Dual signaling by the alpha vbeta 3-integrin activates cytosolic PLA2 in bovine pulmonary artery endothelial cells

Sunita Bhattacharya1, Rashmi Patel2, Namita Sen2,3, Sadiqa Quadri2,3, Kaushik Parthasarathi2,3, and Jahar Bhattacharya2,3

Departments of 1 Pediatrics, 2 Medicine, and 3 Physiology and Cellular Biophysics, College of Physicians and Surgeons and St. Luke's Roosevelt Hospital Center, Columbia University, New York, New York 10019


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Vitronectin, which ligates the alpha vbeta 3-integrin, increases both lung capillary permeability and lung endothelial Ca2+. In stable monolayers of bovine pulmonary artery endothelial cells (BPAECs) viewed with confocal microscopy, multimeric vitronectin aggregated the apically located alpha vbeta 3-integrin. This caused arachidonate release that was inhibited by pretreating the monolayers with the anti-alpha vbeta 3 monoclonal antibody (MAb) LM609. No inhibition occurred in the presence of the isotypic MAb PIF6, which recognizes the integrin alpha vbeta 5. Vitronectin also caused membrane translocation and phosphorylation of cytosolic phospholipase A2 (cPLA2) as well as tyrosine phosphorylation of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK) 2. The cPLA2 inhibitor arachidonyl trifluoromethylketone, the tyrosine kinase inhibitor genistein, and the MAPK kinase inhibitor PD-98059 all blocked the induced arachidonate release. PD-98059 did not inhibit the increase of cytosolic Ca2+ or cPLA2 translocation, although it blocked tyrosine phosphorylation of ERK2. Moreover, although the intracellular Ca2+ chelator MAPTAM also inhibited arachidonate release, it did not inhibit tyrosine phosphorylation of ERK2. These findings indicate that ligation of apical alpha vbeta 3 in BPAECs caused ERK2 activation and an increase of intracellular Ca2+, both conjointly required for cPLA2 activation and arachidonate release. This is the first instance of a tyrosine phosphorylation-initiated "two-hit" signaling pathway that regulates an integrin-induced proinflammatory response.

vitronectin; SC5b-9; arachidonate; cytosolic phospholipase A2


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE alpha  Vbeta 3-integrin, a member of the cytoadhesive family of integrins (9), is richly expressed in lung microvessels (33), but its functional role in pulmonary physiology remains inadequately understood. Because the role of the integrin has been discussed largely in the context of angiogenesis and wound healing (7, 9, 21), its presence in the nonproliferating lung vascular bed under quiescent conditions needs to be better understood. The integrin is luminally expressed in lung microvascular endothelium where it may play a proinflammatory role in lung vascular disease, for example, by increasing capillary permeability (36) and mobilizing endothelial cell (EC) Ca2+ (3). However, other proinflammatory responses may also result.

We considered that alpha vbeta 3 ligation of lung ECs may activate cytosolic phospholipase A2 (cPLA2), which is potentially an important feature of the EC inflammatory response in lung. cPLA2 activation releases free arachidonic acid that may generate prostaglandins and other oxidated compounds and cause Ca2+ mobilization (14, 19, 31). In the family of cytoadhesive integrins, ligation of beta 1-integrins has been shown to induce cPLA2 activation (4, 5, 35, 40, 42). However, these reports concern circulating or proliferating cells and do not represent responses that may be relevant to cells under stable conditions, such as those of the ECs of lung vessels. Furthermore, it is not clear that ligation of the alpha vbeta 3-integrin by soluble ligands leads to cPLA2 activation, nor are the activation mechanisms known.

In confluent EC monolayers, cPLA2 is located in the cytosol, but on activation, it translocates to membranes (32). The translocation is attributable to an increase of EC Ca2+, although activation also requires the enzyme to be phosphorylated (14). Signaling pathways acting through tyrosine phosphorylation (TyrP) may regulate both events because TyrP activates the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) that phosphorylates cPLA2 (14), and it also activates phospholipase Cgamma (PLCgamma ), leading to EC Ca2+ increases (3). Because we have shown that alpha vbeta 3 ligation induces several signaling pathways in lung ECs (2, 3, 36), here we tested the extent to which these pathways interact for cPLA2 activation.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cells, Reagents, and Antibodies

Bovine pulmonary artery endothelial cells (BPAECs; American Type Culture Collection) were maintained as described (2). The following were purchased: fura 2-AM (Molecular Probes), EGTA, EDTA, and genistein (Sigma, St. Louis, MO); arachidonyl trifluoromethylketone (AACOCF3), 1,2-bis(o-amino-5'-methylphenoxy)ethane-N,N,N'N'-tetraacetic acid tetraacetoxymethyl ester (MAPTAM; Calbiochem, La Jolla, CA); the secondary antibody donkey anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA); affinity-purified polyclonal anti-phosphotyrosine rabbit serum (ICN Biomedical, Costa Mesa, CA); protein A/protein G-agarose, polyclonal anti-ERK2 (C-14), and monoclonal anti-cPLA2 (Santa Cruz Biotechnology, Santa Cruz, CA); the inhibitor of MAPK kinase (MEK) 1 inhibitor PD-98059 (New England Biolabs, Beverly, MA); [3H]arachidonic acid (New England Nuclear, Wilmington, DE); and the monoclonal antibody (MAb) PIF6 against the alpha vbeta 5-integrin (Chemicon, CA). The anti-alpha vbeta 3 MAb LM609 was generously provided by D. Cheresh (Scripps Clinic and Research Foundation, La Jolla, CA). Human multimeric vitronectin was purified (2).

