©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Binding of Human Factor VIIa to Tissue Factor Induces Cytosolic Ca Signals in J82 Cells, Transfected COS-1 Cells, Madin-Darby Canine Kidney Cells and in Human Endothelial Cells Induced to Synthesize Tissue Factor (*)

(Received for publication, August 1, 1994; and in revised form, November 30, 1994)

John-Arne Røttingen (1) (2)(§) Tone Enden (2)(§) Eric Camerer (2)(¶) Jens-Gustav Iversen (1) Hans Prydz (2)(**)

From the  (1)Laboratory of Intracellular Signalling, Department of Physiology, Institute of Basic Medical Sciences and the (2)Biotechnology Centre of Oslo, University of Oslo, N-0371 Oslo, Norway

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Tissue factor (TF) is the most potent trigger of blood clotting known. It activates factor VII (FVII) thereby initiating a cascade of proteolytic reactions resulting in thrombin production. The cloning of TF revealed its structural characteristics to be those of a receptor related to the class 2 cytokine receptor superfamily, but until now no intracellular signal has been discovered related to binding of the ligand (FVIIa) to the putative receptor. We have studied possible intracellular signaling effects of the FVIIa-TF interaction by measuring cytosolic free Ca in single fura-2-loaded cells and found that 200 nM FVIIa caused Ca transients in about 30% of human umbilical vein endothelial cells treated with interleukin-1beta to express TF, compared to below 5% in uninduced cells. A gradual increase of the basal Ca level was also caused by binding of FVIIa. In the human bladder carcinoma cell line J82, which has a high constitutive TF activity, similar results were found. An antibody neutralizing TF activity decreased the response rate to control levels. COS-1 cells which do not make TF did not respond to FVIIa as opposed to COS-1 cells expressing TF after transfection with a human TF cDNA construct. The canine kidney cell line MDCK, a constitutive TF producer, responded especially well; up to 100% of the cells examined showed Ca oscillations which were dose dependent with regard to frequency, latency, maximal amplitude, and recruitment of responding cells. The frequency was reduced by inhibition of Ca influx with 100 µM LaCl(3). In confluent MDCK cells the Ca oscillations were synchronous, constituting the first evidence of a synchronous cytosolic Ca oscillator generated by global application of agonist. Thus, TF mediates a cytosolic Ca signal upon interaction with its ligand FVIIa, thereby suggesting a more complex biological role for TF.


INTRODUCTION

Tissue factor (TF) (^1)is the most potent trigger of blood clotting known(1, 2) . TF binds the plasma coagulation factor VII (FVII), a vitamin K-dependent glycoprotein(3) , leading to its proteolytic cleavage and activation (FVIIa). This complex, in the presence of free Ca and phospholipid, will then catalyze the activation of factor X or IX (for reviews see (4, 5, 6) ). The cloning of TF revealed its structural characteristics to be those of a receptor, with a 219-amino-acid extracellular domain, a 23-amino-acid transmembrane part, and a cytoplasmic tail of 21 amino acids(7, 8, 9, 10) . Bazan (11) suggested that TF was structurally related to the class 2 cytokine receptor superfamily which includes the interferon (IFN) alpha/beta and receptors. The receptor concept requires this structural aspect and in addition the existence of a high affinity-high specificity ligand, which when binding to the receptor should result in an intracellular signal. The functional ligand for TF is probably FVII/FVIIa. The structural relationship to known receptors may give a clue to possible signaling effects of the FVIIa-TF interaction. One early signaling event in the IFN--mediated signal is an increase of the cytosolic free Ca concentration ([Ca])(12, 13) . The Ca signal is brought about by both release from internal stores and influx across the plasma membrane. Consequently, we chose to study the effect of FVIIa binding to TF on [Ca] in single cells loaded with the fluorescent Ca indicator fura-2 in a digital imaging system. To look for such a Ca response, we took advantage of the ability of interleukin-1beta (IL-1beta) to induce TF in HUVEC(14) . This allows the use of uninduced HUVEC as controls. TF is expressed at maximum levels after 6 h of treatment with IL-1beta(15) .

We report here the detection of an increase in the intracellular level of Ca on binding of FVIIa to HUVEC induced by IL-1beta to synthesize TF. A similar increase in Ca levels was detected upon binding of FVIIa to TF in the human bladder carcinoma cell line J82 which produces TF constitutively and in monkey kidney fibroblasts (COS-1) stably transfected with a human TF construct. J82 cells treated with an antibody to neutralize TF activity and untransfected COS-1 cells which do not express TF showed no Ca signal upon exposure to FVIIa. The canine kidney cell line MDCK, a constitutive TF producer, responded especially well to FVIIa.


EXPERIMENTAL PROCEDURES

Materials

Collagenase type 1, bovine serum albumin, purified human Factor X (FX) (free of FXa), EGTA, histamine, LaCl(3), and HEPES were obtained from Sigma; trypsin-EDTA, RPMI 1640 (low endotoxin), L-glutamine, penicillin-streptomycin were from Flow (Irvine, Scotland) or Whittaker (Walkerville, MD); Dulbecco's modified Eagle's medium (DMEM), fetal calf serum (FCS), and thapsigargin were from Life Technologies, Inc. (Paisley, Scotland); heparin, recombinant human Factor VIIa, and recombinant human interleukin 1beta were from Novo-Nordisk (Bagsværd, Denmark); endothelial cell growth supplement was from Collaborative Research (Lexington, MA); substrate FXa-1 was from Nycomed (Oslo, Norway); the fluorescent calcium indicator fura-2/AM and the surfactant Pluronic F-127 were from Molecular Probes (Eugene, OR); the non-fluorescent calcium ionophore 4-Br-A23187 was from Calbiochem (San Diego, CA); SKF 96365 was from Biomol Research; Bacto-dextrose was from Difco Laboratories (Detroit, MI); Colorrapid was from Lucerna-Chem (Lucerne, Switzerland); a murine monoclonal antibody against human tissue factor, no. 4507 was from American Diagnostica (Greenwich, CT), and an antibody against von Willebrand factor was from Dakopatts (Copenhagen, Denmark); the mammalian expression vector pcDNA3 was from Invitrogen (San Diego, CA). The HEPES-buffered salt solution (HSS) consisted of 136 mM NACL, 5 MM KCL, 1.2 MM MGCL, 1.2 MM CACL, 11 MMBACTO-DEXTROSE, 10 MMHEPES, PH 7.35. ALL CHEMICALS WERE OF ANALYTICAL QUALITY.

