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Plasminogen activator regulation studies following transfection of human endothelial cells
Fibrinolysk
(1994) 8,
’ 1994 Lmgmpn
SuppI2,
19-21
Group Ltd
Plasminogen
Activator Regulation
R. Hanemaaijer,
J. Arts, L. le Clercq,
Studies Following Transfection
T. Kooistra,
of Human Endothelial
Cells
V. van Hinsbergh
SUMMARY. Transfection of human endothelial cells by the calcium phosphate precipitation method has be-enused to study the gene regulation of plasminogen activator (PA). The method of transfection was optimized, resulting in efficiencies of 1 to 10% and 4 to 20% for human umbilical vein endothelial cells and the hybrid cell line EA.hy926, respectively. Using u-PA-, t-PA- and PAI-l-CAT-promoter constructs a transient CAT-expression was observed. Promoter constructs of different length showed expression of variable amounts of CAT-activity, suggesting a role for enhancer or repressor elements. Stimulation of cells transfected with a 72 bp- or a 2212 bp u-PA-promoter construct with PMA or TNF resulted in induction of only the larger fragment. KEYWORDS. Transfection. Human endothelial cells. Plasminogen activator. Regulation
INTRODUCTION
10% human serum, 10% heat-inactivated newborn calf serum, 150 pg/ml crude endothelial cell growth factor, 5 U/ml heparin, and penicillin/streptomycin. The cells were seeded at low density so that they reached 70-80% confluency after a three days period of growth during which the culture medium was not changed. After this period the culture medium was renewed, and after three hours replaced by culture medium from which HEPES was omitted. Transfections by the calcium phosphate coprecipitation method were then conducted using 1 pg DNA per 10 cm2 dish. Four hours after addition of the DNA-calcium phosphate-precipitate, cells were shocked in 15% glycerol and cultured in medium containing 1 mM sodium butyrate. After 16-48 hours cells transfected with an RSV-promoter-@galactosidase construct were fixed and stained for galactosidase activity in order to determine the transfection efficiency. From cells transfected with different PA-promoter-CAT constructs, extracts were prepared in which CAT (chloramphenicol acetyl transferase) activity was measured. DNA constructs containing different lengths of u-PA promoter fused to a CAT reporter gene were a gift of Dr. F. BIasi.’ DNA constructs containing different lengths of the t-PA promoter fused to a CAT reporter gene were a gift from Dr. R.L. Medcalf.6 PAIpromoter-CAT constructs were made by Dr. P.J. Bosma.’
Plasminogen activators (PAS) play a central role in fibrinolysis and in pericellular proteolysis involved in endothelial migration and angiogenesis. The synthesis of these PAS and their inhibitor PAT-1 are under regulatory control. Stimulation of endothelial cells (EC) by TNFcY increases the synthesis of u-PA and PAI-1, while activation of protein kinase C by the phorbol ester PMA induces t-PA and u-PA synthesis.’ To study gene regulation of PAS and PAI- in human EC, transfection studies are performed using constructs of u-PA-, t-PA- and PAI-l-promoter fragments fused to a reporter gene, such as chloramphenicol acetyl transferase (CAT). Several procedures for transfection of EC have been reported, 2*3 but regulation studies have been hampered by low transfection efficiencies ( < 0.1%) in human EC. In this study we report on an adapted transfection method using the non-infectious calcium phosphate precipitation technique. We show induction of promoter constructs after activation of the cells with TNFQ or PMA.
METHODS Human umbilical vein endothelial cells (HUVEC) or the hybrid endothelial cell line EA.hy9264 were cultured on tibronectin-coated dishes in Ml99 supplemented with Gaubius Laboratory
TNO-PG,
RESULTS Transfection of HUVEC or EA.hy926 improved if the cells were synchronized starvation of the cells before transfection,
P.O. Box 430, 2300 AK Lriden, The
Netherlands
19
was greatly in growth by omittance of
20 Plasminogen Activator Transfection
400
in Endothelial Ceils
, 3 .fl
c 3
800
F e .z
f!