Release of [3H]Arachidonic Acid From BPAECs

Arachidonate release was quantified as the increase of supernatant radioactivity in cells loaded with [3H]arachidonic acid. This is a standard indicator of cPLA2 activation (22, 23, 25). BPAECs grown in 24-well plates were prelabeled with 0.5-1 µCi/ml of [3H]arachidonate (100 Ci/mmol for 18 h at 37°C), washed (serum-free DMEM and 0.2% fatty acid-free albumin for 2 h at 37°C), and exposed to multimeric vitronectin. After 15 min, the collected supernatants (centrifuged at 3,000 rpm for 10 min) and cell lysates were subjected to liquid scintillation counting. The supernatant radioactivity was calculated as a percentage of the total radioactivity (cell lysate plus supernatant). The radioactivity released under experimental conditions is expressed as a percentage of release under baseline conditions. Because of considerable interbatch variation in arachidonate release, baseline secretion was determined for each batch of monolayers. Because inhibitors independently increase arachidonate release (22, 34), in inhibitor studies, we compared these data against inhibitor-treated controls as previously reported by others (22, 23, 34).

Membrane Translocation of cPLA2

BPAECs were treated with multimeric vitronectin (400 µg/ml for 15 min) in both the presence and absence of PD-98059 (10 µmol/l for 15 min for 37°C), then were permeabilized, fixed, blocked, and immunofluorescently stained with anti-cPLA2 MAb and FITC-labeled goat anti-mouse IgG as described (28). The monolayers were imaged with fluorescence confocal microscopy.

Immunofluorescent Labeling of the alpha vbeta 3-Integrin

BPAEC monolayers grown on coverslips were treated with control buffer or multimeric vitronectin (400 µg/ml for 15 min) and fixed. The alpha vbeta 3-integrin was immunofluorescently labeled with the anti-alpha vbeta 3 MAb LM609 (200 µg/ml for 30 min at 37°C) followed by FITC-labeled donkey anti-mouse IgG (30 µg/ml for 30 min at 37°C). Immunofluorescence was detected by confocal or conventional fluorescence microscopy. Fluorescence intensity per unit area (gray levels) was quantified by digital imaging of single cells (MCID-M4, Imaging Research, Brock University, St. Catharines, Canada).

Ca2+ Imaging of Single Endothelial Cells

We used methods previously described by our laboratory (41). BPAEC monolayers were fura 2 loaded by the addition of fura 2-AM (5 µmol/l for 30 min at 20°C) and then were maintained at 37°C during digital imaging. Intracellular Ca2+ concentration ([Ca2+]i) was determined in a 2-µm2 window placed over 340- to 380-nm ratio images of single cells based on appropriate calibrations and a fura 2-Ca2+ dissociation constant (Kd) of 224 nmol/l (10).

Immunoprecipitation and Immunoblotting of ERK2

BPAECs in confluent monolayers were lysed (4°C) in buffer containing 150 mmol/l of NaCl, 50 mmol/l of Tris base, 2 mmol/l of EGTA, 50 mmol/l of NaF, 0.1% SDS, 1% Nonidet P-40, 10 µg/ml of leupeptin, 10 µg/ml of aprotinin, 1 mmol/l of phenylmethylsulfonyl fluoride, and 1 mmol/l of the phosphatase inhibitor sodium orthovanadate, pH 7.5. After the lysates were cleared (14,000 rpm for 15 min), protein concentrations were determined (DC Protein Assay, Bio-Rad, Richmond, CA), and ERK2 was immunoprecipitated and subjected to SDS-PAGE, transfer, and immunoblotting by methods previously described by Bhattacharya et al. (2).

Mobility Shift Assay of cPLA2 Phosphorylation

BPAEC lysates were treated with 4× Laemmli buffer, boiled for 5 min, and subjected to 8% SDS-PAGE (20 mA/gel). Electrophoresis was continued for 3 h after the tracking dye exited the gel to increase separation between the native and phosphorylated isoforms (26). Transfer and immunoblotting were carried out with methods previously described by Bhattacharya et al. (2).

