The sequential proliferation, lineage-specific differentiation, migration and death of the epithelial cells of the intestinal mucosa is a tightly regulated process modulated by a broad range of regulatory peptides, neurotransmitters, bioactive lipids and differentiation signals. Despite its fundamental importance for understanding intestinal homeostasis, wound healing and pathogenesis of human diseases, the signaling mechanisms involved remain incompletely understood. Many gastrointestinal (GI) peptides, neurotransmitters and bioactive lipids initiate their characteristic effects in their target cells through heptahelical G protein-coupled receptors (GPCRs). Protein kinase D (PKD1) is emerging as a key node in GPCR signaling and consequently the understanding of PKD1 regulation and function is of intense interest and potential impact. The studies proposed here will identify new links between GPCR/PKD1 signaling and pivotal pathways that control migration and proliferation of intestinal epithelial cells. The overarching hypothesis to be explored is that PKD1 plays a critical role in the signal transduction pathways leading to intestinal epithelial cell proliferation.
Three Specific Aims are proposed:
Specific Aim 1 : Characterize the role of PKD1 signaling in intestinal epithelial cell proliferation in vivo and in stem cell-derived intestinal organoids.
This Aim will focus on PKD1 signaling in vivo, examining its function in homeostatic turnover via stem/progenitor cell proliferation and repair in response to injury of the intestinal mucosa, using genetically modified mouse models, including PKD1 transgenic mice and stem-cell derived intestinal organoids.
Specific Aim 2) Characterize crosstalk mechanisms between PKD1 and ?-catenin signaling systems in intestinal epithelial cells. The studies proposed in this Aim will characterize an novel crosstalk between GPCR/PKD1 and ?-catenin signaling, leading to PKD1/?- catenin complex formation, ?-catenin translocation to the nucleus, stimulatory phosphorylation at Ser552 and transcriptional activation;
Specific Aim 3) Identify a novel mechanism of PKD1 regulation through PAK-mediated PKD1 phosphorylation at the N-terminal residue Ser203. Based on new preliminary results, we will identify a novel phosphorylation in the N-terminal domain of PKD1 in response to GPCR agonists in intestinal epithelial cells mediated by p21-activated kinases (PAKs), effectors for Rac and Cdc42. The discovery of a PAK/PKD1 cascade uncovers a new point of integration in the signal transduction pathways initiated by GPCR agonists. We anticipate that the mechanistic studies proposed in this application will identify novel upstream pathway(s) and downstream crosstalk mechanisms by which PKD1 regulates the proliferation and migration of intestinal epithelial cells thereby providing the rationale for innovative therapeutic intervention in diseases of the digestive system.

Public Health Relevance

Gastrointestinal (GI) peptides and other chemical messengers that act via G protein-coupled receptors play critical roles in the regulation of cell proliferation and in the pathogenesis of chronic inflammatory processes and cancer. In this application, we propose to elucidate novel mechanisms of regulation and function of protein kinase D1 (PKD1), a key player in transmitting GPCR signals in their target cells. These studies have important implications not only for understanding fundamental regulatory mechanisms in the GI tract but also for identifying PKD1 as a novel target for therapeutic intervention in diseases characterized by either excessive or defective cell proliferation.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK100405-04
Application #
9335342
Study Section
Clinical, Integrative and Molecular Gastroenterology Study Section (CIMG)
Program Officer
Greenwel, Patricia
Project Start
2014-09-01
Project End
2018-07-31
Budget Start
2017-08-01
Budget End
2018-07-31
Support Year
4
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
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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
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Chang, Hui-Hua; Young, Steven H; Sinnett-Smith, James et al. (2015) Prostaglandin E2 activates the mTORC1 pathway through an EP4/cAMP/PKA- and EP1/Ca2+-mediated mechanism in the human pancreatic carcinoma cell line PANC-1. Am J Physiol Cell Physiol 309:C639-49
Young, Steven H; Rey, Osvaldo; Rozengurt, Enrique (2015) Intracellular Ca(2+) oscillations generated via the extracellular Ca(2+)-sensing receptor (CaSR) in response to extracellular Ca(2+) or L-phenylalanine: Impact of the highly conservative mutation Ser170Thr. Biochem Biophys Res Commun 467:1-6
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
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 (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
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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