Endosomal Cysteine Proteases

One set of enzymes actively under investigation are the group of endosomal proteases. Prior studies and inspection of human genome databases indicate there are 12 human endosomal cysteine proteases (summarized in Figure below). Blue text indicates human disease associated with enzyme deficiency.  The remainder are published mouse phenotypes.

The lab has helped to identify and define the function of several of these enzymes and the biological roles of endosomal cysteine proteases continue to be a focus of the lab. 

Under some conditions endosomal proteases spill out into the pericellular environment and mediate matrix degradation.  This may be a normal physiological process as occurs in bone matrix turnover or may be pathogenic, e.g. in emphysema and vascular aneurysms. Regulation of cysteine protease activity in the extracellular space as it relates to matrix destruction and/or cellular injury is an ongoing lab interest. We are currently asking two main questions:  (1) What is the molecular mechanism(s) of lung injury in cathepsin S-dependent,  cytokine-induced experimental emphysema in mice?  (2)  Can we define a degradative “signature” for cathepsin S in vivo during experimental emphysema and if so,  is there evidence for this degradative activity in human disease,  especially COPD.  The answers to these questions should help clarify the role of cathepsin S in COPD and its potential as a therapeutic target.

Proteases and Integrin Function

In addition to degradation of extracellular matrices, extracellular proteases help cells respond to their immediate environment in many ways:  solubilization of matrix-bound and cell surface-bound cytokines, activation of protease cascades leading to modified cellular behavior (for example coagulation factors), and altered cellular adhesion. A developing paradigm (See Figure 2 below) is that activation of proteolytic enzymes couple to and regulate the function of cellular adhesion receptors, coordinating the process of matrix attachment and detachment by cells. Signaling through integrin adhesion receptors regulates protease activity in the pericellular environment and proteases in turn modify the function of integrins.  Generation of bioactive matrix fragments by proteases associated with integrins is also an increasingly recognized common phenomenon. 

The protease system that has been of longstanding interest to the lab is the urokinase/urokinase receptor (uPAR).  Recent studies indicate uPAR is a multifunctional receptor capable of direct interaction with at least 3 protein partners:  urokinase,  vitronectin,  and certain integrins. These interactions are presumed to account for the strong empirical observations linking uPAR expression with cell migration.  Results from our studies of the last several years have directed our focus at understanding how uPAR regulates integrin signaling and under what conditions this is important in vivo. A developing theme is that uPAR is a cis-acting integrin ligand,  engaging integrins near the canonical integrin ligand binding sites for matrix engagement in the integrin head domains. We are currently focused on alpha3beta1 (laminin receptor) and alpha5beta 1(fibronectin receptor), both of which bind uPAR. Interestingly,  in both cases the complex of integrin and uPAR does not block matrix ligand binding but changes the specificity of integrin matrix binding. For example,  uPAR/alpha5beta1 binds fibronectin at a different site (non-RGD) than alpha5beta1 alone (RGD).  

We are currently asking two main questions:  (1) Can we use site-directed mutagenesis of uPAR to separate its urokinase binding activity from its integrin binding capacity? If so, these mutants will be used to define the distinct role of uPAR integrin binding on cellular behavior in vitro and in vivo. (2)  Does uPAR/integrin association affect the metastatic potential of tumor cells? We are using mouse models of lung cancer to test this question. One project is to test the proposition that disruption of the uPAR/integrin binding site(s) is an efficacious way to modify the malignant progression of tumor cells. Additionally, we are examining the function of uPAR and beta1 integrins on primary tumor cells isolated from human adenocarcinomas to test whether data obtained in mice appear consonant with observations in humans.

Pulmonary Fibrosis

Many proteases and integrins are integral to the complex biology of lung matrix remodeling leading to pulmonary fibrosis. A recent direction of the laboratory has been to explore the role of beta1 integrins in this process.  In part this direction is stimulated by work of other investigators which has revealed a role for beta 1 integrins in TGFbeta1 signaling and by our discovery that some integrin mutants we have expressed are resistant to TGFbeta1-mediated epithelial to mesenchymal transition (EMT).  This has led to a more serious attempt to define the role of integrins in EMT, using mouse model systems. Our current questions center around whether EMT occurs in the lung during fibrogenesis and if so what regulates the process.  Our focus is the development of transgenic and conditional knockout systems to reveal the key elements of EMT regulated by integrins in mouse models of pulmonary fibrosis.