Statistics

All data are means ± SE. Differences between groups were tested by paired t-test for two groups and by the Newman-Keuls test for more than two groups. Significance was accepted at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Vitronectin-Induced Arachidonate Release

Addition of vitronectin (15 min at 37°C) to BPAEC monolayers loaded with [3H]arachidonate (see MATERIALS AND METHODS) caused an enhanced, concentration-dependent release of radioactivity (Fig. 1A), indicating the release of arachidonate and its metabolites, which was attributable to cPLA2 activation (22, 23, 25). Vitronectin ligates the integrins alpha vbeta 3 and alpha vbeta 5 on ECs (20). However, when we separately preincubated monolayers with the anti-alpha vbeta 3 MAb LM609 (30 min at 4°C) and the anti-alpha vbeta 5 MAb PIF6, LM609 alone blocked the arachidonate response (Fig. 1B). Because PIF6 is an IgG isotype of LM609, it also provided a negative control for the inhibiting effect of LM609. Hence, the response was alpha vbeta 3 specific and was not attributable to the ligation of other vitronectin-recognizing receptors.


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Fig. 1.   Arachidonate release after the addition of vitronectin (VN) was determined in [3H]arachidonate-loaded confluent bovine pulmonary artery endothelial cell (BPAEC) monolayers. A: effects of different concentrations of VN. B: BPAEC pretreatment (30 min at 4°C) with antibodies PIF6 or LM609 before addition of VN (400 µg/ml). C: pretreatment (15 min at 37°C) with indicated inhibitors before addition of VN (400 µg/ml). PIF6, anti-alpha vbeta 5 monoclonal antibody (MAb); LM609, anti-alpha vbeta 3 MAb; GN, genistein (100 µmol/l); PD, PD-98059 (10 µmol/l); AC, arachidonyl trifluoromethylketone (AACOCF3; 1 µmol/l). Data are means ± SE; n, no. of monolayers. P < 0.01, linear regression in A. *P < 0.01 compared with VN alone in both B and C.

The Signaling Pathway

Protein TyrP. As Bhattacharya and colleagues (2, 3) have shown previously, ligation of the alpha vbeta 3-integrin enhances TyrP in ECs. Here, incubation of monolayers with the tyrosine kinase inhibitor genistein completely blocked vitronectin-induced arachidonate release (Fig. 1C), implicating alpha vbeta 3-induced TyrP mechanisms in the arachidonate response.

MAPK. Because TyrP of MAPK may lead to cPLA2 activation, we determined the role of MAPK in these responses. The MEK-1 inhibitor PD-98059 (23) blocked vitronectin-induced arachidonate release (Fig. 1C). Moreover, as shown by the bands in Fig. 2A, (bottom, lane 2), immunoprecipitation experiments indicated that TyrP of ERK2 was enhanced in vitronectin-treated monolayers compared with that in control monolayers (lane 1). This TyrP response was also inhibited by PD-98059 (compare Fig. 2A, lanes 3 and 4). These results indicate that ERK2 was involved in these vitronectin-induced responses. Bhattacharya et al. (3) previously showed that vitronectin treatment increases [Ca2+]i in BPAEC monolayers. To determine a possible role of [Ca2+]i, we treated monolayers with the [Ca2+]i chelator MAPTAM. MAPTAM decreased baseline TyrP but failed to inhibit the postvitronectin increase of TyrP (Fig. 2A, bottom, compare lanes 5 and 6, and Fig. 2B, bars at left and right). These findings indicate that the enhanced TyrP of ERK2 was not [Ca2+]i dependent.


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Fig. 2.   Tyrosine phosphorylation of extracellular signal-regulated kinase (ERK) 2 in BPAECs. Confluent BPAEC monolayers were pretreated with buffer (-) or the indicated inhibitors (+) [10 µmol/l of PD or 200 µmol/l of 1,2-bis(o-amino-5'-methylphenoxy)ethane-N,N,N'N'-tetraacetic acid tetraacetoxymethyl ester (MAPTAM); MAP] for 15 min before addition of buffer or 400 µg/ml of VN. ERK2 was immunoprecipitated (IP) from lysates, then subjected to SDS-PAGE, transfer, and immunoblotting (IB) with anti-ERK2 and anti-phosphotyrosine (TyrP) antibodies, respectively. A: immunoblots of ERK2 IP (replicated 3 times). B: optical densitometry of immunoblots expressed as tyrosine phosphorylation (P-Tyr)-to-protein ratios for each condition was quantified in 3 separate experiments, and the effects of VN are expressed as percent of control (buffer or inhibitor). Values are means ± SE; n, no. of monolayers. *P < 0.05 compared with VN.

cPLA2. The cPLA2 inhibitor AACOCF3 (1) completely blocked vitronectin-induced arachidonate release (Fig. 1C), providing further evidence for cPLA2 activation. Because this activation is associated with cPLA2 translocation from the cytosol to the membrane (28), we assayed the translocation. We exposed BPAECs to vitronectin. Then, after fixation and permeabilization, we immunofluorescently labeled cPLA2 with target-specific antibodies. The principle of this assay was that membrane-bound cPLA2 is not removed from permeabilized cells during washes and may be detected by immunofluorescence. Although no fluorescence was evident in control monolayers (Fig. 3, left), vitronectin treatment resulted in well-defined fluorescent staining (Fig. 3, right), indicating that alpha vbeta 3 ligation induced membrane translocation of cPLA2.