Tissue Factor

The complete human TF cDNA (263 amino acids) was cloned into the mammalian expression vector pcDNA3. This vector contains the cytomegalovirus promoter for high level expression and the neomycin gene for selection of stable transfectants.

TF activity was measured by a one-stage procoagulant assay (16) using pooled normal human plasma as substrate and by a twostage assay (17) based on the activation of FX by the TFbulletFVIIabulletCabulletmembrane complex and expressed as dOD (0-60 min) at 405 nm from the cleavage of the chromogenic substrate. The results were compared to a standard curve established by serial dilution of a standard human brain TF preparation, which clotted human plasma in 12-13 s (arbitrarily set as 100 units/ml).

Cell Cultures

HUVEC were prepared from human umbilical veins as described(18) . The cells were cultured at 37 °C and 5% CO(2) in 25 cm^2 bottles (Costar, Cambridge, MA), using RPMI 1640 supplemented with 20% native FCS, L-glutamine (380 µg/ml), penicillin (100 IU/ml), and streptomycin (100 µg/ml). Confluent primary and secondary cultures were propagated at a split ratio of 1:3 with addition of endothelial cell growth supplement (10 µg/ml) and heparin (90 µg/ml) to the growth medium. Immunofluorescent detection of Weibel-Palade bodies was used to monitor homogeneity of the cultures. In all experiments tertiary cultures were used. Approximately 2 times 10^5 cells/ml were seeded on glass coverslips placed in a 6-well cluster plate (Costar) supplied with 2 ml of growth medium. The cells were grown for 2 days. Expression of TF in HUVEC was induced by adding rIL-1beta (100 units/ml) to the growth medium, and the cells were then incubated for 6 h at 37 °C under 5% CO(2) in air. Growth medium containing 10% FCS was used during the incubation.

The human bladder carcinoma cell line J82 was obtained from the American Type Culture Collection (Rockville, MD) and cultured in DMEM supplied with 10% FCS, L-glutamine (380 µg/ml), penicillin (100 IU/ml), and streptomycin (100 µg/ml). For experiments approximately 2 times 10^5 cells/ml were seeded on glass coverslips and grown for 2 days.

All transfections of COS-1 cells were done using calcium phosphate coprecipitation procedures (19) with glycerol shock. Cells with stably integrated constructs were selected with 500 µg/ml geneticin (G418). Total TF expression for selection of transfectants was determined by mixing cell homogenates with human plasma and 30 mM Ca and recording the clotting time as described(16) . The cells were cultured in DMEM supplied with 5% FCS, penicillin (100 IU/ml), streptomycin (100 µg/ml), and L-glutamine (380 µg/ml). For experiments approximately 1 times 10^5 untransfected cells/ml and 2 times 10^5 transfected cells were seeded on separate glass coverslips and grown for 2 days (the latter grew more slowly).

The Madin-Darby Canine Kidney (MDCK) type I cell line was obtained from K. Prydz (Oslo) and cultured in DMEM supplied with 5% FCS, L-glutamine (380 µg/ml), penicillin (100 IU/ml), and streptomycin (100 µg/ml) in 30-mm tissue culture dishes (Nunc, Copenhagen, Denmark). For experiments approximately 2 times 10^5 cells/ml were seeded on glass coverslips and grown for 1 day.

Loading of Cells with Fura-2

The coverslip with cells was removed from the well, and a 10-mm diameter polyethylene cylinder was attached with grease (Dow Corning High vacuum grease) to its central part. The cells within the cylinder were washed once with HSS and incubated with 400 µl of fura-2 solution for 30 min at 37 °C. The fura-2 solution was prepared by dissolving 50 µg of fura-2 in 25 µl of Me(2)SO with 10% (w/v) of the dispersing agent Pluronic F-127 for 30 min at room temperature. This solution was diluted with 10 ml HSS giving final concentrations of 5 µM fura-2, 0.25% Me(2)SO, and 0.025% Pluronic F-127. After the incubation the cells were washed once and then incubated with 400 µl HSS.

