Differential Regulation of E-Cadherin Expression by the Soluble Ectodomain and Intracellular Domain of Jagged1.

ABSTRACT

Aberrant Jagged1-mediated Notch activation is linked to cancer and induces epithelial-tomesenchymal transition through the repression of E-cadherin transcription. All three proteins are subject to sequential proteolytic events referred to as regulated intramembrane proteolysis. This process releases soluble protein ectodomains from the cell and, concomitantly, generates intracellular domains capable of nuclear translocation and transcriptional regulation. Aim: To determine the cognate roles of the Jagged1 ectodomain and intracellular domain fragments in the regulation of E-cadherin expression. Methodology: Human embryonic kidney cells were stably transfected with coding DNA constructs analogous to full-length Jagged1, the soluble Jagged1 ectodomain, or the intracellular domain fragment of the protein. Correct construct expression and processing were confirmed by immunoblot analysis of transfectant cell lysates and conditioned culture medium. The effects of the various Jagged1 constructs on endogenous E-cadherin expression and processing were subsequently monitored by immunoblot and RT-qPCR analyses. Results: Both full-length Jagged1 and the soluble Jagged1 ectodomain construct downregulated E-cadherin expression at the protein and RNA level. In contrast, the Jagged1 intracellular domain fragment construct enhanced E-cadherin expression but only at the RNA level. Conclusion: The soluble Jagged1 ectodomain is sufficient for the down-regulation of Ecadherin expression whereas the intracellular domain of the protein does not exhibit such an effect and actually increases E-cadherin RNA expression. These results raise the interesting possibility of E-cadherin regulation in cells distal to the site of soluble Jagged1 ligand generation.