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Fig. 3.   Membrane translocation of cytosolic phospholipase A2 (cPLA2) in lung endothelial cells. BPAEC monolayers were treated with buffer, exposed to 400 µg/ml of VN (15 min at 37°C), and then fixed, permeabilized, and stained with anti-cPLA2 MAb followed by FITC anti-mouse IgG (replicated in 3 separate experiments).

The vitronectin-induced immunofluorescence of cPLA2 was maximal in the perinuclear region, which was to be expected because the enzyme translocates to the nuclear and perinuclear membranes (28). This response was not blocked in monolayers pretreated with PD-98059 (n = 3 monolayers; data not shown). This lack of inhibition was consistent with the notion that MEK activation is not required for cPLA2 translocation because the translocation event is Ca2+ initiated (14).

Because phosphorylation of cPLA2 is essential for its activation (14), we determined whether vitronectin induced cPLA2 phosphorylation in BPAECs. Gel shift analysis showed that vitronectin caused a reduction in the electrophoretic mobility of cPLA2 (Fig. 4, right) compared with buffer-treated control cells (left). The reduced mobility was consistent with cPLA2 phosphorylation as reported by others (22, 23, 26) in other cell types.


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Fig. 4.   Effect of VN on cPLA2 phosphorylation. BPAEC monolayers were treated with buffer or VN (400 µg/ml for 15 min). Lysates were subjected to SDS-PAGE, transfer, and immunoblotting with anti-cPLA2 MAb (replicated 3 times). Note reduced mobility of phosphorylated cPLA2 (cPLA2-P).

Site of alpha vbeta 3 Aggregation in BPAECs

Previously, our laboratory (3) showed that multimeric vitronectin aggregates the alpha vbeta 3-integrin in BPAEC monolayers. To determine the sites of alpha vbeta 3 aggregation, we exposed BPAEC monolayers to vitronectin, then obtained images in the z-axis with a confocal microscope. As shown in Fig. 5A, fluorescent clumps were evident only in the apicalmost sections (right), indicating that aggregation occurred at the luminal surface. In contrast, the basal surface (Fig. 5A, left) showed diffuse fluorescence, indicating the absence of aggregation at the cell-substrate interface. In separate experiments, we confirmed that the several inhibitors used in these experiments did not modify the extent of alpha vbeta 3 aggregation induced by vitronectin (Fig. 5B).


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Fig. 5.   VN-induced alpha vbeta 3 aggregation in BPAECs. A: confocal microscopy of the fluorescently labeled alpha vbeta 3-integrin on a single cell from a VN-treated, fixed BPAEC monolayer. Images show optical sections at basal and apical levels. Arrows, alpha vbeta 3 aggregates (replicated 3 times). B: effects of inhibitors (400 µg/ml of VN, 100 µmol/l of GN, 10 µmol/l of PD, 1 µmol/l of AC, or 200 µmol/l of MAP) on alpha vbeta 3 aggregation. The alpha vbeta 3-integrin was fluorescently labeled after VN treatment in the presence of inhibitors. Fluorescence intensity was quantified by digital imaging in single cells. n, No. of cells imaged. *P < 0.01 compared with effect of control buffer alone (extreme left).

The Role of [Ca2+]i

Bhattacharya et al. (3) reported that ligation of the alpha vbeta 3-integrin by vitronectin increases [Ca2+]i in ECs. Here, pretreatment of BPAECs with the Ca2+ chelator MAPTAM (200 µmol/l) completely blocked vitronectin-induced arachidonate release (Fig. 6A), indicating that the effect was [Ca2+]i dependent. The MEK inhibitor PD-98059 did not block the vitronectin-induced [Ca2+]i increase (Fig. 6B), indicating that ERK2 did not play a role in the [Ca2+]i response.


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Fig. 6.   The role of endothelial intracellular Ca2+ concentration ([Ca2+]i) in arachidonate release. C, control buffer. A: [3H]arachidonate-loaded BPAEC monolayers were treated with control buffer or MAP (15 min at 37°C), and then VN was added. Data are means ± SE; n, no. of monolayers. *P < 0.01 compared with control. B: effects of PD on VN-induced [Ca2+]i responses. BPAEC monolayers were loaded with fura 2-AM, and then [Ca2+]i was determined by ratiometric fluorescence imaging at baseline and after VN addition. Data are means ± SE of peak [Ca2+]i responses; n, no. of BPAEC monolayers. C: arachidonate release vs. [Ca2+]i increase induced by different concentrations of VN. [Ca2+]i data are from a previous study (3). Line drawn by nonlinear regression, P < 0.01.

A comparison of previous vitronectin-induced [Ca2+]i responses in BPAECs (3) with the present data revealed a nonlinear correlation between [Ca2+]i increase and arachidonate release (Fig. 6C). This plot indicated that significant arachidonate release occurred at concentrations of vitronectin that increased [Ca2+]i 20% or more above baseline, showing a [Ca2+]i threshold of 80-100 nmol/l for vitronectin-induced arachidonate release.