Measurement of Cytosolic Ca in Single Cells

The imaging and registration software for measuring cytosolic Ca concentrations in single cells has been developed in our laboratory(20) . The excitation device (PTI Delta-scan 1, Hamburg, Germany) consisted of a water cooled Xe-lamp (150 watts), two monochromators set at 345 and 385 nm, a chopper with two rotating mirrors (150 revolutions/min), and fiber optical light summation. Two electronic shutters were set at the end of the monochromators to avoid photo bleaching by limiting the exposure of the cells to excitation light. A Nikon Diaphot-TMD inverted microscope was set in an incubator box thermostatically regulated at 35-37 °C. The coverslip with the well was set in a chamber that was mounted on the stage upon a Fluor 40 X oil immersion objective (numerical aperture 1.3). 100 µl of ligand was injected into the well. Fluorescence images were collected by a Hamamatsu CCD video camera (C3077) and an intensifier head (C2400-8) (Hamamatsu, Japan) interfaced to the microscope by a Nikon 0.9-2.25 zoom lens. The images were stored on video tape by a Sony u-matic SP video recorder and simultaneously digitized by a frame grabber (512 times 512 pixel/frame, 8-bit resolution) controlled by a computer (DFI 386/33 MHz 12 Mb RAM) which also synchronized the chopper speed with the signal from the video camera. The cells were illuminated for 100 ms at each wavelength followed by a 200-ms period with the shutters closed resulting in a ratio image frequency of 2.5 Hz. At the end of the experiment, the cells were stained to reveal their nuclei(20) . These images were also recorded and a time series of [Ca](c) were sampled from the video tape by measuring the fluorescence from user-selected squares that covered the cell nuclei. The cytosolic Ca concentration was calculated using the equation(21) :

R is the ratio between fluorescence intensity at 345 and 385 nm excitation. R(min) and R(max) signify this ratio when fura-2 is depleted of and saturated with calcium, respectively. The calcium dissociation constant K(d) of fura-2 is 224 nM(21) , and beta is the ratio of fluorescence between Ca-depleted and Ca-saturated fura-2 at 385 nm excitation. The autofluorescence values found by measuring fluorescence from non-fura-2-loaded cells were less than 10% of the total fluorescence and were subtracted from the fluorescence values before calculating the ratio.

Calibration of Cytosolic Ca

A new indirect intracellular calibration method developed in our laboratory (22) was used to calculate the calibration constants (beta, R(min), R(max)). This method to estimate beta is advantageous over standard methods, since in the latter, changes in cell shape, focus plane, or loss of indicator may alter the measured fluorescence intensity due to the long interval between depletion and saturation of fura-2. beta can thus be calculated from the formula:

where alpha is the ratio of parallel changes in the fluorescence intensities at wavelengths 345 and 385 nm (345/385), which has a constant negative value independent of changes in the fluorescence signals. By analyzing Ca responses from several cells with a marked and rapid rise in [Ca](c) and without changes in cell shape, an average value for alpha could be calculated. R(min) was measured by means of cells depleted of Ca by incubating them for 30 min with a Ca-free HSS buffer containing 1 µM 4-Br-A23187 and 5 mM EGTA. R(max) was measured by incubating the same cells for another 20 min after addition of 10 mM CaCl(2).

Quantitative and Statistical Analysis

The fluorescence data sampled from the video tape were treated by a program (LICS) written in our laboratory. This program quantitatively analyses Ca signals from case control or dose-response experiments. The fluorescence and Ca signals were smoothed by excluding higher frequencies than 0.33 and 0.20 Hz, respectively, by using a Hamming window low pass filter. Cells showing spontaneous responses before addition of stimulants were excluded from the analyses. LICS exported the data to STATISTICA/w (StatSoft) for performing the statistical analyses. The data are presented as means and their standard error (S.E.). The statistical significance of the difference between groups was estimated by analysis of variance and the paired t test for dependent samples.


RESULTS

Effects of FVIIa Binding to TF in HUVEC and J82 Cells

TF is constitutively produced in J82 cells with high surface availability. In HUVEC TF synthesis reaches a maximum level about 6 h after addition of recombinant IL-1beta(15) . In the present experiments, the total TF activity of J82 cells averaged 12-14 units/mg cell protein, in HUVEC values for total TF activity increased from nominally none to 1-2 units/mg upon IL-1beta stimulation. The surface available activity on intact cells is more than 90% in J82 cells (23) and about 25% in IL-1beta-treated HUVEC(24) . To investigate the effect of FVIIa binding to TF, [Ca](c) was measured in single HUVEC. A rather high concentration of FVIIa (200 nM) was used since the local concentration of this signaling ligand in vivo may be considerably higher than the normal plasma concentration (10 nM). A complex set of Ca responses was observed. Some cells showed distinct Ca transients. However, such transients occurred in FVIIa-treated cells, in control cells treated with HSS, and in FVIIa-treated HUVEC without IL-1beta pretreatment. To evaluate any differences between these groups of cells, their Ca responses were averaged (Fig. 1). IL-1beta-pretreated HUVEC clearly responded to the addition of FVIIa (final concentration 200 nM) with an increase in the Ca level (Fig. 1A), whereas non-pretreated HUVEC (not carrying surface available TF) showed no difference from the controls (Fig. 1B). This effect of FVIIa was confirmed when IL-1beta-pretreated HUVEC were used as their own controls, supplied first with FVIIa and then HSS, or vice versa (Fig. 1C).


Figure 1: Average Ca responses after binding of FVIIa to TF on the surface of HUVEC and J82 cells. Panel A, HUVEC incubated with IL-1beta (100 units/ml) for 6 h at 37 °C. The cells were exposed to either FVIIa (200 nM) (solid line) or HSS as control (dotted line) at 30 s as indicated by the arrow. Panel B, HUVEC not pretreated with IL-1beta were exposed to FVIIa or HSS as in panel A. Panel C, HUVEC pretreated with IL-1beta were first exposed to 200 nM FVIIa at 60 s and then HSS as control at 180 s (solid line) or vice versa (dotted line). Panel D, J82 cells treated like the HUVEC in panel C. n denotes the number of single cells investigated. Note different scaling.



To see whether FVIIa binding to TF could generate a similar response in other cell types, we investigated the effects of FVIIa in the J82 cell line and found a similar result (Fig. 1D). The average Ca signals following the addition of FVIIa (Fig. 1) most frequently showed only moderate increase of [Ca](c) (20-40 nM). This small average response might either be due to a gradual increase of the basal Ca concentration in all cells or to recruitment of single cell responses.

