The sequential proliferation, lineage-specific differentiation, migration, and death of the epithelial cells of the colonic mucosa is a tightly regulated process modulated by a broad range of regulatory peptides, differentiation signals and luminal stimuli. Despite its fundamental importance for understanding the pathogenesis of human diseases, including inflammatory bowel diseases (IBD) and colorectal cancer (CRC), the signaling mechanisms involved remain incompletely understood. In this context, GTP-binding (G) protein-coupled receptors (GPCRs) and their intracellular signal transduction pathways play a critical role in the regulation of multiple functions of the digestive system, including cell proliferation, inflammation and promotion of CRC. The extracellular calcium-sensing receptor (CaSR), a member of the GPCR family, is prominently expressed by epithelial cells of the gastrointestinal (GI) tract but its function and mechanism in the regulation of normal and/or abnormal intestinal epithelial cell proliferation in vivo remains unknown. We demonstrated that CaSR stimulation by extracellular Ca2+ inhibits the proliferation of colon-derived epithelial cells. Our new preliminary studies obtained with a novel CaSR intestinal-specific knock out mouse show that the genetic ablation of the CaSR strikingly increases colonic crypt proliferation. Further preliminary results with colon-derived cells in culture demonstrate that CaSR stimulation strikingly decreases 2-catenin phosphorylation at Ser-552 and Ser-675, two amino acid residues that regulate 2-catenin localization and transcriptional activity. CaSR-mediated 2-catenin dephosphorylation at Ser-552 and Ser-675 coincided with its translocation from the nucleus to the plasma membrane and with 2-catenin-mediated transcription inhibition. Based on our preliminary results, the overarching hypothesis of this proposal is that CaSR signaling inhibits colon epithelial cell proliferation via down-regulation of b-catenin signaling. We plan to explore this hypothesis by pursuing the following Specific Aims: 1). Characterize the role of the CaSR in colon epithelial cell proliferation, differentiation, and cancer using a novel CaSR GI tract-specific knock-out mouse model;2) Identify the signal transduction pathways that mediate the decrease in b-catenin phosphorylation at Ser- 552 and Ser-675 in response to CaSR stimulation in colon-derived epithelial cells. 3). Characterize b- catenin sub-cellular distribution and transcriptional activity in response to CaSR stimulation in colon- derived epithelial cells. We anticipate that the mechanistic studies proposed with genetically modified mice and human epithelial cells in culture will demonstrate that the CaSR inhibits colonic epithelial cell proliferation via negative crosstalk with the b-catenin signaling. These studies will also establish a robust rationale to explore protective effects of CaSR activation (e.g. via dietary Ca2+) in preventing CRC, one of the most common malignancies in the general population as well as in the US veteran population.
The mechanistic studies proposed in this application on the function of the extracellular calcium-sensing receptor (CaSR) using genetically modified mice and human colon- derived cells in culture are of importance to define the role of the CaSR in the regulation of intestinal epithelial cell proliferation and consequently, are of high significance for Colorectal Cancer (CRC) and Inflammatory Bowel Disease (IBD). The completion of the proposed studies will provide a robust intellectual framework for targeting the colonic CaSR in the chemoprevention of CRC in US veterans as well as in the US population at large.
|Chang, Jen-Kuan; Ni, Yang; Han, Liang et al. (2017) Protein kinase D1 (PKD1) phosphorylation on Ser203 by type I p21-activated kinase (PAK) regulates PKD1 localization. J Biol Chem 292:9523-9539|
|Hao, Fang; Xu, Qinhong; Zhao, Yinglan et al. (2017) Insulin Receptor and GPCR Crosstalk Stimulates YAP via PI3K and PKD in Pancreatic Cancer Cells. Mol Cancer Res 15:929-941|
|Wang, Jia; Han, Liang; Sinnett-Smith, James et al. (2016) Positive cross talk between protein kinase D and ?-catenin in intestinal epithelial cells: impact on ?-catenin nuclear localization and phosphorylation at Ser552. Am J Physiol Cell Physiol 310:C542-57|
|Zhou, G; Yu, J; Wang, A et al. (2016) Metformin Restrains Pancreatic Duodenal Homeobox-1 (PDX-1) Function by Inhibiting ERK Signaling in Pancreatic Ductal Adenocarcinoma. Curr Mol Med 16:83-90|
|Wang, Jia; Sinnett-Smith, James; Stevens, Jan V et al. (2016) Biphasic Regulation of Yes-associated Protein (YAP) Cellular Localization, Phosphorylation, and Activity by G Protein-coupled Receptor Agonists in Intestinal Epithelial Cells: A NOVEL ROLE FOR PROTEIN KINASE D (PKD). J Biol Chem 291:17988-8005|
|Soares, Heloisa P; Ming, Ming; Mellon, Michelle et al. (2015) Dual PI3K/mTOR Inhibitors Induce Rapid Overactivation of the MEK/ERK Pathway in Human Pancreatic Cancer Cells through Suppression of mTORC2. Mol Cancer Ther 14:1014-23|
|Rozengurt, Enrique (2014) Mechanistic target of rapamycin (mTOR): a point of convergence in the action of insulin/IGF-1 and G protein-coupled receptor agonists in pancreatic cancer cells. Front Physiol 5:357|
|Sinnett-Smith, James; Ni, Yang; Wang, Jia et al. (2014) Protein kinase D1 mediates class IIa histone deacetylase phosphorylation and nuclear extrusion in intestinal epithelial cells: role in mitogenic signaling. Am J Physiol Cell Physiol 306:C961-71|
|Rozengurt, Enrique; Soares, Heloisa P; Sinnet-Smith, James (2014) Suppression of feedback loops mediated by PI3K/mTOR induces multiple overactivation of compensatory pathways: an unintended consequence leading to drug resistance. Mol Cancer Ther 13:2477-88|
|Ni, Yang; Sinnett-Smith, James; Young, Steven H et al. (2013) PKD1 mediates negative feedback of PI3K/Akt activation in response to G protein-coupled receptors. PLoS One 8:e73149|
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