SC5b-9

Multimeric vitronectin is the alpha vbeta 3-ligating constituent of the soluble complement complex SC5b-9 (2). Figure 7 shows that in BPAEC monolayers, SC5b-9 caused a concentration-dependent arachidonate release (P < 0.01), indicating that soluble vitronectin-containing complement complexes are capable of causing arachidonate release from endothelial cells.


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Fig. 7.   Arachidonate release in lung endothelial cells by SC5b-9. Arachidonate release was determined in [3H]arachidonate-loaded BPAEC monolayers after the addition of SC5b-9, the VN-containing complement complex. Data are means ± SE; n, no. of monolayers. *P < 0.01.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In previous experiments, our laboratory determined that ligation of the alpha vbeta 3-integrin in lung endothelial monolayers activates cytosolic tyrosine kinases, one consequence of which is the TyrP of focal adhesion proteins (2) and another of which is the induction of the PLCgamma -inositol 1,4,5-trisphosphate pathway that increases [Ca2+]i (3). However, the proinflammatory importance of these signaling pathways was not clear. Now we show that ligating alpha vbeta 3 with vitronectin caused genistein-inhibitable arachidonate release that was attributable to membrane translocation and phosphorylation of cPLA2. Thus TyrP due to alpha vbeta 3 ligation in ECs constitutes a sufficient mechanism for cPLA2-induced arachidonate release, indicating a role for alpha vbeta 3 in augmenting and sustaining inflammatory processes (14).

Unexpectedly, the arachidonate release was inhibited by the blockade of two different signaling pathways. Thus in the presence of the [Ca2+]i chelator MAPTAM and the MEK inhibitor PD-98059, each given separately, the vitronectin-induced arachidonate release was blocked. The inhibition seen with MAPTAM confirms the acknowledged role of [Ca2+]i in arachidonate activation (14). Importantly, the PD-98059-mediated inhibition of ERK2 TyrP and arachidonate release indicated that MAPK activation was concomitantly required for cPLA2 activation. Moreover, MAPTAM did not block TyrP of ERK2, and PD-98059 did not inhibit the [Ca2+]i increase, indicating that these blockers did not cross inhibit the two arms of the pathway. Hence, we conclude that cPLA2 activation that follows alpha vbeta 3 ligation in lung ECs was induced by a "two-hit" mechanism resulting from the convergence of tyrosine kinase pathways that both increased [Ca2+]i and activated MAPK, respectively.

Signaling responses of cytoadhesive integrins have been extensively reported in the context of cell-matrix interactions and cell cycle progression (9). Thus alpha vbeta 3-induced MAPK activation in ECs occurs in angiogenesis (7, 21). Integrin-induced responses attributable to fibroblast spreading on a fibronectin matrix are associated with MAPK activation and arachidonate release (4). Despite their qualitative similarity to the data shown here, the relevance of these reported responses to stable, nonproliferating ECs has remained unclear. In this regard, three new conclusions may be drawn from our present findings. First, by confocal microscopy, we obtained definitive evidence that vitronectin aggregated the lumen-facing alpha vbeta 3-integrin. Considered in the context of the TyrP and [Ca2+]i responses that occur within 1 min of alpha vbeta 3 ligation (2, 3), this finding suggests that luminal integrin ligation initiates the signaling cascade. The signaling may be further regulated by processes causing translocation of vitronectin from the apical to the basal surfaces of ECs (17). Second, because our experiments were conducted in stable monolayers, it is clear that ECs do not have to be in cell cycle progression to support integrin-induced MAPK activation. Third, the two-hit mechanism discussed above has not been reported previously and may be characteristic of responses attributable to the luminal alpha vbeta 3-integrin.

Although understanding of the signaling pathways induced by the alpha vbeta 3-integrin in stable ECs remains inadequate, our laboratory previously reported (2) that ligation of the integrin causes focal adhesion kinase (FAK) and Shc TyrP in ECs. Because the association of FAK and Shc with Grb2 leads to activation of Ras and subsequently of ERK MAPK (29), our finding that ERK MAPK TyrP was enhanced by alpha vbeta 3 ligation suggests that the present signaling was routed through the Grb2-Ras pathway. Figure 8 summarizes our view that after soluble ligands bind and aggregate, the alpha vbeta 3-integrin TyrP pathways first bifurcate into the PLCgamma right-arrow inositol 1,4,5-trisphosphate and FAK right-arrow Shc right-arrow Grb2 directions, then subsequently converge through [Ca2+]i increase and MAPK activation on cPLA2 activation and release of arachidonate products. However, these considerations remain speculative because we did not obtain direct determinations.


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Fig. 8.   Signaling pathways for alpha vbeta 3-induced arachidonate release. FAK, focal adhesion kinase; MAPK, mitogen-activated protein kinase; PLC-gamma , phospholipase Cgamma .

