Transcriptional PHILIP
regulation
A, MARSDEN
of the endothelin-1
AND BARRY
gene by TNF-a
M. BRENNER
Renal Division, Department of Medicine, St. Michael’s Hospital, University of Toronto, Toronto, Ontario M5S lA8, Canada; and Renal Division, Department of Medicine, Brigham and Women’s Hospital and the Harvard Center for the Study of Kidney Diseases, Harvard Medical School, Boston, Massachusetts 02115 Marsden, Philip A., and Barry M. Brenner. Transcriptional regulation of the endothelin-1 gene by TNF-cu. Am. J. Physiol. 262 (Cell Physiol. 31): C854-C861, 1992.-Cytokines, such as tumor necrosis factor-a (TNF-a), induce profound alterations in endothelial cell phenotype and are implicated in the organ dysfunction that characterizes septic shock. We explored whether TNF-a! modulates cellular expression of endothelin-1 (ET-l). ET-l is a potent vasoconstrictor peptide released by endothelial, vascular smooth muscle, and mesangial cells that could function as a paracrine/autocrine regulator of vascular tone and proliferation. We found that TNF-ar induced release of ET-l from bovine aortic endothelial cells (BAEC) in a time- and concentration-dependent manner. Rates of ET-1 release were maximal over l-8 h and declined to, or below, baseline values after 16 h. When measured at 8 h, TNF-a! augmented ET-l release over the range 0.1-250 rig/ml (threshold, 0.1 rig/ml; 50% effective dose, 1.6 & 1.2 rig/ml; maximal effect, 100 rig/ml). The increase in secretion was accompanied by a corresponding increase in the transcriptional rate of the ET-l gene resulting in augmented preproendothelin-1 mRNA transcript levels. TNF-a-stimulated increases in ET-1 gene transcription were not dependent on new protein synthesis. Actinomycin D chase experiments suggested that enhanced stability of preproendothelin-1 mRNA could not account for the increase in ET-1 transcript levels. TNF-cr increased ET-1 release and preproendothelin-1 mRNA content in bovine renal artery and bovine glomerular capillary endothelial cells, demonstrating that the TNF-ar effect was evident in endothelial cells derived from a variety of sources. Furthermore, augmented ET-1 expression in response to TNF-a! was evident in bovine glomerular mesangial cells. Enhanced production of ET-1 in vascular beds may follow cytokine activation and contribute to the organ dysfunction observed in septic shock. aorta; cytokines; endothelium; glomerulus; gene expression; kidney; mesangial; molecular biology; septic shock; vascular smooth muscle; vasoconstriction; vasomotor tone FACTOR-~ (TNF-a)playsakeyrolein the pathobiology of septic shock (1,29). Animals infused with TNF-a! developed multiple organ system dysfunction that is characteristic of gram-negative sepsis or lipopolysaccharide (LPS) administration (30). The mechanism(s) whereby TNF-a! induces organ dysfunction, however, is not understood. Vascular endothelium, located at the interface between blood and extravascular tissues, is now appreciated to regulate many fundamental aspects of organ function, including blood coagulation, trafficking of cells, vascular wall metabolism, transcapillary permeability, and remodeling of the underlying vascular tissues, among others. It has recently been recognized that the endothelium contributes to the control of vascular tone and local blood flow by releasing potent vasodilatory mediators, namely prostacyclin (PGI& and endothelium-derived relaxing factor (EDRF), and impressive vasoconstrictor TUMORNECROSIS
C854
0363-6143/92
$2.00 Copyright
mediators (23, 31). Endothelin-1 (ET-l), a vasoconstrictor peptide purified from the conditioned medium of porcine aorta endothelial cells (12, 34), is an endothelium-derived vasconstrictor and represents one member of a family of structurally related peptides (13). The three endothelins ET-l, ET-2, and ET-3 consist of 21 amino acids and contain two intramolecular disulfide bonds. Current evidence suggests that endothelial cells express ET-l (3, 34) but do not express ET-2 or ET-3. Mature human ET-l is derived from a 212-amino acid precursor, preproendothelin-1, via a %-amino acid intermediate “big ET-l” (15). Mature bovine ET-1 is identical to human ET-l yet is derived from a 202-amino acid precursor via a 39amino acid intermediate (8). Conversion of big ET-1 to mature ET-l requires unique processing at Trp-21 to Val-22 via a peptidase termed “endothelin-converting enzyme.” Endothelin-converting enzyme appears to represent a critical physiological regulator of ET-1 activity in that human big ET-l requires conversion to the mature