA2 (30 µg/ml) plus GRGDSP (panel g) for 30 min with occasional shaking. Flow cytometric analyses were performed using the FITC-labeled anti-β1 integrin mAb, AG89, as described under "Experimental Procedures." Data shown are representative of three individual experiments. C and D, confocal microscopic observation of active β1 integrins on K562 (C) or WI38VA13 (D) cell surfaces. Cells were plated on FN-coated cover glasses for 1 h in the absence (left panels, Control) or presence (right panels, +TNIIIA2) of TNIIIA2 (25 µg/ml), fixed, washed to remove unadhered cells and then incubated with FITC-AG89 (C) or FITC-HUTS-4 (D). Similar results were obtained in three independent experiments. Scale bars, 50 µm.

 


Figure 4
View larger version (35K):
[in this window]
[in a new window]

  		FIGURE 4.
Exposure of the matricryptic site by processing of rA1–4 protein with MMP-2. rA1–4 was processed with trypsin or MMP-2 as described under "Experimental Procedures" and then examined for cell adhesion assay (A) or flow cytometric analysis of β1 integrin activation (B) using K562 cells. A, effects of TNIIIA2 (20 µg/ml), rA1–4 (50 µg/ml), and rA1–4 processed with trypsin or MMP-2 on K562 cell adhesion (attachment) to FN without Mn2+ were investigated as in Fig. 3. Each point represents the mean ± S.E. of triplicate determinations. One of two individual experiments is shown. B, effects of rA1–4 (50 µg/ml) or rA1–4 processed with MMP-2 (12 h) on β1 integrin activation in K562 cells were examined as in Fig. 3. Similar results were obtained in two independent experiments.

 
To confirm whether the active site of TNIIIA2 is exposed on the TN-C molecule, we examined the effects of a recombinant protein composed of the FN-III repeats A1–4 (rA1–4) (supplemental Fig. S1) on K562 cell attachment to the FN substrate. K562 cell attachment to FN was not affected by rA1–4, while processing of rA1–4 with MMP-2, but not trypsin, caused a significant increase in K562 cell attachment to FN, even without Mn2+ (Fig. 4A). The MMP-2 digest of rA1–4 also weakly induced β1 integrin activation (Fig. 4B). Thus, the active site appears to be buried within the TN-C molecule, but exposed by processing with MMP-2.

Involvement of Syndecan-4 in Expression of TNIIIA2 Activity—Assuming the presence of a membrane receptor mediating TNIIIA2 activity, we attempted to detect a cell surface molecule, with specific binding affinity toward TNIIIA2, by affinity labeling. We did not detect such a molecule by using conventional cross-linking chemicals containing succinimide, carbodiimide, or azide groups (data not shown). These can covalently connect peptide with protein based on its reactivity against a primary amine. However, we successfully detected the molecule by photoaffinity labeling, using a photosensitive TNIIIA2 derivative containing the benzoyl group, biotinylated TNIIIA2-Bpa, which strongly reacts with macromolecules without primary amines, such as sugars (24).

By photoaffinity labeling of WI38VA13 cells using this photoreactive probe, a high molecular mass band (>200 kDa) and two bands of 110 and 70 kDa, were detected with avidin-conjugated peroxidase (Fig. 5A). Among these, only the high molecular mass band resulted from specific binding with biotinylated TNIIIA2-Bpa (Fig. 5A, lane 2), because the 110- and 70-kDa bands were visualized nonspecifically, even in the absence of biotinylated TNIIIA2-Bpa (Fig. 5A, lane 1). The high molecular mass band tagged with biotinylated TNIIIA2-Bpa was precipitated with avidin-immobilized beads and analyzed by immunoblot analysis. A high molecular mass band was also visualized by anti-HS mAb (Fig. 5B, lanes 1 and 2). When the affinity labeling was done in the presence of heparin, the band disappeared (Fig. 5, lanes 3 of A and B). TNIIIA2 was shown to bind to heparin with relatively high affinity (Kd = 1.1 x 10-7 M), compared with chondroitin sulfate (Kd = 4.9 x 10-7 M) and hyaluronic acid (Kd > 10-5) (supplemental Fig. S3). Furthermore, β1 integrin activation in response to TNIIIA2 was abolished in the presence of heparin (supplemental Fig. S3). These results suggest that TNIIIA2 expresses its activity through specific binding to cell surface HSPG via its HS chain. Interestingly, β1 integrin was also detected within precipitates with avidin beads, but not within control precipitates, as a band migrating at a position different from that of high molecular mass HSPG (Fig. 5B, lanes 4 and 5). This suggests an induced association of β1 integrin with cell surface HSPG in response to TNIIIA2.

