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

A cell-based assay system for monitoring COLIA2 [alpha 2(1) collagen gene] promoter activity was developed to determine the influence of activated COLIA2 promoter in human dermal fibroblast cells. A pLuc-COLIA2 promoter plasmid that expresses the luciferase reporter gene in response to COLIA2 promoter activity was constructed. The pLuc-COLIA2 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. COLIA2 promoter activities were measured by luciferase reporter gene assay using a luminescence detection method. Fibroblast cell transfectants treated with TNF alpha (tumour necrosis factor alpha), known to be an inhibitor of COLIA2 promoter expression, showed a reduction of COLIA2 promoter activity in a concentration-dependent manner, whereas TGF-ss (transforming growth factor-fl), known to be a stimulator of COLIA2 promoter expression, increased COLIA2 activity in a concentration-dependent manner. This assay system could be used to quantitatively measure COLIA2 promoter activity in human dermal fibroblast cells and allow the screening of anti-wrinkle agents from various synthetic chemicals and natural products.