Ellen Puré, PhD
Grace Lansing Lambert Professor of Biomedical Science
Chair, Department of Biomedical Sciences
Cancer Biology Program
Cellular and molecular basis of inflammation and fibrosis
Key words: inflammation, fibrosis, extracellular matrix, mouse genetics, ageing, dynamic macromolecular complexes, cell polarity, alternative splicing
This laboratory is studying the cellular and molecular basis of inflammation and fibrosis, with a particular focus on the role of stromal cells and extracellular matrix (ECM), in the context of chronic inflammatory diseases and cancer. The molecular pathways currently being studied include the adhesion receptor CD44 and its principle ligand, hyaluronan, and fibroblast activation protein (FAP), a stromal cell surface protease. Studies of CD44 and FAP are being conducted in mouse models of cancer, cardiovascular disease and pulmonary fibrosis using conditional CD44 knockout mice and FAP-null mice generated in the lab. Also the FAP promoter has been exploited to generate mice that can be used to non-invasively image reactive stromal cells in fibrotic lesions and epithelial-derived tumors, to conditionally ablate reactive stromal cells, and to manipulate gene expression specifically in fibrotic lesions and tumor stromal cells. We are studying the impact of matrix modification on cell behavior directly through regulation of receptor mediated signal transduction as well as through modulation of tissue stiffness. We are also exploring the function of CD44 and FAP in human disease.
SELECTED RECENT PUBLICATIONS
Cichy, J. and E. Puré. 2000. Oncostatin M and Transforming Growth Factor-ß1 induce post-translational modification and hyaluronan binding to CD44 in lung-derived epithelial tumor cells. J. Biol. Chem. 275:18061-18069.
Puré, E. and C.A. Cuff. 2001. A crucial role for CD44 in inflammation. Trends in Molecular Medicine. 7:213-221.
Cuff, C., D. Kothapalli, I. Azonobi, S. Chun, Y. Zhang, R. Belkin, C. Yeh, A. Secreto, R.K. Assoian, D.J. Rader and E. Puré. 2001. The adhesion receptor CD44 promotes atherosclerosis by mediating inflammatory cell recruitment and vascular cell activation. J. Clin. Invest. 108:1031-1040.
Teder, P., R. W. Vandivier, D. Jiang, J. Liang, L. Cohn, E. Puré, P.M. Henson and P.W. Noble. 2002. Resolution of lung inflammation by CD44. Science 296:155-158.
Cichy, J., R. Bals, J. Potempa, A. Mani and E. Puré. 2002. Proteinase-mediated release of epithelial cell-associated CD44: Extracellular CD44 complexes with components of cellular matrices. J. Biol. Chem. 277:44440-44447.
Cichy, J. and E.Puré. 2003. The liberation of CD44. J. Cell Biol.161:839-843.
Acharya, P.S., A.M. Zukas, V. Chandan, A.-L.A. Katzenstein and E. Puré. 2006. Fibroblast activation protein:
serine protease expressed at the remodeling interface in idiopathic pulmonary fibrosis. Human Pathology 37:352-360.
Middleton, M.K., A.M. Zukas, T. Rubinstein, L.Zhao, M. Jacob, P. Zhu, L. Zhao, I. Blair and E. Puré. 2006. Identification of 12/15-lipoxygenase as a suppressor of myeloproliferative disease. J. Exp. Med. 203:2529-2540.
Kothapalli, D., L. Zhao, E.A. Hawthorne, Y. Cheng, E. Lee, E. Puré and R.K. Assoian. 2007. Hyaluronan and
CD44 antagonize mitogen-dependent cyclin D1 expression in mesenchymal cells. J. Cell Biol. 176:535-44.
Zhao, L., J.A. Hall, N. Levenkova, M.K. Middleton, A.M. Zukas, E. Lee, D.J. Rader, J.J. Rux and E. Puré. 2007. CD44 regulates vascular gene expression in a pro-atherogenic environment. Arterioscler. Thromb. and Vascular. Biol. 27:886-892.
