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[Cancer Research 62, 1541-1548, March 1, 2002]
© 2002 American Association for Cancer Research

Tumor Biology

Identification of Heat Shock Protein 60 as a Molecular Mediator of {alpha}3ß1 Integrin Activation

Heba O. Barazi, Longen Zhou, Nancy Smyth Templeton, Henry C. Krutzsch and David D. Roberts1

Laboratory of Pathology, National Cancer Institute, NIH, Bethesda, Maryland 20892 [H. O. B., L. Z., H. C. K., D. D. R.], and Center for Cell and Gene Therapy and Department of Molecular & Cellular Biology, Baylor College of Medicine, Houston, Texas 77030 [N. S. T.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The {alpha}3ß1 integrin is involved in the adhesion of metastatic breast cancer cells to the lymph nodes and to osteoblasts in the bone. Regulation of the affinity or avidity of integrins for their ligands may result from conformational changes induced by changes in the microenvironment of the integrin. Two surface proteins, 55 and 32 kDa, coimmunoprecipitated with the {alpha}3ß1 integrin from breast carcinoma cells. The 55-kDa protein preferentially associated with the active form of the {alpha}3ß1 integrin. The protein was identified as HSP60 using two-dimensional electrophoresis and mass spectrometry and confirmed by reimmunoprecipitation of the integrin immune complex with an anti-HSP60 antibody. In cell spreading assays on a thrombospondin-1 substrate, addition of exogenous-recombinant HSP60 was sufficient to specifically activate {alpha}3ß1 integrin but not to activate function of {alpha}2ß1, {alpha}vß3, {alpha}4ß1, or {alpha}5ß1 integrins. Furthermore, mizoribine, an HSP60-binding drug, blocked activation of the {alpha}3ß1 integrin induced by insulin-like growth factor 1 (IGF1) or exogenous recombinant HSP60 and inhibited the association of HSP60 with the integrin. Additionally, inhibiting the surface expression of endogenous HSP60 by nonactin inhibited activation of the {alpha}3ß1 integrin by IGF1. These data demonstrate that HSP60 binding is sufficient to activate {alpha}3ß1 integrin function and suggest that association of endogenous HSP60 with {alpha}3ß1 integrin is necessary for IGF1-induced activation.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The {alpha}3ß1 integrin plays a role in normal branching morphogenesis in mammary epithelia (1) , as well as in tumorigenesis and metastasis of breast carcinoma. Inhibition of invasiveness of the breast cancer cell line, MDA-MB435, by the tumor suppressor gene product maspin correlates with reduced expression of the {alpha}3ß1 integrin (2) . In animal models, breast cancer cell lines with higher expression of the {alpha}3ß1 integrin show preferential metastasis to the bone (3) . Engagement of the integrin with one of its ligands, TSP12 (4, 5, 6) , inhibited proliferation and induced differentiation of small cell lung carcinoma (7) . Furthermore, the {alpha}3ß1 integrin has been shown to be responsible for mediating attachment of breast cancer cells to the two major metastatic sites, lymph nodes (8) and bone (9) .

Integrins can relay signals from the extracellular environment into the cell by virtue of their linkage to the cellular cytoskeleton and by their association with cytoplasmic signaling molecules (10, 11, 12) . In such a capacity, integrins influence many fundamental physiological functions in the cell, including motility, proliferation, apoptosis, and differentiation.

Alteration of integrin expression and/or activity is associated with the invasive phenotypes of cancer cells (13 , 14) , e.g., metastasis of breast carcinoma requires detachment of cells from the site of origin, mediated by the loss of cell adhesion. The cells then migrate toward, invade into, and extravasate from the circulatory system to establish metastases (15 , 16) . All of these steps require the recycling of integrins between their active and inactive states. Therefore, a fundamental understanding of the regulation of integrin activity is necessary for designing better antimetastatic treatments.

The ligand binding activities of integrins are modulated through changes in their affinity or avidity. Avidity is regulated by clustering of integrin molecules on the plane of the plasma membrane in response to interaction of the integrin with its target extracellular matrix ligand (17 , 18) . Affinity, on the other hand, involves the intrinsic ability of the individual integrin molecule to bind its extracellular matrix target. Regulation of integrin affinity is generally believed to involve conformational changes (19, 20, 21) , which may be induced by biochemical modification of the molecule or association or dissociation from regulatory molecules. There are only a few examples in the literature of biochemical modifications of integrins that alter function. In vitro, the ILK phosphorylates a peptide derived from the cytoplasmic domain of the ß1 integrin subunit (22) . Although overexpression of ILK down-regulates ß1 integrin adhesion (23) , no direct relationship has been shown between phosphorylation of the integrin by ILK and integrin activity. In some hematopoietic cells and in platelets, the ß3 integrin subunit has been shown to be phosphorylated on tyrosine 747 (24 , 25) , an amino acid involved in the regulation of the integrin activity (26) .

In the absence of strong evidence implicating biochemical modification of integrins in their regulation, the alternative model is that association or dissociation with other molecules could induce conformational changes in integrins. Integrins associate with a variety of cytoskeletal (27, 28, 29, 30, 31) and signaling molecules (32) . A few of these identified proteins were demonstrated to interact directly with an integrin and to regulate its function, e.g., the cytoplasmic protein calreticulin binds the cytoplasmic domain of the {alpha}2 subunit and is believed to stabilize the integrin in a high-affinity state (33 , 34) .

To better understand the regulation of {alpha}3ß1-integrin activity, we examined this process in breast carcinoma cells. The integrin is relatively inactive in both MDA-MB 231 and MDA-MB 435 cells, but its activity is inducible by growth signals, such as serum and IGF1 (5) . Using these cells, we now show that the active integrin associates preferentially with a heat shock protein HSP60 on the cell surface. Remarkably, exogenous addition of this chaperonin can directly activate the {alpha}3ß1 integrin, and its induced expression on the cell surface is necessary for activation of {alpha}3ß1 integrin after treatment with IGF1.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Monoclonal Antibodies.
TS2/16 (anti-ß1; Ref. 35 ) and 4F2 (anti-CD98) were each purified by protein G affinity chromatography (Pierce) from conditioned media of the respective hybridomas (American Type Culture Collection) grown in PFHM-II medium (Life Technologies, Inc.). P1B5 (anti-{alpha}3ß1) and P1D6 (anti-{alpha}5ß1) were both purchased from Life Technologies, Inc. Goat anti-HSP60 antibody was purchased from Santa Cruz Biotechnology, Inc. Antibody 6D7 (anti-{alpha}2ß1) was provided by Dr. Harvey Gralnick (Hematology Service, NIH).

Cell Lines and Reagents.
The breast carcinoma cell lines MDA-MB231 and MDA-MB435 (American Type Culture Collection) were propagated weekly in RPMI 1640 containing 10% FCS. OH1 small cell lung carcinoma cells were propagated in RPMI 15% FCS as described previously (7) . PT, Nonactin, and Miz were purchased from Sigma Chemical Co. IGF1 was purchased from Bachem. TSP1 was purified from the supernatant of thrombin-activated platelets as described (36) . Bovine type I collagen was purchased from Becton Dickinson, and human vitronectin was from Sigma Chemical Co. Fibronectin was purified from human plasma (NIH Blood Bank) as described (37) . Recombinant human HSP60 produced in Escherichia coli was purchased from StressGen. Purified {alpha}3ß1 integrin was obtained from Chemicon. MBP-invasin (1–497) was expressed in E. coli and purified as described (38) . The protein was labeled with 125I in the presence of Iodogen (Pierce) as described previously (7) .