0
20
40
time after
60 transfection
80
100
600
-72~CAT
120
m
Control
0
TNFa
0
PMA
,;
-2212-CAT
(hours)
Fig. 1 CAT-activity in human EC transfected with the -2212-u-PApromoter construct. Cells were harvested at different times following transfection. CAT-activity was measured using [“Cl-butyryl-CoA as the labelled substrate or using a CAT-specific Elisa (Boehringer Mannheim).
HEPES from the culture medium, and addition of sodium butyrate to the culture medium following transfection. Without these modifications a transfection efficiency of 0.1% was observed. Using the modified procedure the transfection efficiency was improved to 1 to 10% for HUVEC and 4 to 20% for EA.hy926. Omittance of butyrate resulted in a 2- to 4-fold and a 2- to IO-fold lower transfection efficiency in HUVEC and EA.hy926, respectively. After transfection of the u-PA promoter-CAT constructs, CAT-activity was measured in cell extracts. In both types of EC a transient expression was observed,with a peak activity between 16 and 48 hours after transfection (Fig. 1). A similar transient expression was obtained with t-PA- or PAI-l-promoter constructs. In HUVEC the basal expression of u-PA-, t-PA- and PAL1 promoter constructs was high, indicating a strong promoter activity. However, the endogenous expression of u-PA and t-PA protein was at least one order of magnitude lower relative to the PAT-l expression. This suggests that in EC in vitro repressors play a role in the u-PA- and t-PA gene regulation. Transfection with different u-PA promoter CAT-reporter constructs, containing u-PA Promoter fragments with lengths, varying from 72 to 2212 basepairs, resulted in expression of variable amounts of CAT activity, depending on the length of the promoter fragment. In both cell types a similar pattern was observed for the different constructs: the -1592 u-PA promoter fragment showed lower expression than the -2212 promoter fragment and the -72 promoter fragment, suggesting a role for repressor and enhancer elements in u-PA expression. Similar results were found after transfection of these constructs in tumor cells.’ After stimulation of HUVECs or EA.hy926 cells with TNFa, an increase in u-PA and PAI-I was observed, both at protein, mRNA and transcriptional level. Incubation of EC with the protein kinase C activator PMA induced an increase in both plasminogen activators.
Fig. 2 Induction of CAT-activity by TNFar or PMA in EA.hy926 cells transfected with CAT-constructs containing the -72- or -2212 u-PA promoter fragments. Following transfection of the cells the culture medium was replaced by culture medium containing 1 mM sodium butyrate and in addition IO nM PMA or 10 nglml TNFar. AtIer 24 hours cell lysates were prepared for CAT analysis.
After PMA stimulation of HUVEC or EA.hy926 transfected with different u-PA promoter constructs, only cells transfected with the construct containing the -2212 u-PA promoter fragment showed a 15fold and 6-fold induction in CAT activity, whereas with the shorter constructs no induction of CAT-activity was observed (Fig. 2). When similar experiments were performed with TNFar, EA.hy926 cells showed a 2-fold induction in CAT activity. None of the constructs were activated by TNFa in HUVEC. The low stimulatory effect of PMA and TNFa in HUVEC may be related to the high basal expression of the promoter-CAT constructs, or that the cells are slightly activated after the transfection procedure.
DISCUSSION For a better understanding of gene regulation it is desirable to be able to transfect cells with DNA constructs, containing promoter fragments of the gene of interest fused to a reporter gene. Using a modified calcium phosphate transfection method we were able to obtain high transfection efficiencies in human EC. Both synchronization of the cell growth and the addition of sodium butyrate greatly improved the transfection efficiency. Overexpression of genes in human endothelial cells is possible by this technique, which may contribute to the understanding of the expression and role of EC products. In this study a transient expression of u-PA-/t-PA-/and PAI-l-promoter-CAT constructs was observed. Promoter fragments of variable lengths showed a different activity, suggesting the presence of activator and repressor elements in the gene regulation of these proteins. Following activation of the transfected cells with PMA both in HUVEC and in EA.hy926 an induction of the -2212-u-PA-promoter construct was observed, whereas no induction was present using the -72-promoter construct.
