Proc. Nati. Acad. Sci. USA Vol. 89, pp. 8150-8154, September 1992 Medical Sciences
Glucose and glucosamine regulate growth factor gene expression in vascular smooth muscle cells (glucose regulation/diabetes mellitus/transcnptional relaton/atheroscleross)
DONALD A. MCCLAIN*t*, ANDREW J. PATERSON*, MARK D. Roos*, XIPING WEI*, AND JEFFREY E. KUDLOW* *Departments of Medicine and Cell Biology and tVeterans Administration Medical Center, University of Alabama, Birmingham, AL 35294 Communicated by Edmond H. Fischer, June 1, 1992
growth factor (bFGF; refs. 5 and 6). In addition, because the promoter for the TGFa gene has been cloned and characterized (7, 8), we studied in more detail the transcriptional regulation of the TGFa gene. In other cells, the expression of TGFa has been shown to be regulated at both the transcriptional and the translational levels (4, 7), and the regions of the TGFa promoter that confer hormonal regulation have been identified (7). We show in this paper that the mRNAs for both TGFa and bFGF accumulate significantly in vascular smooth muscle cells exposed to high glucose concentrations. The effect on the TGFa mRNA appears to be transcriptional and is conferred by a proximal element within the TGFa promoter. Based on dose-response analysis, this regulation by glucose appears to be mediated by one of its metabolites, glucosamine. These studies establish the TGFa promoter as a model to investigate the regulation of gene expression by this metabolic pathway. The effects ofglucose and its metabolites on the expression of growth factors and perhaps other genes could play a role in the pathogenesis of the vascular complications of diabetes mellitus.
We have investigated the regulation of the ABSTRACT expression of two growth factors found in vascular smooth muscle, transforming growth factor a (TGFa) and basic fibroblast growth factor (bFGF). Cells cultured in medium containing 30 mM glucose exhibited a 2-fold increase in TGFa mRNA and a 3-fold increase in bFGF mRNA compared with cells grown in normal (5.5 mM) glucose. Glucosamine was more potent than glucose, leading to a 6-fold increase in TGFa mRNA. TGFa protein levels were also increased by glucosamine treatment, and the predominant species present was the membrane-bound precursor form of TGFa. To examine further the regulation of growth factors by sugars, cultured rat aortic smooth muscle cells were transfected with a plasmid construct consisting of a 1.2-kilobase-pair fragment of the TGFa promoter linked to a luciferase reporter gene. Increasing the concentration of glucose in the culture medium from 5.5 mM to 30 mM led to a rapid, 1.7-fold increase in the activity of the TGFa promoter. Glucosamine was much more potent than glucose in this stimulation, with 2 mM gluamine causing a 12-fold increase in TGFa promoter activity. Insulin had no effect on luciferase activity in either the presence or the absence of added sugars. The glucose response element of the TGFa gene maps to a 130-base-pair segment that includes three potential binding sites for the transcription factor Spl. We conclude that high glucose concentrations such as are reached in diabetes mellitus can stimulate the transcription of the genes for growth factors in vascular smooth muscle cells. This signaling pathway apparently involves the metabolism of glucose to glucosamine. This effect could be representative of nutritional regulation of a family of genes and could contribute to the toxicity of hyperglycemia and the vascular complications of diabetes.
