JAM‐A signals through the Hippo pathway to regulate intestinal epithelial proliferation

Abstract

The intestinal mucosa is lined by a columnar epithelial monolayer that serves as a dynamic interface, regulating barrier function and homeostasis. Perturbation of homeostasis, including changes in intestinal epithelial cell (IEC) proliferation and migration, can give rise to pathological states including ulcerative colitis and cancer. Regulation of proliferation and migration is a complex process coordinated, in part, by signaling events emanating from tight junction proteins at cell-cell contacts. One tight junction protein that has been linked to regulation of cellular proliferation is Junctional Adhesion Molecule A (JAM-A). JAM-A forms homodimers in cis and trans through interactions between its extracellular immunoglobulin-like domains. Loss of JAM-A results in enhanced IEC proliferation that has been linked to loss of homophilic interactions and Akt-dependent, beta-catenin signaling. However, JAM-A contributions to other signaling pathways that regulate cell proliferation are poorly understood. Since the Hippo signaling pathway is a well-known regulator of cellular proliferation, differentiation, and apoptosis, we examined whether loss of JAM-A alters Hippo pathway activity. We assessed activity of the Hippo transcriptional coactivator, Yes-1 Associated Protein (YAP), in JAM-A deficient IEC by gene-reporter activity assay and immunoblotting and observed significant increases, suggesting that JAM-A may interact with Hippo pathway constituents upstream of YAP. Immunolabeling studies with monolayers of IEC revealed association of JAM-A with two Hippo pathway constituents, Large Tumor Suppressor Kinase (LATS1) and Merlin. Co-immunoprecipitation experiments confirmed LATS1 and Merlin association with JAM-A. Moreover, phosphoprotein specific immunoblotting studies on JAM-A deficient IEC revealed decreased Hippo pathway activity characterized by decreased LATS1 activity and subsequent increased YAP activity. Such findings are consistent with JAM-A signaling upstream of Merlin and LATS1 and coordinating downstream Hippo signaling. Since there is evidence suggesting that dimerization of JAM-A is a required signal that suppresses IEC proliferation, we performed experiments using an exogenous overexpression system to study effects of a dimerization deficient JAM-A mutant on Hippo signaling. Phosphoprotein specific immunoblotting and EdU incorporation revealed that overexpression of full-length JAM-A resulted in activation of Hippo signaling, suppressing cell proliferation compared to empty vector control cells. However, overexpression of a dimerization defective JAM-A mutant protein lacking the distal immunoglobulin loop (DL1) was unable to activate Hippo signaling and suppress cell proliferation. Based on these observations, we propose that homodimerization of JAM-A functions as a cell-cell contact sensor, initiating downstream Hippo signaling to suppress IEC proliferation. These findings have important implications for understanding effects of altered proliferation on mucosal wound repair, as well as in morbidity associated with certain cancers which have been reported to express decreased levels of JAM-A protein.