Protein kinase c regulates cytokine-induced tissue factor transcription and procoagulant activity in human endothelial cells

Protein kinase C regulates cytokine-induced tissue factor transcription and procoagulant activity in human endothelial cells CHRISTI M. TERRY and KARLEEN S. CALLAHAN SALTLAKECITY, UTAH

Interleukin-l~ (IL-I=) and tumor necrosis factor-= (TNF-~) induce tissue factor in endothelium, which results in activation of the coagulation cascade. Despite extensive investigation, in which various stimuli that induce tissue factor have been defined, the intracellular processes that control tissue factor expression are not well understood. It has been proposed that protein kinase C regulates tissue factor expression primarily because phorbol myristate acetate, the protein kinase C activator, induces tissue factor expression. In this study we examined whether IL- I ~ - or TNF-=-stimulated tissue factor production is regulated through a protein kinase C - d e p e n d e n t mechanism. Northern blot analysis showed that cytokine-induced tissue factor mRNA was significantly reduced in human umbilical vein endothelial cells treated with calphostin C, a specific protein kinase C inhibitor. Tissue factor functional activity was decreased in the presence of calphostin C as well. Calphostin C also inhibited phorbol myristate acetate-induced tissue factor expression. In contrast, calphostin C did not alter cytokine induction of E-selectin or prostacyclin release. Because calcium stimulates protein kinase C binding to the membrane and its resulting catalytic activity, human umbilical vein endothelial cells were exposed to IL-I= or TNF-= in the presence of calcium ionophore A23187. A23187 had little effect alone but significantly augmented cytokine stimulation of tissue factor mRNA. Okadaic acid, a phosphatase inhibitor, increased cytokine-induced tissue factor mRNA compared with cytokine alone, which suggests that a phosphorylation event is important in tissue factor expression. These results indicate that protein kinase C is involved in cytokine activation of endothelial cell tissue factor expression. (J lAB CLIN MED 1996:127;81-93)

Abbreviations: DMSO = dimethyl sulfoxide; dPPA = 12-deoxy phorbol ]3-phenyl acetate 20acetate; HUVEC = human umbilical vein endothelial cells; IL-]~ = interleukin-l~; PMA = phorbol myristate acetate; PMN = polymorphonuclear leukocyfes; TNF-~ = tumor necrosisfactor-~ From the Departments of Pharmacology and Toxicology, the Veterans Affairs Medical Center, and the Department of Internal Medicine, University of Utah, Salt Lake City, Utah. Supported by DVA Medical Research Funds (K.S.C.), by the Utah affiliate of the American Heart Association (K.S.C.), by United States Public Health Service Grant No. GM07579 (C.M.T.), and by a fellowship from the American Foundation for Pharmaceutical Education (C.M.T.). Submitted for publication Apr. 21, 1995; revision submitted .Aug. 23, 1995; accepted Aug. 26, 1995. Reprint requests: Karleen S. Callahan, PhD, Department of Internal Medicine, Pulmonary Division, University of Utah School of Medicine, 50 N. Medical Dr,, Salt Lake City, UT 84132. 0022-2143/96 $5.00 + 0 5/1/68838

issue factor is a t r a n s m e m b r a n e glycoprotein that plays a central role in blood coagulation. W h e n expressed on the surface of cells such as endothelium, this protein is a cofactor for the conversion of circulating factor V I I to its active form. T h e proteolytic tissue factor/factor V I I a complex then activates both coagulation factors I X and X, which leads to thrombin generation and subsequent fibrin deposition. Endothelial cells lining the blood vessels are normally n o n t h r o m b o g e n i c and do not constitutively express tissue factor. However, u p o n physical injury or inflammatory insult, procoagulant activity in the

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form of tissue factor can be induced. Tissue factor is induced in endothelium by several agents, including the cytokines IL-I= 1 and TNF-oL,2 as well as by bacterial endotoxin 3 and immune complexes. 4'5 Although the mediators that induce tissue factor in endothelial cells have been well described, the molecular mechanisms that control this induction are not as clearly understood. Protein kinase C, which comprises a family of serine/threonine-specific isoenzymes, has been implicated in TNF-e~ and IL-loL actions in endothelium. 6-9 The involvement of protein kinase C in TNF-oL and IL-lc~ effects is suggested largely because exposure of endothelial cells to PMA, a potent protein kinase C activator, results in the expression of many proteins, including tissue factor, 1°'11 which are also induced by IL-le~ and TNF-~. Thus, it has been proposed that protein kinase C is an important signaling molecule that regulates tissue factor expression. However, prior investigations of this hypothesis have yielded contradictory results. For instance, one investigation in H U V E C demonstrated an augmentation of functional tissue factor activity by a variety of protein kinase C inhibitors in response to TNF-e~ and IL-I~ stimulation, 12 whereas Pettersen et a1.13 observed an apparent biphasic response, with low doses of protein kinase C inhibitors slightly increasing TNF-o~and IL-le~-induced tissue factor activity and higher doses decreasing procoagulant activity. When examining these previous studies, it should be taken into consideration that nonspecific protein kinase C inhibitors were used. Interpretation of these results may be complicated by the fact that the commonly used inhibitors, staurosporine, sphingosine, and H7, which are nucleotide analogs, also significantly inhibit other kinases such as the cyclic adenosine monophosphate- and cyclic guanosine monophosp h a t e - d e p e n d e n t protein kinases 14 and Ca+Z/cal modulin-dependent protein kinases, is For example, it has recently been reported that cyclic adenosine monophosphate attenuates tissue factor expression in HUVEC, 16 which indicates that nonspecific inhibitors may be having unintentional effects, a6 Although these prior studies have examined the effect of protein kinase C inhibition on tissue factor protein, a role for protein kinase C in TNF-~ or IL-le~ induction of endothelial tissue factor messenger R N A was not reported. Investigations into the effect of protein kinase C inhibition on tissue factor transcriptional regulation would help delineate at which molecular level this enzyme acts. The present study was designed to investigate the effect of protein kinase C inhibition on tissue factor m R N A levels and tissue factor functional activity. In