Immunoprecipitation.
Cells grown in 10-cm dishes were labeled using a 1 mg/ml solution of EZ-Link Sulfo-NHS-LC-Biotin (Pierce) at 4°C for 1.5 h. On lysis in RIPA buffer [50 mM Tri (pH 7.5), 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 1 mM EGTA, and 1 mM NaF supplemented with 10 µg/ml each of the following protease inhibitors: antipain, pepstatin A, chymostatin, leupeptin, aprotinin, soybean trypsin inhibitor, and 1 mM phenylmethylsulfonyl fluoride], the lysate was precleared by centrifugation. Equal volumes with equal protein concentrations are immunoprecipitated using the indicated antibody prebound to either antimouse IgG agarose (Sigma Chemical Co.) or protein A-agarose (Boehringer Mannheim) as indicated. The immune complexes were washed three times with TBS [140 mM NaCl, 20 mM Tris (pH 7.5), 0.1% Tween 20], eluted with sample buffer containing 10% ß-mercaptoethanol, heated, and fractionated on precast SDS gels (Bio-Rad). After transfer to PVDF membrane, the proteins were detected using horseradish peroxidase-streptavidin (Pierce) and visualized using chemiluminescent substrate (Pierce).

For sequential immunoprecipitation, the immune complexes were washed three times with TBS, eluted with RIPA buffer containing 4% SDS, and heated. The samples were then diluted 10-fold with RIPA buffer and reimmunoprecipitated and analyzed as described above.

Two-dimensional Gel Electrophoresis.
Proteins were processed for two-dimensional electrophoresis using the IPGphor IEF system (Amersham Pharmacia Biotech) as described by the manufacturer. Essentially, immune complexes were washed with TBS as above followed by a single wash with water and elution using IEF lysis solution (8 M urea, 4% NP40, and 40 mM Tris base). Equal amounts of proteins as determined by one-dimensional gel analysis were mixed with rehydration stock solution (8 M urea, 2% NP40, 2% IPG buffer, and bromphenol blue) and incubated with the IPG strip (pH 4–7 linear) overnight. The proteins were then focused for 16,000 V/h and processed for the second dimension on a 12.5% SDS-acrylamide slab gel. After transfer to PVDF membrane, the protein spots were detected using horseradish peroxidase-streptavidin (Pierce) and visualized using chemiluminescent substrate (Pierce).

Biotinylated Protein Purification and LCMS Identification.
MDA-MB-231 cells were surface labeled with biotin, lysed in RIPA, and precleared as described above. The biotinylated proteins were purified using the UltraLink Immobilized Monomeric avidin column as per manufacturer’s instructions with some modifications. Basically, the bound proteins were washed with PBS containing 0.02% NP40 and eluted using 2 mM biotin containing 0.02% NP40. Eluted proteins were then lyophilized, resuspended in IEF lysis buffer, and processed for two-dimensional electrophoresis as described above. To visualize the proteins, the slab gel was stained with Colloidal Coomassie G250 blue stain using the Novex staining kit as described in the instructions. For LCMS determination, the desired protein spots were excised from the gel and placed in microfuge tubes. The gel pieces were washed twice with a methanol ammonium bicarbonate buffer, dried in vacuo, then treated with trypsin overnight. The resulting peptides were extracted, then separated and analyzed on a Finnigan LCQ LCMS system. The resulting run files were analyzed using Sequest database searching software.

Cell Spreading Assay.
Freshly passed cells were serum starved overnight in RPMI 1640 containing 1% FCS. Cells were then detached by incubating with PBS containing 2.5 mM EDTA for 5–10 min, collected by centrifugation, resuspended in RPMI media containing 0.1% BSA, and layered onto bacteriological polystyrene substrates coated with the indicated proteins (39) . After a 60-min incubation in the presence or absence of the indicated protein or drug, the cells were washed to remove nonadherent cells and then fixed with 1% glutaraldehyde.

Cell Binding Assay.
125I-MBP-invasin binding to OH-1 cells was determined as described previously (7) except that 5 x 105 cells/tube were incubated with the indicated proteins for 1 h at 37°C in Dulbecco’s PBS containing 0.1% BSA. Unbound label was separated from the cells by centrifugation through oil.

HSP60 Integrin-binding Assay.
Immulon-2 Removawell strips (Dynatech Laboratories) were coated with 50 µl of {alpha}3ß1 integrin (5 µg/ml) in Dulbecco’s PBS by incubating overnight at 4°C. The wells were aspirated and blocked by incubating with 1% BSA in buffer containing 50 mM Tris (pH 7.4), 50 mM KCl, 10 mM MgCl2, and 1 mM EDTA for 30 min at room temperature. The wells were then incubated with 50 µl of 125I-HSP60 (0.5 mg/ml) in binding buffer containing 50 mM Tris (pH 7.4), 50 mM KCl, 10 mM MgCl2, 1 mM EDTA, 1 mM DTT, and 0.1% BSA in the presence or absence of 5 mM Miz for 1 h at room temperature and washed three times with PBS. The bound radioactivity was quantified with a gamma counter (Packard Instruments).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Activated {alpha}3ß1 Integrin Preferentially Associates with a 55-kDa Surface Protein.
We hypothesized that {alpha}3ß1 integrin responds to activation signals by disassociating or associating with a regulatory cell surface molecule. To test this, we compared anti-ß1 integrin-subunit immune complexes from surface labeled breast cancer cells where the integrin was either inactivated or activated. We showed previously that both FCS and IGF1 can specifically induce the activation of {alpha}3ß1 integrin in serum-starved breast cancer cells as determined by cell spreading on TSP1 (5) . Furthermore, PT induces activation of {alpha}3ß1 integrin in MDA-MB 231 cells but inhibits {alpha}3ß1 integrin activity in MDA-MD 435 breast cancer cells (5) . Immunoprecipitated ß1 integrin subunit from both MDA-MB 231 and MDA-MB435 breast cancer cell lines migrated with a molecular mass of 130 kDa and was associated with a surface protein with a molecular mass of 55 kDa (Fig. 1)Citation . A 32-kDa protein was also coimmunoprecipitated with ß1 integrin subunit from MDA-MB 231 but not from MDA-MB 435 cells. Notably, the amount of 55-kDa protein coimmunoprecipitated with the ß1 integrin was increased in immune complexes derived from cells where {alpha}3ß1 integrin was activated (5) . Those included MDA-MB 231 and MDA-MB 435 cells treated with FCS (Fig. 1Citation , Lanes 3 and 7) or IGF1 (Fig. 1Citation , Lanes 4 and 8) and MDA-MB 231 cells treated with PT (Fig. 1Citation , Lane 1). In cells where {alpha}3ß1 integrin activity was reduced (5) , such as serum-starved MDA-MB 231 (Fig. 1Citation , Lane 2) and MDA-MB 435 cells (Fig. 1Citation , Lane 6) or PT-treated MDA-MB 435 cells, the level of 55-kDa protein in the immune complex was also decreased. The differential association of the 55-kDa protein with the ß1 integrin subunit correlated closely with the activation status of the integrin and clearly reflected the opposing effects that PT had on activation of the integrin in MDA-MB 231 versus MDA-MB 435 breast cancer cells (5) . Taken together, these data suggest a role for the 55-kDa protein in mediating ß1 integrin activation.



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  		Fig. 1. Detection of surface proteins coimmunoprecipitated with ß1 integrins from breast cancer cells. MDA-MB 231 (Lanes 1–4) or MDA-MB 435 (Lanes 5–8) cells were serum starved overnight and then treated for 30 min with 200 µg/ml PT (Lanes 1 and 5), no treatment (-, Lanes 2 and 6), 10% FCS (Lanes 3 and 7), or 10 nM IGF1 (Lanes 4 and 8). Surface-biotinylated cell lysates, normalized by total protein, were then immunoprecipitated using the anti-ß1 antibody (TS2/16). The immune complex was fractionated on a 5–12% gradient SDS-polyacrylamide gel, transferred onto PVDF, and visualized using streptavidin conjugated to horseradish peroxidase followed by chemiluminescent substrate. The migration of molecular weight markers is indicated (Mr x 10-3). Arrowhead, the position of the integrin chains; arrow, the position of the 55-kDa protein.