To distinguish between responding and non-responding cells, an increase of more than 50 nM from the pretreated basal to the maximal Ca level was used as a criterion. All true ``responders'' will probably exceed this limit. Observing cells for 120-150 s after agonist addition, the response rate was calculated for each well (18-36 predetermined cells were analyzed in each well) (Fig. 2). The fraction of responding cells in IL-1beta-pretreated HUVEC exposed to 200 nM FVIIa was 21% and in J82 cells 17%, the controls varied from 1 to 5%. By observing HUVEC and J82 cells for a longer period, a higher response rate (30-35%) was found due to latency of the responses. The number of control cells responding did not increase significantly.


Figure 2: Number of HUVEC and J82 cells responding with Ca transients to addition of FVIIa. Ca transients induced by binding of FVIIa to TF were observed for 120-150 s after addition of FVIIa (200 nM) or HSS as control. An increase of [Ca] of more than 50 nM was the criterion for a response. The response rate of each well was calculated. The mean response rate of the wells is expressed as percent. The bars represent standard error of the mean (S.E.) of n different wells. Asterisks indicate a significant difference from control (p < 0.01). The data are from two to four different cultures.



The Ca signals showed a heterogeneous pattern with single transients, a train of transients (oscillations), or a spike followed by an elevated level with superimposed oscillations (Fig. 3). The spontaneous transients in HUVEC controls (upper panel) reached the same maximal level as the FVIIa-induced transients (centerpanel) (265 ± 10 nM compared to 279 ± 6 nM). In HUVEC we could thus not easily distinguish between spontaneous and FVIIa evoked Ca signals with regard to size of peaks, only with regard to number of cells responding. This was the case also for the Ca transients in J82 cells in which the maximal Ca level reached 241 ± 5 nM, significantly lower than in HUVEC (p < 0.001).


Figure 3: Ca transients in single cells. Upper panel, spontaneous Ca transients in IL-1beta-treated HUVEC after addition of HSS. Centerpanel, Ca transients in IL-1beta-treated HUVEC after addition of FVIIa (200 nM). Lower panel, Ca transients in J82 cells after addition of FVIIa (200 nM). FVIIa or HSS was added at 30 s as indicated by the arrows.



FVIIa addition also increased the basal Ca concentration. In non-responding cells the difference between the Ca levels at 5 and 140 s after FVIIa addition was calculated. The basal prestimulatory Ca level was not used in order to avoid the effect of possible application artifacts. When the increase of the basal Ca level exceeded 50 nM, the cells were excluded, irrespective of the rate of change in Ca levels. These data may thus represent a slight underestimate of this part of the Ca response. FVIIa raised basal Ca significantly more than in control cells both in IL-1beta-treated HUVEC and in J82 cells. In HUVEC not treated with IL-1beta, there was no significant difference between the FVIIa-stimulated cells and controls (Table 1).



The IL-1beta-treated cells had a significantly (p < 0.001) higher basal level (132 ± 1 nM) than the untreated HUVEC (106 ± 2 nM) suggesting that the IL-1beta treatment alone increased basal Ca levels. Of IL-1beta-pretreated HUVEC exposed to FVIIa and not responding with Ca transients, 18% responded with an increase of basal Ca of more than 15 nM, whereas only 3-6% of controls showed such a response. Similar results were found for the J82 cells. Although the 15 nM limit was arbitrary, these results indicated that a significant number of the FVIIa-treated cells contributed to the increase of basal Ca, thus bringing the total number of responding cells up to 40-50% according to our criteria.

The Role of Extracellular Ca

It was difficult to evaluate the role of extracellular Ca in the FVIIa induced Ca signals, since binding of FVIIa to TF requires Ca and is reversible upon Ca removal. In a Ca-free medium with 0.5 mM EGTA, no responses were seen in J82 cells. Influx of Ca can be inhibited by La, but again 100 µM La reduced TF clotting activity to about 15% of control and the percentage of responding cells from 31 ± 5% to 6 ± 2%, suggesting reduced binding of FVIIa to TF under these conditions. Delayed addition of 5 mM EGTA or 100 µM LaCl(3) (180 and 90 s after addition of FVIIa, respectively) also reduced the number of responding cells (8 ± 1% and 6 ± 3%), which nevertheless was somewhat higher than in controls. These results confirm that the external Ca level through its necessity for ligand binding influences the number of responding cells and suggest that Ca influx may not be essential for obtaining FVIIa-induced Ca transients.

To study the role of Ca influx more directly, we then used an inhibitor of receptor-mediated Ca influx, SKF 96365(25) , that did not interfere with the FVIIa-TF interaction. The effects of SKF 96365 were studied in both HUVEC and J82 cells and proved to be very complex. In HUVEC Ca influx induced by 100 µM histamine was blocked at concentrations of SKF 96365 from 30 to 50 µM (data not shown). In addition SKF 96365 induced an increase of the basal Ca level. In IL-1beta-treated HUVEC pretreated with SKF 96365 for 3-5 min the Ca level was 148 ± 1 nM, significantly higher (p < 0.001) than in controls (132 ± 1 nM).

In J82 cells incubation with 30 µM SKF 96365 for 3-5 min resulted in a total inhibition of the Ca responses normally induced by 10 µM histamine or by 1.6 µM thapsigargin, an inhibitor of the Ca ATPases of the intracellular Ca stores(26) . This inhibition was not caused by inhibition of Ca influx since no free Ca (in the presence 0.5 mM EGTA) was present in the positive controls. By direct application of 30 µM SKF 96365, we observed a Ca response mimicking the thapsigargin response in Ca-free medium (data not shown). This effect was dose-dependent. Altogether, the SKF 96365 effects in J82 cells could be explained by a combination of inhibition of Ca influx and depletion of the internal Ca stores.

