Mo1885 Role of the Hippo-YAP Pathway in the Regulation of DNA Synthesis by cAMP in Intestinal Epithelial Cells and Swiss 3T3 Fibroblasts

Abstract

Background: The mammalian Hippo pathway, comprised by the Mst1/2 and Lats1/2 kinase cascade, has been implicated in the regulation of organ size in response to cell-cell contact and diffusible molecules, including those that act via G protein-coupled receptors (GPCRs). The transcription co-activator Yes-associated protein (YAP) is a major downstream effector of the Hippo pathway. The precise role of YAP in intestinal epithelial cell proliferation, regeneration and cancer remains controversial, as evidence for both growth-stimulatory and growth-suppressive roles of YAP in the gut have been presented. Given the fundamental role of the Hippo-YAP pathway in cell regulation, it is vital to define the function of YAP in intestinal epithelial cells. Results: In order to identify a model system to explore GPCRmediated regulation of the Hippo pathway in intestinal epithelial cells, we determined the localization of endogenous YAP1/2 in response to the Gq-coupled receptor agonist angiotensin II (ANGII) in IEC-6 and IEC-18 cells. ANGII is a regulatory peptide generated in the GI tract increasingly recognized to play a role in GI mucosal inflammation and carcinogenesis. In un-stimulated IEC-18 or IEC-6 cells, YAP1/2 was located in the nucleus and cytoplasm. Stimulation of these cells with ANG II induced a rapid and dramatic cytoplasmic relocalization of YAP1/2. Pretreatment with the selective AT1 antagonist Losartan completely prevented ANG II-induced YAP1/2 re-localization, verifying that this unexpected effect was mediated by the high affinity Gq-coupled ANG II receptor. The re-localization of YAP1/2 in response to ANG II was dose-dependent and could be detected as early 15 min after stimulation and subsided after 2-4 h of stimulation. Similar findings were made with IEC6 cells. These novel results indicate that ANG II induces biphasic re-distribution of YAP1/ 2 in intestinal epithelial cells characterized by strong and transient cytoplasmic localization followed by subsequent nuclear re-localization. We next determined whether ANG II induces YAP phosphorylation at Ser127 and Ser391 in IEC-18 cells, highly conserved residues located within a consensus Lats1/2 sequence (HXRXXS). Lysates of IEC-18 cells stimulated with ANGII for various times were analyzed by Western blotting with antibodies that detect the phosphorylated state of YAP1 at Ser127 and at Ser391. We found that ANGII induced a rapid (detectable within 15 min) and transient (persisted for 2 h) increase in YAP phosphorylation in intestinal epithelial IEC-18 cells. Conclusion: These novel results demonstrated that induces YAP re-localization and phosphorylation at residues targeted by Lats1/2 in intestinal epithelial cells. As ANG II is a potent mitogen for IEC-18 cells, we hypothesize that YAP acts sequentially in the cytoplasm and nucleus to promote proliferation in intestinal epithelial cells.