X-amino acid peptide for biologic activity. Endothelial cells do not contain a storage pool of ET-l; secretion of endothelial ET-1 is regulated primarily at the level of transcription (33). Exogenous signals that modify endothelial cell expression of preproendothelin-1 mRNA include changes in hemodynamic shear stress (35), hypoxia (US), transforming growth factor-p (19), calcium mobilizing agonists such as thrombin (34) or bradykinin (25), and the tumor promoter phorbol ester (33). The central role of TNF-a! in the pathogenesis of septic shock and the capacity for TNF-a! to induce profound alterations of endothelial cell phenotype led us to examine the effect of TNF-a! on endothelial ET-l gene expression. Thus the aims of the current study were to determine whether TNF-a! modulates endothelial expression of this potent vasoconstrictor peptide and to define the mechanisms implicated in the TNF-a effect. We provide evidence that TNF-a! induced ET-1 release from endothelial cells, augmented ET-1 gene transcription and steady-state levels of preproendothelin- 1 mRNA transcript, but did not modify the stability of preproendothelin-1 mRNA. Furthermore, TNF-a-induced increases in ET-l release and preproendothelin-1 mRNA were evident in endothelial cells derived from a variety of sources. Consistent with observations suggesting that ET-1 may be expressed in vascular smooth muscle and mesangial cells (10, 26, 38), TNF- a! also stimulated endothelin release and preproendothelin-1 mRNA accumulation in bovine glomerular mesangial cells. METHODS Materials. Cell culture media and balanced salt solutions were purchased from GIBCO (Grand Island, NY), low endo-
0 1992 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on November 12, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
TRANSCRIPTIONAL
REGULATION
toxin defined supplemented bovine calf serum (SCS) from Hyclone Labs (Logan, UT), endothelial mitogen from Biomedical Technologies (Stoughton, MA), cell culture plates from Costar (Cambridge, MA), Sep-Pak Cl8 extraction columns from Waters Associates (Milford, MA), and X-OMAT AR X-ray film from Eastman Kodak (Rochester, NY). Human recombinant TNF-a (rhTNF-a; sp act 9.8 x IO” U/mg) was a gift of Knoll Pharmaceuticals (Whippany, NJ); human recombinant interleukin-l/3 (rhIL-l/3; sp act 5.0 x lo8 U/mg) was from R. & D. Systems (Minneapolis, MN). L-[4,5-3H(N)]leucine (53 Ci/ mmol), [5,6-3H]uridine (38 Ci/mmol), [ ao3*P] dCTP (3,000 Ci/ mmol), [CY-“*P]UTP (3,000 Ci/mmol), and ‘*?-ET-l (2,200 Ci/ mmol) were obtained from New England Nuclear (Wilmington, DE). ET-1 was from Peninsula Laboratories (Belmont, CA); guanidinium thiocyanate from Fluka BioChemika (Buchs, Switzerland); molecular-grade phenol from Boehringer Mannheim Biochemicals (Indianapolis, IN); and LPS (Escherichia coli serotype 026:B6, phenol extracted), bovine thrombin, cycloheximide, actinomycin D, and all other reagents were pur- chased from Sigma Chemical (St. Louis, MO). Cell isolation ati culture. Bovine aortic endothelial cells (BAEC) and bovine renal artery endothelial cells (BRAE) were isolated from the thoracic aorta and main renal artery, respectively, using published methods (24). Briefly, primary cultures of endothelial cell clones were initiated on loo-mm tissue culture plates that had been precoated with gelatin (0.2 g/dl), isolated with cloning cylinders, detached with trypsin-EDTA, and passaged at cloning density onto gelatin (0.2 g/d&coated loo-mm plates. To obtain homogeneous endothelial cell cultures, single clones were isolated a second time with cloning cylinders to establish subcloned populations of cells. Individual clones were examined for angiotensin I-converting enzyme activity, expression of factor VIII-related antigen, and uniform uptake of fluorescent acetylated low-density lipoprotein (LDL) as described (24). Fluorescence-activated cell sorting (FACS) of clones labeled with fluorescent acetylated LDL confirmed that endothelial cell clones represented homogeneous populations of cells that were both uniformly labeled and clearly distinguishable from cloned populations of vascular smooth muscle or mesa&al cells. Endothelial cells were fed every 48 h with RPM1 1640 medium supplemented with 2 mM L-glutamine, 15% low endotoxin SCS, 100 U/ml penicillin, and 100 pg/ml streptomycin. Cells were utilized at passages 5-10. Studies were routinely performed on confluent monolayers lo-14 days after passage that had placed in serum-deplete medium for 48 h. Working concentrations of rhTNF-a, rhIL-l& and culture medium contained