We then incubated IL-1beta-pretreated HUVEC with 50 µM SKF 96365 or HSS as control for 3-5 min before addition of FVIIa. The response rates for the cells exposed to 200 nM FVIIa were significantly higher than controls (Table 2). There was no significant difference between the FVIIa-induced Ca transients in HUVEC incubated with or without SKF 96365. As expected, J82 cells incubated with 30 µM SKF for 3-5 min showed almost no responses. These results for HUVEC support the view that Ca influx is not necessary for the FVIIa-induced Ca transients.



In the same experiments the increase of the basal Ca concentration was calculated in cells that did not show a peak response, i.e. an increase of more than 50 nM. In both SKF 96365 and HSS-pretreated HUVEC, FVIIa induced a significantly higher increase of the basal Ca level than in controls (Table 2). The lower difference between FVIIa and control and the total higher increase in SKF 96365-pretreated cells may be explained by a continued increase in Ca induced by SKF 96365. Although SKF 96365 inhibited the Ca responses in J82 cells, there was a high and significant increase of the basal Ca level in the FVIIa-stimulated cells compared to control (Table 2), indicating a rather high number of responsive J82 cells. Since SKF 96365 inhibited Ca influx and depleted the internal Ca stores in J82 cells, the increase of basal Ca was probably mediated by a membrane Ca flux not inhibited by SKF 96365.

Test of Specificity Examined by Antibodies and Transfected COS-1 Cells

FVIIa induces Ca transients in IL-1beta pretreated, but not in untreated HUVEC, and extracellular Ca is necessary for the FVIIa-induced Ca transients in HUVEC as well as in J82 cells. Since there is no TF activity in untreated HUVEC and FVIIa binding to TF is Ca dependent, both of these observations suggest an effect of FVIIa binding mediated through the TF molecule acting as a receptor. To exclude nonspecific effects or effects of FVIIa binding to other sites (27) , we investigated the effect of a monoclonal antibody that neutralizes TF activity. We also carried out experiments using COS-1 cells that do not normally express TF as well as COS-1 lines stably transfected with and expressing the gene for human TF. We pretreated J82 cells for 30 min with a monoclonal antibody to human TF (hTFab) and with an irrelevant monoclonal antibody (murine antibody to human von Willebrand factor (vWFab)) as a control. Simultaneously, the cells were incubated with fura-2 and then washed once. Incubation with hTFab resulted in a 75-80% decrease of total TF activity, while the number of cells responding with Ca transients decreased from 22% in the vWFab-treated controls to 8% (p = 0.01) (Fig. 4).


Figure 4: Response rates in antibody pretreated J82 cells and transfected and untransfected COS-1 cells. Ca transients induced by binding of FVIIa to TF were observed for 300 s after addition of 200 nM FVIIa. J82 cells were incubated with vWF or hTF antibodies for 30 min and then washed once. COS-1 cells stably transfected with hTF cDNA or untransfected cells were used. An increase of more than 50 nM was the criterion for a response. The number of responding cells in each well was calculated and expressed as percent of total cells. The bars represent standard error of the mean (S.E.) of n different wells. Asterisks indicate a significant difference from control (p leq 0.01). The data are from two different cultures.



COS-1 cells were stably transfected with a human TF cDNA construct giving a total TF activity of 8-10 units/mg cell protein, of which about 10% was available for interaction with FVIIa on the cell surface as revealed by the two-stage assay on intact cells. In untransfected COS-1 cells there was no detectable activity. Stimulating the hTF-transfected COS-1 cells with 200 nM FVIIa, we observed a response rate of 19% compared to 2% in untransfected COS-1 cells (p = 0.004) (Fig. 4).

Although the number of cells responding with Ca transients clearly was different, no significant difference in the increase of basal Ca levels was detected between J82 cells incubated with the hTFab and the vWFab. In the COS-1 cells, both groups revealed a slight increase during the experiment, but the hTF-transfected cells had a significantly higher (p < 0.001) increase than the untransfected cells. Altogether, these experiments show that the effects of FVIIa in HUVEC and J82 cells depended upon available TF and are thus mediated by the direct ligand-receptor interaction of FVIIa and TF.

Ca Oscillations Induced by FVIIa Binding to TF in MDCK Cells

MDCK cells synthesize TF constitutively and express about 10% of their total activity on their surface under the conditions applied. Canine TF reacts apparently well with recombinant human FVIIa as indicated by the total TF activity of MDCK homogenates (3-4 units/mg cell protein). When stimulating MDCK cells with various concentrations of FVIIa (2-200 nM), heterogeneous response patterns with Ca oscillations of variable frequencies were observed. Individual cells responded with very different oscillation patterns (Fig. 5).


Figure 5: FVIIa-induced Ca oscillations in single MDCK cells. Examples of Ca oscillations in MDCK cells treated with 10 nM (upper panel) and 200 nM (lower panel) FVIIa at 30 s as indicated by the arrows.



By averaging the responses of single MDCK cells at each concentration of FVIIa, an obvious dose dependence was observed (Fig. 6). A similar dose dependence was observed when the percentage of responding single cells was calculated (Table 3). With control MDCK cells an average of 7% responded with spontaneous Ca transients within 180 s after addition of vehicle, comparable to the spontaneous response rates observed in HUVEC and J82 cells. At the physiological concentration of FVIIa in plasma (10 nM), 78% of the cells responded, and at 200 nM approximately all the MDCK cells revealed a Ca signal.


