During inflammation, epithelial cells of the intestine are exposed to proteases from inflammatory cells (e.g. mast cell tryptase), the epithelium (extrapancreatic trypsins) and the circulation (coagulation factors). However, nothing is known about the biological actions of these proteases or the mechanisms of their effects. This proposal examines the hypothesis that proteases that are generated during inflammation signal to intestinal epithelial cells by cleaving protease-activated receptors (PARs); activation of PARs increases paracellular permeability and thereby exacerbates inflammation. This hypothesis is tested in cell-based model systems (cultured colonocytes) and intact animals. The proposal focuses on PAR2, a receptor for tryptase, trypsins and coagulation factors Vlla and Xa.
Aim 1 will investigate the importance of receptor trafficking in colonocytes to the initiation and termination of PAR2 signaling.
Aim 1 A will examine the formation of a PAR2 """"""""signaling module"""""""" in endosomes that determines the function of mitogen activated protein kinases and thereby plays a major role in signaling in colonocytes.
Aim 1 B will investigate the mechanisms that terminate PAR2 signaling. Since proteolytic activation is irreversible, efficient mechanisms must terminate PAR2 signaling. Experiments will examine the role of the E3 ubiquitin ligase c-Cbl in ubiquitination of PAR2 and will determine the importance of ubiquitination for lysosomal trafficking of PAR2.
Aim 2 will investigate the mechanisms by which proteases regulate paracellular permeability of colonocytes in culture.
Aim 2 A will determine if proteases that are generated during inflammation can signal to colonocytes by cleaving and activating PAR2.
Aim 2 B will investigate the signal transduction mechanisms by which PAR2 regulates the formation of tight junctions.
Aim 3 will investigate the role of proteases and PAR2 in the control of paracellular permeability and inflammation in animal models of colitis.
Aim 3 A will characterize the proteases that are generated in the inflamed colon and will determine if proteases increase mucosal permeability and cause inflammation by activating PAR2.
Aim 3 B will define the contribution of PAR2 and tryptase in colitis using PAR2 knockout animals and specific inhibitors. Thus, aim 1 will provide basic information about mechanisms of signal transduction by G-protein coupled receptors, and aims 2 and 3 identify novel mechanisms by which proteases and their receptors regulate epithelial permeability and mucosal inflammation.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-GMPB (01))
Program Officer
Hamilton, Frank A
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of California San Francisco
Schools of Medicine
San Francisco
United States
Zip Code
Pelayo, Juan-Carlos; Veldhuis, Nicholas A; Eriksson, Emily M et al. (2014) Localisation and activation of the neurokinin 1 receptor in the enteric nervous system of the mouse distal colon. Cell Tissue Res 356:319-32
Haerteis, Silke; Krappitz, Annabel; Krappitz, Matteus et al. (2014) Proteolytic activation of the human epithelial sodium channel by trypsin IV and trypsin I involves distinct cleavage sites. J Biol Chem 289:19067-78
Poole, Daniel P; Amadesi, Silvia; Veldhuis, Nicholas A et al. (2013) Protease-activated receptor 2 (PAR2) protein and transient receptor potential vanilloid 4 (TRPV4) protein coupling is required for sustained inflammatory signaling. J Biol Chem 288:5790-802
Alemi, Farzad; Kwon, Edwin; Poole, Daniel P et al. (2013) The TGR5 receptor mediates bile acid-induced itch and analgesia. J Clin Invest 123:1513-30
Alemi, Farzad; Poole, Daniel P; Chiu, Jonathan et al. (2013) The receptor TGR5 mediates the prokinetic actions of intestinal bile acids and is required for normal defecation in mice. Gastroenterology 144:145-54
Veldhuis, Nicholas A; Lew, Michael J; Abogadie, Fe C et al. (2012) N-glycosylation determines ionic permeability and desensitization of the TRPV1 capsaicin receptor. J Biol Chem 287:21765-72
Law, Ivy Ka Man; Murphy, Jane E; Bakirtzi, Kyriaki et al. (2012) Neurotensin-induced proinflammatory signaling in human colonocytes is regulated by ?-arrestins and endothelin-converting enzyme-1-dependent endocytosis and resensitization of neurotensin receptor 1. J Biol Chem 287:15066-75
Haerteis, Silke; Krappitz, Matteus; Bertog, Marko et al. (2012) Proteolytic activation of the epithelial sodium channel (ENaC) by the cysteine protease cathepsin-S. Pflugers Arch 464:353-65
Hasdemir, Burcu; Mahajan, Shilpi; Bunnett, Nigel W et al. (2012) Endothelin-converting enzyme-1 actions determine differential trafficking and signaling of corticotropin-releasing factor receptor 1 at high agonist concentrations. Mol Endocrinol 26:681-95
Lyo, Victoria; Cattaruzza, Fiore; Kim, Tyson N et al. (2012) Active cathepsins B, L, and S in murine and human pancreatitis. Am J Physiol Gastrointest Liver Physiol 303:G894-903

Showing the most recent 10 out of 115 publications