A correlation between TGF-β1-mediated JAM-A downregulation and cell invasiveness

Abstract

Transforming growth factor-beta (TGF-β) and junctional adhesion molecule-A (JAM-A) have been indicated as key regulators of cell migration, and an inverse relationship of JAM-A expression and the invasiveness has been reported in breast cancer cells. Whether TGF- β1 signaling induces JAM-A downregulation leading to cancer cell invasion has not been investigated. In this study, we report that TGF- β1 significantly downregulates JAMA expression at both mRNA and protein levels in MCF-7 cells that is slightly migratory and normally expresses high level of JAM-A. We showed that TGF-β1 downregulates JAM-A mRNA level via transcriptional regulation and the promoter region responsible to TGF-β1 stimulation is located between nt -367 and -1. In addition, TGF-β1 exerted its negative effect on JAMA expression via post-translational control. There was a significant reduction (60% reduction) in JAM-A protein levels in cyclohexamide-pretreated TGF-β1-treated cells, indicating that TGF-β1 promotes JAM-A protein turnover. We showed that JNK inhibitor, but not Smad3 and Smad4 siRNA, could effectively abolish TGF-β1-mediated JAM-A protein degradation, although Smad activation alone could abolish JAM-A degradation in the absence of TGF-β1 stimulation. Furthermore, clathrin siRNA abolished TGF-β1-mediated JAM-A degradation, indicating that the degradation is mediated via clathrin-dependent endocytosis. Reduction of JAM-A in MCF-7 cells upon TGF-β1 treatment led to enhanced invasion in the invasion assays. Taken together, these results indicate a strong correlation between TGF-β1-mediated JAM-A downregulation and cell invasiveness.