Decrease in the Amount of Focal Adhesion Kinase (p125 FAK ) in Interleukin-1 (cid:98) -stimulated Human Umbilical Vein Endothelial Cells by Binding of Human Monocytic Cell Lines*

Monocytes in the blood circulation migrate across en- dothelial 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-1 (cid:98) (IL-1 (cid:98) )-stimulated human umbilical vein endothelial cells (HUVEC) induced a de- crease in the amount of focal adhesion kinase (p125 FAK ) 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, (cid:97) -catenin, and talin. A maximum decrease in the amount of p125 FAK was observed 15–30 min after addition of THP-1 cells to HUVEC, after which the level of p125 FAK gradually re-covered. A reduction in the density of actin stress fibers in IL-1 (cid:98) -activated HUVEC was observed in parallel with the decrease in p125 FAK . The p125 FAK decrease was partially inhibited by preventing THP-1


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-1␤ (IL-1␤)-stimulated human umbilical vein endothelial cells (HUVEC) induced a decrease in the amount of focal adhesion kinase (p125 FAK )
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, ␣-catenin, and talin. A maximum decrease in the amount of p125 FAK was observed 15-30 min after addition of THP-1 cells to HUVEC, after which the level of p125 FAK gradually recovered. A reduction in the density of actin stress fibers in IL-1␤-activated HUVEC was observed in parallel with the decrease in p125 FAK . The p125 FAK 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 p125 FAK 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.
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 media-tors 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-1␤ (IL-1␤)-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 (p125 FAK ), 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. tic 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 g/ml streptomycin.
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 g/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-1␤ or TNF-␣ 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 l of an extraction buffer (1% Triton X-100, 1% Nonidet P-40, 150 mM NaCl, 2 mM Na 3 VO 4 , 10 mM NaF, 2 mM phenylmethylsulfonyl fluoride, 250 g/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 l of the lysates were then transferred to new tubes as the whole cell lysate. The residual lysates were centrifuged at 13,000 ϫ g for 30 min, and the supernatants were collected and used as the cell extract. Four hundred l 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 g of anti-Tyr(P) 4G10 for 2 h or with 4 g of anti-p125 FAK 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-l 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 l of a 2 ϫ SDS-sample buffer (100 mM Tris, 5% SDS, 30% glycerol, 5% 2-mercaptoethanol, pH 6.8), and boiled for 5 min. After centrifugation, 20 l 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 ϫ SDS-sample buffer (150 mM Tris, 7.5% SDS, 45% glycerol, 7.5% 2-mercaptoethanol, pH 6.8), and 20 l 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% NaN 3 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 In-terMed, 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-1␤ for 5 h and were subsequently layered with 1.5 ϫ 10 4 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 Ϫ20°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-1␤ or TNF-␣ 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 g/ml streptomycin) before addition of isotope-labeled THP-1 cells. THP-1 cells were labeled with 51 CrO 4 (Amersham) at 37°C for 1 h. After washing three times with the culture medium, 5 ϫ 10 4 labeled THP-1 cells suspended in the assay medium were added to each well in 100-l volumes. In inhibition experiments using adhesion-blocking antibodies, 51 Cr-labeled THP-1 cells incubated with 50 g/ml mAbs to adhesion molecules for 60 min at rt and washed twice were used. After mild centrifugation at 40 ϫ 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 p125 FAK Level in IL-1␤-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-1␤ 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-1␤-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-1␤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, p125 FAK was selected as a probable candidate for the tyrosinephosphorylated 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-p125 FAK 2A7 and probed with anti-Tyr(P) PY20. As shown in Fig. 1B, anti-p125 FAK 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 p125 FAK .
The residual immunoprecipitates obtained by immunoprecipitation with anti-p125 FAK 2A7 were probed with anti-p125 FAK C-20. As shown in Fig. 1C, the pattern of immunoblots detected with polyclonal anti-p125 FAK C-20 was almost identical to the pattern that was obtained with anti-Tyr(P) PY20, indicating that the decrease in p125 FAK band resulted from a decrease in the amount of p125 FAK protein itself and not from tyrosine de-phosphorylation of the protein. To further clarify the reason for the decrease in p125 FAK , changes in the amount of p125 FAK in whole cell lysates, cell extracts, and in insoluble fractions were investigated by direct immunoblotting with an-ti-p125 FAK C-20. As shown in Fig. 1D, decreased p125 FAK levels induced by monocytic cell treatment was observed in whole cell lysates. In addition, the pattern of p125 FAK levels in cell extracts was identical to that in whole cell lysates, and no p125 FAK 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 p125 FAK induced by monocytic cell seeding resulted from a decrease in p125 FAK protein and not from a decrease in solubility of the protein. However, obvious candidates for the degradation products of p125 FAK could not be observed. The decrease in p125 FAK was observed not only in THP-1-treated HUVEC but also in U937treated 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-1␤-activated HUVEC other than p125 FAK were reduced by THP-1 seeding or not. Talin present at focal contacts (8) such as is p125 FAK , VCAM-1 expressed on the cell surface of cytokineactivated endothelial cells (9), and ␣-catenin co-localized at the sites of intercellular junctions with cadherin and ␤-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 ␣-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 p125 FAK . 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).
VCAM-1 and ␣-catenin were not detected in whole cell lysates obtained from 2 ϫ 10 6 THP-1 cells alone, although talin was faintly detectable (lane 4). In subsequent experiments we included ␣-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 p125 FAK in IL-1␤treated HUVEC-To further characterize the decrease in the amount of p125 FAK , the kinetics of the changes in p125 FAK after  addition of THP-1 cells were investigated. As shown in Fig. 3, the decrease in p125 FAK 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-1␤-stimulated HUVEC. Although the amount of p125 FAK did not return to initial levels, a tendency for recovery of p125 FAK was observed (lane 10) 4 h from THP-1 cell seeding. Interestingly, the p125 FAK degradation was not observed in unstimulated HUVEC (lanes 1-3).
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-1␤ 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-1␤-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-1␤ (Fig. 4, F-H). The changes were observed from 30 min after seeding THP-1 cells and continued for at least 2 h.

