Decrease in the Amount of Focal Adhesion Kinase (p125FAK) in Interleukin-1beta -stimulated Human Umbilical Vein Endothelial Cells by Binding of Human Monocytic Cell Lines*

(Received for publication, November 19, 1996, and in revised form, May 12, 1997)

Kanso Iwaki Dagger §, Kunihiro Ohashi Dagger , Masao Ikeda Dagger , Katsuhiko Tsujioka , Fumihiko Kajiya par and Masashi Kurimoto Dagger

From the Dagger  Fujisaki Institute, Hayashibara Biochemical Laboratories, Inc., 675-1 Fujisaki, Okayama 702, the  Department of Physiology, and the par  Department of Medical Engineering, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-01, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Monocytes in the blood circulation migrate across endothelial cell monolayers lining the blood vessels and infiltrate into the underlying tissues in inflammation. However, little is known about the mechanisms by which leukocytes migrate across the endothelial barrier after binding and what molecules participate in the process. Addition of the human monocytic cell line THP-1 to interleukin-1beta (IL-1beta )-stimulated human umbilical vein endothelial cells (HUVEC) induced a decrease in the amount of focal adhesion kinase (p125FAK) protein, a tyrosine kinase localized at focal contacts and essential for cell attachment to the extracellular matrix, whereas little change was observed in the amount of other molecules associated with cell adhesion such as vascular cell adhesion molecule-1, alpha -catenin, and talin. A maximum decrease in the amount of p125FAK was observed 15-30 min after addition of THP-1 cells to HUVEC, after which the level of p125FAK gradually recovered. A reduction in the density of actin stress fibers in IL-1beta -activated HUVEC was observed in parallel with the decrease in p125FAK. The p125FAK decrease was partially inhibited by preventing THP-1 binding to HUVEC using a mixture of antibodies to adhesion molecules. We suggest that the decrease in p125FAK triggered by binding of monocytes in inflammation facilitates the transendothelial migration of the monocytes by altering the adhesiveness of endothelial cells to the extracellular matrix.


INTRODUCTION

In the early stages of inflammation, monocytes and other leukocytes in the blood circulation migrate across endothelial cell monolayers lining the blood vessels and enter the perivascular tissues. The migration of leukocytes involves multiple steps, and various types of adhesion molecules participate in these processes, including selectins mediating initial tethering and rolling of leukocytes over the endothelial cells, and integrins on leukocytes interacting with adhesion molecules belonging to the immunoglobulin superfamily expressed on the endothelial cells (1, 2). In acute inflammation, the expression and activation of adhesion molecules are regulated by mediators such as thrombin, inflammatory cytokines, and chemokines (2).

Although many observations have focused on the molecules participating in the events from tethering to adhesion of leukocytes to endothelial cells, little is known about the mechanisms whereby leukocytes migrate across the endothelial barrier after binding and which molecules participate in the process.

Platelet/endothelial cell adhesion molecule-1 (PECAM-1)1 is one of the adhesion molecules that is concentrated at intercellular junctions between endothelial cells (3). Anti-PECAM-1 monoclonal antibody (mAb) or soluble PECAM-1 inhibits the transmigration of leukocytes through endothelial cell monolayers in vitro without interfering with the leukocyte's potential to adhere tightly to the apical surface of endothelial cells (4). For neutrophils, integrin-associated protein (CD47) present on both neutrophils and endothelial cells is supposed to be essential for invasion (5). Activation of intercellular adhesion molecule-1 (ICAM-1) by binding of T cells has been reported to transduce a signal into endothelial cells, which induces tyrosine phosphorylation of the actin-binding protein cortactin, indicating alterations in the cytoskeleton (6). These findings suggesting the possibility that binding itself induces changes in endothelial cells leading to relaxation of interendothelial cell junctions are significant.