Fibrinolysis
This indicates that the induction by PMA of the large construct is specific and that this promoter fragment contains a PMA responsive element. However, promoter regulation studies in transfected HUVEC were often hampered by a variable degree of cell activation and a limited period during which the promoter-activation studies could he done. In conclusion, it is shown that human EC can be transfected with a high efficiency using non-infectious methods, which may aid studies on the regulation of proteins involved in tibrinolysis.
3.
4.
5.
6.
REFERENCES 1.
2.
Van Hinsbergh tibrinolysis and Sci 667:151-162 Descheemaeker Collen D 1992
V W M 1992 Impact of endothelial activation on local proteolysis in tissue repair. Ann N Y Acad K A, Nelles L, Moreau H, Strandberg L, Ny T, Transfection of human endothelial cells with
7.
21
plasminogen activator inhibitor type-l promoter/reporter gene fragment reveals phorbol ester induction of gene expression. Fibrinolysis 6: 256-262 Powell I T, Klaasse Bos J M, Van Mourik J A 1992 The uptake and expression of the factor VIII and reporter genes by vascular cells. FEBS Lett 303: 173-177 Edge11 C J, McDonald C C, Graham J B 1983 Permanent cell line expressing human factor VIII-related antigen established by hybridization. Proc Nat1 Acad Sci USA 80: 3734-3737 Verde P, Boast S, Robbiati F, Blasi F 1988 An upstream enhancer and a negative element in the 5’ flanking region of the human utokinase plasminogen activator gene. Nucl Acid Res 16: 10699-10715 Medcalf R L, Ruegg M, Schleuning W-D 1990 A DNA motif related to the CAMP-responsive element and an exon-located activator protein-2 binding site in the human tissue-type plasminogen activator gene promoter cooperate in basal expression and convey activation by phorbol ester and CAMP. J Biol Chem 265: 14618-14626 Bosma P J 1991 Structure and expression of the human plasminogen activator inhibitor I gene. Thesis, Leiden, The Netherlands
(1994) 8,
’ 1994 Lmgmpn
SuppI2,
19-21
Group Ltd
Plasminogen
Activator Regulation
R. Hanemaaijer,
J. Arts, L. le Clercq,
Studies Following Transfection
T. Kooistra,
of Human Endothelial
Cells
V. van Hinsbergh
SUMMARY. Transfection of human endothelial cells by the calcium phosphate precipitation method has be-enused to study the gene regulation of plasminogen activator (PA). The method of transfection was optimized, resulting in efficiencies of 1 to 10% and 4 to 20% for human umbilical vein endothelial cells and the hybrid cell line EA.hy926, respectively. Using u-PA-, t-PA- and PAI-l-CAT-promoter constructs a transient CAT-expression was observed. Promoter constructs of different length showed expression of variable amounts of CAT-activity, suggesting a role for enhancer or repressor elements. Stimulation of cells transfected with a 72 bp- or a 2212 bp u-PA-promoter construct with PMA or TNF resulted in induction of only the larger fragment. KEYWORDS. Transfection. Human endothelial cells. Plasminogen activator. Regulation
INTRODUCTION
10% human serum, 10% heat-inactivated newborn calf serum, 150 pg/ml crude endothelial cell growth factor, 5 U/ml heparin, and penicillin/streptomycin. The cells were seeded at low density so that they reached 70-80% confluency after a three days period of growth during which the culture medium was not changed. After this period the culture medium was renewed, and after three hours replaced by culture medium from which HEPES was omitted. Transfections by the calcium phosphate coprecipitation method were then conducted using 1 pg DNA per 10 cm2 dish. Four hours after addition of the DNA-calcium phosphate-precipitate, cells were shocked in 15% glycerol and cultured in medium containing 1 mM sodium butyrate. After 16-48 hours cells transfected with an RSV-promoter-@galactosidase construct were fixed and stained for galactosidase activity in order to determine the transfection efficiency. From cells transfected with different PA-promoter-CAT constructs, extracts were prepared in which CAT (chloramphenicol acetyl transferase) activity was measured. DNA constructs containing different lengths of u-PA promoter fused to a CAT reporter gene were a gift of Dr. F. BIasi.’ DNA constructs containing different lengths of the t-PA promoter fused to a CAT reporter gene were a gift from Dr. R.L. Medcalf.6 PAIpromoter-CAT constructs were made by Dr. P.J. Bosma.’