Affinity chromatography, using the TNIIIA2-immobilized SG beads, was performed to isolate HSPG which had specific binding affinity to TNIIIA2. Immunoblot analysis of the purified material using the anti-HS mAb, showed a diffuse band at around 200 kDa (Fig. 5C, lane 2). This purified HSPG(s) was treated with heparitinase I and chondroitinase ABC to identify it based on its core protein. Immunoblot analysis using an anti-{Delta}HS mAb (3G10) showed a major intense band of 30 kDa and several weak bands, including a high molecular mass band migrating near the original HSPG band (Fig. 5C, lane 3). The major band of 30 kDa was recognized by either of two different mAbs against syndecan-4 core protein (lanes 4 and 5), but not syndecan-1 (lane 6). This suggests that, among the cell surface HSPGs, syndecan-4 may have the greatest involvement in TNIIIA2-induced β1 integrin activation, although involvement of other types of HSPG cannot be excluded completely.

To verify the involvement of syndecan-4/HSPG in the expression of TNIIIA2 activity, we examined the effect of HS chain degradation on TNIIIA2-induced β1 integrin activation. Treatment of WI38VA13 cells with heparitinase I abrogated β1 integrin activation in response to TNIIIA2 (Fig. 6A). We then examined the effect of syndecan-4 knockdown by RNA interference (RNAi). Flow cytometric and immunoblot analyses showed that syndecan-4 expression on WI38VA13 cells was reduced by about 70% upon introduction of siRNA targeted against syndecan-4 core protein (Fig. 6B). In cells transfected with control siRNA, TNIIIA2 clearly induced β1 integrin activation (Fig. 6C, panel b) and cell adhesion (Fig. 6D, panel b), while syndecan-4 knockdown resulted in a remarkable reduction of that activation (Fig. 6C, panel d), with a concomitant decrease in cell adhesion/spreading to FN (Fig. 6D, panel d). These results suggest that the syndecan-4 core protein, and its extracellular HS chains are required for β1 integrin activation by TNIIIA2.


Figure 5
View larger version (34K):
[in this window]
[in a new window]

  		FIGURE 5.
Detection and isolation of a cell surface molecule which specifically binds to TNIIIA2 by photoaffinity labeling and affinity chromatography using TNIIIA2-immobilized SG-beads. A, photoaffinity labeling of WI38VA13 cells without (lane 1) or with biotinylated TNIIIA2-Bpa (lanes 2 and 3) was performed in either in the absence (lanes 1 and 2) or presence (lane 3) of heparin (20 µg/ml), as described under "Experimental Procedures." Cell lysates were subjected to immunoblot analysis using avidin-peroxidase to detect a biotinylated-TNIIIA2-Bpa-linked molecule(s). B, WI38VA13 cells tagged with biotinylated TNIIIA2-Bpa (Bi-TNIIIA2) under the indicated conditions were dissolved, precipitated with avidin-immobilized beads, and then subjected to immunoblot analysis using anti-HS antibody (lanes 1–3) or anti-β1 integrin mAb (DE9) (lanes 4 and 5). C, sample purified by affinity chromatography using the TNIIIA2-immobilized SG-beads was subjected to immunoblot analysis using normal mouse IgM (lane 1) or anti-HS IgM mAb (lane 2). Immunoblot analysis of the purified sample treated with heparitinase I (0.1 milliunits/ml) and chondroitinase ABC (1 units/ml) was performed using anti-{Delta}HS (lane 3), anti-syndecan-4 (D16 in lane 4, N19 in lane 5), or anti-syndecan-1 (lane 6) mAb. Data shown are representative of two individual experiments.