this work, a recently available selective inhibitor of protein kinase C, calphostin C (hereon referred to as calphostin), was used. Calphostin, unlike other commonly used inhibitors, derives its specificity from the fact that it acts at the diacylglycerol binding site, which is unique to protein kinase C. 17 In addition to the calphostin studies, experiments examining the effect of increased intracellular calcium or inhibition of phosphatase activity on tissue factor transcription were also carried out. Information obtained from this study regarding the intracellular events involved in endothelial tissue factor expression is of interest, because inappropriate expression of tissue factor occurs in several pathologic conditions involving disordered hemostasis, such as the adult respiratory distress syndrome, disseminated intravascular coagulation, and neoplasias. 18 METHODS Cell culture. HUVEC were isolated and cultured as previously described. 19 Cells were grown to confluency in 75 cm2 plastic flasks in endothelial cell growth media (EGM, Clonetics Corp., San Diego, Calif.). Confluent cells were harvested with trypsin and transferred to 75 cma flasks for RNA extraction experiments and 6-well plastic plates (35 mm diameter) for tissue factor activity determinations. Confluent, first-passage cells were used for all experiments. During agonist treatment, cultures were observed for any sign of cell injury, and, if cell injury was evident, the flasks with these cultures were excluded from the experiment.

Cell treatment with calphostin and cytokine or PMA.

Confluent HUVEC were incubated at 37° C, 5% CO2/ 95% air in serumless Neuman Tytell (Gibco BRL, Grand Island, N.Y.) with either human recombinant IL-lc~ (25 to 50 U/ml) (Boehringer Mannheim Corp., Indianapolis, Ind.) or human recombinant TNF-c~ (500 U/ml) (Genzyme, Cambridge, Mass.) for 1 to 5 hours in the absence or presence of 0.1 ixmol/L to 1 ~mol/L calphostin (Kamiya Biomedical Co., Thousand Oaks, Calif., or LC Laboratories, Woburn, Mass.). Calphostin stock was dissolved in DMSO at a concentration of 1 mmol/L. HUVEC treated with the highest concentration of DMSO alone (0.1%) revealed no effect on any of the cellular responses investigated in this study. In experiments with PMA, cells were incubated with 0.1 izg/ml of PMA (Sigma Chemical Co., St. Louis, Mo.) for 2 hours in the absence or presence of 0.5 ixmol/L or 1.0 txmol/L calphostin. The PMA stock was dissolved in DMSO at a concentration of l mg/ml. In some experiments 1.0 ixmol/L calphostin was used because dose-response studies revealed potency differences between a new lot and the previous lot of calphostin that was used in the majority of experiments. A report indicated that calphostin requires light for full activation; therefore, cells were preincubated with calphostin under room light for 15 minutes before the addition of agonist. 2°

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Cell treatment with calcium ionophore or okadaic acid. Confluent endothelial cells were exposed for 3 hours

