J. Biochem. 108, 839-845 (1990)
Up-Regulation of Thrombomodulin in Human Umbilical Vein Endothelial Cells In Vitro1 Kazunori Hirokawa and Nobuo Aoki' The First Department of Medicine, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113 Received for publication, June 28, 1990
Thrombomodulin (TM) is an endothelial cell membrane glycoprotein which forms a 1 : 1 complex with thrombin. Activation of protein C by thrombin is accelerated several thousandfold after formation of the thrombin-TM complex (1). Activated protein C is a protease and with its cofactor protein S (2) acts as an anti-coagulant by cleaving the activated coagulation factors V (3) and VIII (4). The physiological importance of the TM-protein C system in prevention of thrombosis is supported by the following observations: recurrent venous thromboembolism occurs in kindred with a quantitative and/or qualitative deficiency of protein C (5) or protein S (6); activated protein C injected into baboons acts as an anticoagulant and prevents death from septic shock (7); and purified TM participates in defense against thrombin-induced thrombosis in mice (8). In addition, endotoxin, TNF, and IL-1/9 suppress surface TM activity and induce the synthesis and expression of the coagulation cofactor tissue factor in endothelial cells [9-11). This loss of normal hemostasis may provide a cellular environment for development of a hypercoagulable state followed by thrombosis and/or disseminated intravascular coagulation (DIC) in sepsis, chronic inflammation, and cancer. It was reported that the induced loss of surface TM 1 Presented in part at the Seminar on Thrombosis, Nagoya, November 8, 1989, and at the 52nd annual meeting of the Japan Hematological Society, Tokyo, March 31, 1990. 1 To whom correspondence should be addressed. Abbreviations: 4a-PDD, 4a-phorbol 12,13-didecanoate; 4/SPDD, 4^-phorbol 12,13-didecanoate; 4/S-PMA (PMA), 4/S-phorbol 12myristate 13-acetate; 8-BrcAMP, 8-bromo cAMP; 8-(4-CPT) cAMP, 8-(4-cyclophenylthio) cAMP; dBcAMP, dibutyryl cAMP; Dil-AcLDL, l,l'-dioctadecyl-l-3,3,3',3'-tetramethylindocarbocyanine percholate; HUVECs, human umbilical vein endothelial cells; IL-1/9, interleukin-1/9; TM, thrombomodulin; TNF, tumor necrosis factor.
Vol. 108, No. 5, 1990
cofactor activity results from internalization of TM {12, 13) and may {14, 15) or may not {16, 17) be accompanied with changes in TM mRNA levels. TM expression, thus, is susceptible to cellular regulatory mechanisms and presumably can also be up-regulated, although such results have not been reported. The current studies were designed to investigate agents that may enhance TM expression and activity in endothelial cells. Such increased TM activity and resultant increase in protein C activation might prevent vascular thrombosis in pathological states associated with various diseases. In this study, we measured surface TM activity, surface-expressed TM antigen, and TM mRNA levels in HUVECs. Our results demonstrate that phorbol esters, forskolin, and analogs of cAMP can up-regulate TM expression. MATERIALS AND METHODS Materials—Lipopolysaccharide (LPS) extracted from Escherichia coli serotype 026:B6, 4/9-phorbol 12-myristate 13-acetate (PMA), 4/S-phorbol 12,13-didecanoate (4/9-PDD), 4«-phorbol 12,13-didecanoate (4^-PDD), forskolin, cycloheximide, sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), porcine mucosal heparin, human a--thrombin (4,000 NIH U/mg protein), and bovine serum albumin (BSA) were purchased from Sigma (St. Louis, MO). Recombinant human interleukin1/3 (IL-1/3) was purchased from Cistron Technology (Pine Brook, NJ). Recombinant human tumor necrosis factor (TNF) was purchased from Genzyme (Boston, MA). Iscove's modified Dulbecco's medium (IMDM), fetal calf serum (FCS), peniciUin-streptomycin, and 0.05% trypsin0.02% EDTA in phosphate-buffered saline (trypsin-EDTA) were purchased from GIBCO (Grand Island, NY). Col-
839
Downloaded from https://academic.oup.com/jb/article-abstract/108/5/839/813775 by university of winnipeg user on 22 January 2019