To delineate the mechanism whereby monocytes can transmigrate through the endothelium during inflammation, we first investigated the changes in protein phosphorylation patterns of interleukin-1beta (IL-1beta )-stimulated human umbilical vein endothelial cells (HUVEC) overlayered with human monocytic THP-1 cells and found that the addition of THP-1 cells induces a decrease in the amount of a phosphorylated 120-130-kDa protein(s) in HUVEC. In this study, we show that the decreased protein is focal adhesion kinase (p125FAK), a tyrosine kinase present at focal contact sites, and we discuss the possible involvement of this alteration in the process of leukocyte migration at sites of inflammation.


EXPERIMENTAL PROCEDURES

Cell Lines

HUVEC were purchased from Kurabo (Osaka, Japan) and were cultured on gelatin-coated culture flasks (Iwaki glass, Tokyo, Japan) with EGM-UV medium (Kurabo). Human monocytic THP-1 cells (Japanese Cancer Research Resources Bank, Tokyo, Japan), monoblastic U937 cells (American Type Culture Collection, Rockville, MD), promyelocytic HL-60 cells (Fujisaki Cell Center, Hayashibara Biochemical Labs., Inc., Okayama, Japan), and T leukemic MOLT-16 cells (Fujisaki Cell Center) were maintained in RPMI 1640 (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% fetal bovine serum (Life Technologies, Inc.), 10 mM HEPES, 100 units/ml penicillin, and 50 µg/ml streptomycin.

Reagents

Human IL-1beta was purchased from Genzyme (Cambridge, MA). Human natural tumor necrosis factor-alpha (TNF-alpha ) (specific activity of 2 × 106 Japan reference unit/mg) was prepared in our laboratory (7). Mouse monoclonal anti-phosphotyrosine (anti-Tyr(P)) PY20, anti-Tyr(P) 4G10, anti-p125FAK 2A7, and anti-alpha 4 integrin SG/73 were purchased from Seikagaku Corp. (Tokyo, Japan). Rabbit polyclonal anti-p125FAK C-20 antibody and goat polyclonal anti-vascular cell adhesion molecule-1 (VCAM-1) C-19 antibody were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-beta 2 integrin mAb MEM 48 was purchased from R&D Systems (Abingdon, UK), and anti-sialyl Lex CSLEX1 (IgM) was purchased from Becton Dickinson (Bedford, MA). Anti-talin mAb TA205 was purchased from Genosys (Cambridge, UK). Rabbit polyclonal anti-alpha -catenin was purchased from Sigma.

Preparation of Whole Cell Lysates, Cell Extracts, and Insoluble Fractions of HUVEC

Six-cm culture dishes (Falcon 3002, Becton Dickinson) were coated with 4 ml of a 100 µg/ml solution of gelatin (Iwaki Glass) in phosphate-buffered saline (PBS) for 2 h, and HUVEC were grown to confluency on the coated dishes. HUVEC were stimulated with IL-1beta or TNF-alpha for 5 h and subsequently overlayered with various human leukemic cells for the indicated times at different cell densities.

After washing the mixed cultures of HUVEC and leukemic cells with PBS, the cells were lysed with 500 µl of an extraction buffer (1% Triton X-100, 1% Nonidet P-40, 150 mM NaCl, 2 mM Na3VO4, 10 mM NaF, 2 mM phenylmethylsulfonyl fluoride, 250 µg/ml leupeptin, 2 mM EDTA, 50 mM Tris, pH 7.5) with the aid of a cell scraper. The lysates were stood on ice for 30 min with occasional mixing. One hundred µl of the lysates were then transferred to new tubes as the whole cell lysate. The residual lysates were centrifuged at 13,000 × g for 30 min, and the supernatants were collected and used as the cell extract. Four hundred µl of extraction buffer solutions containing 1% SDS were added to each remaining pellet and were dissolved by vigorous pipetting. These fractions were defined as the insoluble fraction.