to 10 or 25 U/ml human recombinant IL-lc~ or 500 U/ml human recombinant TNF-c~ in the absence or presence of 1.0 ~mol/L of the calcium ionophore A23187 (stock concentration of 0.5 × 10 3 mol/L in DMSO) (Calbiochem Corp., La Jolla, Calif.) at 37 ° C, 5% CO 2. In experiments examining the role of phosphorylation in tissue factor expression, H U V E C were exposed to IL-lc~ (50 U/ml) or TNF-c~ (500 U/ml) for 2 or 3 hours, respectively, in the presence or absence of the phosphatase inhibitor okadaic acid (100 nmol/L) (LC Laboratories). 21 Okadaic acid stock solution (1 mmol/L) was dissolved in DMSO. Northern blot analysis. R N A was extracted from cells by an acid guanidinium thiocyanate-phenol-chloroform procedure according to the methods described by Chomczynski and Sacchi. 22 R N A was quantitated by ultraviolet absorbance at 260 nm, and 10 or 20 txg of denatured total R N A was electrophoresed on a 1% agarose/formaldehyde denaturing gel. 23 The R N A was transferred to nylon membranes (Hybond N, Amersham Corp., Arlington Heights, Ill.) overnight by capillary transfer in 20X SSC or by vacuum blotting for 90 minutes in 10X SSC (model 785, Bio-rad, Melville, N.Y.). R N A was fixed to the membrane by baking under vacuum at 80° C or by ultraviolet crosslinking (Stratalinker, Stratagene, La Jolla, Calif.). Membrane hybridizations were carried out with a radiolabeled cDNA tissue factor probe or radiolabeled tissue factor R N A transcripts, cDNA tissue factor probes were labeled with c~-32p-deoxycytidine triphosphate (3000 Ci/ mmol, Amersham Corp.) with use of a random primerlabeling kit (Boehringer Mannheim) or Prime-it (Stratagene). Radiolabeled R N A transcripts (a-32p-cytid~ne triphosphate, 800 Ci/mmol, Amersham Corp.) were produced by in vitro transcription with use of SP6 polymerase following the manufacturer's protocol (Riboprobe Gemini Transcription Kit, Promega Corp., Madison, Wis.). Hybridizations were carried out overnight at 42 ° C when using the cDNA probe and at 60° C when using the R N A probe. The membranes were then washed under stringent conditions, placed between intensifying screens, and exposed to Kodak X-OMAT A R film (Eastman Kodak Company, Rochester, N.Y.) at - 7 0 ° C for 1 hour to overnight. Lane loading equivalencies were determined by hybridizing the membranes with either actin or CHO B probe, levels of which were not changed by the various cell treatments94 Membranes were first stripped of tissue factor label by pouring boiling water over the membrane and shaking at room temperature for approximately 10 minutes. Autoradiographic images from the tissue factor label and the actin or CHO B label were scanned with a laser densitometer (Ultrascan XL Enhanced Laser Densitometer, LKB Bromma), which converted the densities of the bands to relative absorbance units. The 2.2 kb transcript of tissue factor was used for the scanning densitometry analysis. The densities of the CHO-B or actin bands were used to correct the tissue factor m R N A density values.

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These normalized values were plotted in graph form (Figs. 1, B and 2, B). In Fig. 3 the cytokine alone absorbance values were set as 100% stimulation, and the mean absorbance values for the calphostin treatments were reported as a percentage of the maximum stimulation. In Table I, means and standard errors of the means were calculated from the normalized densitometric absorbance units for each treatment group. Probes. Human tissue factor probes were a generous gift from Dr. James H. Morrissey of the Oklahoma Medical Research Foundation, Oklahoma City, Okla. The cDNA tissue factor insert that was used to make random primer labeled cDNA probes is a 641 base pair fragment extending from the mature N-terminus of tissue factor to the Eco RI site just prior to the transmembrane d o m a i n Y This fragment was provided to us inserted in pUCSRS, a derivative of pUC8. The tissue factor cDNA used for production of labeled R N A transcripts is an approximately 1700 base pair fragment of tissue factor containing the transmembrane domain coding sequence and part of the 3'-noncoding sequence with the Alu-repeat deleted. This fragment was provided to us cloned into p G E M 7 Z f ( + ) vector (Promega, Madison, Wis.) for in vitro transcription. Tissue factor activity'determination. To determine the effect of protein kinase C inhibition on tissue factor functional clotting activity, H U V E C were incubated with 500 U/ml TNF-c~ or 25 U/ml IL-lc~ in the absence or presence of 1.0 txmol/L calphostin for 5 hours. After incubation, cells were scraped up in 0.5 ml of phosphate-buffered saline solution, pelleted immediately at 4° C at 12,000 g for 5 minutes, and the supernatant discarded. The pellets were kept frozen at - 7 0 ° C until the clotting assay was carried out. Tissue factor activity was determined with use of a one-stage recalcification clotting assay with normal, pooled human plasma (Sigma Chemical Co., St. Louis, Mo.), as previously described. 26 Briefly, 25 mmol/L HEPES in normal saline solution was added to the frozen pellets, which were then thawed at 4 ° C. The cell pellets were lysed by three 10-second periods of sonication while on ice, and 100 ixl of disrupted cell suspension was assayed in duplicate for clotting activity. A standard curve was generated with rabbit brain thromboplastin (Sigma Chemical Co.). PMN a d h e r e n c e assay. Confluent H U V E C in 12-well plates were incubated for 3.5 hours with TNF-e~ (500 U/ml) in the absence or presence of 0.5 ixmol/L calphostin. After incubation with TNF-e~, the media was removed and 5 × 105 lllIn-oxine-labeled PMN were added to each well of endothelial cells and incubated for 5 minutes. 27 Supernatant from each well was then transferred to individual borosilicate glass tubes, the monolayer was washed once with Hanks' balanced salt solution, and the wash was combined with the supernatant fraction. This fraction measured nonadherent PMN per well. To measure adherent PMN, 1% ammonium hydroxide solution was placed on the cells for 30 minutes and then transferred into a separate glass tube. Any remaining radioactivity in each