 


Figure 6
View larger version (22K):
[in this window]
[in a new window]

  		FIGURE 6.
Syndecan-4 participates in TNIIIA2-induced β1 integrin activation. A, requirement of HS chains for TNIIIA2-induced β1 integrin activation. WI38VA13 cells treated with or without heparitinase I (0.1 milliunits/ml) at 37 °C for 30 min were mixed with TNIIIA2 (25 µg/ml) and then examined for β1 integrin activation as in Fig. 2. Mean fluorescence intensity (MFI) is expressed in parentheses. Data are representative of three individual experiments. B–D, effect of syndecan-4 knockdown on expression of TNIIIA2 activity. WI38VA13 cells were transfected with control siRNA or syndecan-4 siRNA and then examined as follows. B, syndecan-4 expression levels in cells were evaluated by flow cytometry (upper panel) and immunoblot analysis (lower panel). Upper panel, thin line, without Ab; solid line, control siRNA with anti-syndecan4 (5G9); shaded histogram, syndecan-4 siRNA with anti-syndecan4 (5G9). Lower panel, cells were dissolved with the buffer containing 4 M urea, dialyzed, treated with heparitinase I and chondroitinase ABC, and then subjected to immunoblot analysis using anti-syndecan-4 core protein (5G9) as described under "Experimental Procedures." The syndecan-4 expression is knocked down over 70% by syndecan-4 siRNA as determined densitometrically. Similar results were obtained in three independent experiments. C and D, cells transfected with control siRNA (panels a and b) or syndecan-4 siRNA (panels c and d) were treated with (panels b and d) or without (panels a and c) TNIIIA2 (25 µg/ml) and then examined for β1 integrin activation using AG89 (C) or cell adhesion to FN (D). Similar results were obtained in three independent experiments. Scale bars, 100 µm.

 
The cytoplasmic region of syndecan-4 and its effector enzyme PKC{alpha} play an indispensable role in the syndecan-4-based signaling for focal contact and actin stress-fiber formation (25). We overexpressed syndecan-4 core protein or its mutant (S4{Delta}R), with complete deletion of the cytoplasmic domain (Fig. 7A). Overexpression of wild type syndecan-4 caused a significant increase in β1 integrin activation by TNIIIA2 (Fig. 7B, panel b). Unexpectedly, overexpression of S4{Delta}R did not reduce, but rather increased, β1 integrin activation in response to TNIIIA2 (Fig. 7B, panel c). Additionally, treating the cells with a specific PKC{alpha} inhibitor Gö6976 did not influence TNIIIA2-induced β1 integrin activation (Fig. 7C). These results suggest that activation of β1 integrin by TNIIIA2 may be dependent on cell surface HSPG including syndecan-4, but independent of its cytoplasmic region.


Figure 7
View larger version (26K):
[in this window]
[in a new window]