Our model of cPLA2 activation involving [Ca2+]i increase and MAPK activation may be relevant in the context of small increases of EC [Ca2+]i. Although the Ca2+ dependence of cPLA2 activation has been discussed in relation to large [Ca2+]i increases, e.g., those in which [Ca2+]i increases to micromolar levels (11, 18) in lung microvessels, EC [Ca2+]i increases are relatively modest. Thus an increase in lung vascular pressure or an intravascular infusion of tumor necrosis factor-alpha increases EC [Ca2+]i in lung capillaries by only 100 nmol/l (12, 13). These modest increases are nevertheless proinflammatory because they increase the expression of the leukocyte adhesion receptor P-selectin (13). Perhaps MAPK activation sensitizes the effect of a modest [Ca2+]i increase such that cPLA2 activation occurs in the absence of large and potentially deleterious elevations of [Ca2+]i. A consideration of our previous and present vitronectin data indicates that significant arachidonate release occurred after [Ca2+]i increased 20% above baseline (Fig. 6C), suggesting that the sensitizing effect of MAPK activation may occur at [Ca2+]i increases in the range of 100 nmol/l.

Our findings bear on the potential importance of the alpha vbeta 3-integrin as a significant proinflammatory receptor in the lung. Inflammatory consequences of alpha vbeta 3 ligation may be considerable because the lung vascular bed extensively expresses the integrin both on the luminal and abluminal aspects of ECs (33). Vitronectin induces alpha vbeta 3 aggregation on the luminal surface of ECs. This may be relevant to responses generated by circulating ligands such as the vitronectin-containing complement product SC5b-9 (2) or the thrombin-antithrombin complex (39) that is released in conditions such as bypass surgery or sepsis (8, 15). We previously showed (2) that SC5b-9 ligates the alpha vbeta 3-integrin and induces TyrP of endothelial proteins. Here, we show that SC5b-9 causes arachidonate release similar to that caused by vitronectin. This finding reaffirms our view that ligation of the luminal alpha vbeta 3 of ECs may constitute a proinflammatory mechanism in conditions associated with complement activation.

In conclusion, the binding of the alpha vbeta 3-integrin constitutes a basis for cPLA2 activation and arachidonate release from lung ECs that may occur under pathophysiological conditions. A role of the integrin in lung inflammation requires consideration because it not only directs the migration of neutrophils (24) and promotes removal of apoptotic cells (27, 38), but through arachidonate release, it may lead to increased vascular permeability (37), vasoconstriction (30), gene regulation (16), and alveolocapillary cross talk (12). Released arachidonate may generate feedback effects by itself, inducing Ca2+ influx (19, 31) and further activating ERK (6), thereby amplifying proinflammatory signaling and further augmenting lung injury.


    ACKNOWLEDGEMENTS

This study was supported by National Heart, Lung, and Blood Institute Grants HL-36024, HL-57556 (both to J. Bhattacharya), and HL-54157 (to S. Bhattacharya).


    FOOTNOTES

Address for reprint requests and other correspondence: S. Bhattacharya, St. Luke's-Roosevelt Hospital Center, 1000 10th Ave, New York, NY 10019 (E-mail: sb80{at}columbia.edu).

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.

Received 17 August 2000; accepted in final form 30 November 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ackermann, EJ, Conde-Frieboes K, and Dennis EA. Inhibition of macrophage Ca2+-independent phospholipase A2 by bromoenol lactone and trifluoromethyl ketones. J Biol Chem 270: 445-450, 1995[Abstract/Free Full Text].

2.   Bhattacharya, S, Fu C, Bhattacharya J, and Greenberg S. Soluble ligands of the alpha vbeta 3-integrin mediate enhanced tyrosine phosphorylation of multiple proteins in adherent bovine pulmonary artery endothelial cells. J Biol Chem 270: 16781-16787, 1995[Abstract/Free Full Text].

3.   Bhattacharya, S, Ying X, Fu C, Patel R, Kuebler W, Greenberg S, and Bhattacharya J. alpha vbeta 3-Integrin induces tyrosine phosphorylation-dependent Ca2+ influx in pulmonary endothelial cells. Circ Res 86: 456-462, 2000[Abstract/Free Full Text].

4.   Clark, EA, and Hynes RO. Ras activation is necessary for integrin-mediated activation of extracellular signal-regulated kinase 2 and cytosolic phospholipase A2 but not for cytoskeletal organization. J Biol Chem 271: 14814-14818, 1996[Abstract/Free Full Text].

5.   Cybulsky, AV, Carbonetto S, Cyr MD, McTavish AJ, and Huang Q. Extracellular matrix-stimulated phospholipase activation is mediated by beta 1-integrin. Am J Physiol Cell Physiol 264: C323-C332, 1993[Abstract/Free Full Text].

6.   Dulin, NO, Alexander LD, Harwalkar S, Falck JR, and Douglas JG. Phospholipase A2-mediated activation of mitogen-activated protein kinase by angiotensin II. Proc Natl Acad Sci USA 95: 8098-8102, 1998[Abstract/Free Full Text].