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Fig. 1. Inhibitory effect of calphostin on IL-le~induction of tissue factor mRNA. A, Northern blot analysis of HUVEC incubated with IL-I~ (25 U/ml) in the absence or presence of 1.0 txmol/Lcalphostin for 1, 2, and 3 hours; 20 ~xg total RNA was loaded per lane. Control lanes contain RNA from cells exposed to media alone. The calphostin lane contains RNA from cells exposed to calphostin alone. Tissue factor mRNA was detected with random primer c~32P-deoxycytidinetriphosphate-labeled cDNA for human tissue factor. B, Densitometric analysis of autoradiograph (A) with normalization to actin mRNA as described in the Methods section.

well was scraped up in 1% ammonium hydroxide with a cotton swab and added to the glass tube that contained the adherent cell fraction. Radioactivity was measured for each fraction on a gamma counter, and adherence was calculated by dividing adherent cpm by total cpm for each individual well. E-selectin western blotting. Endothelial cells were exposed to TNF-c~ (500 U/ml) for 5 hours after a 15-minute preincubation with either 0.5 ixmol/L calphostin or media alone. Media was then removed and cells were washed twice with ice-cold phosphate buffered saline solution and analyzed as previously described, a8 Prostacyclin 12 measurement. Prostacyclin was measured by radioimmunoassay of its major metabolite, 6-keto PGFla, as discussed in detail previously.29 SIChromium release injury assay. Cell injury was assessed in HUVEC exposed to IL-lc~ or TNF-c~ in the absence or presence of 0.5 ixmol/L calphostin by a 51Chromium release method as previously described. 3° Specific 51Chromium release was calculated by dividing test cpm minus control cpm by maximal cpm minus control cpm and multiplying by 100.

Statistical analysis. The means and standard error of the means in Figs. 3 and 4 and in Table I were calculated from the absorbance units obtained from scanning densitometry of autoradiographic images of Northern blots. A pooled t-test on the raw data was used for determination of statistical significance. A Dixon test for outliers was performed on extreme values.

RESULTS Effect of calphostin on cytokine induction of tissue factor mRNA. E x p e r i m e n t s to e x a m i n e the effect of protein kinase C i n h i b i t i o n o n IL-lo~ a n d TNF-c~ induction of tissue factor m R N A were carried out with use of N o r t h e r n blot analysis. Figs. 1, A, a n d 2, A, are representative a u t o r a d i o g r a p h s from these experiments. As shown in these figures, the tissue factor probes hybridized to a p r i m a r y 2.2 kb transcript a n d to two m i n o r transcripts at approximately 3.4 kb. T h e p r e s e n c e of these transcripts is similar to that r e p o r t e d in H U V E C by others s m a n d prob-

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Fig. 2. Inhibitory effect of calphostin on q['NF-a induction of tissue factor mRNA. A, Northern blot analysis of HUVEC incubated with TNF-c~ (500 U/ml) in the absence or presence of 1.0 Ixmol calphostin for 1, 2, 3, and 4 hours; 20 Ixg total RNA was loaded per lane. Control lanes contain RNA from cells exposed to media alone. The calphostin lane contains RNA from cells exposed to calphostin alone. Tissue factor mRNA was detected with random primer c~32p-deoxycytidine triphosphate-labeled cDNA for human tissue factor. B, Densitometric analysis of autoradiograph (A) with normalization to actin mRNA as described in the Methods section.

ably arise through alternative splicing. 32 Exposure of HUVEC to IL-IoLcauses a time-dependent increase in tissue factor mRNA, as reported by others, with maximal levels seen at 2 hours, as shown in Fig. 1, A. Cells stimulated with IL-Ie~ in the presence of the protein kinase C inhibitor, calphostin, show decreased levels of tissue factor mRNA. The degree of inhibition of message is most notable at 2 hours, the time point of maximal tissue factor induction by IL-le~. Control cells that were exposed to media alone and cells exposed to calphostin alone show little detectable tissue factor mRNA. In this same experiment, tissue factor mRNA band intensities were quantified by densitometric analysis and normalized to actin mRNA band intensities to correct for any lane loading differences. The quantitative tissue factor mRNA data are shown in Fig. 1, B. Calphostin exposure results in a marked decrease in ILa-induced tissue factor mRNA. Similar results were obtained when TNF-oL was used as the agonist. Fig. 2, A shows that TNF-eL-

induced increases in tissue factor mRNA are inhibited by calphostin. Once again, the degree of inhibition of message is most notable at the time point where maximal induction normally occurs, that is, at 3 hours. Fig. 2, B illustrates in graph form the effect of calphostin on tissue factor mRNA induction after correction for any lane loading differences. As shown, protein kinase C inhibition results in a substantial decrease in TNF-~-induced tissue factor mRNA at all time points examined. Fig. 3 shows the combined results of several experiments in which HUVEC were exposed to IL-le~ for 2 hours or TNF-a for 3 hours in the presence of either calphostin or media alone. Protein kinase C inhibition results in a 54% _+ 9.8% decrease in TNF-o~-stimulated cells and a 69% -+ 7.7% decrease in tissue factor mRNA in IL-loL-treated HUVEC compared with cells exposed to cytokine alone. Calphostin inhibits cytokine-induced tissue factor mRNA in a concentration-dependent manner. Fig. 4

shows an autoradiograph from a representative experiment where varying concentrations of calphostin