  		FIGURE 7.
TNIIIA2 induces β1 integrin activation independently of syndecan-4 cytoplasmic domains and PKC{alpha}. A and B, NIH3T3 cells were transfected with an empty vector (NIH3T3), wild type syndecan-4 core protein (S4NIH3T3), or syndecan-4 core protein truncated with the cytoplasmic region (S4{Delta}RNIH3T3). Overexpression was confirmed by immunoblotting using anti-syndecan-4 mAb (5G9) (A). NIH3T3 (panel a), S4NIH3T3 (panel b), or S4{Delta}RNIH3T3 (panel c) cells were treated with (shaded histograms) or without (open histograms) TNIIIA2 (25 µg/ml) and then examined for β1 integrin activation as in Fig. 3 using mAb (9EG7) recognizing an active conformation of mouse β1 integrin (B). C, WI38VA13 cells suspended in serum-free medium were treated with a PKC{alpha} inhibitor, Gö6976 (5 µM), for 30 min and incubated in the presence (right panels) or absence (left panels) of TNIIIA2 (25 µg/ml). β1 integrin activation was evaluated by flow cytometry as in Fig. 3. MFI is expressed in parentheses (B and C). In A–C, similar results were obtained in three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
TN-C plays multiple roles in tissue regulation as a matricellular ECM protein, especially in certain pathophysiological situations such as embryonic development, inflammation, and tumorigenesis. A number of studies using isolated recombinant domains of the TN-C molecule show that regions with adhesive and antiadhesive activities exist on TN-C. A previous study using a recombinant large TN-C variant has demonstrated an antiadhesive mechanism as a specific interference of TN-C, with cell binding to the HepII/syndecan-4 site in FN through direct binding of TN-C to the 13th FN-III (26). The research group developed their study and reported that TN-C blocks cell cycle progression of anchorage-dependent fibroblasts on FN, through inhibition of syndecan-4 (27). Midwood et al. (28) demonstrated that TN-C modulates cell behavior by interfering with the binding of FN to the HS chains of syndecan-4, and inhibiting down stream activation of RhoA and FAK. Consequently, TN-C and syndecan-4 co regulate FN signaling and matrix contraction in tissue repair. Thus, TN-C acts as a negative modulator for cell adhesion and function by interfering with cooperative regulation of cell adhesion by integrin {alpha}5β1 and syndecan-4.

In contrast, the results of the present study suggested that the TN-C molecule harbors a matricryptic site that positively modulates cell adhesion to FN. A synthetic TN-C peptide, TNIIIA2, had the ability to induce activation of β1 integrin, as determined by active β1 integrin-specific mAbs AG89, HUTS-4, and 9EG7. We have observed that TNIIIA2 protects normal fibroblasts from anoikis-like apoptosis by stimulating the PI3-K/Akt/Bcl-2 pathway.3 This pathway is known to be immediately downstream of integrin {alpha}5β1 in anchorage-dependent cell survival signaling (29). The result further provides evidence of its relevance to integrin function. Therefore, we conclude that conformational change in β1 integrin in response to TNIIIA2 caused its functional activation.

It has generally been considered that integrin activation is regulated by "inside-out" signals from the cell interior. This signaling switch is triggered by an extracellular soluble agonist such as ADP, thrombin, phorbor ester, or certain chemokines. However, several artificial factors not found in vivo are able to induce β1 integrin activation. These include divalent cations such as Mn2+ and Mg2+ and anti-β1 integrin mAbs, including TS2/16 and 12G10, which directly bind to the extracellular domain of β1 integrin. It is possible that some physiological process targeting the extracellular region of β1 integrin may also be involved in β1 integrin activation. Cell surface molecules known to modulate integrin activity include tetraspan/TM4SF, CD87/uPAR, and CD47/IAP, which associate with integrins via their extracellular domains. For example, integrin {alpha}3β1 constitutively associates with CD151 via the {alpha}3 chain at amino acid residues 570–705, corresponding to the "thigh" and "calf" domain of the {alpha}v chain (30). Nishiuchi et al. (31) demonstrated that CD151 serves as a constitutive potentiator for {alpha}3β1 activity, through a direct association with this integrin. They speculate that the laterally associated CD151 acts as a "prop" to stabilize the extended high affinity conformation of {alpha}3β1 and render the bending unfavorable.

The syndecan family may provide another example of the regulatory roles of ectodomains in cell adhesion signaling (32–34), although a number of studies have shown the importance of the transmembrane and cytoplasmic domains (35). A site within the ectodomain of syndecan-4 interacts with an unidentified surface molecule to promote adhesion without requiring the cytoplasmic domain (36). The integrin {alpha}vβ3 was shown to be dependent on syndecan-1 for activation and to mediate signals required for human mammary carcinoma cell spread on vitronectin (37). Recently, Whiteford and Couchman (38) have shown that a conserved NXIP motif is required for cell adhesion properties of the syndecan-4 ectodomain. Interestingly, they have also shown that cell adhesion to the syndecan-4 ectodomain involves β1 integrin.