Previous studies have shown that thrombomodulin (TM) on endothelial cells is downregulated by endotoxin, interleukin-1/9 (IL-1/9), and tumor necrosis factor (TNF). This loss of anti-coagulant potential is thought to be related to the hypercoagulable state in sepsis, inflammation, and cancer. The current studies describe up-regulation of TM in human umbilical vein endothelial cells (HUVECs) by several compounds as judged by increased surface cofactor activity, surface TM antigen, and TM mRNA levels. Surface TM activity was increased by active phorbol esters (10~8 M, 24-48 h), analogs of cAMP (1-10 mM, 4 h), and forskolin ( 1 0 6 M, 24-48 h). Up-regulation of TM in HUVECs by 4/9-phorbol 12myristate 13-acetate (PMA) and dibutyryl cAMP (dBcAMP) was due to de novo synthesis of TM protein resulting from increased TM mRNA levels. The results suggest that protein kinase C and protein kinase A may be involved in cellular regulatory mechanisms for TM expression. In addition, PMA effects on surface TM activity are biphasic, with an initial reduction followed by a significant enhacement. Hence, we propose that compounds capable of increasing intracellular cAMP concentrations in HUVECs may be useful in preventing thrombosis by increasing the anti-thrombotic properties of endothelial cells.
840
antithrombin HI (AT EH) and 15 U/ml heparin (25). A Vmax Kinetic Microplate Reader interfaced to the Softmax software (Molecular Devices, Palo Alto, CA) was used to quantitate cleavage of S-2366 at 405 nm. Endothelial cells in each well were counted by the method described by Drysdale et aL (26). Briefly, the cells were stained with 200 /^I/well crystal violet (0.2% in 2% ethanol) for 10 min at 37'C. Multiwell plates were rinsed with tap water and dried, then 300 //I of 1% SDS was added to each well to solubilize the stained cells. One hundred microliters of solubilized lysate from each well was transferred to microtiter plates (Nunc, Denmark) and the absorbance of each well was read at 550 nm. Cell numbers were calculated from a standard curve (Ai60 vs. cell number). Surface TM activity per 105 cells was expressed as a percentage of the control ± SD of four determinations. Data were analyzed by using Student's t test. Flow Cytometry—After incubation for the indicated times with or without agents in 25-cm2 tissue culture flasks (Corning-Iwaki Glass), HUVECs were washed twice with serum-free medium and incubated at 37'C for 30 min with 0.02% EDTA in phosphate-buffered saline. Cells were scraped off with a rubber policeman, collected, and washed twice with buffer A. HUVECs were incubated for 60 min at room temperature with either pre-immune mouse IgG (Cappel, Organon Teknika, West Chester, PA) or with the KA-4 or KA-2 anti-human TM monoclonal antibodies, whose characteristics were previously reported (27). After being washed with buffer A three times, HUVECs were incubated for 60 min at room temperature with goat antimouse IgG antibody conjugated with FITC (Cappel). After further washing three times, the intensity of fluorescence was monitored by an EPICS 751J (Coulter Electronics, FL). Northern Blotting — Total cytoplasmic RNA was prepared from confluent HUVECs grown in 150 cm2 tissue culture flasks using a total RNA isolation kit. Isolated RNA was heat-denatured and fractionated on formaldehydecontaining agarose gels. The gels were then stained with 1.0 / / g / m l ethidium bromide for 30 min and washed with distilled water four times for 30 min each. The RNA was then transferred to a HyBond-N filter (Amersham International, Amersham, U.K.) by standard capillary blotting techniques in transfer buffer (28). The RNA was crosslinked to the nylon membrane by UV illumination. The probe employed for hybridization was a "P-labeled 450 bp DNA fragment corresponding to the human TM coding region, nick-translated to a specific activity of > 10" dpm/ fig. Nick-translation was performed using a nick-translation kit and [