Effect of IL-1␤ and TNF-␣ Treatment of HUVEC on the
Decrease in p125 FAK in HUVEC-The decrease in the amount of p125 FAK induced by THP-1 cells was observed in IL-1␤treated HUVEC but not in unstimulated HUVEC. Therefore, we investigated further whether THP-1 cells could induce the decrease in p125 FAK in HUVEC stimulated with inflammatory stimuli other than IL-1␤. HUVEC were stimulated with TNF-␣ for 5 h and were then co-cultured with THP-1 for 30 min. The cells were lysed and subjected to immunoblotting with anti-p125 FAK C-20. As shown in Fig. 5A, decrease in the amount of p125 FAK after addition of THP-1 was observed not only in IL-1␤-activated HUVEC (lanes 2-5) but also in TNF-␣-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. 51 Cr-labeled THP-1 cells were added to activated HUVEC and incubated for 30 min. As shown in Fig. 5B, it was observed that IL-1␤ and TNF-␣ treatment of HUVEC augmented THP-1 binding to HUVEC in a dose-dependent manner, suggesting a correlation between decrease in p125 FAK and binding of THP-1 cells to cytokine-activated HUVEC.
Inhibition of the Decrease in p125 FAK by Adhesion-blocking mAbs-It is well known that IL-1␤ and TNF-␣ 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 p125 FAK or not. THP-1 cells have been reported to express ␤2 integrin, a ␤ subunit of the ␤2 integrin family, ␣4 integrin, an ␣ subunit of very late antigen-4, and sialyl Le x (13). ␤2 integrins, very late antigen-4, and sialyl Le x are known to interact with ICAM-1, VCAM-1, and E-selectin, respectively (14 -18). THP-1 cells were incubated with 50 g/ml anti-␣4, anti-␤2, anti-sialyl Le x , 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-1␤-activated HUVEC. As shown in Fig.  6A, pretreatment of THP-1 cells with a mixture of anti-␣4, anti-␤2, and anti-sialyl Le x inhibited the decrease in p125 FAK in IL-1␤-activated HUVEC (lane 6), whereas treatment with either of these mAbs alone could not inhibit the decrease in p125 FAK (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). 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 p125 FAK 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-1␤-stimulated HUVEC induces a decrease in the amount of the p125 FAK molecule in HUVEC. It has been reported that inhibition of the function of p125 FAK by p41/43 FRNK (pp125 FAK -related non-kinase) blocked the formation of focal contacts, indicating a functional relation between p125 FAK and the formation of focal contacts (25). Moreover, the loss of p125 FAK has been reported to be a prerequisite for cell detachment (26). Taken together, it was considered that the decrease in p125 FAK 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 p125 FAK . 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 p125 FAK (27)(28)(29). Integrity of the actin cytoskeleton has also been reported to be required for the increased phosphorylation of p125 FAK 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 p125 FAK .
It is unclear why the p125 FAK protein level drops so rapidly. Recently, it was reported that p125 FAK is cleaved by calpain, a calcium-dependent cysteine protease, in platelets (31). Therefore, we investigated whether the decrease in p125 FAK 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 M could not affect the decrease in p125 FAK (data not shown). In addition, little change was observed in the amount of talin which interacts with p125 FAK (32) and is cleaved by calpain preferentially (33). From these results, it is unlikely that calpain is responsible for the decrease in p125 FAK . The molecular mechanisms of the decrease in p125 FAK are still inconclusive, even though we have also tried to inhibit the decrease in p125 FAK by other protease inhibitors.
The decrease in p125 FAK in IL-1␤-stimulated HUVEC was

cells by pretreatment with mAbs to adhesion molecules.
A, THP-1 cells were incubated with 50 g/ml mAbs to adhesion molecules for 60 min at rt. After washing, the pretreated THP-1 cells were seeded on IL-1␤-stimulated HUVEC, and the plates were incubated for 30 min. The cells were lysed and subjected to immunoblotting with anti-p125 FAK C-20. Subsequently, ␣-catenin on the same membrane was also probed. The positions of the p125 FAK and ␣-catenin bands are indicated on the right. B, intensities of immunoblotted p125 FAK were quantified by densitometer. C, HUVEC grown on a gelatin-coated 96well microplate were treated with IL-1␤ for 5 h. 51 Cr-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 Ϯ S.D. of quadruplicate wells. Control mAbs, mixture of classmatched irrelevant mAbs.
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 p125 FAK was independent of the extracellular matrix on which the HUVEC were grown. These results indicate that the decrease in the amount of tyrosine-phosphorylated p125 FAK 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 p125 FAK 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-1␤activated HUVEC did not induce a decrease in p125 FAK levels (data not shown). With regard to the counter-receptor(s) on the surface of HUVEC responsible for the transduction of the p125 FAK -modifying signal, although the decrease in the amount of p125 FAK 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 p125 FAK reducing signal induced by other molecule(s) into HUVEC.