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"~

20

Control

Calphostin

TNF

TNF + Calphostln

IL-1

IL-1 + Calphostin

Fig. 3. Inhibitory effect of calphostin on IL-I~ and TNF-c~ induction of tissue factor m R N A in combined studies. H U V E C were exposed to IL-le~ (25 or 50 U/ml) or TNF-c~ (500 U/ml) in the absence or presence of 0.5 ~mol/L calphostin. As controls, cells were exposed to media (control) or calphostin alone. Northern blot analysis was carried out followed by densitometric analysis and normalization as described in the Methods section. The percentage of cytokine induction of tissue factor m R N A in the presence of calphostin is expressed as the m e a n _+ SEM from an n = 5 for TNF-~x and n = 7 for IL-I~. The differences between the m e a n s of cytokine-treated cells and cytokine plus calphostin-treated cells are significant (p = 0.02, e~ = 0.05, for TNF; p = 0.001, c~ = 0.01 for I t - l ) .

Table I. Effect of okadaic acid on cytokineinduced tissue factor mRNA levels IL-1 c~ 0.80 ± 0.13 (n = 4) TNF-c~ 1.14 -+ 0.33 (n = 5) Control 0.005 -+ 0.004 (n = 5)

+Okadaic acid 2.53 ± 0.50 (n = 6) +Okadaic acid 2.27 _+ 1.35 (n 6) ÷Okadaic acid 0.005 ± 0.002 (n = 3)

HUVEC were exposed to IL-lc~ (50 U/ml) or TNF-c~(500 U/ml) for 2 or 3 hours, respectively, in the presence or absence of 100 nmol/L okadaic acid. Control ceils were exposed to Neuman-Tytell media alone. Okadaic acid alone represents cells exposed to either 100 or 500 nmol/L okadaic acid alone for 2 hours. Values reported are the mean (_+SEM)of the absorbance units from scanning densitometry of Northern blot autoradiograpic images as described in the Methods section.

were used to inhibit tissue factor m R N A induction by IL-I~. Calphostin at 0.5 ~mol/L decreased tissue factor m R N A by 76%, calphostin at 0.25 ~mol/L attenuated by 46%, whereas 0.1 txmol/L calphostin had no significant inhibitory effect on tissue factor mRNA, which demonstrates that this agent acts in a concentration-dependent manner. Similar results were obtained with TNF-~x as the inducer of tissue factor m R N A (data not shown). These inhibitory concentrations of calphostin are comparable with those reported for other protein kinase C-mediated responses in endothelial cells. 33-35 TNF-e~ and lL-l=-induced tissue factor procoagulant activity is inhibited by calphostin. Because our results

showed that protein kinase C inhibition caused a

decrease in cytokine-induced tissue factor mRNA, it was of interest to determine if HUVEC procoagulant activity would be similarly affected. H U V E C were exposed to I L - l a or TNF-oL for 5 hours in the absence or presence of calphostin. Tissue factor procoagulant activity was then determined by a onestage clotting assay. As shown in Fig. 5, tissue factor activity is very low in H U V E C incubated with media or calphostin alone, whereas H U V E C incubated with agonist show the expected increase in procoagulant activity. However, H U V E C incubated with agonist in the presence of calphostin showed a more than 50% decrease in tissue factor activity compared with agonist alone. This decrease of functional procoagulant activity is similar to work by Pettersen et al. 13 with use of other nonselective protein kinase C inhibitors, and Herbert et al. 36 in bovine aortic endothelial cells. These results, combined with those from the Northern blot analysis, demonstrate that protein kinase C inhibition causes both a decrease in tissue factor m R N A and functional protein activity. Calphostin does not effect P M N adherence. It has been reported that TNF-~x induction of leukocyte adhesion molecules on the surface of endothelium does not involve protein kinase C. 37 TO determine whether calphostin's inhibition of TNF-c~-induced tissue factor activity was a selective action or an all-inclusive inhibition of TNF-a-induced protein, we examined neutrophil adherence to stimulated endothelial cells. H U V E C were incubated with TNF-~ (500 U/ml) for 3.5 hours in the absence or

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Fig. 4. Calphostin inhibition of IL-le~-induced tissue factor mRNA is concentration dependent. Northern blot analysis of HUVEC exposed to IL-lc~ (25 U/ml) for 2 hours in the presence of 0.5 txmol/L, 0.25 ixmot/L or 0.1 ~mol/L calphostin. The control lane contains RNA from cells exposed to media alone. Each lane contains 20 p~g of total RNA. The blot was probed with c~32p-cytidine triphosphate-labeled tissue factor riboprobe.