regulation
A, MARSDEN
of the endothelin-1
AND BARRY
gene by TNF-a
M. BRENNER
Renal Division, Department of Medicine, St. Michael’s Hospital, University of Toronto, Toronto, Ontario M5S lA8, Canada; and Renal Division, Department of Medicine, Brigham and Women’s Hospital and the Harvard Center for the Study of Kidney Diseases, Harvard Medical School, Boston, Massachusetts 02115 Marsden, Philip A., and Barry M. Brenner. Transcriptional regulation of the endothelin-1 gene by TNF-cu. Am. J. Physiol. 262 (Cell Physiol. 31): C854-C861, 1992.-Cytokines, such as tumor necrosis factor-a (TNF-a), induce profound alterations in endothelial cell phenotype and are implicated in the organ dysfunction that characterizes septic shock. We explored whether TNF-a! modulates cellular expression of endothelin-1 (ET-l). ET-l is a potent vasoconstrictor peptide released by endothelial, vascular smooth muscle, and mesangial cells that could function as a paracrine/autocrine regulator of vascular tone and proliferation. We found that TNF-ar induced release of ET-l from bovine aortic endothelial cells (BAEC) in a time- and concentration-dependent manner. Rates of ET-1 release were maximal over l-8 h and declined to, or below, baseline values after 16 h. When measured at 8 h, TNF-a! augmented ET-l release over the range 0.1-250 rig/ml (threshold, 0.1 rig/ml; 50% effective dose, 1.6 & 1.2 rig/ml; maximal effect, 100 rig/ml). The increase in secretion was accompanied by a corresponding increase in the transcriptional rate of the ET-l gene resulting in augmented preproendothelin-1 mRNA transcript levels. TNF-a-stimulated increases in ET-1 gene transcription were not dependent on new protein synthesis. Actinomycin D chase experiments suggested that enhanced stability of preproendothelin-1 mRNA could not account for the increase in ET-1 transcript levels. TNF-cr increased ET-1 release and preproendothelin-1 mRNA content in bovine renal artery and bovine glomerular capillary endothelial cells, demonstrating that the TNF-ar effect was evident in endothelial cells derived from a variety of sources. Furthermore, augmented ET-1 expression in response to TNF-a! was evident in bovine glomerular mesangial cells. Enhanced production of ET-1 in vascular beds may follow cytokine activation and contribute to the organ dysfunction observed in septic shock. aorta; cytokines; endothelium; glomerulus; gene expression; kidney; mesangial; molecular biology; septic shock; vascular smooth muscle; vasoconstriction; vasomotor tone FACTOR-~ (TNF-a)playsakeyrolein the pathobiology of septic shock (1,29). Animals infused with TNF-a! developed multiple organ system dysfunction that is characteristic of gram-negative sepsis or lipopolysaccharide (LPS) administration (30). The mechanism(s) whereby TNF-a! induces organ dysfunction, however, is not understood. Vascular endothelium, located at the interface between blood and extravascular tissues, is now appreciated to regulate many fundamental aspects of organ function, including blood coagulation, trafficking of cells, vascular wall metabolism, transcapillary permeability, and remodeling of the underlying vascular tissues, among others. It has recently been recognized that the endothelium contributes to the control of vascular tone and local blood flow by releasing potent vasodilatory mediators, namely prostacyclin (PGI& and endothelium-derived relaxing factor (EDRF), and impressive vasoconstrictor TUMORNECROSIS
C854
0363-6143/92
$2.00 Copyright
mediators (23, 31). Endothelin-1 (ET-l), a vasoconstrictor peptide purified from the conditioned medium of porcine aorta endothelial cells (12, 34), is an endothelium-derived vasconstrictor and represents one member of a family of structurally related peptides (13). The three endothelins ET-l, ET-2, and ET-3 consist of 21 amino acids and contain two intramolecular disulfide bonds. Current evidence suggests that endothelial cells express ET-l (3, 34) but do not express ET-2 or ET-3. Mature human ET-l is derived from a 212-amino acid precursor, preproendothelin-1, via a %-amino acid intermediate “big ET-l” (15). Mature bovine ET-1 is identical to human ET-l yet is derived from a 202-amino acid precursor via a 39amino acid intermediate (8). Conversion of big ET-1 to mature ET-l requires unique processing at Trp-21 to Val-22 via a peptidase termed “endothelin-converting enzyme.” Endothelin-converting enzyme appears to represent a critical physiological regulator of ET-1 activity in that human big ET-l requires conversion to the mature X-amino