Plasminogen activators (PAS) play a central role in fibrinolysis and in pericellular proteolysis involved in endothelial migration and angiogenesis. The synthesis of these PAS and their inhibitor PAT-1 are under regulatory control. Stimulation of endothelial cells (EC) by TNFcY increases the synthesis of u-PA and PAI-1, while activation of protein kinase C by the phorbol ester PMA induces t-PA and u-PA synthesis.’ To study gene regulation of PAS and PAI- in human EC, transfection studies are performed using constructs of u-PA-, t-PA- and PAI-l-promoter fragments fused to a reporter gene, such as chloramphenicol acetyl transferase (CAT). Several procedures for transfection of EC have been reported, 2*3 but regulation studies have been hampered by low transfection efficiencies ( < 0.1%) in human EC. In this study we report on an adapted transfection method using the non-infectious calcium phosphate precipitation technique. We show induction of promoter constructs after activation of the cells with TNFQ or PMA.
METHODS Human umbilical vein endothelial cells (HUVEC) or the hybrid endothelial cell line EA.hy9264 were cultured on tibronectin-coated dishes in Ml99 supplemented with Gaubius Laboratory
TNO-PG,
RESULTS Transfection of HUVEC or EA.hy926 improved if the cells were synchronized starvation of the cells before transfection,
P.O. Box 430, 2300 AK Lriden, The
Netherlands
19
was greatly in growth by omittance of
20 Plasminogen Activator Transfection
400
in Endothelial Ceils
, 3 .fl
c 3
800
F e .z
f!
0
20
40
time after
60 transfection
80
100
600
-72~CAT
120
m
Control
0
TNFa
0
PMA
,;
-2212-CAT
(hours)
Fig. 1 CAT-activity in human EC transfected with the -2212-u-PApromoter construct. Cells were harvested at different times following transfection. CAT-activity was measured using [“Cl-butyryl-CoA as the labelled substrate or using a CAT-specific Elisa (Boehringer Mannheim).
HEPES from the culture medium, and addition of sodium butyrate to the culture medium following transfection. Without these modifications a transfection efficiency of 0.1% was observed. Using the modified procedure the transfection efficiency was improved to 1 to 10% for HUVEC and 4 to 20% for EA.hy926. Omittance of butyrate resulted in a 2- to 4-fold and a 2- to IO-fold lower transfection efficiency in HUVEC and EA.hy926, respectively. After transfection of the u-PA promoter-CAT constructs, CAT-activity was measured in cell extracts. In both types of EC a transient expression was observed,with a peak activity between 16 and 48 hours after transfection (Fig. 1). A similar transient expression was obtained with t-PA- or PAI-l-promoter constructs. In HUVEC the basal expression of u-PA-, t-PA- and PAL1 promoter constructs was high, indicating a strong promoter activity. However, the endogenous expression of u-PA and t-PA protein was at least one order of magnitude lower relative to the PAT-l expression. This suggests that in EC in vitro repressors play a role in the u-PA- and t-PA gene regulation. Transfection with different u-PA promoter CAT-reporter constructs, containing u-PA Promoter fragments with lengths, varying from 72 to 2212 basepairs, resulted in expression of variable amounts of CAT activity, depending on the length of the promoter fragment. In both cell types a similar pattern was observed for the different constructs: the -1592 u-PA promoter fragment showed lower expression than the -2212 promoter fragment and the -72 promoter fragment, suggesting a role for repressor and enhancer elements in u-PA expression. Similar results were found after transfection of these constructs in tumor cells.’ After stimulation of HUVECs or EA.hy926 cells with TNFa, an increase in u-PA and PAI-I was observed, both at protein, mRNA and transcriptional level. Incubation of EC with the protein kinase C activator PMA induced an increase in both plasminogen activators.