Immunoprecipitation and Immunoblotting

The cell extracts were incubated with 1 µg of anti-Tyr(P) 4G10 for 2 h or with 4 µg of anti-p125FAK 2A7 for 16-18 h at 4 °C with continuous mixing. Protein G-Sepharose (Pharmacia, Uppsala, Sweden) was washed twice with Tris-buffered saline (150 mM NaCl, 10 mM Tris, pH 7.4) and once with a washing buffer (1% Triton X-100, 1% Nonidet P-40, 150 mM NaCl, 50 mM Tris, pH 7.5), and the resin pellet was resuspended in washing buffer. The resins from 100-µl volumes of 50% suspensions were mixed with the HUVEC lysates and were incubated for 2 h at 4 °C with continuous mixing. The resins were washed three times with washing buffer and then resuspended in 40 µl of a 2 × SDS-sample buffer (100 mM Tris, 5% SDS, 30% glycerol, 5% 2-mercaptoethanol, pH 6.8), and boiled for 5 min. After centrifugation, 20 µl of the supernatants were subjected to electrophoresis on a 7.5% polyacrylamide gel in the presence of SDS and transferred to nitrocellulose filters. In the case of direct immunoblotting, samples were treated with half their volume of 3 × SDS-sample buffer (150 mM Tris, 7.5% SDS, 45% glycerol, 7.5% 2-mercaptoethanol, pH 6.8), and 20 µl of the treated samples were subjected to electrophoresis.

After blocking nonspecific binding with Block Ace (Yukijirushi, Sapporo, Japan), the filters were probed with the antibody of interest for 2-3 h at room temperature (rt), followed by either horseradish peroxidase-labeled rabbit anti-mouse Igs (Dako Japan, Kyoto, Japan), horseradish peroxidase-labeled swine anti-rabbit Igs (Dako, Japan), or a Vectastain ABC-PO kit for goat IgG (Vector Laboratories, Burlingame, CA) for 2-3 h at rt. Washing of the membranes was performed with Tris-buffered saline containing 0.05% Tween 20. The bands were visualized with the enhanced chemiluminescence detection system (Amersham, Buckingamshire, United Kingdom) as directed by the manufacturer. In the case of reprobing the same membranes with a different first antibody, the horseradish peroxidase of the already bound second antibody was inactivated by treating the filters with Block Ace supplemented with 0.1% NaN3 for 16-18 h at rt. Quantification of the density of the detected blots was performed by scanning densitometry using ImageMaster DTS (Pharmacia).

Fluorescence Microscopy

Polystyrene chamber slides (Nippon InterMed, Tokyo, Japan) were coated with gelatin for 2 h. HUVEC were plated on the slides and cultured to confluency. HUVEC were then stimulated with 0.5 ng/ml IL-1beta for 5 h and were subsequently layered with 1.5 × 104 THP-1 cells at different periods. After removing the supernatant and washing, the cells were fixed with a mixture of acetone and methanol (1:1 v/v) for 20 min at -20 °C, and after washing with PBS, the cells were incubated with 2.5 units/ml of rhodamine phalloidin (Wako Pure Chemical Industries, Osaka, Japan) for 1 h at rt. After washing, the slides were mounted using 50% glycerol in PBS and observed under a fluorescence microscope (model BHF, Olympus, Tokyo, Japan).

Cell Adhesion Assay

Ten thousand HUVEC were seeded in each well of gelatin-coated 96-well culture plates (Iwaki Glass) and cultured for 48 h. Confluent cultures of HUVEC were stimulated with IL-1beta or TNF-alpha at 37 °C for 5 h and then washed once with assay medium (RPMI 1640 supplemented with 0.1% bovine serum albumin (Armour Pharmaceutical, Kankakee, IL), 10 mM HEPES, 100 units/ml penicillin, and 50 µg/ml streptomycin) before addition of isotope-labeled THP-1 cells. THP-1 cells were labeled with 51CrO4 (Amersham) at 37 °C for 1 h. After washing three times with the culture medium, 5 × 104 labeled THP-1 cells suspended in the assay medium were added to each well in 100-µl volumes. In inhibition experiments using adhesion-blocking antibodies, 51Cr-labeled THP-1 cells incubated with 50 µg/ml mAbs to adhesion molecules for 60 min at rt and washed twice were used. After mild centrifugation at 40 × g for 1 min, the plates were incubated at 37 °C for 30 min. The nonadherent cells were removed by washing twice with the assay medium, and the adherent THP-1 cells were lysed with 1 N NaOH. Radioactivity from samples of supernatants from each well and the original THP-1 cell suspension was determined by gamma counter, and the percentage of THP-1 cells adhering to HUVEC in each well was calculated.