Figure 6: Average Ca responses after stimulation of MDCK cells with FVIIa. MDCK cells were stimulated with different doses (0-200 nM) of FVIIa, or HSS as control, at 30 s as indicated by the arrow. The values were normalized as percent of the starting basal Ca level. The line symbols are control (n = 118) (line a), 2 nM (n = 96) (line b), 10 nM (n = 87) (line c), 20 nM (n = 101) (line d), 200 nM (n = 118) (line e). n is the number of cells making up each averaged response. The results are from three different cultures.





We then examined whether the maximal amplitude, frequency, and latency of the Ca oscillations were dependent on the concentration of FVIIa. Cells that responded with a Ca increase of more than 100 nM were analyzed. The 100 nM level was chosen to exclude small spontaneous transients. By calculating the difference between basal and maximal Ca levels, we found that the amplitude increased with increasing FVIIa concentrations (Table 3). The frequency was calculated for cells responding with at least three Ca spikes with fixed intervals after addition of FVIIa, while the latency, i.e. the time from addition of ligand to the beginning of the first Ca increase was calculated for all responding cells. When MDCK cells were observed for 180-300 s after addition of FVIIa, the frequency increased while the latency decreased with increasing FVIIa concentrations (Table 3). Their inverse relationship was confirmed by a significant negative correlation (r = -0.25, p < 0.05). Individual MDCK cells were able to alter their firing frequency upon repeated additions of increasing doses of FVIIa. When treating cells first with 20 nM and then after 180 s with 200 nM FVIIa, the mean frequency changed from 0.99 ± 0.05 to 1.50 ± 0.08 min (n = 29). Correspondingly, the frequency increased from 0.73 ± 0.05, via 0.87 ± 0.06 to 1.01 ± 0.07 min when the cells were subsequently stimulated with 10, 50, and 200 nM FVIIa at a 270-300-s interval (n = 24).

The role of extracellular Ca for the FVIIa-induced Ca oscillations is difficult to ascertain because the binding of ligand (FVIIa) to receptor (TF) requires Ca. We therefore used La which blocks Ca influx and which inhibits the FVIIabulletTF complex formation to a lesser degree than removal of extracellular Ca by EGTA addition. When treating the cells with 200 nM FVIIa for 270 s, allowing complex formation with TF and Ca oscillations to start, and then adding 100 µM LaCl(3), the frequency of the oscillations decreased from 1.19 ± 0.09 to 0.92 ± 0.09 min (n = 30, p = 0.03).

Cells that responded with a biphasic Ca signal with superimposed Ca oscillations changed to a simpler oscillatory pattern after addition of La (Fig. 7, upper panels), while cells with base-line oscillations only decreased their frequency (lower panel). These results indicated that Ca influx contributed to the oscillatory signal although influx was neither necessary for generation nor maintenance of the oscillations. However, inhibition of Ca influx reduced their frequency.


Figure 7: Ca oscillations in single cells after inhibition of Ca influx. MDCK cells were stimulated with 200 nM FVIIa at 30 s, and 100 µM LaCl(3) was added at 300 s as indicated by the arrows.



Synchronized CaOscillations

When MDCK cells were grown to confluence, the Ca oscillations in some wells seemed to be synchronized, even to the extent that almost all cells responded with synchronized Ca transients. The averaged Ca response of such wells looked like a single cell Ca response (Fig. 8, upper panel). This could not be explained as an instrumental artifact since the single cell responses showed clear individual differences (lower panel). The synchrony is elucidated by digital imaging of the [Ca](c) level at different times in the oscillatory response (Fig. 8, pseudo-color images). This synchrony must be mediated through an intercellular signal, since FVIIa-induced oscillations regularly were asynchronous in cell layers that had not reached confluence (Fig. 5).


Figure 8: Synchronized Ca oscillations induced by FVIIa in MDCK cells. MDCK cells grown to confluence were stimulated with 200 nM FVIIa at 30 s and then observed for 300 s. Application of FVIIa is indicated by the arrows. Upper panel, an average Ca response of 108 single cells in the same well. A-E indicate the time points of the pseudo-colorimages. The line below the time axis indicates the time interval of the images in Fig. 9. Lower panel, examples of three different single cell responses in the same well. Pseudo-color image: A-E, Ca images taken at the points indicated in the upper panel. F, an image of the MDCK cells stained by Colorrapid. The pseudo-color scale indicates the Ca concentrations.




Figure 9: The onset of a synchronized Ca transient. The pseudo-color images show an upper right segment of the well in Fig. 8. The 12 Ca images are taken at a 1.2-s interval from 252.8 to 266 s as indicated in the upper panel of Fig. 8.



We analyzed one well in greater detail to see if ``leading'' cells could be detected that started their transients before the others, thereby initiating a signal that established responses in their neighboring cells. Fig. 9shows Ca images at the start of a Ca transient. Two or three cells seem to increase their Ca levels before the others and may be such leading cells. A full sequence of Ca images, including several subsequent synchronous Ca transients, indicated that some cells, especially the first cell responding in the upper left corner of Fig. 9, started an intercellular Ca wave, apparently generating a series of individual synchronous Ca transients in adjacent cells. These observations show that the Ca oscillations induced by the binding of FVIIa to TF in MDCK cells can set up a synchronous Ca signal when the cells are grown to confluence.