 
Because {alpha}3ß1, {alpha}2ß1, and {alpha}5ß1 integrins are expressed in MDA-MB 231 and MDA-MB 435 cells but are differentially activated (5 , 40, 41, 42) , we examined the association of the 55-kDa protein with these ß1 integrins (Fig. 2)Citation . The 55-kDa protein coimmunoprecipitated with {alpha}3ß1 integrin from surface-labeled MDA-MB 231 and MDA-MB 435 cells. Additionally, the 55-kDa protein also preferentially associated with {alpha}3ß1 integrin derived from cells treated with the activators FCS and IGF1 (Fig. 2Citation , Lanes 2 and 3 and 5 and 6). The differential association of the 55-kDa protein with {alpha}3ß1 integrin in PT-treated MDA-MB 231 and MDA-MB 435 cells also correlated with the opposing effects of PT for activating the integrin in these cells (data not shown). The activation-dependent association of the 55-kDa protein with ß1 and {alpha}3 subunits in both cell lines was highly reproducible. A 32-kDa protein also coimmunoprecipitated with {alpha}3ß1 integrin derived from MDA-MB 231 but not MDA-MB 435 cells. This protein also showed preferential association with the active integrin subunit from MDA-MB 231 cells (Fig. 2Citation , Lanes 2 and 3). However, because the protein was not present in immune complexes derived from MDA-MB 435 cells, we did not study this protein further.



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  		Fig. 2. Detection of surface proteins coimmunoprecipitated with {alpha}3ß1 integrin from breast cancer cells. MDA-MB 231 (Lanes 1–3) or MDA-MB 435 (Lanes 4–5) cells were serum starved overnight and then either left untreated (-, Lanes 1 and 4) or treated for 30 min with 10% FCS (Lanes 2 and 5) or 10 nM IGF1 (Lanes 3 and 6). Surface biotinylated cell lysates, normalized by total protein, were then immunoprecipitated using the anti-{alpha}3 antibody (P1B5). The immune complex was fractionated on a 12% SDS-polyacrylamide gel, transferred onto PVDF, and visualized using streptavidin conjugated to horseradish peroxidase followed by chemiluminescent substrate. The migration of molecular weight markers is indicated (Mr x 10-3). Arrowhead, the position of the integrin chains; arrow, the position of the 55-kDa protein.

 
Purification and Identification of the {alpha}3ß1-associated 55-kDa Surface Protein.
ß1 immune complexes from MDA-MB 231 cells were analyzed using two-dimensional gel electrophoresis, and the location of the 55-kDa protein on the gel was determined. Biotin-labeled proteins from total cell lysates of MDA-MB 231 cells were purified by avidin affinity chromatography. The spots on two-dimensional gel of avidin-purified surface proteins corresponding to the location of the 55-kDa protein were excised, and the protein was identified as HSP60 by LCMS identification of four tryptic peptides (Table 1)Citation .


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  		Table 1 HSP60 tryptic peptide fragments identified by LCMS analysis of the 55-kDa corresponding protein spot from a two-dimensional gel of avidin-purified proteins

 
Confirmation and Specificity of the HSP60 and {alpha}3ß1 Integrin Association.
To confirm that the integrin-associated 55-kDa protein is HSP60, we used sequential immunoprecipitation to detect the HSP60 protein in an anti-{alpha}3 integrin subunit immune complex. Immune complexes of the {alpha}3, {alpha}2, and {alpha}5 integrin subunits all contained coimmunoprecipitated surface proteins (Fig. 3A)Citation . The {alpha}2 integrin subunit coimmunoprecipitated with a surface-exposed protein migrating with a molecular mass of 53 kDa (Fig. 3ACitation , Lane 2), whereas the {alpha}5 integrin subunit coimmunoprecipitated with a surface-exposed protein migrating with a molecular mass of 58 kDa (Fig. 3ACitation , Lane 4). Two surface-exposed proteins were immunoprecipitated with the HSP60 antibody (Ref. 43 ; Fig. 3ACitation , Lane 1). When these immune complexes were reimmunoprecipitated with anti-HSP60 antibody, the protein was detected in the {alpha}3 but not in the {alpha}2 or {alpha}5 integrin subunit immune complexes (Fig. 3B)Citation . These results confirm the identity of the 55-kDa protein in {alpha}3ß1 immunoprecipitates as HSP60 and suggest that this association with {alpha}3ß1 integrin is specific.



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  		Fig. 3. Reimmunoprecipitation of HSP60 from an {alpha}3 integrin immune complex. In A, equal volumes with equal amount of surface-biotinylated proteins from MDA-MB 231 cells were immunoprecipitated with goat anti-HSP60 antibody (Lane 1), 6D7 antibody against the {alpha}2 integrin subunit (Lane 2), P1B5 antibody against the {alpha}3 integrin subunit (Lane 3), or P1D6 against the {alpha}5 integrin subunit (Lane 4). Immune complexes were fractionated on a 10% SDS-polyacrylamide gel and detected as described in the legend to Fig. 1Citation . In B, immune complexes generated above were reimmunoprecipitated using the goat anti-HSP60 antibody and fractionated on a 10% SDS gel. The surface proteins were visualized as described above. The migration of molecular weight markers is indicated (Mr x 10-3). Arrow, the migration of HSP60.

 
Recombinant HSP60 Regulates {alpha}3ß1 Integrin Activity.
Association of the {alpha}3ß1 integrin with HSP60 could be either a cause or an effect of activation. To determine the role of HSP60 in activating {alpha}3ß1 integrin, we tested the effect of recombinant HSP60 on the ability of {alpha}3ß1 integrin to mediate cell spreading (Fig. 4)Citation . Both serum-starved MDA-MB 231 and starved MDA-MB 435 cells exhibited low spreading activity on TSP1, which is specifically mediated by the {alpha}3ß1 integrin (Refs. 5 and 6 ; Fig. 4Citation ). As described previously (5) , IGF1 induced spreading of both cell lines on TSP1 (Fig. 4)Citation . Notably, recombinant HSP60 also induced cell spreading of both cell lines on TSP1 (Fig. 4, A and B)Citation , which is mediated by the {alpha}3ß1 integrin (5) . This induction is specific for the {alpha}3ß1 integrin, because recombinant HSP60 did not induce spreading of either cell line on type I collagen, a largely {alpha}2ß1 integrin-specific matrix for these cells (9) . In addition, recombinant HSP60 did not induce MDA-MB 231 cell spreading on fibronectin (Fig. 4A)Citation , an {alpha}5ßl ligand (44) , nor did it induce spreading of either cell line on vitronectin (Fig. 4B and data not shown)Citation , which is an {alpha}vß3- and {alpha}vß5-specific ligand for these cells (45) .



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  		Fig. 4. Regulation of cell spreading by exogenous recombinant HSP60. MDA-MB 231 (A) or MDA-MB 435 (B) cells were serum starved overnight, then lifted and overlaid on substrates coated with 50 µg/ml TSP1, 10 µg/ml fibronectin, 10 µg/ml vitronectin, and 5 µg/ml type I collagen. Cell spreading was allowed to proceed in the absence or presence of either 10 µM IGF1 or 5 µg/ml recombinant HSP60. After 60-min incubation, the number of spread cells per mm2 was determined. The mean ± SD for triplicate determinations is presented.