Up-Regulation of Thrombomodulin in Human Umbilical Vein Endothelial Cells In Vitro1 Kazunori Hirokawa and Nobuo Aoki' The First Department of Medicine, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113 Received for publication, June 28, 1990
Thrombomodulin (TM) is an endothelial cell membrane glycoprotein which forms a 1 : 1 complex with thrombin. Activation of protein C by thrombin is accelerated several thousandfold after formation of the thrombin-TM complex (1). Activated protein C is a protease and with its cofactor protein S (2) acts as an anti-coagulant by cleaving the activated coagulation factors V (3) and VIII (4). The physiological importance of the TM-protein C system in prevention of thrombosis is supported by the following observations: recurrent venous thromboembolism occurs in kindred with a quantitative and/or qualitative deficiency of protein C (5) or protein S (6); activated protein C injected into baboons acts as an anticoagulant and prevents death from septic shock (7); and purified TM participates in defense against thrombin-induced thrombosis in mice (8). In addition, endotoxin, TNF, and IL-1/9 suppress surface TM activity and induce the synthesis and expression of the coagulation cofactor tissue factor in endothelial cells [9-11). This loss of normal hemostasis may provide a cellular environment for development of a hypercoagulable state followed by thrombosis and/or disseminated intravascular coagulation (DIC) in sepsis, chronic inflammation, and cancer. It was reported that the induced loss of surface TM 1 Presented in part at the Seminar on Thrombosis, Nagoya, November 8, 1989, and at the 52nd annual meeting of the Japan Hematological Society, Tokyo, March 31, 1990. 1 To whom correspondence should be addressed. Abbreviations: 4a-PDD, 4a-phorbol 12,13-didecanoate; 4/SPDD, 4^-phorbol 12,13-didecanoate; 4/S-PMA (PMA), 4/S-phorbol 12myristate 13-acetate; 8-BrcAMP, 8-bromo cAMP; 8-(4-CPT) cAMP, 8-(4-cyclophenylthio) cAMP; dBcAMP, dibutyryl cAMP; Dil-AcLDL, l,l'-dioctadecyl-l-3,3,3',3'-tetramethylindocarbocyanine percholate; HUVECs, human umbilical vein endothelial cells; IL-1/9, interleukin-1/9; TM, thrombomodulin; TNF, tumor necrosis factor.
Vol. 108, No. 5, 1990
cofactor activity results from internalization of TM {12, 13) and may {14, 15) or may not {16, 17) be accompanied with changes in TM mRNA levels. TM expression, thus, is susceptible to cellular regulatory mechanisms and presumably can also be up-regulated, although such results have not been reported. The current studies were designed to investigate agents that may enhance TM expression and activity in endothelial cells. Such increased TM activity and resultant increase in protein C activation might prevent vascular thrombosis in pathological states associated with various diseases. In this study, we measured surface TM activity, surface-expressed TM antigen, and TM mRNA levels in HUVECs. Our results demonstrate that phorbol esters, forskolin, and analogs of cAMP can up-regulate TM expression. MATERIALS AND METHODS Materials—Lipopolysaccharide (LPS) extracted from Escherichia coli serotype 026:B6, 4/9-phorbol 12-myristate 13-acetate (PMA), 4/S-phorbol 12,13-didecanoate (4/9-PDD), 4«-phorbol 12,13-didecanoate (4^-PDD), forskolin, cycloheximide, sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), porcine mucosal heparin, human a--thrombin (4,000 NIH U/mg protein), and bovine serum albumin (BSA) were purchased from Sigma (St. Louis, MO). Recombinant human interleukin1/3 (IL-1/3) was purchased from Cistron Technology (Pine Brook, NJ). Recombinant human tumor necrosis factor (TNF) was purchased from Genzyme (Boston, MA). Iscove's modified Dulbecco's medium (IMDM), fetal calf serum (FCS), peniciUin-streptomycin, and 0.05% trypsin0.02% EDTA in phosphate-buffered saline (trypsin-EDTA) were purchased from GIBCO (Grand Island, NY). Col-
839
Downloaded from https://academic.oup.com/jb/article-abstract/108/5/839/813775 by university of winnipeg user on 22 January 2019
