cDNA microarray technology provides a novel approach to identify individual target genes and survey global genetic programs under the control of specific signalling pathways in mammalian systems. Smad2 and Smad3 are highly homologous members of the receptor-regulated subfamily of Smad proteins with a central role in TGF-β signalling and target gene regulation.

The purpose of this study is to identify genes that are regulated by TGF-β through activation of Smad2 or Smad3, or through Smad-independent pathways. Using cDNA microarray technology developed at AECOM, we have profiled the expression of 9,000 genes in wild-type mouse embryonic fibroblasts (MEFs+/+), Smad2−/− MEFs or Smad3−/− MEFs at baseline and following exposure to TGF-β1 or activin for 1 hour, 4 hours and 10 hours, respectively. Cells are cultured in exponential growth conditions. Total RNA from MEFs+/+ and matched test samples (Smad2−/− or Smad3−/− MEFs) is fluorescently labelled with either Cye3- or Cye5-dUTP. Probes are then co-hybridized to genes gridded on the array and single-channel fluorescence intensities are measured. Results are expressed as a normalized ratio (Cye3/Cye5) in which deviations from 1 indicate increased (>1), or decreased (<1) levels of gene expression in Smad2−/− or Smad3−/− MEFs relative to MEFs+/+. Using microarray data mining and analysis tools (microarray analysis tools or MAT) developed at AECOM, we have identified a large number of novel genes (expressed sequence tags) that are differentially regulated in Smad2−/− or Smad3−/− compared with MEFs+/+, depending on genotype, cytokine treatment and time. MAT is used for global survey (cluster analysis) of gene expression profiles in this genetically defined model system.

In conclusion, using a high-throughput cDNA microarray approach, we have identified gene modules differentially expressed in Smad2- and Smad3-deficient fibroblasts compared with wild-type fibroblasts in response to TGF-β. Stratification of gene activation profiles according to specific signalling mediators should provide new insights into the mechanisms that determine biological specificity of TGF-β.