 
The effect of recombinant HSP60 on the activity of {alpha}3ß1 was dose dependent. Spreading of MDA-MB 231 cells on TSP1 was significantly enhanced at 1 µg/ml exogenous HSP60, whereas maximal induction was achieved using 5 µg/ml recombinant HSP60 (Fig. 5A)Citation . No change was observed in the activity of {alpha}2ß1-dependent spreading on type I collagen over the range of recombinant HSP60 protein concentrations used (Fig. 5A)Citation . Additionally, activation of {alpha}3ß1 integrin by recombinant HSP60 increased with increasing TSP1 concentration (Fig. 5B)Citation . The cell spreading response was not TSP concentration dependent in the absence of HSP60 (Fig. 5B)Citation .



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  		Fig. 5. HSP60-induced cell spreading and ligand binding to {alpha}3ß1 integrin. A, dose dependence of HSP60-induced cell spreading. Spreading of MDA-MB 231 cells onto TSP1 (50 µg/ml) and type I collagen (5 µg/ml) was determined in the absence or presence of the indicated concentration of recombinant HSP60. The mean ± SD for triplicate determination is presented. B, TSP1 dose response of HSP60-induced cell spreading. Spreading of MDA-MB 231 cells onto TSP1 (12.5, 25, or 50 µg/ml) was determined in the absence or presence of 5 µg/ml recombinant HSP60. The mean ± SD for triplicate determination is presented. C, effect of HSP60 on MBP-invasin binding to OH-1 cells. A suspension of OH-1 small cell lung carcinoma cells (5 x 105) was incubated with 125I-MBP-invasin alone (Control), in the presence of 5 µg/ml HSP60, or with 5 µg/ml TS2/16 for 1 h at 37°C.

 
To directly assess the effect of exogenous HSP60 on the ligand-binding activity of {alpha}3ß1 integrin, we used a small cell lung carcinoma cell line, OH-1, that lacks most ß1 integrins except {alpha}3ß1 and for which we have previously demonstrated interaction with the high-affinity ligand invasin to be mediated primarily by {alpha}3ß1 integrin (7) . Furthermore, the OH-1 cells maintain {alpha}3ß1 integrin in a partially inactive state that responds to physiological and pharmacological activators (7) . A recombinant invasin fragment was radiolabeled with 125I to measure binding of this soluble integrin ligand to a suspension of OH-1 cells. Binding of MBP-invasin was significantly and reproducibly increased in the presence of exogenous HSP60, and in some experiments, the stimulation was equivalent to that induced by the ß1 integrin-activating antibody TS2/16 (Fig. 5C)Citation .

Functional experiments described in this report have implicated surface-exposed HSP60 in IGF1-induced {alpha}3ß1 integrin activation. In turn, growth factors rapidly up-regulated HSP60 on the cell surface (Fig. 6)Citation . Treatment of MDA-MD-231 cells with FCS (Fig. 6)Citation or IGF1 (data not shown) resulted in surface expression of HSP60 (Fig. 6Citation , Lane 4) and increased association of surface HSP60 with the {alpha}3ß1 integrin (Fig. 6Citation , Lane 2).



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  		Fig. 6. Up-regulation of surface-exposed HSP60 protein by growth factors. MDA-MB 231 cells were serum starved overnight, then either left untreated (-, Lanes 1 and 3) or treated for 15 min with 10% FCS (+, Lanes 2 and 4). Surface-biotinylated cell lysates, normalized by total protein, were then immunoprecipitated using TS2/16 antibody (anti-ß1, Lanes 1 and 2) or anti-HSP60 (Lanes 3 and 4). The immune complexes were fractionated on a 12% SDS-polyacrylamide gel, transferred onto PVDF, and visualized using streptavidin conjugated to horseradish peroxidase followed by chemiluminescent substrate. The migration of molecular weight markers is indicated (Mr x 10-3). These results are representative of three independent experiments.

 
The immunosuppressant Miz specifically binds to HSP60 and inhibits its chaperonin activity (46) . Miz inhibited the {alpha}3ß1 integrin-mediated spreading on TSP1 induced by either IGF1 or exogenous recombinant HSP60 (Fig. 7A)Citation . The drug, however, had no effect on the activity of {alpha}2ß1 integrin as tested by cell spreading on type I collagen (Fig. 7A)Citation . The inhibitory effect of Miz on adhesion to TSP1 was also specific for that induced by HSP60, in that MDA-MB-231 cell spreading on TSP1 stimulated by antibody TS2/16 was not inhibited by Miz (Fig. 7B)Citation . Therefore, Miz is probably inhibiting spreading through its binding to HSP60 rather than through indirect effects on cell spreading, resulting from other pharmacological targets of this drug (47) . Coincident with inhibiting the {alpha}3ß1 integrin-mediated spreading on TSP1, Miz also inhibited IGF1-induced association of endogenous surface-exposed HSP60 with the ß1-integrin subunit immune complex (Fig. 7C)Citation . Miz alone had no effect on the adhesion and spreading of unstimulated MDA-MB-231 cells to TSP (data not shown).



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  		Fig. 7. Inhibition by Miz of HSP60-induced activation of {alpha}3ß1 integrin and HSP60-{alpha}3ß1 integrin association. In A, MDA-MB 231 cell spreading on 50 µg/ml TSP1 or 5 µg/ml collagen type I was determined as described previously. Spreading was allowed to proceed in the absence or presence of 10 µM IGF1, 10 nM IGF1 plus 5 mM Miz (IGF1 + Miz), 5 µg/ml HSP60, or 5 µg/ml HSP60 plus 5 mM Miz (HSP60 + Miz). In B, MDA-MB-231 cell spreading on a TSP1 substrate (mean ± SD) was quantified in untreated cells (control) or with the indicated combinations of HSP60 (3 µg/ml), Miz (0.5 mM), and the ß1 integrin-activating antibody TS2/16 (5 µg/ml). In C, serum-starved MDA-MB 231 cells were activated for 20 min with 10 nM IGF1 (Lanes 2 and 4) in the absence (Lanes 1 and 2) or presence (Lanes 3 and 4) of 0.5 mM Miz. After surface biotinylation, cell lysates, normalized by total protein, were immunoprecipitated with anti-ß1 integrin subunit antibody (TS2/16), fractionated on 10% SDS-polyacrylamide gel, and transferred to membrane. Proteins were detected using chemiluminescent substrate as described in the "Materials and Methods" section. The migration of molecular weight markers is indicated. The experiment shown is an independent representative experiment. D, effect of Miz on HSP60-stimulated MBP-invasin binding to OH-1 cells. A suspension of OH-1 small cell lung carcinoma cells (5 x 105) was incubated with 125I-MBP-invasin in the presence of 5 µg/ml HSP60, 5 µg/ml HSP60 + 5 mM Miz (HSP60 + Miz), and 5 µg/ml antibody TS2/16 for 1 h at 37°C, respectively. Specific binding was determined by subtracting basal invasin binding and is presented as mean ± SD. E, inhibition of HSP60 and integrin binding by Miz. Recombinant 125I-labeled HSP60 (25 ng) was incubated with immobilized purified {alpha}3ß1 integrin (250 ng) in the absence (Control) or the presence of 5 mM Miz. Specific binding was determined by subtracting nonspecific binding of 125I-HSP60 to wells without integrin and is presented as mean ± SD.