RESULTS

The p125FAK Level in IL-1beta -stimulated HUVEC Is Decreased by Co-culture with Monocytic Cell Lines

First we investigated the changes in the tyrosine phosphorylation levels of molecules in HUVEC after adding human leukemic THP-1, U937, HL-60, or MOLT-16 cells. To simulate the activated state of blood vessels in inflammation, HUVEC were pretreated with IL-1beta for 5 h. After treating HUVEC with leukemic cells, the cells were lysed with the extraction buffer, and cell extracts were prepared. Tyrosine phosphorylation patterns were assessed by immunoprecipitation with anti-Tyr(P) 4G10 and subsequent immunoblotting with anti-Tyr(P) PY20. As shown in Fig. 1A, several tyrosine-phosphorylated proteins were observed in IL-1beta -stimulated HUVEC in the absence of the leukemic cell lines (lane 1). In the case of co-culture with THP-1 cells, a decrease in tyrosine-phosphorylated 120-130-kDa proteins in IL-1beta -stimulated HUVEC was very obvious (lanes 2 and 3). Considering the molecular size and the levels of expression of the phosphorylated molecule(s) observed in our experiments, p125FAK was selected as a probable candidate for the tyrosine-phosphorylated 120-130-kDa protein observed in HUVEC. To confirm the identity of the protein(s) banding at 120-130 kDa, cell extracts from HUVEC co-cultured with leukemic cell lines were immunoprecipitated with anti-p125FAK 2A7 and probed with anti-Tyr(P) PY20. As shown in Fig. 1B, anti-p125FAK 2A7 immunoprecipitated a 120-130 kDa protein, and the amount of immunoprecipitated molecule(s) was reduced by THP-1 co-culture in a manner depending on the number of seeded THP-1 cells (Fig. 1B, lanes 2 and 3). This indicates that a component of the tyrosine-phosphorylated 120-130-kDa band is identical to p125FAK.


Fig. 1. Decreased p125FAK in IL-1beta -stimulated HUVEC by co-culture with monocytic cell lines. Confluent HUVEC cultures on gelatin-coated culture dishes were treated with 0.5 ng/ml IL-1beta for 5 h and were subsequently overlayered with 6 × 105 cells/2 ml or 2 × 106 cells/2 ml of the human leukemic cells for 30 min. A, the cells were lysed with extraction buffer, and the cell extracts were subjected to immunoprecipitation with anti-Tyr(P) 4G10. The immunoprecipitates were then immunoblotted with anti-Tyr(P) PY-20. Molecular markers (kDa) are indicated on the left, and the tyrosine-phosphorylated 120-130-kDa bands (arrow) are indicated on the right. B, the cells were lysed with extraction buffer, and the cell extracts were subjected to immunoprecipitation with anti-p125FAK 2A7. One-half of the immunoprecipitates were immunoblotted with anti-Tyr(P) PY-20. The position of the p125FAK band is indicated on the right. C, the other half of the immunoprecipitates were immunoblotted with anti-p125FAK C-20. The position of the p125FAK band is indicated on the right. D, the cells were lysed with the extraction buffer, and the whole cell lysates were immunoblotted directly with anti-p125FAK C-20. The position of the p125FAK band is indicated on the right. IP, immunoprecipitation; IB, immunoblotting.
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The residual immunoprecipitates obtained by immunoprecipitation with anti-p125FAK 2A7 were probed with anti-p125FAK C-20. As shown in Fig. 1C, the pattern of immunoblots detected with polyclonal anti-p125FAK C-20 was almost identical to the pattern that was obtained with anti-Tyr(P) PY20, indicating that the decrease in p125FAK band resulted from a decrease in the amount of p125FAK protein itself and not from tyrosine de-phosphorylation of the protein. To further clarify the reason for the decrease in p125FAK, changes in the amount of p125FAK in whole cell lysates, cell extracts, and in insoluble fractions were investigated by direct immunoblotting with anti-p125FAK C-20. As shown in Fig. 1D, decreased p125FAK levels induced by monocytic cell treatment was observed in whole cell lysates. In addition, the pattern of p125FAK levels in cell extracts was identical to that in whole cell lysates, and no p125FAK was detected in the insoluble fractions under the same detection conditions (data not shown). From these results, we assumed that the decrease in the amount of p125FAK induced by monocytic cell seeding resulted from a decrease in p125FAK protein and not from a decrease in solubility of the protein. However, obvious candidates for the degradation products of p125FAK could not be observed. The decrease in p125FAK was observed not only in THP-1-treated HUVEC but also in U937-treated HUVEC (Fig. 1C, lanes 4 and 5), although no change was detected in HUVEC treated with HL-60 and MOLT-16 (Fig. 1C, lanes 6-9).