MATERIALS AND METHODS Materials and Cell Culture. Cell culture media and supplies were from GIBCO/BRL. Routine reagents and sugars were from Sigma. Rat aortic smooth muscle cells were prepared from aortas from 250- to 300-g male Sprague-Dawley rats (9). The cells were cultured routinely in Dulbecco's modified Eagle's medium (DMEM) (low glucose, 1 mg/ml)/10%o fetal bovine serum (FBS), passed every 5 days at 1:10, and used for up to six passages. Plasmid Constructs and Electroporation. The plasmid constructs containing 5' flanking sequences of the TGFa gene subcloned upstream of the luciferase gene have been described (7). After trypsinization and washing, -2 x 107 cells were combined with 30 pg of supercoiled plasmid that had been twice purified on CsCl gradients. Electroporation was done in DMEM/10%o FBS at 280 V and 500 OF in a Bio-Rad Gene Pulser. Cells were then plated in 60-mm culture dishes (Costar), with =5 x 105 cells per dish. The next day the cells were refed with either DMEM (5.5 mM D-glucose)/10%/ FBS or the same medium that contained only 1 mM D-glucose. The cells were assayed for luciferase activity 3 days after refeeding. Sugars were added at various times prior to assay from 1 M stocks that had been filter-sterilized. Glucosamine stocks were prepared freshly before each use. Luciferase and Glucose Transport Assays. Luciferase activity was measured in detergent extracts of cells in the presence of ATP and luciferin in an LKB luminometer (7).
Vascular smooth muscle cell proliferation is an early event in the pathogenesis of atherosclerosis, and growth factors have been hypothesized to play an important role in the control of this process (1). For example, platelet-derived growth factor is expressed in atherosclerotic lesions and is found in macrophages in all phases of the development of those lesions (2). Growth factors synthesized by the vascular smooth muscle cells themselves may also be involved in the pathogenesis of vascular disease. Thus, the exposure of vascular smooth muscle cells to growth factors could be determined by the plasma concentration of a growth factor, by delivery from blood-derived cells, or by the production rate of the growth factor by cells within the vessel wall. To examine whether the synthesis of growth factors might be sensitive to environmental conditions, we studied two growth factors known to be expressed in vascular smooth muscle cells, transforming growth factor a (TGFa; refs. 3 and 4) and basic fibroblast
Abbreviations: TGFa, transforming growth factor a; bFGF, basic fibroblast growth factor; FBS, fetal bovine serum. tTo whom reprint requests should be addressed at: Division of Endocrinology, DH 777, Department of Medicine, University of Alabama, UAB Station, Birmingham, AL 35294.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 8150
Medical Sciences: McClain
et
al.
Background luminescence was subtracted from all readings and was
Glucose and glucosamine regulate growth factor gene expression in vascular smooth muscle cells (glucose regulation/diabetes mellitus/transcnptional relaton/atheroscleross)
DONALD A. MCCLAIN*t*, ANDREW J. PATERSON*, MARK D. Roos*, XIPING WEI*, AND JEFFREY E. KUDLOW* *Departments of Medicine and Cell Biology and tVeterans Administration Medical Center, University of Alabama, Birmingham, AL 35294 Communicated by Edmond H. Fischer, June 1, 1992
growth factor (bFGF; refs. 5 and 6). In addition, because the promoter for the TGFa gene has been cloned and characterized (7, 8), we studied in more detail the transcriptional regulation of the TGFa gene. In other cells, the expression of TGFa has been shown to be regulated at both the transcriptional and the translational levels (4, 7), and the regions of the TGFa promoter that confer hormonal regulation have been identified (7). We show in this paper that the mRNAs for both TGFa and bFGF accumulate significantly in vascular smooth muscle cells exposed to high glucose concentrations. The effect on the TGFa mRNA appears to be transcriptional and is conferred by a proximal element within the TGFa promoter. Based on dose-response analysis, this regulation by glucose appears to be mediated by one of its metabolites, glucosamine. These studies establish the TGFa promoter as a model to investigate the regulation of gene expression by this metabolic pathway. The effects ofglucose and its metabolites on the expression of growth factors and perhaps other genes could play a role in the pathogenesis of the vascular complications of diabetes mellitus.
