The Role of Notch Signaling in Mechanical-Tension Regulation of H2-Calponin Gene

Abstract

The essential role of mechanical signaling in regulating the cellular function of living organisms has been widely recognized. However, how mechanical signals are transduced in cells to regulate gene expression and other biochemical activities is not well understood. Our previous studies have demonstrated that the gene encoding h2-calponin (Cnn2) is regulated by mechanical tension (Hossain et al., JBC 280:42442-53, 2005). The data indicated that cis-regulatory element(s) located between −1.6-kb and −1.4-kb upstream of the mouse Cnn2 gene is responsible for the mechanical tension-regulation. The present study quantitatively studied the regulation of h2-calponin gene by mechanical tension in the cytoskeleton focusing on this region. Potential transcriptional regulatory factor binding sites within the −1.6 to −1.4-kb region were explored using reporter gene constructs in cells cultured on high and low stiffness substrates or in comparison between floating and adherent cultures. The results indicated a role of HES1 downstream of the Notch signaling pathway. While floating cultures of C2C12 myoblasts showed a significant decrease in h2-calponin expression compared to the adherent cultures, treatments with a Notch pathway inhibitor DAPT were able to minimize this low cytoskeleton tension-dependent inhibition of Cnn2 expression in a dose dependent manner. The Notch signaling pathway has been implicated in the mechanical tension-regulation of cells and our results suggest that it plays a role in the regulation of Cnn2 gene. The current hypothesis is that the Notch pathway modulates h2-calponin gene expression via the HES1 site in the −1.6kb and −1.4-kb region of the Cnn2 promoter where it acts to inhibit expression when cells are experiencing decreased mechanical tension. Further studies are underway to further elucidate the mechanotransduction mechanism that regulates h2-calponin expression.