 
The inhibitory activity of Miz on {alpha}3ß1-integrin activation was confirmed in the invasin binding assay using OH-1 cells (Fig. 7D)Citation . Miz reproducibly inhibited the enhanced binding of invasin to OH-1 cells induced by adding exogenous HSP60. To confirm the ability of Miz to directly block interaction of HSP60 with {alpha}3ß1 integrin, we examined its effect on binding of 125I-labeled HSP60 to purified {alpha}3ß1 integrin immobilized on plastic (Fig. 7E)Citation . Miz strongly inhibited specific binding of HSP60 to the integrin. Taken together, these data demonstrate that HSP60 can directly bind to {alpha}3ß1 integrin and is sufficient to activate {alpha}3ß1 integrin function in intact cells. They also suggest that association of endogenous HSP60 with {alpha}3ß1 integrin is necessary for IGF1-induced activation.

To further confirm the role of surface-exposed HSP60 in {alpha}3ß1-integrin activity, we used the potassium ionophore nonactin to inhibit surface expression of HSP60. Nonactin inhibits import of mitochondrial proteins (48) , which is a prerequisite to HSP60 maturation and surface expression (43) . MDA-MB 231 cells required at least 4 h of nonactin treatment to abolish all detectable surface expression of HSP60 protein (Fig. 8ACitation , Lane 3). Concomitantly, a 4-h nonactin treatment of MDA-MB 231 cells reduced {alpha}3ß1-mediated and IGF1-induced cell spreading on TSP1 relative to that of untreated cells (Fig. 8B)Citation . Nonactin treatment, however, did not interfere with the ability of {alpha}3ß1 integrin to be activated by exogenous recombinant HSP60 and with {alpha}2ß1 integrin-mediated cell spreading on type I collagen (Fig. 8B)Citation . Thus, surface expression of HSP60 requires mitochondrial function and is necessary for IGF1-induced activation of {alpha}3ß1 integrin.



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  		Fig. 8. Inhibition of HSP60 surface expression and {alpha}3ß1-integrin activation by nonactin. In A, MDA-MB 231 cells were treated with 10 µg/ml nonactin in RPMI 1640 containing 1% FCS. Treatment was stopped after 2 (Lane 1), 3 (Lane 2), or 4 (Lane 3) h followed by surface labeling and cell lysis. HSP60 was immunoprecipitated from each lysate and normalized by total protein, and the resulting immunocomplexes were fractionated on 10% SDS-polyacrylamide gels, transferred to membrane, and visualized by chemiluminescent substrate. In B, nonactin treatment inhibits {alpha}3ß1-integrin activation. MDA-MB 231 cells were either treated or not treated for 4 h with nonactin as indicated above. Induction of both treated and untreated MDA-MB 231 cell spreading on TSP1 by 10 nM IGF1 or 3 µg/ml recombinant HSP60 was determined in the presence or absence of 10 µg/ml nonactin. As a control, cell spreading on type I collagen was also assayed under the same conditions. Shown is the mean ±SD for triplicate determinations.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Human HSP60 is known commonly as a mitochondrial protein involved in protein folding, transport, and secretion (49, 50, 51) . However, recent studies clearly demonstrate extramitochondrial locations for the protein (reviewed in Ref. 52 ), including foci on the endoplasmic reticulum (53) , the cell surface (54, 55, 56) , and in unidentified vesicles and cytoplasmic granules (43) . No clear function has been identified for HSP60 in any of these sites. This work describes a role for cell surface-expressed HSP60. We show that surface-exposed HSP60 specifically mediates activation of the {alpha}3ß1 integrin and that this function requires the association of HSP60 with a noncovalent complex containing the integrin.

The primary sequence of HSP60 does not contain a significant stretch of hydrophobic sequence that could act as a membrane-spanning domain (57 , 58) . Therefore, it is likely that the protein localizes to the membrane as a peripheral membrane protein by associating with intrinsic membrane proteins, including {alpha}3ß1 integrin. Our observation that surface detection of HSP60 and its association with the integrin is sensitive to cycles of freeze and thaw (data not shown) is consistent with it being only a peripheral membrane protein.

By virtue of its association with p21Ras (59) and proteins of the A- and L-amino acid transport systems (58 , 60) , HSP60 has been implicated as functioning in signal transduction and amino acid transport. The role of HSP60 in both these systems is believed to extend beyond assisting in protein folding and maturation, e.g., overexpression of Ras does not result in increased association with HSP60 (59) . This observation excludes a primary role of HSP60 in folding and maturation of p21Ras (59) . In other reports, interaction of extracellular HSP60 with the macrophage cell surface receptors CD14 (61) and Toll-like recepetor-4 complex (62) elicited a signaling pathway that resulted in activation of these cells. Furthermore, surface-exposed HSP60, together with integrin, mediate internalization of Staphylococcal aureus by epithelial cells (63) . These studies and others demonstrate that HSP60 takes part in some nonchaperonin functions.

Because the association of HSP60 with {alpha}3ß1 integrin is rapidly regulated at the cell surface, it is unlikely that this association is simply a chaperonin function necessary for the maturation of the integrin. Rather, HSP60 interacts with the mature low activity integrin and induces an activated state. As such, HSP60 may modulate the conformation of {alpha}3ß1 to regulate protein function rather than maturation. By analogy, HSP60 was shown to modulate folding of src kinase, thereby altering the activity of this protein (64) .

HSP60 is probably not the sole inducer of {alpha}3ß1-integrin activation. Residual inducible activity of the integrin remains even after blocking HSP60 association using the pharmacological agent Miz or the ammonium and potassium ionophore nonactin (Figs. 7Citation and 8Citation ). Although these drugs may have other biochemical effects on the cell, Miz inhibited HSP60 association with the {alpha}3ß1 integrin on the cell surface and with purified integrin in vitro. Furthermore, both drugs prevented IGF1-mediated activation of {alpha}3ß1 without interfering with the activity of the {alpha}2ß1 integrin or with cell spreading induced by antibody activation of {alpha}3ß1 integrin. Nonactin inhibited activation of {alpha}3ß1 mediated by IGF1, but not that mediated by exogenous HSP60, showing that this drug does not inhibit any signal downstream from the integrin. These observations suggest that these drugs specifically inhibit HSP60-dependent activation of {alpha}3ß1 integrin.

HSP60 association with {alpha}3ß1 integrin is induced by IGF1 and FCS. PTs differentially modulate {alpha}3ß1-integrin activity in two breast cancer cell lines. These signals appear to induce export to the cell surface and perhaps a biochemical modification of HSP60 that triggers its association with the integrin. By analogy, HSP60 interacts with histone 2B in the plasma membrane, and this interaction is dependent on phosphorylation of both HSP60 and the histone (65) .

On the cell surface, {alpha}3ß1 interacts with two detectable surface-exposed proteins. We have identified one of them as HSP60. The other migrated with a molecular mass of 32 kDa and is possibly CD151, a member of the tetraspanin 4 superfamily of proteins (66 , 67) . Although we cannot exclude the possibility that the 32-kDa protein is mediating HSP60 interaction with the integrin, it appears that the interaction of HSP60 with the integrin is independent of this protein for the following reasons. HSP60 binds to purified {alpha}3ß1 integrin, and this interaction is inhibited by mixoribine, which also inhibited function. The amount of 32-kDa protein in the complex does not change with the amount of HSP60 in the complex. In fact, anti-ß1 integrin immune complexes from cells treated with PT have reduced amounts of 32-kDa protein, whereas HSP60 protein amount is increased. Furthermore, the 32-kDa protein is not present in the MDA-MB435 cell line, indicating that at least in this cell line, the 32-kDa protein is not necessary to mediate HSP60’s association with the integrin. We are in the process of analyzing the mechanism of the interaction of HSP60 and the integrin.