DISCUSSION

We have shown that the binding of FVIIa to TF induces a Ca signal in four different cell types, HUVEC induced to synthesize TF, J82 and MDCK cells constitutively expressing TF, and COS-1 cells stably transfected with human TF cDNA. TF thus fulfills the last requirement for being a true receptor, i.e. binding of its ligand causes an intracellular signal. The Ca signals were either Ca transients produced mainly by intracellular release of Ca or a continuous gradual increase of the basal Ca concentration generated by an altered membrane Ca flux. The Ca oscillations in MDCK cells were dependent on the concentration of FVIIa with respect to frequency, latency, maximal amplitude, and number of responding cells, and the frequency of the oscillations was reduced when Ca influx was blocked by La. Frequency coded Ca transients were first observed in hepatocytes (28) and is now accepted as a general principle for hormone-induced Ca signals. The amplitude of the oscillations has been reported to be constant, independent of agonist concentrations(29) , whereas we observed increasing maximal peak [Ca](c) levels with increasing FVIIa concentration. The notion that these signals are mediated by the ligand-receptor interaction of FVIIa and TF is supported by the relationship between cellular TF activity and the Ca signal, i.e. no signal in uninduced HUVEC or untransfected COS-1 cells, the dependence on external Ca of both FVIIa-TF binding and the intracellular Ca signal, and the blockage of the signal by a TF neutralizing antibody. Binding sites for FVIIa other than TF have been found on HUVEC(27) , but they have lower affinity, and our experiments rule out these sites as possible signal transducers.

Inhibition of Ca influx by SKF 96365 in HUVEC and the delayed addition of EGTA in J82 cells suggest that the Ca transients were mainly caused by release from internal Ca stores, although an influx component was not excluded. We could not detect any difference between spontaneous and FVIIa-induced transients in these cell types, indicating that they might be generated by the same mechanisms or that FVIIa, instead of setting up its own Ca transients, just increased the probability of spontaneous firing. Spontaneous Ca transients evoked by release from internal pools have also been reported in other non-excitable cells(30, 31) .

A cellular Ca signal induced by hormones or other extracellular signaling mediators is usually evoked by the activation of phospholipase C (PLC) which hydrolyzes phosphatidylinositol (4, 5) -bisphosphate to diacylglycerol and inositol(1, 4, 5) -trisphosphate (for review, see (32) ). There exist two main routes for activation of PLC. The G-protein-linked receptors activate the PLC-beta isoenzyme, whereas tyrosine kinase receptors (e.g. growth factor receptors) or tyrosine kinase-linked receptors (e.g. T-cell receptor, cytokine receptors) stimulate the form of PLC. Because of its single transmembrane domain, it seemed likely that TF induces a Ca signal by activation of PLC-. TF does not have an intrinsic tyrosine kinase activity but might be coupled to a tyrosine kinase. However, immunoblotting using anti-phosphotyrosine antibodies revealed no difference in the phosphorylation pattern between unstimulated and FVIIa-stimulated cells (data not shown).

SKF 96365, one inhibitor of receptor-mediated Ca entry, has recently been shown to have some side effects. Like thapsigargin it blocked the endosomal Ca ATPase in thymic lymphocytes (33) , and in HUVEC it activated a non-selective cation channel resulting in Ca influx(34) . Our results indicate a thapsigargin-like effect of SKF 96365 in J82 cells, and the increased basal Ca level in SKF 96365 pretreated HUVEC might be produced through the non-selective cation channel. A plausible mechanism for the opening of this channel is by emptying of internal Ca stores as suggested by the capacitative model(35) . The effect of SKF 96365 on inhibition of Ca influx and on emptying of Ca stores leading to Ca influx is therefore probably occurring in all cell types but at different concentrations of SKF 96365 and with different relative potency. The effects of this inhibitor must therefore be thoroughly investigated before use in any cell type.

The lack of inhibition of SKF 96365 on the gradual increase of basal Ca in J82 cells indicates that the source of this Ca was extracellular since SKF 96365 had depleted the internal stores as witnessed by the deficient histamine and thapsigargin responses. Neither did SKF 96365 inhibit this phenomenon in HUVEC, suggesting that altered membrane Ca flux in these cells was not through the normal receptor-operated channels inhibited by SKF 96365. Several possible mechanisms can be responsible for such increased Ca permeability of the plasma membrane, which has also been reported in IFN--stimulated human neutrophils(36) . It might be a potentiation of the capacitative Ca entry as seen with the protein kinase C activator, phorbol 12-myristate 13-acetate, in the insulin-secreting cell line RINm5F(37) . Another possible mechanism is decreased efflux of Ca either by the Ca-ATPase or the Na/Ca exchanger.

On the basis of the mechanisms involved, Ca oscillations have been characterized as either membrane or cytosolic oscillators (38) . Membrane oscillators arise from opening of plasma membrane Ca channels, either the voltage-operated channels or the channels opened by emptying of the internal stores (capacitative entry (35) ). Since the FVIIa-evoked Ca oscillations were not abolished by inhibition of Ca influx, they must primarily be driven by periodic release from the internal stores, i.e. a cytosolic oscillator. Such oscillators may be described as sinusoidal transients where the [Ca](c) oscillates upon a sustained elevated level or base-line transients where [Ca](c) reaches the resting level in between each spike. We observed both sinusoidal and base-line oscillations. The sinusoidal ones were evoked at the maximal concentration of FVIIa and their frequency seemed to be constant. In neutrophils we have earlier found that sinusoidal oscillations from a large number of cells tend to assemble around one characteristic frequency(22) . Similar observations in lacrimal cells have been explained by oscillating protein kinase C activity(39) .

In wells with confluent MDCK cells, we observed synchronous Ca oscillations in 50-150 cells. Sage and co-workers (40) reported that bradykinin induced synchronized oscillations in monolayers of endothelial cells. Similar observations on adjacent cells from different tissues have since been reported(41, 42, 43) . A common feature seems to be that these oscillations are dependent on the presence of external Ca or Ca influx. Functional gap junctions between the cells appear to be required for synchronization(44, 45) . A membrane oscillator controlled by electrically transmitted signals between the cells seems to be the most likely explanation of these findings.