Huang,Q., K. Gumireddy, M. Schrier, C. le Sage, R. Nagel, S. Nair, D.A. Egan, A. Li, G. Huang, A.J. Klein-
Szanto, P.A. Gimotty, D. Katsaros, G. Coukos, L. Zhang, E. Puré and R. Agami. 2008. The microRNAs miR-373 and miR-520C promote tumor migration and invasion. Nature Cell Biology. 10:202-210.
Acharya, P.S., S. Majumdar, M. Jacob, J. Hayden, P. Mrass, W. Weninger, R.K. Assoian and E. Puré. 2008. Fibroblast migration is mediated by CD44-dependent TGF-ß activation. J. Cell Sci. 121: 1393-1402.
Zhao, L., E. Lee, A.M. Zukas, M.K. Middleton, M. Kinder, P.S. Acharya, R. Yahil, J.A. Hall, D.J. Rader and E. Puré. 2008. CD44 expressed on both bone marrow-derived and non-bone marrow-derived cells promotes atherogenesis in apoE-deficient mice. Arterioscler, Thromb. Vasc. Biol. 28:1283-1289.
Kothapalli, D., J. Flowers, T. Xu, E. Puré and R.K. Assoian. 2008. Differential activation of Erk and Rac mediates the proliferative and anti-proliferative effects of hyaluronan and CD44. J. Biol. Chem. 283:31823-31829.
Mrass, P., I. Kinjyo, S.L. Reiner, E. Puré and W. Weninger. 2008. Fine-tuning of effector T cell migration by CD44 regulates tumor surveillance. Immunity. 29:971-985 (accompanying Preview).
Puré, E. and R.K. Assoian. 2009. Rheostatic signaling by CD44 and hyaluronan. Cellular Signalling. 21:651-655.
Jacob, M., L.A. Todd, R.S. Majumdar, Y. Li, K.I. Yamamoto and E. Puré. 2009. Endogenous cAbl regulates receptor endocytosis. Celullar Signalling, 28:1308-1316
Jacob, M. and E. Puré. Stromal cells & tumor milieu: PDGF et al. In: Cancer Genome and Tumor microenvironment Thomas-Tikhonenko, Andrei (Ed.) 2009. In Press.
ROTATION PROJECTS FOR 2009-2010
Impact of the tumor microenvironment on disease progression in cancer.
1. CD44 has been implicated in tumor growth and metastasis. However, the roles of tumor cell and host cell CD44 have not been distinguished and the mechanisms by which CD44 regulates tumor progression are not known. The impact of tumor cell and host cell CD44 will be studied using a conditional knockout allele of CD44 that by crossing to appropriate tissue specific cre recombinase-expressing mice, will provide the first opportunity to dissect the role of CD44 expressed on various cell types including epithelial-derived tumor cells, infiltrating inflammatory cells, and reactive tumor stromal fibroblasts, on tumor progression.
2. FAP is a cell surface serine protease that is specifically induced on reactive fibroblasts in settings of irreversible fibrosis and in epithlelial-derived tumors. Its restricted expression pattern and the potential of its enzymatic activity to modify extracellular matrix and thereby the tumor microenvironment, has generated interest in the potential of FAP as a therapeutic target. We demonstrated that tumor initiation/growth is inhibited in FAP-deficient mice and in wild-type mice treated with a FAP inhibitor and we are delineating the mechanisms by which FAPa promotes tumorigenesis. In addition, using knock-in technology we are generating mice that express fluorescent/ luminescent proteins, a conditional suicide gene, cre recombinase, and an enzymatically dead mutant of FAP, under the control of the endogenous FAP promoter. Rotations projects will utilize these genetically engineered mice to a) non-invasively image stromal cells during the course of tumor progression using bioluminescence and, by multiphoton microscopy, the interactions between reactive stromal cells, matrix and tumor cells; b) conditionally ablate reactive stromal fibroblasts to determine their requirement to support different stages of tumor progression; c) manipulate gene expression specifically in tumor stromal cells; and d) determine the impact of FAP enzymatic activity on the extracellular matrix and tumor progression.
