A cell-based assay system for high-throughput screening of anti-wrinkle agents in human dermal fibroblast transfectant cells

Biotechnol Appl Biochem. 2007 May;47(Pt 1):27-31. doi: 10.1042/BA20060122.

Abstract

A cell-based assay system for monitoring COL1A2 [alpha2(I) collagen gene] promoter activity was developed to determine the influence of activated COL1A2 promoter in human dermal fibroblast cells. A pLuc-COL1A2 promoter plasmid that expresses the luciferase reporter gene in response to COL1A2 promoter activity was constructed. The pLuc-COL1A2 promoter plasmid and pCI-neo plasmid containing the NPT (neomycin phosphotransferase) gene for Geneticin resistance in host cells were co-transfected into human dermal fibroblast cells. COL1A2 promoter activities were measured by luciferase reporter gene assay using a luminescence detection method. Fibroblast cell transfectants treated with TNFalpha (tumour necrosis factor alpha), known to be an inhibitor of COL1A2 promoter expression, showed a reduction of COL1A2 promoter activity in a concentration-dependent manner, whereas TGF-beta (transforming growth factor-beta), known to be a stimulator of COL1A2 promoter expression, increased COL1A2 activity in a concentration-dependent manner. This assay system could be used to quantitatively measure COL1A2 promoter activity in human dermal fibroblast cells and allow the screening of anti-wrinkle agents from various synthetic chemicals and natural products.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cells, Cultured
  • Collagen / genetics
  • Collagen / metabolism*
  • Collagen Type I
  • Fibroblasts / drug effects
  • Fibroblasts / metabolism*
  • Genes, Reporter
  • Humans
  • Luciferases / genetics
  • Promoter Regions, Genetic
  • Skin Aging*
  • Transfection
  • Transforming Growth Factor beta1 / pharmacology
  • Tumor Necrosis Factor-alpha / pharmacology

Substances

  • Collagen Type I
  • Transforming Growth Factor beta1
  • Tumor Necrosis Factor-alpha
  • Collagen
  • Luciferases