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Calphosfin

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Fig. 5. Calphostin inhibits cytokine-stimulated tissue factor procoagulant activity. H U V E C were exposed to I L - l a (25 U/ml) or TNF-c~ (500 U/ml) for 5 hours in the presence or absence of 1.0 ~mol/L calphostin. Tissue factor activity was determined with use of a one-stage recalcification clotting assay. Each bar represents the m e a n _+ SD from replicate wells.

presence of 0.5 ixmol/L calphostin. Cells were then exposed to radiolabeled neutrophils and the percentage of adherence was determined as described in the Methods section. Cells exposed to medium alone showed minimal PMN adherence (2.2% _+ 0.1%). However, PMN adherence was greatly increased in cells exposed to T N F - a (48% _+ 0.5%) and in cells exposed to TNF-o~ in the presence of calphostin (45% _+ 1.3%) (data presented as mean plus or minus standard error, n = 4). In addition, the endothelial cell expression of the early adhesion protein, E-selectin, was examined with use of West-

ern blots. These experiments confirmed the previous adherence results as TNF-a-induced E-selectin protein levels were not significantly affected by calphostin (data not shown). Thus calphostin has very little effect on the expression of TNF-oL-induced E-selectin, which indicates that its effects on tissue factor procoagulant activity is not a nonspecific inhibition of all TNF-oL-induced proteins. Colphosfin inhibits PMA induction of tissue factor mRNA.

Calphostin acts at the diacylglycerol binding site of protein kinase C to inhibit this enzyme. 17'z8 The tumor-promoting agent PMA also acts at this site to

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100-

80-

60-

40-

20-

0-

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Control

Caiphosfin

PMA

+ Caiphoslin

Fig. 6. Calphostin inhibits PMA-induction of TF m R N A . H U V E C were exposed to 0.1 I~g/ml P M A for 2 hours in the absence or presence of 0.5 ixmol/L or 1.0 ~mol/L calphostin. As controls, cells were exposed to media (control) or calphostin alone (Calphostin). Northern blot analysis was carried out followed by densitometric analysis and normalization as described in the Methods section. The percentage of P M A induction of tissue factor m R N A in the presence of calphostin is expressed as the m e a n _+ SEM from an n = 8 for each treatment except for "control" or "calphostin" alone, which were n = 3. The difference between the m e a n s of PMA-treated cells and P M A plus calphostin-treated cells was significant (p = 0.002,

c~ = 0.01). activate the enzyme. To further investigate calphostin's ability to inhibit protein kinase C, experiments were carried out to determine whether calphostin could inhibit PMA induction of tissue factor mRNA. Tissue factor mRNA was examined at 2 hours, which has been shown to be the time point of maximal stimulation with PMA. 8 Similar to the results observed in cytokine-stimulated HUVEC, calphostin attenuated PMA-induced tissue factor mRNA as shown in Fig. 4. Of note, calphostin attenuation of PMA-induced tissue factor mRNA was greater than that observed in the cytokine studies. Ternisien et al. 39 previously showed that calphostin inhibits PMA-induced tissue factor functional activity in monocytes.

marked increase in tissue factor mRNA compared with cytokine alone. Of note, A23187 itself caused only negligible enhancement of tissue factor mRNA compared with control. The dose of A23187 used was previously found to neither induce tissue factor functional activity nor elicit any cell damagel 4° A notable augmentation of I L - I ~ induced tissue factor mRNA by A23187 was consistently seen when these studies were repeated (i.e., a 4-fold increase in several experiments with IL-lo~ as the stimulus). These data again support a role for protein kinase C in cytokine induction of tissue factor mRNA.

Calcium ionophore increases TNF-~IIL-I~ induction of tissue factor mRNA. Conventional protein kinase C

be expected to increase tissue factor mRNA if a protein kinase is participating in TNF-~ or IL-la signaling of tissue factor expression. Okadaic acid, a phosphatase inhibitor, was used to investigate this possibility. Okadaic acid is an inhibitor of both protein phosphatase 1 and protein phosphatase 2A, although it is more potent against protein phosphatase 2A. 21 Table I illustrates that tissue factor mRNA levels were markedly increased in HUVEC exposed to okadaic acid plus cytokine compared with cells incubated with cytokine alone.

isoforms (~, 131, 13II, "y) are regulated by calcium and lipid second messengers. Therefore, we investigated the effect of increased calcium on tissue factor mRNA. If conventional isoforms are involved in tissue factor expression, an elevation in intracellular calcium that increases protein kinase C activation should enhance tissue factor mRNA levels in response to cytokine. In these experiments, HUVEC were exposed to IL-loL or TNF-oL in the presence or absence of the calcium ionophore, A23187, at a concentration known to increase intracellular calcium in endothelial cells. The results are shown in Fig. 7. Exposure to either cytokine in the presence of A23187 resulted in a

Okadaic acid enhances cytokine induction of tissue factor mRNA. I n h i b i t i o n o f dephosphorylation w o u l d

Calphostin does not effect prostacyclin synthesis or cause nonspecific cell injury. It is possible that cal-

phostin caused decreased tissue factor mRNA and procoagulant activity through an effect on the cytokine receptors or by causing nonspecific cell injury

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+ A23187

Fig. 7. A23187 enhances cytokine induction of TF mRNA. Northern blot analysis of HUVEC exposed for 3 hours to TNF-e~ (500 U/ml) or IL-lc~ (10 U/ml) in the absence or presence of A23187 (1.0 0~mol/L). Each lane contains 20 ~xg of total RNA. The control lane contains RNA from cells exposed to media alone, and the A23187 lane contains ionophore alone.

undetectable by visual inspection. IL-I~ and TNF-oL stimulate H U V E C to release prostacyclin, and IL1eL-induced release of prostacyclin is a non-protein kinase C mediated effect.41 Therefore, we examined whether calphostin had any effect on cytokine-mediated prostacyclin release in our experiments. Results of these experiments are shown in Table II. Both TNF-a and IL-Io~ exposure resulted in increased prostacyclin release compared with media alone. Calphostin did not affect the ability of the cytokines to induce prostacyclin release. These results suggest that calphostin does not affect TNF or IL-1 receptor function. In addition, the observed decreases in tissue factor m R N A were not a result of general cell injury, because chromium release from cells in media alone and treatment groups were very similar at 3 hours, as shown in Table II, and as late as 6 hours (data not shown).

