Transcriptional activation of transforming growth factor-beta1 in mesangial cell culture by high glucose concentration

Kidney Int. 1998 Oct;54(4):1107-16. doi: 10.1046/j.1523-1755.1998.00119.x.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Base Sequence
  • Binding Sites / genetics
  • Cell Line
  • Chloramphenicol O-Acetyltransferase / genetics
  • DNA / genetics
  • DNA / metabolism
  • Diabetic Nephropathies / etiology
  • Genes, Reporter
  • Glomerular Mesangium / drug effects*
  • Glomerular Mesangium / metabolism*
  • Glucose / pharmacology*
  • Humans
  • Kinetics
  • Mice
  • Nuclear Proteins / metabolism
  • Promoter Regions, Genetic / drug effects
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Transcriptional Activation / drug effects
  • Transfection
  • Transforming Growth Factor beta / genetics*
  • Transforming Growth Factor beta / metabolism

Substances

  • Nuclear Proteins
  • RNA, Messenger
  • Transforming Growth Factor beta
  • DNA
  • Chloramphenicol O-Acetyltransferase
  • Glucose