Several aspects of tumor progression and development, including proliferation, modulation of differentiation (68 , 69) , metastasis (8 , 9) , and invasion (70) , have been shown to be dependent on {alpha}3ß1-integrin activity. Inhibition of proliferation and induction of differentiation of small cell lung carcinoma cells by TSP1 is mediated by {alpha}3ß1 integrin (7) . Induction of {alpha}3ß1-integrin activity results in enhanced motility and adhesion of breast cancer cells (5) . On the other hand, engagement of {alpha}3ß1 integrin stimulates endothelial cell proliferation and angiogenesis (71) . Therefore, therapeutics that selectively inhibit {alpha}3ß1-integrin activation or ligand binding may inhibit tumor progression by disrupting function of this integrin both in tumor cells and tumor vasculature.


   FOOTNOTES
 
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.

1 To whom requests for reprints should be addressed, at Building 10 Room 2A33, 10 Center Drive MSC 1500, NIH Bethesda, MD 20892-1500. Phone: (301) 496-6264; Fax: (301) 402-0043; E-mail: droberts{at}helix.nih.gov. Back

2 The abbreviation used are: TSP1, human thrombospondin-1; IPG, immobilized pH gradient; MBP, maltose binding protein; IGF1, insulin-like growth factor 1; ILK, integrin-linked kinase; PT, pertussis toxin; IEF, isoelectric focusing; HSP60, heat shock protein 60; LCMS, liquid chromatography and mass spectrometry; PVDF, polyvinylidene difluoride; RIPA, radioimmunoprecipitation assay; Miz, mizoribine; TBS, Tris-buffered saline. Back