Table II. Calphostin does not alter cytokine-

stimulated PGI2 release or induce cell injury Treatment Control IL-lc~ IL-lc~ + calphostin TNF-c~ TNF-c~ + calphostin

H202

PGI 2 ng/well 0.52 3.13 2.57 1.03 1.16

+_ 0.08 ± 0.34 ± 0.31 ± 0.33 +_ 0.32 ND

% Specific 51Chromium release 12.9 13.4 14.0 13.9 14.1 54.6

+ 2.0 ± 1.8 £ 1.9 £ 2.2 __ 1.7 + 6.53

ND, Not determined. HUVEC were exposed to IL-lc~ (50 U/ml) or TNF-c~ (500 U/ml) in absence or presence of 0.5 ixmol/L caiphostin for 5 hours in the case of PGI2 analysis and for 3 hours in the case of chromium release assay. As controls, cells were exposed to media alone (control) or to 10 mmol/L H202.Data shown are mean _+ standard error from a minimum of three experiments with replicates of four wells each. The means of cytekine-treated cells and cytokine plus calphostin-treated cells are not significantly different as determined by statistical analysis.

DISCUSSION

The purpose of this study was to determine if protein kinase C, a protein phosphorylation enzyme, acts as a signal transducer in TNF-a and IL-I~ induction of tissue factor in human endothelial cells. In particular, the role protein kinase C might play in the transcriptional regulation of the tissue factor gene was examined. Prior investigations have focused on protein kinase C involvement in cytokine induction of tissue factor protein. However, thorough investigation of protein kinase C participation in the induction of tissue factor at the message level has not previously been performed. By measuring cytokine-induced tissue factor m R N A levels and functional activity in the presence or absence of the specific protein kinase C inhibitor, calphostin, we show here that protein kinase C is an important

mediator in cytokine-induced expression of tissue factor at both the message and protein level. In this study, cytokines caused a significant increase in tissue factor message and protein (as measured by procoagulant activity), and these increases were inhibited by calphostin. The inhibition of tissue factor m R N A was not a result of nonspecific cell injury caused by calphostin exposure because this agent did not cause significant 51Chromium release (Table II). In addition, calphostin's inhibitory actions were not global, because two non-protein kinase C mediated cytokine effects, stimulation of PMN adhesion and production of prostacyclin37'41 were unaffected by calphostin exposure (see Results section and Table II). The fact that TNF-oL and I L - l a were able to elicit these varied responses in

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the presence of calphostin argues that the observed decreases in tissue factor were not a result of calphostin affecting interaction of these cytokines with their respective receptors. Although we were able to achieve significant decreases (50% to 60%) in cytokine-induced tissue factor activity and mRNA, we were unable to obtain complete inhibition. However, these observations are still consistent with the hypothesis that protein kinase C participates in cytokine induction of tissue factor. It is well established that protein kinase C consists of a family of isoforms that differ in substrate specificity, tissue distribution, and calcium and diacylglycerol dependence (for review, see reference 42). Of the isoforms involved in TNF-~ or IL-I~ functions, some may be less sensitive to calphostin inhibition.43 Because calphostin acts at the diacylglycerol binding site and because some isoforms of protein kinase C have been shown to be unresponsive to diacylglycerol (and phorbol esters) or to respond with varying kinetics, the scenario given for the inability of calphostin to effect full inhibition is plausible. 42'44 This conjecture is further supported by our data on PMA induction of tissue factor mRNA (Fig. 4). Calphostin's inhibition of PMA induction of tissue factor mRNA was consistently greater (approximately 80% inhibition) than its inhibition of cytokine induction of tissue factor message (approximately 55%). Because PMA activates protein kinase C directly by binding to the diacylglycerol site of the protein, this compound should only be activating the protein kinase C species responsive to diacylglycerol. Thus, during TNF-~ and IL-lo~ induction of tissue factor, some protein kinase C isoforms that are not activated by PMA or inhibited by calphostin may be activated or up-regulated. HUVEC have been shown to constitutively express the oL, [3, and e protein kinase C isoforms.45 It is not known whether TNF-~ or IL-I~ induce expression of these or other nonconstitutive isoforms in endothelium. Although it has been reported that a [31-selective protein kinase C agonist, dPPA, mimics TNF-a induction of adhesion proteins in EC, 46 dPPA's specificity for the [31 isozyme is controversial. 47-49 Thus, we are currently attempting to identify the different protein kinase C isoforms that may be involved in TNF-a and IL-lc~ induction of tissue factor message and protein. An alternative explanation for why calphostin may not be able to completely inhibit tissue factor expression is that TNF-e~ and IL-I~ may activate other signaling mediators, in addition to protein kinase C, that participate in tissue factor expression.