acid peptide for biologic activity. Endothelial cells do not contain a storage pool of ET-l; secretion of endothelial ET-1 is regulated primarily at the level of transcription (33). Exogenous signals that modify endothelial cell expression of preproendothelin-1 mRNA include changes in hemodynamic shear stress (35), hypoxia (US), transforming growth factor-p (19), calcium mobilizing agonists such as thrombin (34) or bradykinin (25), and the tumor promoter phorbol ester (33). The central role of TNF-a! in the pathogenesis of septic shock and the capacity for TNF-a! to induce profound alterations of endothelial cell phenotype led us to examine the effect of TNF-a! on endothelial ET-l gene expression. Thus the aims of the current study were to determine whether TNF-a! modulates endothelial expression of this potent vasoconstrictor peptide and to define the mechanisms implicated in the TNF-a effect. We provide evidence that TNF-a! induced ET-1 release from endothelial cells, augmented ET-1 gene transcription and steady-state levels of preproendothelin- 1 mRNA transcript, but did not modify the stability of preproendothelin-1 mRNA. Furthermore, TNF-a-induced increases in ET-l release and preproendothelin-1 mRNA were evident in endothelial cells derived from a variety of sources. Consistent with observations suggesting that ET-1 may be expressed in vascular smooth muscle and mesangial cells (10, 26, 38), TNF- a! also stimulated endothelin release and preproendothelin-1 mRNA accumulation in bovine glomerular mesangial cells. METHODS Materials. Cell culture media and balanced salt solutions were purchased from GIBCO (Grand Island, NY), low endo-
0 1992 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on November 12, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
TRANSCRIPTIONAL
REGULATION
toxin defined supplemented bovine calf serum (SCS) from Hyclone Labs (Logan, UT), endothelial mitogen from Biomedical Technologies (Stoughton, MA), cell culture plates from Costar (Cambridge, MA), Sep-Pak Cl8 extraction columns from Waters Associates (Milford, MA), and X-OMAT AR X-ray film from Eastman Kodak (Rochester, NY). Human recombinant TNF-a (rhTNF-a; sp act 9.8 x IO” U/mg) was a gift of Knoll Pharmaceuticals (Whippany, NJ); human recombinant interleukin-l/3 (rhIL-l/3; sp act 5.0 x lo8 U/mg) was from R. & D. Systems (Minneapolis, MN). L-[4,5-3H(N)]leucine (53 Ci/ mmol), [5,6-3H]uridine (38 Ci/mmol), [ ao3*P] dCTP (3,000 Ci/ mmol), [CY-“*P]UTP (3,000 Ci/mmol), and ‘*?-ET-l (2,200 Ci/ mmol) were obtained from New England Nuclear (Wilmington, DE). ET-1 was from Peninsula Laboratories (Belmont, CA); guanidinium thiocyanate from Fluka BioChemika (Buchs, Switzerland); molecular-grade phenol from Boehringer Mannheim Biochemicals (Indianapolis, IN); and LPS (Escherichia coli serotype 026:B6, phenol extracted), bovine thrombin, cycloheximide, actinomycin D, and all other reagents were pur- chased from Sigma Chemical (St. Louis, MO). Cell isolation ati culture. Bovine aortic endothelial cells (BAEC) and bovine renal artery endothelial cells (BRAE) were isolated from the thoracic aorta and main renal artery, respectively, using published methods (24). Briefly, primary cultures of endothelial cell clones were initiated on loo-mm tissue culture plates that had been precoated with gelatin (0.2 g/dl), isolated with cloning cylinders, detached with trypsin-EDTA, and passaged at cloning density onto gelatin (0.2 g/d&coated loo-mm plates. To obtain homogeneous endothelial cell cultures, single clones were isolated a second time with cloning cylinders to establish subcloned populations of cells. Individual clones were examined for angiotensin I-converting enzyme activity, expression of factor VIII-related antigen, and uniform uptake of fluorescent acetylated low-density lipoprotein (LDL) as described (24). Fluorescence-activated cell sorting (FACS) of clones labeled with fluorescent acetylated LDL confirmed that endothelial cell clones represented homogeneous populations of cells that were both uniformly labeled and clearly distinguishable from cloned populations of vascular smooth muscle or mesa&al cells. Endothelial cells were fed every 48 h with RPM1 1640 medium supplemented with 2 mM L-glutamine, 15% low endotoxin SCS, 100 U/ml penicillin, and 100 pg/ml streptomycin. Cells were utilized at passages 5-10. Studies were routinely performed on confluent monolayers lo-14 days after passage that had placed in serum-deplete medium for 48 h. Working concentrations of rhTNF-a, rhIL-l& and culture medium contained