We have investigated the regulation of the ABSTRACT expression of two growth factors found in vascular smooth muscle, transforming growth factor a (TGFa) and basic fibroblast growth factor (bFGF). Cells cultured in medium containing 30 mM glucose exhibited a 2-fold increase in TGFa mRNA and a 3-fold increase in bFGF mRNA compared with cells grown in normal (5.5 mM) glucose. Glucosamine was more potent than glucose, leading to a 6-fold increase in TGFa mRNA. TGFa protein levels were also increased by glucosamine treatment, and the predominant species present was the membrane-bound precursor form of TGFa. To examine further the regulation of growth factors by sugars, cultured rat aortic smooth muscle cells were transfected with a plasmid construct consisting of a 1.2-kilobase-pair fragment of the TGFa promoter linked to a luciferase reporter gene. Increasing the concentration of glucose in the culture medium from 5.5 mM to 30 mM led to a rapid, 1.7-fold increase in the activity of the TGFa promoter. Glucosamine was much more potent than glucose in this stimulation, with 2 mM gluamine causing a 12-fold increase in TGFa promoter activity. Insulin had no effect on luciferase activity in either the presence or the absence of added sugars. The glucose response element of the TGFa gene maps to a 130-base-pair segment that includes three potential binding sites for the transcription factor Spl. We conclude that high glucose concentrations such as are reached in diabetes mellitus can stimulate the transcription of the genes for growth factors in vascular smooth muscle cells. This signaling pathway apparently involves the metabolism of glucose to glucosamine. This effect could be representative of nutritional regulation of a family of genes and could contribute to the toxicity of hyperglycemia and the vascular complications of diabetes.
MATERIALS AND METHODS Materials and Cell Culture. Cell culture media and supplies were from GIBCO/BRL. Routine reagents and sugars were from Sigma. Rat aortic smooth muscle cells were prepared from aortas from 250- to 300-g male Sprague-Dawley rats (9). The cells were cultured routinely in Dulbecco's modified Eagle's medium (DMEM) (low glucose, 1 mg/ml)/10%o fetal bovine serum (FBS), passed every 5 days at 1:10, and used for up to six passages. Plasmid Constructs and Electroporation. The plasmid constructs containing 5' flanking sequences of the TGFa gene subcloned upstream of the luciferase gene have been described (7). After trypsinization and washing, -2 x 107 cells were combined with 30 pg of supercoiled plasmid that had been twice purified on CsCl gradients. Electroporation was done in DMEM/10%o FBS at 280 V and 500 OF in a Bio-Rad Gene Pulser. Cells were then plated in 60-mm culture dishes (Costar), with =5 x 105 cells per dish. The next day the cells were refed with either DMEM (5.5 mM D-glucose)/10%/ FBS or the same medium that contained only 1 mM D-glucose. The cells were assayed for luciferase activity 3 days after refeeding. Sugars were added at various times prior to assay from 1 M stocks that had been filter-sterilized. Glucosamine stocks were prepared freshly before each use. Luciferase and Glucose Transport Assays. Luciferase activity was measured in detergent extracts of cells in the presence of ATP and luciferin in an LKB luminometer (7).
Vascular smooth muscle cell proliferation is an early event in the pathogenesis of atherosclerosis, and growth factors have been hypothesized to play an important role in the control of this process (1). For example, platelet-derived growth factor is expressed in atherosclerotic lesions and is found in macrophages in all phases of the development of those lesions (2). Growth factors synthesized by the vascular smooth muscle cells themselves may also be involved in the pathogenesis of vascular disease. Thus, the exposure of vascular smooth muscle cells to growth factors could be determined by the plasma concentration of a growth factor, by delivery from blood-derived cells, or by the production rate of the growth factor by cells within the vessel wall. To examine whether the synthesis of growth factors might be sensitive to environmental conditions, we studied two growth factors known to be expressed in vascular smooth muscle cells, transforming growth factor a (TGFa; refs. 3 and 4) and basic fibroblast
Abbreviations: TGFa, transforming growth factor a; bFGF, basic fibroblast growth factor; FBS, fetal bovine serum. tTo whom reprint requests should be addressed at: Division of Endocrinology, DH 777, Department of Medicine, University of Alabama, UAB Station, Birmingham, AL 35294.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 8150
Medical Sciences: McClain
et
al.
Background luminescence was subtracted from all readings and was