Received 3/15/01. Accepted 12/28/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Stahl S., Weitzman S., Jones J. C. R. The role of laminin-5 and its receptors in mammary epithelial cell branching morphogenesis. J. Cell Sci., 110: 55-63, 1997.[Abstract]
  2. Seftor R. E., Seftor E. A., Sheng S., Pemberton P. A., Sager R., Hendrix M. J. Maspin suppresses the invasive phenotype of human breast carcinoma. Cancer Res., 58: 5681-5685, 1998.[Abstract/Free Full Text]
  3. Engebraaten O., Fodstad O. Site-specific experimental metastasis patterns of two human breast cancer cell lines in nude rats. Int. J. Cancer, 82: 219-225, 1999.[Medline]
  4. DeFreitas M. F., Yoshida C. K., Frazier W. A., Mendrick D. L., Kypta R. M., Reichardt L. F. Identification of integrin {alpha}3ß1 as a neuronal thrombospondin receptor mediating neurite outgrowth. Neuron, 15: 333-343, 1995.[Medline]
  5. Chandrasekaran S., Guo N., Rodrigues R. G., Kaiser J., Roberts D. D. Pro-adhesive and chemotactic activities of thrombospondin-1 for breast carcinoma cells are mediated by {alpha}3ß1 integrin and regulated by insulin-like growth factor-1 and CD98. J. Biol. Chem., 274: 11408-11416, 1999.[Abstract/Free Full Text]
  6. Krutzsch H. C., Choe B. J., Sipes J. M., Guo N., Roberts D. D. Identification of an {alpha}3ß1 integrin recognition sequence in thrombospondin-1. J. Biol. Chem., 274: 24080-24086, 1999.[Abstract/Free Full Text]
  7. Guo N., Templeton N. S., Al-Barazi H., Cashel J. A., Sipes J. M., Krutzsch H. C., Roberts D. D. Thrombospondin-1 promotes {alpha}3ß1 integrin-mediated adhesion and neurite-like outgrowth and inhibits proliferation of small cell lung carcinoma cells. Cancer Res., 60: 457-466, 2000.[Abstract/Free Full Text]
  8. Tawil N. J., Gowri V., Djoneidi M., Nip J., Carbonetto S., Brodt P. Integrin {alpha}3ß1 can promote adhesion and spreading of metastatic breast carcinoma cells on the lymph node stroma. Int. J. Cancer, 66: 703-710, 1996.[Medline]
  9. Lundstrom A., Holmbom J., Lindqvist C., Nordstrom T. The role of {alpha}2 ß1 and {alpha}3ß1 integrin receptors in the initial anchoring of MDA-MB-231 human breast cancer cells to cortical bone matrix. Biochem. Biophys. Res. Commun., 250: 735-740, 1998.[Medline]
  10. Schoenwaelder S. M., Burridge K. Bidirectional signaling between the cytoskeleton and integrins. Curr. Opin. Cell Biol., 11: 274-286, 1999.[Medline]
  11. Coppolino M. G., Dedhar S. Bi-directional signal transduction by integrin receptors. Int. J. Biochem. Cell Biol., 32: 171-188, 2000.[Medline]
  12. Giancotti F. G., Ruoslahti E. Integrin signaling. Science (Wash. DC), 285: 1028-1032, 1999.[Abstract/Free Full Text]
  13. Clezardin P. Recent insights into the role of integrins in cancer metastasis. Cell Mol. Life Sci., 54: 541-548, 1998.[Medline]
  14. Sanders R. J., Mainiero F., Giancotti F. G. The role of integrins in tumorigenesis and metastasis. Cancer Investig., 16: 329-344, 1998.[Medline]
  15. Price J. T., Bonovich M. T., Kohn E. C. The biochemistry of cancer dissemination. Crit. Rev. Biochem. Mol. Biol., 32: 175-253, 1997.[Medline]
  16. Morris V. L., Schmidt E. E., MacDonald I. C., Groom A. C., Chambers A. F. Sequential steps in hematogenous metastasis of cancer cells studied by in vivo videomicroscopy. Invasion Metastasis, 17: 281-296, 1997.[Medline]
  17. Diamond M. S., Springer T. A. The dynamic regulation of integrin adhesiveness. Curr. Biol., 4: 506-517, 1994.[Medline]
  18. Kucik D. F., Dustin M. L., Miller J. M., Brown E. J. Adhesion-activating phorbol ester increases the mobility of leukocyte integrin LFA-1 in cultured lymphocytes. J. Clin. Investig., 97: 2139-2144, 1996.[Medline]
  19. O’Toole T. E., Katagiri Y., Faull R. J., Peter K., Tamura R., Quaranta V., Loftus J. C., Shattil S. J., Ginsberg M. H. Integrin cytoplasmic domains mediate inside-out signal transduction. J. Cell Biol., 124: 1047-1059, 1994.[Abstract/Free Full Text]
  20. O’Toole T. E., Mandelman D., Forsyth J., Shattil S. J., Plow E. F., Ginsberg M. H. Modulation of the affinity of integrin {alpha}IIb ß3 (GPIIb-IIIa) by the cytoplasmic domain of {alpha}IIb. Science (Wash. DC), 254: 845-847, 1991.[Abstract/Free Full Text]
  21. Hughes P. E., Diaz-Gonzalez F., Leong L., Wu C., McDonald J. A., Shattil S. J., Ginsberg M. H. Breaking the integrin hinge. A defined structural constraint regulates integrin signaling. J. Biol. Chem., 271: 6571-6574, 1996.[Abstract/Free Full Text]
  22. Hannigan G. E., Leung-Hagesteijn C., Fitz-Gibbon L., Coppolino M. G., Radeva G., Filmus J., Bell J. C., Dedhar S. Regulation of cell adhesion and anchorage-dependent growth by a new ß1-integrin-linked protein kinase. Nature (Lond.), 379: 91-96, 1996.[Medline]
  23. Wu C. Integrin-linked kinase and PINCH: partners in regulation of cell-extracellular matrix interaction and signal transduction. J. Cell Sci., 112: 4485-4489, 1999.[Abstract]
  24. Blystone S. D., Lindberg F. P., Williams M. P., McHugh K. P., Brown E. J. Inducible tyrosine phosphorylation of the ß3 integrin requires the {alpha}V integrin cytoplasmic tail. J. Biol. Chem., 271: 31458-31462, 1996.[Abstract/Free Full Text]
  25. Law D. A., Nannizzi-Alaimo L., Phillips D. R. Outside-in integrin signal transduction. {alpha}IIb ß3-(GP IIb IIIa) tyrosine phosphorylation induced by platelet aggregation. J. Biol. Chem., 271: 10811-10815, 1996.[Abstract/Free Full Text]
  26. Blystone S. D., Williams M. P., Slater S. E., Brown E. J. Requirement of integrin ß3 tyrosine 747 for ß3 tyrosine phosphorylation and regulation of {alpha}vß3 avidity. J. Biol. Chem., 272: 28757-28761, 1997.[Abstract/Free Full Text]
  27. Chen H. C., Appeddu P. A., Parsons J. T., Hildebrand J. D., Schaller M. D., Guan J. L. Interaction of focal adhesion kinase with cytoskeletal protein talin. J. Biol. Chem., 270: 16995-16999, 1995.[Abstract/Free Full Text]
  28. Otey C. A., Pavalko F. M., Burridge K. An interaction between {alpha}-actinin and the ß1 integrin subunit in vitro. J. Cell Biol., 111: 721-729, 1990.[Abstract/Free Full Text]
  29. Lo S. H., Weisberg E., Tensin C. L. B. A potential link between the cytoskeleton and signal transduction. Bioessays, 16: 817-823, 1994.[Medline]
  30. Sharma C. P., Ezzell R. M., Arnaout M. A. Direct interaction of filamin (ABP-280) with the ß 2-integrin subunit CD18. J. Immunol., 154: 3461-3470, 1995.[Abstract]
  31. Burridge K., Molony L., Kelly T. Adhesion plaques: sites of transmembrane interaction between the extracellular matrix and the actin cytoskeleton. J. Cell Sci. Suppl., 8: 211-229, 1987.[Medline]
  32. Yamada K. M., Geiger B. Molecular interactions in cell adhesion complexes. Curr. Opin. Cell Biol., 9: 76-85, 1997.[Medline]
  33. Coppolino M., Leung-Hagesteijn C., Dedhar S., Wilkins J. Inducible interaction of integrin {alpha}2ß1 with calreticulin. Dependence on the activation state of the integrin. J. Biol. Chem., 270: 23132-23138, 1995.[Abstract/Free Full Text]
  34. Zhu Q., Zelinka P., White T., Tanzer M. L. Calreticulin-integrin bidirectional signaling complex. Biochem. Biophys. Res. Commun., 232: 354-358, 1997.[Medline]
  35. Hemler M. E., Sanchez-Madrid F., Flotte T. J., Krensky A. M., Burakoff S. J., Bhan A. K., Springer T. A., Strominger J. L. Glycoproteins of 210,000 and 130,000 m.w. on activated T cells: cell distribution and antigenic relation to components on resting cells and T cell lines. J. Immunol., 132: 3011-3018, 1984.[Abstract]
  36. Roberts D. D., Cashel J., Guo N. Purification of thrombospondin from human platelets. J. Tiss. Cult. Meth., 16: 217-222, 1994.
  37. Akiyama S. K., Yamada K. M. The interaction of plasma fibronectin with fibroblastic cells in suspension. J. Biol. Chem., 260: 4492-4500, 1985.[Abstract/Free Full Text]
  38. Eble J. A., ucherpfennig K. W., Gauthier L., Dersch P., Krukonis E., Isberg R. R., Hemler M. E. Recombinant soluble human {alpha}3ß1 integrin: purification, processing, regulation, and specific binding to laminin-5 and invasin in a mutually exclusive manner. Biochemistry, 37: 10945-10955, 1998.[Medline]
  39. Roberts D. D., Sherwood J. A., Ginsburg V. Platelet thrombospondin mediates attachment and spreading of human melanoma cells. J. Cell Biol., 104: 131-139, 1987.[Abstract/Free Full Text]
  40. Coopman P. J., Thomas D. M., Gehlsen K. R., Mueller S. C. Integrin {alpha}3ß1 participates in the phagocytosis of extracellular matrix molecules by human breast cancer cells. Mol. Biol. Cell, 7: 1789-1804, 1996.[Abstract]
  41. van der P., Vloedgraven H., Papapoulos S., Lowick C., Grzesik W., Kerr J., Robey P. G. Attachment characteristics and involvement of integrins in adhesion of breast cancer cell lines to extracellular bone matrix components. Lab. Investig., 77: 665-675, 1997.[Medline]
  42. Doerr M. E., Jones J. I. The roles of integrins and extracellular matrix proteins in the insulin-like growth factor I-stimulated chemotaxis of human breast cancer cells. J. Biol. Chem., 271: 2443-2447, 1996.[Abstract/Free Full Text]
  43. Soltys B. J., Gupta R. S. Immunoelectron microscopic localization of the 60-kDa heat shock chaperonin protein (Hsp60) in mammalian cells. Exp. Cell Res., 222: 16-27, 1996.[Medline]
  44. Akiyama S. K., Olden K., Yamada K. M. Fibronectin and integrins in invasion and metastasis. Cancer Metastasis Rev., 14: 173-189, 1995.[Medline]
  45. Felding-Habermann B., Cheresh D. A. Vitronectin and its receptors. Curr. Opin. Cell Biol., 5: 864-868, 1993.[Medline]
  46. Itoh H., Komatsuda A., Wakui H., Miura A. B., Tashima Y. Mammalian HSP60 is a major target for an immunosuppressant mizoribine. J. Biol. Chem., 274: 35147-35151, 1999.[Abstract/Free Full Text]
  47. Turka L. A., Dayton J., Sinclair G., Thompson C. B., Mitchell B. S. Guanine ribonucleotide depletion inhibits T cell activation. Mechanism of action of the immunosuppressive drug mizoribine. J. Clin. Investig., 87: 940-948, 1991.
  48. Anderson L. Identification of mitochondrial proteins and some of their precursors in two-dimensional electrophoretic maps of human cells. Proc. Natl. Acad. Sci. USA, 78: 2407-2411, 1981.[Abstract/Free Full Text]
  49. Fink A. L. Chaperone-mediated protein folding. Physiol. Rev., 79: 425-449, 1999.[Abstract/Free Full Text]
  50. Sigler P. B., Xu Z., Rye H. S., Burston S. G., Fenton W. A., Horwich A. L. Structure and function in GroEL-mediated protein folding. Annu. Rev. Biochem., 67: 581-608, 1998.[Medline]
  51. Bukau B., Horwich A. L. The Hsp70 and Hsp60 chaperone machines. Cell, 92: 351-366, 1998.[Medline]
  52. Soltys B. J., Gupta R. S. Mitochondrial-matrix proteins at unexpected locations: are they exported?. Trends Biochem. Sci., 24: 174-177, 1999.[Medline]
  53. Soltys B. J., Falah M., Gupta R. S. Identification of endoplasmic reticulum in the primitive eukaryote Giardia lamblia using cryoelectron microscopy and antibody to Bip. J. Cell. Sci., 109: 1909-1917, 1996.[Abstract]
  54. Fisch P., Malkovsky M., Kovats S., Sturm E., Braakman E., Klein B. S., Voss S. D., Morrissey L. W., DeMars R., Welch W. J., et al Recognition by human V {gamma} 9/V {delta} 2 T cells of a GroEL homolog on Daudi Burkitt’s lymphoma cells. Science (Wash. DC), 250: 1269-1273, 1990.[Abstract/Free Full Text]
  55. Soltys B. J., Gupta R. S. Cell surface localization of the 60 kDa heat shock chaperonin protein (hsp60) in mammalian cells. Cell Biol. Int., 21: 315-320, 1997.[Medline]
  56. Xu Q., Schett G., Seitz C. S., Hu Y., Gupta R. S., Wick G. Surface staining and cytotoxic activity of heat-shock protein 60 antibody in stressed aortic endothelial cells. Circ. Res., 75: 1078-1085, 1994.[Abstract/Free Full Text]
  57. Jindal S., Dudani A. K., Singh B., Harley C. B., Gupta R. S. Primary structure of a human mitochondrial protein homologous to the bacterial and plant chaperonins and to the 65-kilodalton mycobacterial antigen. Mol. Cell. Biol., 9: 2279-2283, 1989.[Abstract/Free Full Text]
  58. Woodlock T. J., Chen X., Young D. A., Bethlendy G., Lichtman M. A., Segel G. B. Association of HSP60-like proteins with the L-system amino acid transporter. Arch. Biochem. Biophys., 338: 50-56, 1997.[Medline]
  59. Ikawa S., Weinberg R. A. An interaction between p21ras and heat shock protein hsp60, a chaperonin. Proc. Natl. Acad. Sci. USA, 89: 2012-2016, 1992.[Abstract/Free Full Text]
  60. Jones M., Gupta R. S., Englesberg E. Enhancement in amount of P1 (hsp60) in mutants of Chinese hamster ovary (CHO-K1) cells exhibiting increases in the A system of amino acid transport. Proc. Natl. Acad. Sci. USA, 91: 858-862, 1994.[Abstract/Free Full Text]
  61. Kol A., Lichtman A. H., Finberg R. W., Libby P., Kurt-Jones E. A. Cutting edge: heat shock protein (HSP) 60 activates the innate immune response: CD14 is an essential receptor for HSP60 activation of mononuclear cells. J. Immunol., 164: 13-17, 2000.[Abstract/Free Full Text]
  62. Ohashi K., Burkart V., Flohe S., Kolb H. Cutting edge: heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex. J. Immunol., 164: 558-561, 2000.[Abstract/Free Full Text]
  63. Dziewanowska K., Carson A. R., Patti J. M., Deobald C. F., Bayles K. W., Bohach G. A. Staphylococcal fibronectin binding protein interacts with heat shock protein 60 and integrins: role in internalization by epithelial cells. Infect. Immun., 68: 6321-6328, 2000.[Abstract/Free Full Text]
  64. Abdel-Ghany M., el-Gendy K., Zhang S., Racker E. Control of src kinase activity by activators, inhibitors, and substrate chaperones. Proc. Natl. Acad. Sci. USA, 87: 7061-7065, 1990.[Abstract/Free Full Text]
  65. Khan I. U., Wallin R., Gupta R. S., Kammer G. M. Protein kinase A-catalyzed phosphorylation of heat shock protein 60 chaperone regulates its attachment to histone 2B in the T lymphocyte plasma membrane. Proc. Natl. Acad. Sci. USA, 95: 10425-10430, 1998.[Abstract/Free Full Text]
  66. Yauch R. L., Berditchevski F., Harler M. B., Reichner J., Hemler M. E. Highly stoichiometric, stable, and specific association of integrin {alpha}3ß1 with CD151 provides a major link to phosphatidylinositol 4-kinase, and may regulate cell migration. Mol. Biol. Cell, 9: 2751-2765, 1998.[Abstract/Free Full Text]
  67. Yanez-Mo M., Alfranca A., Cabanas C., Marazuela M., Tejedor R., Ursa M. A., Ashman L. K., de Landazuri M. O., Sanchez-Madrid F. Regulation of endothelial cell motility by complexes of tetraspan molecules CD81/TAPA-1 and CD151/PETA-3 with {alpha}3ß1 integrin localized at endothelial lateral junctions. J. Cell Biol., 141: 791-804, 1998.[Abstract/Free Full Text]
  68. Symington B. E., Carter W. G. Modulation of epidermal differentiation by epiligrin and integrin {alpha}3ß1. J. Cell Sci., 108: 831-838, 1995.[Abstract]
  69. Gout S. P., Jacquier-Sarlin M. R., Rouard-Talbot L., Rousselle P., Block M. R. RhoA-dependent switch between {alpha}2ß1 and {alpha}3ß1 integrins is induced by laminin-5 during early stage of HT-29 cell differentiation. Mol. Biol. Cell, 12: 3268-3281, 2001.[Abstract/Free Full Text]
  70. Sugiura T., Berditchevski F. Function of {alpha}3ß1-tetraspanin protein complexes in tumor cell invasion. Evidence for the role of the complexes in production of matrix metalloproteinase 2 (MMP-2). J. Cell Biol., 146: 1375-1389, 1999.[Abstract/Free Full Text]
  71. Chandrasekaran L., He C. Z., Al-Barazi H., Krutzsch H. C., Iruela-Arispe M. L., Roberts D. D. Cell contact-dependent activation of {alpha}3ß1 integrin modulates endothelial cell responses to thrombospondin-1. Mol. Biol. Cell, 11: 2885-2900, 2000.[Abstract/Free Full Text]