The present study suggests that syndecan-4 may be involved in β1 integrin activation induced by TNIIIA2. This TNIIIA2-induced β1 integrin activation required the extracellular region, not the cytoplasmic domains, of syndecan-4 molecule. Although the precise mechanisms underlying TNIIIA2-induced β1 integrin activation through syndecan-4 remains to be established, a direct interaction between the extracellular regions of syndecan-4 and β1 integrin may induce conformational change in β1 integrin. Indeed, the photoaffinity labeling experiment suggested a physical association of syndecan-4 with β1 integrin on cell surface (see Fig. 5B). It may be that a physical association with the syndecan-4 ectodomain may force β1 integrin to alter its conformation into a functionally active state. Alternatively, syndecan-4 may stabilize the active conformation of β1 integrin, as CD151 acts on integrin {alpha}3β1 (31). Thus, TNIIIA2 induces a net increase in the number of activated β1 integrins on cell surfaces, resulting in enhanced adhesion of adherent and nonadherent cell types. However, adherent cell types, such as the WI38VA13 and NIH3T3 cells used in this study, can attach and spread spontaneously on FN without requiring TNIIIA2 stimulation, although a prolonged time is needed to allow complete spreading. TNIIIA2 may contribute to adhesion of nonadherent cell types, such as hematopoietic cells whose integrins are in their inactive states, rather than to that of adherent cell types. As well as transient expression of TN-C in tissues bearing pathological conditions, such as inflammation and tumorigenesis, significant constitutive expression of TN-C has been observed in adult lymphoid tissues, such as bone marrow, thymus, spleen, and lymph nodes (39, 40), while little is known about a physiological role of TN-C expressed in the lymphoid tissues. A TNIIIA2-related matricyptic site/matricryptin may play a critical role in lymphocyte homing and in leukocyte infiltration, as both require integrin activation. In any case, the present study revealed a new route for β1 integrin activation via a matricryptic site on the TN-C molecule. Further investigation is needed to define how syndecan-4 ligation with TNIIIA2 can induce conformational change in β1 integrin necessary for its functional activation.

Our proposal that β1 integrin is activated through its lateral association with syndecan-4, does not exclude the importance of cell adhesion signaling via the cytoplasmic domains of syndecan-4 in focal adhesion formation and in cytoskeletal organization. Taking into account that the active site of TNIIIA2 appears to be cryptic in the TN-C molecule, it should be assumed that TNIIIA2 works mainly in certain pathophysiological states, in which exposure of matricryptic sites happens frequently, as discussed below.

The ECM generates a variety of signals for cell regulation. Recent studies have shown that some of those signals are derived from biologically active cryptic sites of ECM molecules (9–11). In our study, the TNIIIA2-related matricryptic site was exposed by MMP-2. Siri et al. (41) have shown that large variants of TN-C are much more susceptible to MMP-2, and that MMP-2 completely digests a single FN-III repeat inside the splicing area. It should be noted that TN-C, especially its variants containing the alternative splicing domains, is highly expressed in developing tissues and in pathological tissues (20, 42, 43, 44), where exposure of the matricryptic sites and release of matricryptins occurs frequently. Moreover, expression of syndecan-4 is also up-regulated under similar pathophysiological situations (45). Considering the similarities in specific expression pattern between TN-C and syndecan-4, it is tempting to speculate that the TNIIIA2-related matricryptic site/matricryptin may act, in conjunction with syndecan-4, as a decisive factor for the progression and/or termination of tissue injury. It was reported that TN concentrations in tissues may reach 0.2–2.0 mg/ml (4), which corresponds to the low mM range (46). Thus, significant interactions with syndecan-4 may be expected if the matricryptic site is exposed, based on the binding affinity of TNIIIA2 for HS chain such as heparin (Kd = 1.1 x 10-7 M), and even considering the controlled expression of the alternative splicing region. It is important to examine whether the TNIIIA2-related matricryptic site is exposed at its functional level in tissues.