J Lab Clin Med January 1996

Much interest has been generated by recent work that has shown that TNF can activate sphingomyelinase to produce the second messenger, ceramide. Ceramide causes activation of the nuclear transcription factor, NF-KB, 5° and the tissue factor gene has been shown to contain an NF-KB binding site 51 and a KB-like site. 5z The recently described KB-like site has been implicated in both TNF and IL-1 induction of the tissue factor gene. In this study, a pharmacologic inhibitor of protein kinase C was chosen to examine protein kinase C involvement in tissue factor induction. Another means for assessing protein kinase C involvement in cell functions is through down-regulation of protein kinase C by extended exposure to PMA. As discussed earlier, however, the ability of PMA to downregulate protein kinase C can differ markedly between cell types, and some isoforms are not functionally downregulated by prolonged PMA exposure. 53-55 Atypical isoforms that are not activated by PMA have been discovered as well. 42'44 Measurement of translocation of protein kinase C to the membrane fraction has also been used to examine protein kinase C activation. However, several reports indicate that translocation of enzyme to the plasma or nuclear membrane is not always necessary for activation.46'56-5s Thus, in the face of such incongruities, it was believed that PMA down-regulation and protein kinase C translocation studies would not adequately address the question of protein kinase C involvement in induction of tissue factor by TNF-c~ or IL-loL. Once the protein kinase C isoforms involved in tissue factor regulation are identified, such techniques may be more useful. Okadaic acid, a diarrhetic shellfish toxin, is a specific inhibitor of protein phosphatase 1 and 2A and a potent tumor promotor, a property shared with protein kinase C activators such as the phorbol esters (for review see reference 59). In fact, it has been suggested that okadaic acid acts as a tumor promoter by increasing the phosphorylation of the same proteins as phorbol esters. 59'6° Because protein phosphatase 1 and 2A, which specifically dephosphorylate serine and/or threonine residues, are thought to be the primary phosphatases that reverse the actions of protein kinase C, we examined the effect of okadaic acid on cytokine induction of tissue factor mRNA. In these studies, okadaic acid caused notable increases in TNF-~ and IL-I~ induction of tissue factor mRNA. Although these data show that protein phosphatase 1, 2A, or both are involved in cytokine regulation of tissue factor, the current results do not delineate among these possibilities. In any event, because okadaic acid specifically inhibits

J Lab Clin M e d V o l u m e 127, N u m b e r 1

the removal of phosphate groups from serine-threonine residues, these experiments suggest that tissue factor expression is regulated in a positive manner through phosphorylation events. As previously discussed, our other results suggest this phosphorylation is occurring via protein kinase C. The activity of a number of protein kinase C isoforms is calcium-dependent. Therefore, increasing intracellular calcium might be expected to increase tissue factor mRNA levels if a calcium-dependent protein kinase C isoform is involved. When cells were exposed to the calcium ionophore A23187 in the presence of cytokine, enhanced induction of tissue factor mRNA was observed, even in the absence of any significant induction by ionophore alone (Fig. 7). A recent study reported similar results wherein coincubation with A23187 enhanced the levels of tissue factor mRNA in LPS-stimulated HUVEC. 61 The fact that increased intracellular calcium amplifies the induction of tissue factor message by cytokine does not definitively prove that protein kinase C is involved, because a host of other factors such as calcium-dependent proteases are very likely also being affected. However, it supports our previous data, especially because ionophore alone had no effect. This observation is of particular interest in light of our previous report that showed that ionophore decreased the ability of cytokine to induce tissue factor functional activity in HUVEC. 4° The inhibitory effect of increased Ca +2 on functional activity in that study was attributed to activation of the calcium-binding protein, calmodulin. Calmodulin inhibition increased cytokine-induced tissue factor procoagulant activity in intact cells as measured with use of a chromogenic assay for factor Xa generation. In summary, increased calcium enhances induction of tissue factor mRNA levels, possibly by activating protein kinase C. However, calcium increases also activate calmodulin, which appears to inhibit functional tissue factor expression by an as yet unknown mechanism. In conclusion, the data indicate that protein kinase C is involved in tissue factor mRNA induction by TNF-o~ and IL-loL, but also that tissue factor expression is regulated by many mechanisms and at different molecular levels. Clearly, further investigations are required to delineate the intricacies of this complex signaling pathway. We thank Ms. Jennifer Clikeman, Ms. Wenhua Li, and Ms. Fahima Rahman for expert technical assistance. We also thank Vijay Mudor for carrying out the E-selectin Western blots and Dr. Thomas McIntyre for providing the radiolabeled PMNs. In addition, we thank the labor and delivery nursing staff of St.

Terry a n d Callahan

91

Mark's Hospital in Salt Lake City, Utah, for providing the umbilical cords used in this study. REFERENCES

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