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Neuro-oncol, July 1, 2005; 7(3): 260 - 278.
[Abstract] [PDF]

Home page
 J. Cell Sci.Home page
G. Pfister, C. M. Stroh, H. Perschinka, M. Kind, M. Knoflach, P. Hinterdorfer, and G. Wick
Detection of HSP60 on the membrane surface of stressed human endothelial cells by atomic force and confocal microscopy
J. Cell Sci., April 15, 2005; 118(8): 1587 - 1594.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
B. J. Johnson, T. T. T. Le, C. A. Dobbin, T. Banovic, C. B. Howard, F. d. M. L. Flores, D. Vanags, D. J. Naylor, G. R. Hill, and A. Suhrbier
Heat Shock Protein 10 Inhibits Lipopolysaccharide-induced Inflammatory Mediator Production
J. Biol. Chem., February 11, 2005; 280(6): 4037 - 4047.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. J. Calzada, D. S. Annis, B. Zeng, C. Marcinkiewicz, B. Banas, J. Lawler, D. F. Mosher, and D. D. Roberts
Identification of Novel {beta}1 Integrin Binding Sites in the Type 1 and Type 2 Repeats of Thrombospondin-1
J. Biol. Chem., October 1, 2004; 279(40): 41734 - 41743.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
J. L. Wampler, K.-P. Kim, Z. Jaradat, and A. K. Bhunia
Heat Shock Protein 60 Acts as a Receptor for the Listeria Adhesion Protein in Caco-2 Cells
Infect. Immun., February 1, 2004; 72(2): 931 - 936.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y.-x. Shan, T.-L. Yang, R. Mestril, and P. H. Wang
Hsp10 and Hsp60 Suppress Ubiquitination of Insulin-like Growth Factor-1 Receptor and Augment Insulin-like Growth Factor-1 Receptor Signaling in Cardiac Muscle: IMPLICATIONS ON DECREASED MYOCARDIAL PROTECTION IN DIABETIC CARDIOMYOPATHY
J. Biol. Chem., November 14, 2003; 278(46): 45492 - 45498.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
D. R. Clemmons and L. A. Maile
Minireview: Integral Membrane Proteins that Function Coordinately with the Insulin-Like Growth Factor I Receptor to Regulate Intracellular Signaling
Endocrinology, May 1, 2003; 144(5): 1664 - 1670.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
B. Billack, D. E. Heck, T. M. Mariano, C. R. Gardner, R. Sur, D. L. Laskin, and J. D. Laskin
Induction of cyclooxygenase-2 by heat shock protein 60 in macrophages and endothelial cells
Am J Physiol Cell Physiol, October 1, 2002; 283(4): C1267 - C1277.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
D. G. Stupack and D. A. Cheresh
Get a ligand, get a life: integrins, signaling and cell survival
J. Cell Sci., January 10, 2002; 115(19): 3729 - 3738.
[Abstract] [Full Text] [PDF]

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