Decreased Protein Levels in HUVEC by Co-culture with THP-1 Cells

We investigated whether levels of proteins in IL-1beta -activated HUVEC other than p125FAK were reduced by THP-1 seeding or not. Talin present at focal contacts (8) such as is p125FAK, VCAM-1 expressed on the cell surface of cytokine-activated endothelial cells (9), and alpha -catenin co-localized at the sites of intercellular junctions with cadherin and beta -catenin (10), were probed with their respective antibodies on the same transferred membrane. As shown in Fig. 2, no changes in the amount of VCAM-1 and alpha -catenin were observed in whole cell lysates of HUVEC co-cultured with THP-1 cells. In the case of talin, a slight decrease was observed, and a possible degradation fragment of approximately 200 kDa was identified. However, the extent of the decrease in talin was far less than that observed in p125FAK. In U937-treated HUVEC, patterns for the probed proteins were almost the same as those observed in HUVEC treated with THP-1 cells (Fig. 2).


Fig. 2. Decreased levels of proteins in HUVEC induced by co-culture with monocytic cell lines. Confluent HUVEC cultures on gelatin-coated culture dishes were treated with 0.5 ng/ml IL-1beta for 5 h and were subsequently overlayered with 6 × 105 cells/2 ml or 2 × 106 cells/2 ml THP-1 or U937 cells for 30 min. The cells were lysed with extraction buffer, and the whole cell lysates were immunoblotted directly with anti-p125FAK C-20, anti-talin, anti-VCAM-1, or anti-alpha -catenin antibodies on the same transferred membrane. Molecular markers (kDa) are indicated on the left, and the positions of the proteins of interest are indicated on the right.
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VCAM-1 and alpha -catenin were not detected in whole cell lysates obtained from 2 × 106 THP-1 cells alone, although talin was faintly detectable (lane 4). In subsequent experiments we included alpha -catenin as a control to show that equal amounts of HUVEC protein were included in each sample of our assays.

Kinetics of the Decrease in the Amount of p125FAK in IL-1beta -treated HUVEC

To further characterize the decrease in the amount of p125FAK, the kinetics of the changes in p125FAK after addition of THP-1 cells were investigated. As shown in Fig. 3, the decrease in p125FAK was detected from 5 min after the addition of THP-1 cells (lane 5) and reached a maximum 15-30 min later (lanes 6 and 7) in IL-1beta -stimulated HUVEC. Although the amount of p125FAK did not return to initial levels, a tendency for recovery of p125FAK was observed (lane 10) 4 h from THP-1 cell seeding. Interestingly, the p125FAK degradation was not observed in unstimulated HUVEC (lanes 1-3).