Fig. 2 Induction of CAT-activity by TNFar or PMA in EA.hy926 cells transfected with CAT-constructs containing the -72- or -2212 u-PA promoter fragments. Following transfection of the cells the culture medium was replaced by culture medium containing 1 mM sodium butyrate and in addition IO nM PMA or 10 nglml TNFar. AtIer 24 hours cell lysates were prepared for CAT analysis.
After PMA stimulation of HUVEC or EA.hy926 transfected with different u-PA promoter constructs, only cells transfected with the construct containing the -2212 u-PA promoter fragment showed a 15fold and 6-fold induction in CAT activity, whereas with the shorter constructs no induction of CAT-activity was observed (Fig. 2). When similar experiments were performed with TNFar, EA.hy926 cells showed a 2-fold induction in CAT activity. None of the constructs were activated by TNFa in HUVEC. The low stimulatory effect of PMA and TNFa in HUVEC may be related to the high basal expression of the promoter-CAT constructs, or that the cells are slightly activated after the transfection procedure.
DISCUSSION For a better understanding of gene regulation it is desirable to be able to transfect cells with DNA constructs, containing promoter fragments of the gene of interest fused to a reporter gene. Using a modified calcium phosphate transfection method we were able to obtain high transfection efficiencies in human EC. Both synchronization of the cell growth and the addition of sodium butyrate greatly improved the transfection efficiency. Overexpression of genes in human endothelial cells is possible by this technique, which may contribute to the understanding of the expression and role of EC products. In this study a transient expression of u-PA-/t-PA-/and PAI-l-promoter-CAT constructs was observed. Promoter fragments of variable lengths showed a different activity, suggesting the presence of activator and repressor elements in the gene regulation of these proteins. Following activation of the transfected cells with PMA both in HUVEC and in EA.hy926 an induction of the -2212-u-PA-promoter construct was observed, whereas no induction was present using the -72-promoter construct.
Fibrinolysis
This indicates that the induction by PMA of the large construct is specific and that this promoter fragment contains a PMA responsive element. However, promoter regulation studies in transfected HUVEC were often hampered by a variable degree of cell activation and a limited period during which the promoter-activation studies could he done. In conclusion, it is shown that human EC can be transfected with a high efficiency using non-infectious methods, which may aid studies on the regulation of proteins involved in tibrinolysis.
3.
4.
5.
6.
REFERENCES 1.
2.
Van Hinsbergh tibrinolysis and Sci 667:151-162 Descheemaeker Collen D 1992
V W M 1992 Impact of endothelial activation on local proteolysis in tissue repair. Ann N Y Acad K A, Nelles L, Moreau H, Strandberg L, Ny T, Transfection of human endothelial cells with
7.
21
plasminogen activator inhibitor type-l promoter/reporter gene fragment reveals phorbol ester induction of gene expression. Fibrinolysis 6: 256-262 Powell I T, Klaasse Bos J M, Van Mourik J A 1992 The uptake and expression of the factor VIII and reporter genes by vascular cells. FEBS Lett 303: 173-177 Edge11 C J, McDonald C C, Graham J B 1983 Permanent cell line expressing human factor VIII-related antigen established by hybridization. Proc Nat1 Acad Sci USA 80: 3734-3737 Verde P, Boast S, Robbiati F, Blasi F 1988 An upstream enhancer and a negative element in the 5’ flanking region of the human utokinase plasminogen activator gene. Nucl Acid Res 16: 10699-10715 Medcalf R L, Ruegg M, Schleuning W-D 1990 A DNA motif related to the CAMP-responsive element and an exon-located activator protein-2 binding site in the human tissue-type plasminogen activator gene promoter cooperate in basal expression and convey activation by phorbol ester and CAMP. J Biol Chem 265: 14618-14626 Bosma P J 1991 Structure and expression of the human plasminogen activator inhibitor I gene. Thesis, Leiden, The Netherlands