We previously found that FN had a matricryptic site suppressing cell adhesion. In sharp contrast to TNIIIA2, peptide FNIII14 containing this matricryptic site is capable of inactivating β1 integrins (12, 15). It is interesting that the adhesive ECM protein FN has a matricryptic site that negatively modulates β1 integrin-mediated cell adhesion and conversely, the representative antiadhesive ECM protein TN-C has a site that positively modulates it. This implies that FN and TN-C harbor active sites that function in opposition to their parental ECM proteins. These matricryptic sites of FN and TN-C may act as a negative feedback loop for preventing excessive cellular responses to these ECM proteins during tissue remodeling.


   FOOTNOTES
 
* This work was supported by Grant-in-aid 17590074 for Scientific Research provided by the Ministry of Education, Science, and Culture of Japan, and by the Vehicle Racing Commemorative Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3 and Table S1. Back

1 To whom correspondence should be addressed: Dept. of Molecular Patho-Physiology, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda-Shi, Chiba 278-8510, Japan. Tel./Fax: 81-4-7121-3619; E-mail: fukai{at}rs.noda.tus.ac.jp.

2 The abbreviations used are: TN-C, tenascin-C; FN, fibronectin; ECM, extracellular matrix; FN-III, fibronectin type III-like; mAb, monoclonal antibody; HUVEC, human umbilical vein endothelial cells; PI3-K, phosphatidylinositol 3-kinase; MMP, matrix metalloproteinase; rA1–4, recombinant protein containing FN-III A1–4 repeat; GAG, glycosaminoglycan; HSPG, heparan sulfate proteoglycan; Hep II, heparin-binding domain II of fibronectin; MFI, mean fluorescence intensity; RNAi, RNA interference; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. Back