Previous studies have shown that thrombomodulin (TM) on endothelial cells is downregulated by endotoxin, interleukin-1/9 (IL-1/9), and tumor necrosis factor (TNF). This loss of anti-coagulant potential is thought to be related to the hypercoagulable state in sepsis, inflammation, and cancer. The current studies describe up-regulation of TM in human umbilical vein endothelial cells (HUVECs) by several compounds as judged by increased surface cofactor activity, surface TM antigen, and TM mRNA levels. Surface TM activity was increased by active phorbol esters (10~8 M, 24-48 h), analogs of cAMP (1-10 mM, 4 h), and forskolin ( 1 0 6 M, 24-48 h). Up-regulation of TM in HUVECs by 4/9-phorbol 12myristate 13-acetate (PMA) and dibutyryl cAMP (dBcAMP) was due to de novo synthesis of TM protein resulting from increased TM mRNA levels. The results suggest that protein kinase C and protein kinase A may be involved in cellular regulatory mechanisms for TM expression. In addition, PMA effects on surface TM activity are biphasic, with an initial reduction followed by a significant enhacement. Hence, we propose that compounds capable of increasing intracellular cAMP concentrations in HUVECs may be useful in preventing thrombosis by increasing the anti-thrombotic properties of endothelial cells.
840
antithrombin HI (AT EH) and 15 U/ml heparin (25). A Vmax Kinetic Microplate Reader interfaced to the Softmax software (Molecular Devices, Palo Alto, CA) was used to quantitate cleavage of S-2366 at 405 nm. Endothelial cells in each well were counted by the method described by Drysdale et aL (26). Briefly, the cells were stained with 200 /^I/well crystal violet (0.2% in 2% ethanol) for 10 min at 37'C. Multiwell plates were rinsed with tap water and dried, then 300 //I of 1% SDS was added to each well to solubilize the stained cells. One hundred microliters of solubilized lysate from each well was transferred to microtiter plates (Nunc, Denmark) and the absorbance of each well was read at 550 nm. Cell numbers were calculated from a standard curve (Ai60 vs. cell number). Surface TM activity per 105 cells was expressed as a percentage of the control ± SD of four determinations. Data were analyzed by using Student's t test. Flow Cytometry—After incubation for the indicated times with or without agents in 25-cm2 tissue culture flasks (Corning-Iwaki Glass), HUVECs were washed twice with serum-free medium and incubated at 37'C for 30 min with 0.02% EDTA in phosphate-buffered saline. Cells were scraped off with a rubber policeman, collected, and washed twice with buffer A. HUVECs were incubated for 60 min at room temperature with either pre-immune mouse IgG (Cappel, Organon Teknika, West Chester, PA) or with the KA-4 or KA-2 anti-human TM monoclonal antibodies, whose characteristics were previously reported (27). After being washed with buffer A three times, HUVECs were incubated for 60 min at room temperature with goat antimouse IgG antibody conjugated with FITC (Cappel). After further washing three times, the intensity of fluorescence was monitored by an EPICS 751J (Coulter Electronics, FL). Northern Blotting — Total cytoplasmic RNA was prepared from confluent HUVECs grown in 150 cm2 tissue culture flasks using a total RNA isolation kit. Isolated RNA was heat-denatured and fractionated on formaldehydecontaining agarose gels. The gels were then stained with 1.0 / / g / m l ethidium bromide for 30 min and washed with distilled water four times for 30 min each. The RNA was then transferred to a HyBond-N filter (Amersham International, Amersham, U.K.) by standard capillary blotting techniques in transfer buffer (28). The RNA was crosslinked to the nylon membrane by UV illumination. The probe employed for hybridization was a "P-labeled 450 bp DNA fragment corresponding to the human TM coding region, nick-translated to a specific activity of > 10" dpm/ fig. Nick-translation was performed using a nick-translation kit and [