7.   Eliceiri, BP, Klemke R, Stromblad S, and Cheresh DA. Integrin alpha vbeta 3 requirement for sustained mitogen-activated protein kinase activity during angiogenesis. J Cell Biol 140: 1255-1263, 1998[Abstract/Free Full Text].

8.   Fitch, JC, Rollins S, Matis L, Alford B, Aranki S, Collard CD, Dewar M, Elefteriades J, Hines R, Kopf G, Kraker P, Li L, O'Hara R, Rinder C, Rinder H, Shaw R, Smith B, Stahl G, and Shernan SK. Pharmacology and biological efficacy of a recombinant, humanized, single-chain antibody C5 complement inhibitor in patients undergoing coronary artery bypass graft surgery with cardiopulmonary bypass. Circulation 100: 2499-2506, 1999[Abstract/Free Full Text].

9.   Giancotti, FG, and Ruoslahti E. Intergrin signaling. Science 285: 1028-1032, 1999[Abstract/Free Full Text].

10.   Grynkiewicz, G, Poenie M, and Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440-3450, 1985[Abstract].

11.   Hirabayashi, T, Kume K, Hirose K, Yokomizo T, Iino M, Itoh H, and Shimizu T. Critical duration of intracellular Ca2+ response required for continuous translocation and activation of cytosolic phospholipase A2. J Biol Chem 274: 5163-5169, 1999[Abstract/Free Full Text].

12.   Kuebler, WM, Parthasarathi K, Wang PM, and Bhattacharya J. A novel signaling mechanism between gas and blood compartments of the lung. J Clin Invest 105: 905-913, 2000[Abstract/Free Full Text].

13.   Kuebler, WM, Ying X, Singh B, Issekutz AC, and Bhattacharya J. Pressure is proinflammatory in lung venular capillaries. J Clin Invest 104: 495-502, 1999[Abstract/Free Full Text].

14.   Leslie, CC. Properties and regulation of cytosolic phospholipase A2. J Biol Chem 272: 16709-16712, 1997[Free Full Text].

15.   Lin, RY, Astiz ME, Saxon JC, Saha DC, and Rackow EC. Alterations in C3, C4, factor B, and related metabolites in septic shock. Clin Immunol Immunopathol 69: 136-142, 1993[ISI][Medline].

16.   Mariani, TJ, Sandefur S, Roby JD, and Pierce RA. Collagenase-3 induction in rat lung fibroblasts requires the combined effects of tumor necrosis factor-alpha and 12-lipoxygenase metabolites: a model of macrophage-induced, fibroblast-driven extracellular matrix remodeling during inflammatory lung injury. Mol Biol Cell 9: 1411-1424, 1998[Abstract/Free Full Text].

17.   Memmo, LM, and McKeown-Longo P. The alpha vbeta 3-integrin functions as an endocytic receptor for vitronectin. J Cell Sci 111: 425-433, 1998[Abstract/Free Full Text].

18.   Millanvoye-Van Brussel, E, David-Dufilho M, Pham TD, Iouzalen L, and Aude Devynck M. Regulation of arachidonic acid release by calcium influx in human endothelial cells. J Vasc Res 36: 235-244, 1999[ISI][Medline].

19.   Oike, M, Droogmans G, and Nilius B. Mechanosensitive Ca2+ transients in endothelial cells from human umbilical vein. Proc Nat Acad Sci USA 91: 2940-2944, 1994[Abstract].

20.   Panetti, TS, and McKeown-Longo PJ. The alpha vbeta 3-integrin receptor regulates receptor-mediated endocytosis of vitronectin. J Biol Chem 268: 11492-11495, 1993[Abstract/Free Full Text].

21.   Penta, K, Varner JA, Liaw L, Hidai C, Schatzman R, and Quertermous T. Del1 induces integrin signaling and angiogenesis by ligation of alpha vbeta 3. J Biol Chem 274: 11101-11109, 1999[Abstract/Free Full Text].

22.   Pyne, NJ, Tolan D, and Pyne S. Bradykinin stimulates cAMP synthesis via mitogen-activated protein kinase-dependent regulation of cytosolic phospholipase A2 and prostaglandin E2 release in airway smooth muscle. Biochem J 328: 689-694, 1997[ISI][Medline].

23.   Qiu, Z, Gijon MA, de Carvalho MS, Spencer DM, and Leslie CC. The role of calcium and phosphorylation of cytosolic phospholipase A2 in regulating arachidonic acid release in macrophages. J Biol Chem 273: 8203-8211, 1998[Abstract/Free Full Text].

24.   Rainger, GE, Buckley CD, Simmons DL, and Nash GB. Neutrophils sense flow-generated stress and direct their migration through alpha vbeta 3-integrin. Am J Physiol Heart Circ Physiol 276: H858-H864, 1999[Abstract/Free Full Text].