3 F. Fukai, unpublished data. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Erickson, H. P. (1993) Curr. Opin. Cell Biol. 5, 869-876[CrossRef][Medline] [Order article via Infotrieve]
  2. Chiquet-Ehrismann, R., and Chiquet, M. (2003) J. Pathol. 200, 488-499[CrossRef][Medline] [Order article via Infotrieve]
  3. Hsia, H. C., and Schwarzbauer, J. E. (2005) J. Biol. Chem. 280, 26641-26644[Free Full Text]
  4. Lightner, V. A., Slemp, C. A., and Erickson, H. P. (1990) Ann. N. Y. Acad. Sci. 580, 260-274[Medline] [Order article via Infotrieve]
  5. Chiquet-Ehrismann, R., Kalla, P., Pearson, C. A., Beck, K., and Chiquet, M. (1988) Cell 53, 383-390[CrossRef][Medline] [Order article via Infotrieve]
  6. Bourdon, M. A., and Ruoslahti, E. (1989) J. Cell Biol. 108, 1149-1155[Abstract/Free Full Text]
  7. Joshi, P., Chung, C. Y., Aukhil, I., and Erickson, H. P. (1993) J. Cell Sci. 106, 389-400[Abstract]
  8. Murphy-Ullrich, J. E. (2001) J. Clin. Investig. 107, 785-790[Free Full Text]
  9. Stetler-Stevenson, W. G. (1996) Am. J. Pathol. 148, 1345-1350[Medline] [Order article via Infotrieve]
  10. Sage, E. H. (1997) Trends Cell Biol. 7, 182-186[CrossRef][Medline] [Order article via Infotrieve]
  11. Davis, G. E., Bayless, K. J., Davis, M. J., and Meininger, G. A. (2000) Am. J. Pathol. 156, 1489-1498[Abstract/Free Full Text]
  12. Fukai, F., Hasebe, S., Ueki, M., Mutoh, M., Ohgi, C., Takahashi, H., Takeda, K., and Katayama, T. (1997) J. Biochem. (Tokyo) 121, 189-192[Medline] [Order article via Infotrieve]
  13. Watanabe, K., Takahashi, H., Habu, Y., Kamiya-Kubushiro, N., Kamiya, S., Nakamura, H., Yajima, H., Ishii, T., Katayama, T., Miyazaki, K., and Fukai, F. (2000) Biochemistry 39, 7138-7144[CrossRef][Medline] [Order article via Infotrieve]
  14. Fukai, F., Mashimo, M., Akiyama, K., Goto, T., Tanuma, S., and Katayama, T. (1998) Exp. Cell Res. 242, 92-99[CrossRef][Medline] [Order article via Infotrieve]
  15. Kamiya, S., Kato, R., Wakabayashi, M., Tohyama, T., Eanami, I., Ueki, M., Yajima, H., Ishii, T., Nakamura, H., Katayama, T., Takagi, J., and Fukai, F. (2002) Biochemistry 41, 3270-3277[CrossRef][Medline] [Order article via Infotrieve]
  16. Kato, R., Ishikawa, T., Kamiya, S., Oguma, F., Ueki, M., Goto, S., Nakamura, H., Katayama, T., and Fukai, F. (2002) Clin. Cancer Res. 8, 2455-2462[Abstract/Free Full Text]
  17. Hayashi, R., Miura, S., Saito, Y., Fukai, F., and Kodama, H. (2004) Peptide Science 2003, 289-292
  18. Dorman, G., and Prestwich, G. D. (1994) Biochemistry 33, 5661-5673[CrossRef][Medline] [Order article via Infotrieve]
  19. Fukai, F., Suzuki, H., Suzuki, K., Tsugita, A., and Katayama, T. (1991) J. Biol. Chem. 266, 8807-8813[Abstract/Free Full Text]
  20. Tsunoda, T., Inada, H., Kalembeyi, I., Imanaka-Yoshida, K., Sakakibara, M., Okada, R., Katsuta, K., Sakakura, T., Majima, Y., and Yoshida, T. (2003) Am. J. Pathol. 162, 1857-1867[Abstract/Free Full Text]
  21. Shimizu, N., Sugimoto, K., Tang, J., Nishi, T., Sato, I., Hiramoto, M., Aizawa, S., Hatakeyama, M., Ohba, R., Hatori, H., Yoshikawa, T., Suzuki, F., Oomori, A., Tanaka, H., Kawaguchi, H., Watanabe, H., and Honda, H. (2000) Nat. Biotechnol. 18, 877-881[CrossRef][Medline] [Order article via Infotrieve]
  22. Takagi, J., Isobe, T., Takada, Y., and Saito, Y. (1997) J. Biochem. (Tokyo) 121, 914-921[Abstract/Free Full Text]
  23. Luque, A., Gomez, M., Puzon, W., Takada, Y., Sanchez-Madrid, F., and Cabanas, C. (1996) J. Biol. Chem. 271, 11067-11075[Abstract/Free Full Text]
