Transcriptional Regulation of Mouse H2-Calponin Gene by Mechanical Tension

Abstract

The importance of mechanical signaling in life process has long been recognized, however, it remains to be established that how mechanical signals is transduced in cells to regulate gene expression. Our previous studies have demonstrated that the gene encoding h2-calponin (Cnn2) is regulated by mechanical tension (Hossain et al., JBC, 2005). To investigate the regulatory mechanism, mouse genomic DNA containing the Cnn2 promoter was cloned and 5’-serial deletion constructs were made in reporter plasmid vectors. By transfective expression in NIH3T3 fibroblasts and HEK293 cells that express endogenous h2-calponin, we tested the activity of the epigenetic Cnn2 promoter constructs and their responsiveness to the stiffness of cultural substrates. The results showed that while the −0.6-kb construct had only near background activity, significant levels of transcriptional activities were observed for the −1.0-kb, −1.6-kb and −2.1-kb promoter constructs. The −1.6-kb and −2.1-kb constructs were both responsive to substrate stiffness that produce different mechanical tensions in the cytoskeleton, whereas the −1.0-kb construct showed no such regulation. The dada indicated that cis-regulatory element(s) located between −1596 and −1016 of the mouse Cnn2 gene is responsible for the mechanical tension-regulated expression of h2-calponin. By establishing a novel experimental system to study mechanical cell signaling, the characterization of Cnn2 promoter activity allows us to identify trans-regulatory factors involved in the cellular signaling pathway that transmits mechanical tension stimuli to the regulation of h2-calponin gene expression.