Paolo Mignatti - US grants

DESCRIPTION (provided by applicant): Our extensive preliminary work has generated the unexpected hypothesis that the well-established apoptotic effect of transforming growth factor-beta 1 (TGF-b1) on endothelial cells is mediated by endogenous vascular endothelial growth factor (VEGF). This conclusion is based on our findings that TGF-b1 stimulates endothelial cell expression of VEGF and that monoclonal antibodies to VEGF block TGF-b1-induced apoptosis. Because apoptosis is an essential component of vessel formation, this novel mechanism of endothelial cell apoptosis may have an important role in angiogenesis and vasculogenesis. We therefore propose: 1. To characterize the VEGF receptor(s) and downstream signaling pathways through which endogenous VEGF mediates the apoptotic activity of TGF-b1 on endothelial cells. We will investigate signal transduction pathways activated by endogenous VEGF in response to TGF-b1 treatment, and potential cross-talk between VEGF- and TGF-b1-specific pathways. The use of synthetic inhibitors and dominant negative mutants will afford the identification of the signaling pathway(s) that mediate TGF-b1 apoptotic signal(s). 2. To study the role of VEGF-mediated apoptosis in angiogenesis and vasculogenesis in vitro and in vivo. TGF-b1 induces both apoptosis and vessel formation. Based on our preliminary work, we hypothesize that the angiogenic and vasculogenic activities of TGF-b1 are mediated by VEGF with an autocrine mechanism. We propose to test our hypothesis using in vitro and in vivo models of angiogenesis and vasculogenesis. The results will elucidate the mechanisms through which vessel formation is controlled by the interplay of VEGF and TGF-b1, two potent angiogenesis inducers. A detailed understanding of the mechanisms through which VEGF and TGF-b1 interact can have important implications for the development of pharmacological treatments aimed at the control of angiogenesis.

DESCRIPTION (provided by applicant): The invasion-promoting, tumorigenic membrane-type 1 matrix metalloproteinase (MT1-MMP), a transmembrane proteinase with extracellular catalytic, hemopexin (PEX) and hinge domains, and a short cytoplasmic tail, forms a stoichiometric complex with its physiological protein inhibitor, tissue inhibitor of metalloproteinases-2 (TIMP-2). It is well established that the proteolytic activity of MT1-MMP is required for tumor cell invasion and proliferation, and that TIMP-2 binds to the catalytic domain of MT1-MMP and blocks its proteolytic activity. Our extensive, recently published studies have shown that, in addition to extracellular proteolysis, MT1-MMP and TIMP-2 control cell proliferation and migration through a non-proteolytic mechanism. TIMP-2 binding to MT1-MMP induces activation of Extracellular signal-Regulated Kinase 1/2 (ERK1/2) by a mechanism independent of the MT1-MMP catalytic domain and the inhibitory domain of TIMP- 2. This effect involves TIMP-2 binding to the hemopexin and/or hinge domain and is mediated by the cytoplasmic tail of MT1-MMP. MT1-MMP-mediated activation of ERK1/2 upregulates cell migration and proliferation in vitro independently of extracellular matrix proteolysis. Consistent with this finding, proteolytically inactive MT1-MMP promotes tumor growth in vivo with an effect comparable to that of wild- type MT1-MMP. While these observations do not diminish the well-established importance of the proteolytic interactions involving TIMP-2 and MT1-MMP, our novel, unexpected findings strongly advocate a very important role for the proteolysis-independent signaling mechanism we identified. Because the ERK1/2 signaling pathway controls a variety of tumor cell functions including proliferation and migration, a detailed understanding of the molecular mechanism by which TIMP-2 - MT1-MMP interaction activates this signaling pathway can provide fundamental information for designing novel inhibitors to block tumor progression. Therefore, we propose to explore this novel signaling mechanism in detail by developing the following Specific Aims: 1) To identify the region(s) of the MT1-MMP PEX and/or hinge domains that mediate TIMP-2 binding and ERK1/2 activation, 2) To identify the region(s) of TIMP-2 required for binding to the PEX and/or hinge domain of MT1-MMP;3) To characterize the mechanism of signal transduction from MT1-MMP to the Ras-ERK1/2 pathway;4) To determine the functional role of TIMP-2 - MT1-MMP interaction in tumor growth in vivo. For this purpose we will use state-of-the-art molecular, cellular and immunological techniques and a tumor xenograft model in immunodeficient mice. The results of our study will lead to the understanding of the non-proteolytic role of MT1-MMP and TIMP-2 in tumor growth, and afford the development of novel inhibitors aimed to block MT1-MMP - TIMP-2 interaction and the generation of intracellular signaling that promotes tumor progression. PUBLIC HEALTH RELEVANCE: We found an unexpected, paradigm-shifting mechanism that controls tumor cell proliferation and migration, as well as tumor growth in an experimental in vivo model. Therefore, we propose to study in detail this novel mechanism and its functional role in tumor growth. The knowledge derived from our study will be of fundamental importance for designing novel pharmacological agents to block tumor progression.