Fig. 3. Kinetics of p125FAK decrease induced by THP-1 binding to IL-1beta -treated HUVEC. Confluent cultures of HUVEC on gelatin-coated culture dishes were untreated (lanes 1-3) or treated with 0.5 ng/ml IL-1beta (lanes 4-10) for 5 h and were subsequently overlayered with 1 × 106 THP-1 cells for the indicated times. The cells were lysed with extraction buffer, and the whole cell lysates were immunoblotted directly with anti-p125FAK C-20. Subsequently, alpha -catenin on the same membrane was also probed. The positions of the p125FAK and alpha -catenin bands are indicated on the right.
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Changes in the Cytoskeletal Structure of HUVEC Induced by THP-1 Binding

To investigate whether THP-1 treatment induces changes in the cytoskeletal structure of HUVEC, the HUVEC were stimulated with or without IL-1beta for 5 h and were subsequently overlayered with THP-1 cells. The cells were fixed, stained with rhodamine phalloidin, and observed by fluorescence microscopy. Well organized actin stress fibers were observed in both unstimulated (Fig. 4A) and IL-1beta -stimulated HUVEC (Fig. 4E) before seeding of THP-1 cells. The well developed stress fibers were also observed in unstimulated HUVEC which were overlayered with THP-1 cells (Fig. 4, B-D). In contrast, THP-1 seeding markedly reduced the number, thickness, and length of actin stress fibers in HUVEC preactivated with IL-1beta (Fig. 4, F-H). The changes were observed from 30 min after seeding THP-1 cells and continued for at least 2 h.


Fig. 4. Changes in cytoskeletal structure induced by THP-1 binding. Confluent cultures of HUVEC on gelatin-coated plastic chamber slides were incubated in the absence (A-D) or presence (E-H) of 0.5 ng/ml IL-1beta for 5 h. The HUVEC were untreated (A and E) or covered with 1.5 × 104 THP-1 cells for 0.5 h (B and F), 1 h (C and G), and 2 h (D and H). The cells were then fixed and stained with rhodamine phalloidin. In panel F, T represents adherent THP-1 cells.
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Effect of IL-1beta and TNF-alpha Treatment of HUVEC on the Decrease in p125FAK in HUVEC

The decrease in the amount of p125FAK induced by THP-1 cells was observed in IL-1beta -treated HUVEC but not in unstimulated HUVEC. Therefore, we investigated further whether THP-1 cells could induce the decrease in p125FAK in HUVEC stimulated with inflammatory stimuli other than IL-1beta . HUVEC were stimulated with TNF-alpha for 5 h and were then co-cultured with THP-1 for 30 min. The cells were lysed and subjected to immunoblotting with anti-p125FAK C-20. As shown in Fig. 5A, decrease in the amount of p125FAK after addition of THP-1 was observed not only in IL-1beta -activated HUVEC (lanes 2-5) but also in TNF-alpha -activated HUVEC (lanes 7-10) in a manner dependent on the concentration of the cytokines added. We simultaneously investigated the binding of THP-1 cells to HUVEC activated with these inflammatory stimuli. 51Cr-labeled THP-1 cells were added to activated HUVEC and incubated for 30 min. As shown in Fig. 5B, it was observed that IL-1beta and TNF-alpha treatment of HUVEC augmented THP-1 binding to HUVEC in a dose-dependent manner, suggesting a correlation between decrease in p125FAK and binding of THP-1 cells to cytokine-activated HUVEC.


Fig. 5. Effect of IL-1beta and TNF-alpha treatment of HUVEC on the decrease in p125FAK induced by THP-1 and the adherence of THP-1 cells. A, confluent HUVEC cultures on gelatin-coated culture dishes were treated with IL-1beta (lanes 2-5) or TNF-alpha (lanes 7-10) for 5 h. Subsequently, 1 × 106 THP-1 cells were added and incubated for 30 min. The cells were lysed with extraction buffer, and the whole cell lysates were subjected to immunoblotting with anti-p125FAK C-20. Subsequently, alpha -catenin on the same membrane was also probed. The position of the p125FAK and alpha -catenin bands are indicated on the right. B, HUVEC grown on a gelatin-coated 96-well microplate were treated with IL-1beta or TNF-alpha for 5 h. 51Cr-labeled THP-1 cells were added to the activated HUVEC and incubated for 30 min. Nonadherent cells were removed, and the adherence of THP-1 cells was determined. Values shown represent the mean ± S.D. of triplicate wells.
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Inhibition of the Decrease in p125FAK by Adhesion-blocking mAbs

