B B Hoffman1, K Sharma, Y Zhu, F N Ziyadeh. 1. Penn Center for Molecular Studies of Kidney Diseases, Department of Medicine, University of Pennsylvania, Philadelphia 19104-6144, USA.
Abstract
BACKGROUND: Transforming growth factor-beta (TGF-beta) is an important hypertrophic and prosclerotic cytokine in the pathogenesis of diabetic nephropathy. The mechanisms of regulation of the TGF-beta system by high ambient glucose in kidney cells are incompletely defined. This study examined the mechanisms of regulation of TGF-beta1 expression by high glucose in murine mesangial cells (MMCs) in culture. METHODS: MMCs were cultured in either normal (100 mg/dl) or high (450 mg/dl) D-glucose concentration. Total TGF-beta1 protein secretion and bioactivity, mRNA expression and stability, and gene transcription rate were measured; promoter-reporter chloramphenicol acetyltransferase (CAT) assays and electrophoretic mobility shift assay (EMSA) were performed to investigate the presence of putative glucose-response elements. RESULTS: Raising the ambient D-glucose concentration for 72 hours increased TGF-beta1 bioactivity in cell culture medium by 47% and total TGF-beta1 secretion by approximately 90%. Northern analysis demonstrated that the steady-state TGF-beta1 mRNA level was increased nearly twofold after 48 hours of growth in high glucose. This increase was not due to increased stability, as the half-life of the message was approximately five hours in both normal and high glucose conditions. Transcriptional activity of the TGF-beta1 gene (nuclear run-on assay) was increased by 73% in cells grown in high glucose for 24 hours. Transiently transfected MMCs with CAT constructs containing varying lengths of the murine TGF-beta1 promoter demonstrated that high glucose selectively increased the expression of only one of the constructs, pA835. Sequence inspection revealed the presence of a putative glucose responsive element, CACGTG, within this construct. High glucose in MMC culture for 24 hours increased nuclear protein binding to a probe containing this element when analyzed using EMSA. CONCLUSIONS: High glucose stimulates total TGF-beta1 protein production and bioactivity as well as the steady-state level of TGF-beta1 mRNA. The latter effect is due primarily to stimulation of gene transcription rate rather than message stability. Transcriptional activation by high glucose may involve a region in the TGF-beta1 promoter containing a putative glucose-response element.
BACKGROUND: Transforming growth factor-beta (TGF-beta) is an important hypertrophic and prosclerotic cytokine in the pathogenesis of diabetic nephropathy. The mechanisms of regulation of the TGF-beta system by high ambient glucose in kidney cells are incompletely defined. This study examined the mechanisms of regulation of TGF-beta1 expression by high glucose in murine mesangial cells (MMCs) in culture. METHODS: MMCs were cultured in either normal (100 mg/dl) or high (450 mg/dl) D-glucose concentration. Total TGF-beta1 protein secretion and bioactivity, mRNA expression and stability, and gene transcription rate were measured; promoter-reporter chloramphenicol acetyltransferase (CAT) assays and electrophoretic mobility shift assay (EMSA) were performed to investigate the presence of putative glucose-response elements. RESULTS: Raising the ambient D-glucose concentration for 72 hours increased TGF-beta1 bioactivity in cell culture medium by 47% and total TGF-beta1 secretion by approximately 90%. Northern analysis demonstrated that the steady-state TGF-beta1 mRNA level was increased nearly twofold after 48 hours of growth in high glucose. This increase was not due to increased stability, as the half-life of the message was approximately five hours in both normal and high glucose conditions. Transcriptional activity of the TGF-beta1 gene (nuclear run-on assay) was increased by 73% in cells grown in high glucose for 24 hours. Transiently transfected MMCs with CAT constructs containing varying lengths of the murineTGF-beta1 promoter demonstrated that high glucose selectively increased the expression of only one of the constructs, pA835. Sequence inspection revealed the presence of a putative glucose responsive element, CACGTG, within this construct. High glucose in MMC culture for 24 hours increased nuclear protein binding to a probe containing this element when analyzed using EMSA. CONCLUSIONS: High glucose stimulates total TGF-beta1 protein production and bioactivity as well as the steady-state level of TGF-beta1 mRNA. The latter effect is due primarily to stimulation of gene transcription rate rather than message stability. Transcriptional activation by high glucose may involve a region in the TGF-beta1 promoter containing a putative glucose-response element.
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