  24. Sobel, M., Fish, W. R., Toma, N., Luo, S., Bird, K., Mori, K., Kusumoto, S., Blystone, S. D., and Suda, Y. (2001) J. Vasc. Surg. 33, 587-594[CrossRef][Medline] [Order article via Infotrieve]
  25. Thodeti, C. K., Alberchtsen, R., Grauslund, M., Asmar, M., Larsson, C., Takada, Y., Mercurio, A. M., Couchman, J. R., and Wewer, U. M. (2003) J. Biol. Chem. 278, 9576-9584[Abstract/Free Full Text]
  26. Huang, W., Chiquet-Ehrismann, R., Moyano, J. V., Garcia-Pardo, A., and Orend, G. (2001) Cancer Res. 61, 8586-8594[Abstract/Free Full Text]
  27. Orend, G., Huang, W., Olayioye, M. A., Hynes, N. E., and Chiquet-Ehrismann, R. (2003) Oncogene 22, 3917-3926[CrossRef][Medline] [Order article via Infotrieve]
  28. Midwood, K. S., and Schwarzbauer, J. E. (2002) Mol. Biol. Cell 13, 3601-3613[Abstract/Free Full Text]
  29. Lee, J. W., and Juliano, R. L. (2000) Mol. Biol. Cell 11, 1973-1987[Abstract/Free Full Text]
  30. Yauch, R. L., Kazarov, A. R., Desai, B., Lee, R. T., and Hemler, M. E. (2000) J. Biol. Chem. 275, 9230-9238[Abstract/Free Full Text]
  31. Nishiguchi, R., Sanzen, N., Nada, S., Sumida, Y., Wada, Y., Okada, M., Takagi, J., Hasegawa, H., and Sekiguchi, K. (2005) Proc. Natl. Acad. Sci. 102, 1939-1944[Abstract/Free Full Text]
  32. Adams, J. C., Kureishy, N., and Taylor, A. L. (2001) J. Cell Biol. 152, 1169-1182[Abstract/Free Full Text]
  33. Carey, D. J., Stahl, R. C., Cizmeci-Smith, G., and Asundi, V. K. (1994) J. Cell Biol. 124, 161-170[Abstract/Free Full Text]
  34. Liu, W., Litwack, E. D., Stanley, M. J., Langford, J. K., Lander, A. D., and Sanderson, S. D. (1998) J. Biol. Chem. 273, 22825-22832[Abstract/Free Full Text]
  35. Keum, E., Kim, J., Kwon, S., Lim, Y., Han, I., and Oh, E. S. (2004) Biochem. J. 378, 1007-1014[CrossRef][Medline] [Order article via Infotrieve]
  36. McFall, A. J., and Rapraeger, A. C. (1998) J. Biol. Chem. 273, 28270-28276[Abstract/Free Full Text]
  37. Beauvais, D. M., Burbach, B. J., and Rapraeger, A. C. (2004) J. Cell Biol. 167, 171-181[Abstract/Free Full Text]
  38. Whiteford, J. R., and Couchman, J. R. (2006) J. Biol. Chem. 281, 32156-32163[Abstract/Free Full Text]
  39. Mackie, E. J., Thesleff, I., and Chiquet-Ehrismann, R. (1987) J. Cell Biol. 105, 2569-2579[Abstract/Free Full Text]
  40. Ocklind, G., Talts, J., Fässler, R., Mattsson, A., and Ekblom, P. (1993) J. Histochem. Cytochem. 41, 1163-1169[Abstract]
  41. Siri, A., Knauper, V., Verrana, N., Caocci, F., Murpy, G., and Zardi, L. (1995) J. Biol. Chem. 270, 8650-8654[Abstract/Free Full Text]
  42. Mackie, E. J., and Tucker, R. P. (1992) J. Cell Sci. 103, 765-771[Abstract]
  43. Hindermann, W., Berndt, A., Borsi, L., Luo, X., Hyckel, P., Katenkamp, D., and Kosmehl, H. (1999) J. Pathol. 189, 475-480[CrossRef][Medline] [Order article via Infotrieve]
  44. Ghert, M. A., Jung, S. T., Qi, W., Harrelson, J. M., Erickson, H. P., Block, J. A., and Scully, S. P. (2001) Oncology 61, 306-314[CrossRef][Medline] [Order article via Infotrieve]
  45. Gallo, R., Kim, C., Kokenyesi, R., Adzick, N. S., and Bernfield, M. (1996) J. Investig. Dermatol. 107, 676-683[CrossRef][Medline] [Order article via Infotrieve]
  46. Ingham, K. C., Brew, S. A., and Erickson, H. P. (2004) J. Biol. Chem. 279, 28132-28135[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
282/48/34929    most recent
M705608200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Google Scholar
Right arrow Articles by Saito, Y.
Right arrow Articles by Fukai, F.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Saito, Y.
Right arrow Articles by Fukai, F.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.