Selected References

Wei Y, Lukasev M, Simon DI, Bodary SC, Rosenburg S, Doyle MV, and HA Chapman. Regulation of integrin function by the urokinase receptor Science 1996;273:1551-1555.

Gelb BD, Shi GP. Chapman HA, and RJ Desnick. Pycnodysostosis, a lysosomal disease caused by cathepsin K deficiency. Science 1996;273:1236-1238.

Riese R, Wolf P, Bromme D, Natkin L, Villadangos JA, Ploegh H, and HA Chapman. Essential role for cathepsin S in MHC Class II-associated invariant chain processing and antigen presentation. Immunity 1996;4:357-366.

Shi GP, Villadangos J, Dranoff G, Ploegh H, Chapman HA. Cathepsin S required for normal MHC class II peptide loading and germinal center development. Immunity 1999;10:196-206.

Wei Y, Yang X, Quimei Liu, Wilkins JA, and Chapman HA. Role for caveolin and urokinase receptors in integrin-mediated adhesion and signaling. J Cell Biol 1999;144:1285-1294.

Simon DI, Wei Y, Chen Z, Rao NK, Rosenberg S, Chapman HA. Identification of a Urokinase Receptor-Integrin Interaction Site: Promiscuous Regulator of Integrin Function. J Biol Chem 2000;275: 10228-10234.

Wei Y, Ebles JA, Weng Z, Kreidberg JA, and Chapman HA. Urokinase receptors promote beta1 Integrin function through interactions with the integrin alpha3/beta1. Mol Biol Cell 2001; 12:2975-2986.

Riese RJ, Shi GP, Villadangos J, Stetson D, Driessen C, Lennon-Dumenil AM, Chu CL, Naumov Y, Behar SM, Ploegh H, Locksley R, Chapman HA. Regulation of CD1 function and NK1.1+ T cell selction and maturation by Cathepsin S. Immunity 2001; 15:909-9

Zhang F, Tom CC, Kugler MC, Ching TT, Kreidberg JA, Wei Y, Chapman HA. Distinct ligand binding sites in integrin alpha3beta1 regulate matrix adhesion and cell-cell contact.  J Cell Biol. 2003;163(1):177-88.

Chapman HA.  Disorders of Lung Matix Remodeling.  J Clin Invest. 2004;113(2):148-57.

Y. Wei, R.-P. Czekay, L. Robillard, M.C. Kugler, F. Zhang, K.K. Kim, J.-p. Xiong, M.J. Humphries, and H.A. Chapman. Regulation of a5ß1 integrin conformation and function by urokinase receptor binding. J. Cell Biol. 2005; 168: 501:511.

Tang CH, Lee JW, Galvez MG, Robillard L, Mole SE, Chapman HA.  Murine cathepsin F deficiency causes neuronal lipofuscinosis and late-onset neurological disease. Mol Cell Biol 2006 Mar;26(6):2309-16

Kevin K. Kim, Matthias C. Kuglar, Paul J. Wolters, Liliane Robillard, Michael G. Galvez, Alexis N. Brumwell, Dean Sheppard and Chapman HA. Alveolar Epithelial Cell Mesenchymal Transition Develops in vivo during Pulmonary Fibrosis and is Regulated by the Extracellular Matrix. Proc Natl Acad Sci 2006 Aug 29; 103(35): 13180-5.

Ying Wei, Chi-Hui Tang, Feng Zhang, Young Kim, Liliane Robillard, Matthias C. Kugler, Chapman HA.  Urokinase Receptors are required for a5b1 integrin-mediated signaling in tumor cells. J of Biol Chem 282(6):3929-39, 2007.