TRANSCRIPTIONAL ACTIVATION OF TRANSFORMING GROWTH-FACTOR-BETA-1 IN MESANGIAL CELL-CULTURE BY HIGH GLUCOSE-CONCENTRATION

Background. Transforming growth factor-beta (TGF-beta) is an important hypertrophic and prosclerotic cytokine in the pathogenesis of diabeti c nephropathy. The mechanisms of regulation of the TGF-beta system by high ambient glucose in kidney cells are incompletely defined. This st udy examined the mechanisms of regulation of TGF-beta 1 expression by high glucose in murine mesangial cells (MMCs) in culture. Methods. MMC s were cultured in either normal (100 mg/dl) or high (450 mg/dl) D-glu cose concentration. Total TGF-beta 1 protein secretion and bioactivity , mRNA expression and stability, and gene transcription rate were meas ured; promoter-reporter chloramphenicol acetyltransferase (CAT) assays and electrophoretic mobility shift assay (EMSA) were performed to inv estigate the presence of putative glucose-response elements. Results. Raising the ambient D-glucose concentration for 72 hours increased TGF -PI bioactivity in cell culture medium by 47% and total TGF-beta 1 sec retion by approximately 90%. Northern analysis demonstrated that the s teady-state TGF-beta 1 mRNA level was increased nearly twofold after 4 8 hours of growth in high glucose. This increase was not due to increa sed stability, as the half-lift: of the message was approximately five hours in both normal and high glucose conditions. Transcriptional act ivity of the TGF-beta 1 gene (nuclear run-on assay) was increased by 7 3% in cells grown in high glucose for 24 hours. Transiently transfecte d MMCs with CAT constructs containing varying lengths of the murine TG F-PI 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 incr eased nuclear protein binding to a probe containing this element when analyzed using EMSA. Conclusions. High glucose stimulates total TGF-be ta 1 protein production and bioactivity as well as the steady-state le vel of TGF-beta 1 mRNA. The latter effect is due primarily to stimulat ion of gene transcription rate rather than message stability. Transcri ptional activation by high glucose may involve a region in the TGF-bet a 1 promoter containing a putative glucose-response element.