3. Although the requirement for reactive stromal fibroblasts to support tumor survival/growth and progression is increasingly appreciated, the differentiative program involved in the evolution of this cell type within tumors is not well understood. FAP is arguably the best markers for fibroblasts that have undergone the differentiative program giving rise to these functionally unique reactive stromal cells. Characterization of the cis- and transacting-factors that determine the tissue specific and inducible expression of FAP will therefore be studied to gain insight into the molecular basis underlying this differentiative program. Interestingly, deficiency in CD44 appears to prevent the induction of FAP and the mechanism by which CD44 regulates fibroblast differentiation will therefore be investigated. FAP will also be used as a marker to isolate and compare gene expression patterns and epigenetic regulation of gene expression in phenotypically distinct subsets of mouse and human tumor (lung, breast and pancreatic) associated fibroblasts.
4. Cancer is a disease of ageing. The lag is in part explained by the time required to accumulate the multiple oncogenic mutations required for cells to manifest a fully transformed phenotype. Recent data, however, indicate that mutagenic events intrinsic to neoplastic cells are not sufficient to support tumorigenesis and that the properties of the tissue microenvironment represent another critical determining factor. Specifically, a “normal” tissue environment can suppress tumorigenesis. Conversely, manifestation of the neoplastic phenotype of epithelial cells in solid tumors requires a “pro-oncogenic/reactive” microenvironment. We hypothesize that age-related changes in stromal fibroblasts drive the transition from a tumor suppressive microenvironment typical of tissue in young animals, to a pron-oncogenic microenvironment that becomes prevalent in old animals. Our novel mouse models referred to above will be used to 1) To define the phenotypic and functional transition of fibroblasts induced as a function of age under homeostatic conditions as well as conditions of chronic inflammation and fibrosis; and 2) To define the mechanisms by which fibroblasts promote tumorigenesis in aged mice.
1. We showed that global deletion of CD44 impacts mouse models of cardiovascular disease, injury-induced vascular lesions (a model of restenosis), atherosclerosis, and injury-induced pulmonary fibrosis. Furthermore, our data indicate that CD44 regulates vascular smooth muscle cell and fibroblast growth and migration. Biochemical and molecular approaches will be used to dissect the networks that mediate CD44-dependent signaling in smooth muscle cells and fibroblasts in vitro and we have generated mice expressing a conditional knockout allele of CD44 (CD44 cKO) to study the impact of cell type-specific deletion of CD44 on the response to injury and in atherogenesis in vivo. We will also test the hypothesis that the outcome of the activity of CD44 is related to the extent of inflammation in the target tissue environment.
2. In addition to reactive tumor fibroblasts, FAP is also induced in the setting of irreversible fibrosis including in association with radiation-induced and idiopathic pulmonary fibrosis, athero-sclerosis, and liver cirrhosis. Based on its protease activity it has been proposed that FAP may play an important role in the matrix remodeling that characterizes these pathologies. Therefore, we will compare the extent of irradiation-induced lung fibrosis and atherosclerosis in wild-type and FAP-deficient mice. In addition, the same genetic approaches as described above will be used to visualize fibrotic cells, ablate fibrotic cells and to manipulate gene expression in fibrotic lesions and determine the impact of specific gene products on pulmonary fibrosis and atherosclerosis.
CD44 Structure and Function
1. Dynamics of CD44 dependent extra- and intra-cellular macromolecular complexes.
There is a growing appreciation that CD44, as well as other cell surface receptors, can bind multiple ligands and associate with multiple other cell surface, intracellular, pericellular and extracellular matrix components to form varied macromolecular complexes depending on the state of cellular differentiation/activation. The dynamics of these interactions may depend on the extensive alternative splicing known to govern the expression of a large number of variant isoforms of CD44 and are manifested in differential functional outcomes. Our goals are to:
a. dissect the signaling pathways that underlie the transition of CD44 from its low affinity to its high affinity state for hyaluronan; this will include high throughput screens of small molecule libraries for inhibitors of the activation-induced transition to the high affinity state in leukocytes and the pathways required to maintain CD44 in its constitutively active state in tumor cells, and studying the role of variant isoforms of CD44 in complex formation.