It is well known that IL-1beta and TNF-alpha induce the expression of adhesion molecules such as ICAM-1, VCAM-1, and E-selectin on the surface of endothelial cells (9, 11, 12). Therefore, we investigated whether pretreating THP-1 cells with a blocking antibody to the counter-receptors for ICAM-1, VCAM-1, or E-selectin could inhibit the decrease in p125FAK or not. THP-1 cells have been reported to express beta 2 integrin, a beta  subunit of the beta 2 integrin family, alpha 4 integrin, an alpha  subunit of very late antigen-4, and sialyl Lex (13). beta 2 integrins, very late antigen-4, and sialyl Lex are known to interact with ICAM-1, VCAM-1, and E-selectin, respectively (14-18). THP-1 cells were incubated with 50 µg/ml anti-alpha 4, anti-beta 2, anti-sialyl Lex, or a mixture of these three mAbs at rt for 1 h. After washing three times with medium, the antibody-pretreated THP-1 cells were seeded over IL-1beta -activated HUVEC. As shown in Fig. 6A, pretreatment of THP-1 cells with a mixture of anti-alpha 4, anti-beta 2, and anti-sialyl Lex inhibited the decrease in p125FAK in IL-1beta -activated HUVEC (lane 6), whereas treatment with either of these mAbs alone could not inhibit the decrease in p125FAK (lanes 3-5). Fig. 6B shows the result of quantification of the density of the detected blots in Fig. 6A. In the case of cell adhesion assays, only treatment with a mixture of the three mAbs similarly inhibited the adherence of THP-1 cells to activated HUVEC (Fig. 6C, lane 6).


Fig. 6. Inhibition of the decrease in p125FAK and adherence of THP-1 cells by pretreatment with mAbs to adhesion molecules. A, THP-1 cells were incubated with 50 µg/ml mAbs to adhesion molecules for 60 min at rt. After washing, the pretreated THP-1 cells were seeded on IL-1beta -stimulated HUVEC, and the plates were incubated for 30 min. The cells were lysed and subjected to immunoblotting with anti-p125FAK C-20. Subsequently, alpha -catenin on the same membrane was also probed. The positions of the p125FAK and alpha -catenin bands are indicated on the right. B, intensities of immunoblotted p125FAK were quantified by densitometer. C, HUVEC grown on a gelatin-coated 96-well microplate were treated with IL-1beta for 5 h. 51Cr-Labeled and mAb-treated THP-1 cells were added to the activated HUVEC and incubated for 30 min. Nonadherent cells were removed, and the adherence of THP-1 cells was determined. Values shown represent the mean ± S.D. of quadruplicate wells. Control mAbs, mixture of class-matched irrelevant mAbs.
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DISCUSSION

Focal contacts are regions of the cell that come in direct contact with the extracellular matrix, providing anchorage sites for actin stress fibers and forming a link between the extracellular matrix and the actin cytoskeleton (19). The p125FAK molecule is a tyrosine kinase co-localized in focal contacts with several other molecules, such as talin and tensin (20-22), and plays a central role in integrin-mediated signal transduction from the extracellular matrix (20-24). In this study, we showed that binding of THP-1 cells to IL-1beta -stimulated HUVEC induces a decrease in the amount of the p125FAK molecule in HUVEC. It has been reported that inhibition of the function of p125FAK by p41/43FRNK (pp125FAK-related non-kinase) blocked the formation of focal contacts, indicating a functional relation between p125FAK and the formation of focal contacts (25). Moreover, the loss of p125FAK has been reported to be a prerequisite for cell detachment (26). Taken together, it was considered that the decrease in p125FAK in HUVEC indicates a decrease in the function of focal contacts, resulting in decreased strength of attachment of the endothelial cell to the extracellular matrix. The decrease in the adhesiveness of endothelial cells would enable monocytes to migrate beneath the endothelial cells more easily.