25.   Ricupero, D, Taylor L, and Polgar P. Interactions of bradykinin, calcium, G-protein and protein kinase in the activation of phospholipase A2 in bovine pulmonary artery endothelial cells. Agents Actions 40: 110-118, 1993[ISI][Medline].

26.   Sa, G, Murugesan G, Jaye M, Ivashchenko Y, and Fox PL. Activation of cytosolic phospholipase A2 by basic fibroblast growth factor via a p42 mitogen-activated protein kinase-dependent phosphorylation pathway in endothelial cells. J Biol Chem 270: 2360-2366, 1995[Abstract/Free Full Text].

27.   Savill, J, Hogg N, Ren Y, and Haslett C. Thrombospondin cooperates with CD36 and the vitronectin receptor in macrophage recognition of neutrophils undergoing apoptosis. J Clin Invest 90: 1513-1522, 1992[ISI][Medline].

28.   Schievella, AR, Regier MK, Smith WL, and Lin LL. Calcium-mediated translocation of cytosolic phospholipase A2 to the nuclear envelope and endoplasmic reticulum. J Biol Chem 270: 30749-30754, 1995[Abstract/Free Full Text].

29.   Schlaepfer, DD, Jones KC, and Hunter T. Multiple Grb2-mediated integrin-stimulated signaling pathways to ERK2/mitogen-activated protein kinase: summation of both c-Src- and focal adhesion kinase-initiated tyrosine phosphorylation events. Mol Cell Biol 18: 2571-2585, 1998[Abstract/Free Full Text].

30.   Seeger, W, Hartmann R, Neuhoff H, and Bhakdi S. Local complement activation, thromboxane-mediated vasoconstriction, and vascular leakage in isolated lungs. Role of the terminal complement sequence. Am Rev Respir Dis 139: 88-99, 1989[ISI][Medline].

31.   Shuttleworth, TJ. Arachidonic acid activates the noncapacitative entry of Ca2+ during [Ca2+]i oscillations. J Biol Chem 271: 21720-21725, 1996[Abstract/Free Full Text].

32.   Sierra-Honigmann, MR, Bradley JR, and Pober JS. "Cytosolic" phospholipase A2 is in the nucleus of subconfluent endothelial cells but confined to the cytoplasm of confluent endothelial cells and to the nuclear envelope and cell junctions upon histamine stimulation. Lab Invest 74: 684-695, 1996[Medline].

33.   Singh, B, Fu C, and Bhattacharya J. Vascular expression of the alpha vbeta 3-integrin in lung and other organs. Am J Physiol Lung Cell Mol Physiol 278: L217-L226, 2000[Abstract/Free Full Text].

34.   Susztak, K, Mocsai A, Ligeti E, and Kapus A. Electrogenic H+ pathway contributes to stimulus-induced changes of internal pH and membrane potential in intact neutrophils: role of cytoplasmic phospholipase A2. Biochem J 325: 501-510, 1997[ISI][Medline].

35.   Szabo, MC, Teague TK, and McIntyre BW. Regulation of lymphocyte pseudopodia formation by triggering the integrin alpha 4/beta 1. J Immunol 154: 2112-2124, 1995[Abstract/Free Full Text].

36.   Tsukada, H, Ying X, Fu C, Ishikawa S, McKeown-Longo P, Albelda S, Bhattacharya S, Anderson B, and Bhattacharya J. Ligation of the the alpha vbeta 3-integrin increases capillary hydraulic conductivity of rat lung. Circ Res 77: 651-659, 1995[Abstract/Free Full Text].

37.   Vincent, PA, Kreienberg PB, Minnear FL, Saba TM, and Bell DR. Simultaneous measurement of fluid and protein permeability in isolated rabbit lungs during edema. J Appl Physiol 73: 2440-2447, 1992[Abstract/Free Full Text].

38.   Walsh, GM, Sexton DW, Blaylock MG, and Convery CM. Resting and cytokine-stimulated human small airway epithelial cells recognize and engulf apoptotic eosinophils. Blood 94: 2827-2835, 1999[Abstract/Free Full Text].

39.   Wells, MJ, and Blajchman MA. In vivo clearance of ternary complexes of vitronectin-thrombin-antithrombin is mediated by hepatic heparan sulfate proteoglycans. J Biol Chem 273: 23440-23447, 1998[Abstract/Free Full Text].

40.   Whitfield, RA, and Jacobson BS. The beta 1-integrin cytosolic domain optimizes phospholipase A2-mediated arachidonic acid release required for NIH-3T3 cell spreading. Biochem Biophys Res Commun 258: 306-312, 1999[ISI][Medline].

41.   Ying, X, Minamiya Y, Fu C, and Bhattacharya J. Ca2+ waves in lung capillary endothelium. Circ Res 79: 898-908, 1996[Abstract/Free Full Text].

42.   Zhu, X, Munoz NM, Kim KP, Sano H, Cho W, and Leff AR. Cytosolic phospholipase A2 activation is essential for beta 1 and beta 2 integrin-dependent adhesion of human eosinophils. J Immunol 163: 3423-3429, 1999[Abstract/Free Full Text].


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