b. determine the dynamics of CD44-dependent extracellular macromolecular complexes that underlie the varied functions of CD44 in cell adhesion, migration, activation of matrix metalloproteinases and TGFbeta, growth factor receptor signaling.
c. determine the dynamics of the macromolecular complexes formed between the cytoplasmic domain of CD44 and intracellular components including ERM proteins, the tumor suppressor merlin, and the actin cytoskeleton ad their impact on CD44-mediated growth regulation and migration.
2. CD44 in cell polarity.
Cell polarity is critical to a number of cellular functions including migration and asymmetric cell division. We recently demonstrated that CD44 not only regulates the velocity of cell migration but is also required for directional migration of fibroblasts in response to wounding. In T lymphocytes, we demonstrated that CD44 is required for efficient interstitial migration of T cells in tumors and anti-tumor immunity of T cells. Furthermore, we demonstrated that CD44 is required to stabilize the polarity of cells that is required for directional migration. Its role in polarity is dependent on the intracellular domain of CD44 and its interaction with ERM proteins but independent of its extracellular domain and ligand binding. The goal is to define the mechanism by which CD44 stabilizes cell polarity and thereby directional migration of cells and the functional consequences of CD44-mediated polarity.
3. Functional consequences of the proteolytic processing of CD44.
CD44 is subject to proteolytic cleavage of the extracellular domain (by MT1-MMP and ADAM) followed by two presinilin-dependent cleavages of the intramembrane domain by gamma secretase leading to the generation of an intracellular domain fragment that translocates to the nuclease where it regulates gene expression. The functional consequences of the proteolytic processing of CD44 is only partially understood. Further studies will be conducted to define the impact of CD44 cleavage on:
a. cell polarity and cell migration
b. gene expression
James Monslow, Ph.D. - Associate Staff Scientist
Leslie Todd - Research Assistant
Diana Avery - Rotation Student
Mia Krolikoski - Rotation Student
Rachel Blomberg - Rotation Student
Priya Govindaraju - Rotation Student
Allyson Lieberman - Rotation Student
Department of Biomedical Sciences
380 S. University Ave
Room 311 Hill Pavilion
Department of Biomedical Sciences
3800 Spruce Street, 209E Vet
Philadelphia, PA 19104
Kawashiri, M., Y. Zhang, E. Puré and D.J. Rader Combined effects of cholesterol reduction and apolipoprotein A-I expression on atherosclerosis in LDL receptor deficient mice Atherosclerosis : , 2002.Cichy, J., R. Bals, J. Potempa, A. Mani and E. Puré Proteinase- mediated release of CD44 from epithelial cells: Soluble CD44 is associated with components of cellular matrices J. Biol. Chem. 277: 44440-44447, 2002.Zhao, L., C. Cuff, E. Moss, U. Wille, T. Cyrus, E.A. Klein, S. Song, D. Pratico, D. J. Rader, C.A. Hunter, E. Puré and C.D. Funk Selective interleukin-12 synthesis defect in 12/15-lipoxygenase deficient macrophages associated with reduced atherosclerosis in a model of familial hypercholesterolemia J. Biol. Chem. 277: 35350-35356, 2002.Lazaar, A.L., M. Plotnick, I., U. Kucich, I. Crichton, S. Lotfi, S. K.P. Das, S. Kane, J. Rosenbloom, R.A. Panettieri Jr., N.M. Schecter, and E. Puré Mast cell chymase modifies cell-matrix interactions and inhibits mitogen-induced proliferation of human airway smooth muscle cells J. Immunol 169: 1014-1020, 2002.Teder, P., W. Vandivier, D. Jiang, J. Liang, L. Cohn, E. Puré, P.M. Henson and P.W. Noble Resolution of lung inflammation by CD44 Science 296: 155-158, 2002.