A decrease in the density of actin fibers induced by THP-1 was also observed in HUVEC in parallel with the decrease in p125FAK. It has been well documented that the formation of actin stress fibers parallels the formation of focal adhesion and is accompanied by increased tyrosine phosphorylation of p125FAK (27-29). Integrity of the actin cytoskeleton has also been reported to be required for the increased phosphorylation of p125FAK in response to a variety of extracellular stimuli (23, 30). Therefore, it can be postulated that the decrease in actin stress fibers is closely associated with the decrease in p125FAK.

It is unclear why the p125FAK protein level drops so rapidly. Recently, it was reported that p125FAK is cleaved by calpain, a calcium-dependent cysteine protease, in platelets (31). Therefore, we investigated whether the decrease in p125FAK could be prevented by calpeptin, a membrane-permeable inhibitor of calpain, or a cysteine protease inhibitor E-64. However, pretreatment of HUVEC by these inhibitors at a concentration of up to 50 µM could not affect the decrease in p125FAK (data not shown). In addition, little change was observed in the amount of talin which interacts with p125FAK (32) and is cleaved by calpain preferentially (33). From these results, it is unlikely that calpain is responsible for the decrease in p125FAK. The molecular mechanisms of the decrease in p125FAK are still inconclusive, even though we have also tried to inhibit the decrease in p125FAK by other protease inhibitors.

The decrease in p125FAK in IL-1beta -stimulated HUVEC was induced not only by monocytic THP-1 cells but also by monoblastic U937 cells. Furthermore, the decrease induced by THP-1 was also observed in HUVEC grown on collagen type I or fibronectin (data not shown), indicating that the decrease in p125FAK was independent of the extracellular matrix on which the HUVEC were grown. These results indicate that the decrease in the amount of tyrosine-phosphorylated p125FAK might be a commonly observed event in cytokine-activated HUVEC.

The molecules participating in the interactions between THP-1 cells and HUVEC remain to be clarified. The candidate molecule that triggers the decrease in p125FAK is considered to be an adhesion molecule present on THP-1 cells rather than a newly secreted soluble factor induced by interaction of THP-1 cells with activated HUVEC because the cell-free culture supernatant obtained after co-culture of THP-1 cells with IL-1beta -activated HUVEC did not induce a decrease in p125FAK levels (data not shown). With regard to the counter-receptor(s) on the surface of HUVEC responsible for the transduction of the p125FAK-modifying signal, although the decrease in the amount of p125FAK was partially blocked by a mixture of neutralizing mAbs against ICAM-1, VCAM-1, and E-selectin pathways, a direct role for these three adhesion molecules in the transmission of a regulatory signal has yet to be established. It is possible that adhesion molecules such as ICAM-1, VCAM-1, and E-selectin, the expression of which is augmented by inflammatory cytokines, enable THP-1 cells to bind tightly to HUVEC, resulting in the effective transduction of the p125FAK reducing signal induced by other molecule(s) into HUVEC.


FOOTNOTES

*   This study was supported by Special Coordination Funds for Promoting Science and Technology (Joint Research Utilizing Scientific and Technological Potential in Region) of the Science and Technology Agency of the Japanese Government.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom correspondence should be addressed: Fujisaki Institute, Hayashibara Biochemical Laboratories, Inc., 675-1 Fujisaki, Okayama 702, Japan. Tel.: 81-86-276-3141; Fax: 81-86-276-6885.
1   The abbreviations used are: PECAM-1, platelet/endothelial cell adhesion molecule-1; ICAM-1, intercellular adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1; HUVEC, human umbilical vein endothelial cells; p125FAK, focal adhesion kinase; IL-1beta , interleukin-1beta ; TNF-alpha , tumor necrosis factor-alpha ; anti-Tyr(P), anti-phosphotyrosine; mAb, monoclonal antibody; rt, room temperature; PBS, phosphate-buffered saline.

ACKNOWLEDGEMENTS

We thank Drs. Mark Micallef and Tsunetaka Ohta for helpful discussions and for review of the manuscript. We thank Shigeto Yamamoto for preparing the figures.


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