Another type of co-ordinated Ca signals are intercellular Ca waves spreading from a single mechanically stimulated cell(46) . Focal application of hormone, i.e. local stimulation of one single cell, induced synchronized Ca oscillations of pancreatic acini cells, whereas global stimulation resulted in asynchrony (47) in contrast to our observations. Signaling molecules diffusing through gap junctions seem to be responsible for these phenomena with inositol(1, 4, 5) -trisphosphate as the most plausible candidate(48) , although waves have also been reported to be evoked by a diffusible messenger in the absence of gap junctions(49) . In our experiments the FVIIa-induced Ca oscillations were asynchronous when the MDCK cells were not in contact with each other, whereas synchrony was observed in confluent cell layers. This suggests that direct cell to cell contact is required for the co-ordination of the signal. The study on pancreatic acini (47) also revealed oscillations of higher amplitude and increased frequency when the cells were synchronized through gap junctions. Likewise, we observed higher spikes and transients of shorter duration when the Ca signal was co-ordinated after the first transients (Fig. 8). The synchronous oscillations that we observed conform to neither of these types since we observed oscillations when Ca influx was impeded and synchrony was initiated by global application. Consequently, our data show for the first time, to our knowledge, that global application of agonist can induce synchronous base-line Ca oscillations in a confluent cell layer by periodic release from internal stores. We speculate that this synchrony may be co-ordinated by intercellular Ca waves (Fig. 9) by the same mechanisms as for mechanically induced Ca waves and synchronous oscillations evoked by focal application of hormone. Obviously, further investigations are required to address this problem.

In most of our experiments, we have used a rather high concentration of FVIIa (200 nM) compared to the normal plasma concentration (10 nM). This was done partly because recombinant FVIIa may have lower specific activity than endogenous FVIIa and partly because FVIIa will bind to cell surfaces most likely through interaction with phospholipid and proteoglycans and thus be present at a locally increased concentration. (^2)If FVIIa is able to induce a specific signal, its local concentration in vivo must exceed the normal plasma concentration which presumably signifies the lower threshold for the cells to recognize this signal. In HUVEC it may be more likely that the appearance of TF triggers the signal, since FVIIa is present at all times. In MDCK cells significant Ca signals were found also at concentrations (2 nM) below the normal plasma concentration. Why FVIIa-generated Ca transients occur in some cell lines (HUVEC, J82, and COS-1) in only about 30% of the cells is not known. Several possible explanations exist. Studies have shown that whereas FVIIa/TF activity on the cell surface reaches its maximum in about 1 min, the binding of FVIIa to TF is saturated much more slowly(50) . This observation has not been analyzed at the level of individual cells and could obviously be linked to heterogeneity in cellular responses. We also observed a large variation in the response rate in cell cultures in different wells, possibly correlated to cell density. The Ca responses were not correlated to the overall TF activity of the various cell types (J82 and HUVEC showing about the same response rate but 5-10-fold difference in TF activity), but the difference between individual cells has not yet been analyzed. Neither did the lower response rate in HUVEC and J82 cells compared to MDCK cells result from differences in receptor numbers since the plasma membrane TF activity in MDCK cells was in between the levels found in the other cell types. The more than 100-fold difference in sensitivity is therefore probably caused by a variation in the signaling system such as a difference in the coupling to PLC, or that the Ca signals are evoked by partly different mechanisms in the different cell types.

We conclude that binding of FVIIa to TF in HUVEC, J82, and MDCK cells and hTF cDNA-transfected COS-1 cells induces cytosolic Ca signals of different characteristics, comprising Ca oscillations and other transients, as well as increased basal [Ca](c). In the MDCK cells the oscillations are synchronous when the cells are grown to confluence, providing the first evidence of a synchronous cytosolic Ca oscillator generated by global application of agonist. Our work demonstrates for the first time that TF is a true receptor which upon binding of its ligand induces an intracellular signal. The biological effects of these signals are now being studied. They are probably components of a more general response chain. The role of TF in hemostasis suggests that the FVIIa-induced signal may modulate the coagulation cascade by regulating production and secretion of relevant components. The fact that the TF gene is an immediate early gene and a member of the cytokine superfamily, in addition to its signal transducing property, suggests a role for TF also in cell biology.


FOOTNOTES

*
This work was supported by the Research Council of Norway, the Norwegian Council for Cardiovascular Diseases, CLOTART Project Contract BMH 1-CT94-1202, and by the Anders Jahre's Foundation for the Promotion of Science. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipients of research studentships of the Research Council of Norway.

Research fellow of the Norwegian Council for Cardiovascular Diseases.

**
To whom correspondence should be addressed: Biotechnology Centre of Oslo, Gaustadalléen 21, N-0371 Oslo, Norway. Tel.: +47-22958755; Fax: +47-22694130.

(^1)
The abbreviations used are: TF, tissue factor; FVIIa, factor VIIa; HUVEC, human umbilical vein endothelial cells; MDCK, Madin-Darby canine kidney; IFN, interferon; IL-1beta, interleukin-1beta; HSS, HEPES-buffered salt solution; vWF, von Willebrand factor; PLC, phospholipase C; [Ca](c), cytosolic free Ca concentration; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum.

(^2)
S. Pringle and H. Prydz, unpublished results.


ACKNOWLEDGEMENTS

Human TF cDNA was kindly provided by Dr. J. H. Morrissey, MDCK cells by Dr. K. Prydz, and recombinant human Factor VIIa and human interleukin 1beta were kindly donated by Novo-Nordisk. The imaging laboratory was developed by Dr. Jan S. Røtnes, and Sverre H. Huseby wrote much of the analyzing software.


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