DESCRIPTION (provided by applicant): Membrane-type 1 matrix metalloproteinase (MT1-MMP), a transmembrane proteinase with extracellular catalytic, hemopexin-like (PEX) and hinge domains, and a short cytoplasmic tail, has been implicated in a variety of physiological and pathological processes. Unlike the other MMPs, whose genetic deficiency has minor phenotypic effects, the genetic deficiency of MT1-MMP causes severe defects in skeletal development with marked deceleration of postnatal growth, shortening of long bones, defective vascularization of the cartilage and delayed formation of secondary ossification centers. In addition, MT1- MMP-deficient mice show important defects in alveolar development and adult angiogenesis, and die by 3 weeks of age. It has been proposed that the phenotype of these mice results from the lack of the proteolytic activity of MT1-MMP. Our recently published, extensive studies have shown that MT1-MMP mediates the activation of intracellular signaling by a proteolysis-independent mechanism that stimulates cell proliferation and migration in vitro and in vivo. MT1-MMP activation of intracellular signaling is mediated by a specific sequence of the cytoplasmic tail and independent of the MT1-MMP proteolytic activity. While this novel and unexpected finding does not diminish the well-established importance of the proteolytic activity of MT1-MMP, it strongly advocates a very important role for the signaling mechanism we identified, and indicates that the phenotype of MT1-MMP null mice can result at least in part from lack of MT1-MMP-mediated signaling. The signaling mechanism we discovered is a novel, proteolysis-independent activity of MT1-MMP that may have important roles in chondrocyte proliferation in the growth plates, formation of secondary ossification centers, skeletal and alveolar development, and in adult angiogenesis. Therefore, we propose to study the physiological role of MT1-MMP- mediated intracellular signaling by developing the following Specific Aim: To generate and characterize the phenotype of a transgenic mouse with a subtle mutation that abolishes MT1-MMP-mediated intracellular signaling. We propose to generate a strain of mice expressing a mutation in the MT1-MMP cytoplasmic tail that abolishes the signaling capacity without affecting the proteolytic activity of MT1-MMP, and to compare their phenotype to those of wt and MT1-MMP null mice. We expect that expression of this mutant MT1-MMP will result in a phenotype that partially recapitulates the phenotype of MT1-MMP null mice. Therefore, the analysis of the phenotype of our mutant mice will allow us to understand the role of MT1-MMP-mediated signaling in development, normal physiology and pathology. PUBLIC HEALTH RELEVANCE: We found an unexpected, paradigm-shifting mechanism that controls cell proliferation and migration in vitro and in vivo. Therefore, we propose to generate and characterize a genetic model in mice to understand the role of this novel mechanism in development, normal physiology and in pathology. The knowledge derived from our study can provide important insight into skeletal and pulmonary alveolar development and in blood vessel formation (angiogenesis).

ABSTRACT We propose to investigate the role of non-proteolytic functions of membrane-type 1 matrix metalloproteinase (MT1- MMP, MMP-14) in the control of bone formation in vivo. MT1-MMP, a cell-membrane-bound proteinase with an extracellular catalytic site and a 20-amino acid (aa) cytoplasmic tail, degrades a variety of extracellular matrix (ECM) components and plays a key role in postnatal bone formation. The genetic deficiency of MT1-MMP (MT1-MM-/-) in the mouse causes severe osteopenia, dwarfism, faciocranial dysmorphism with lack of closure of the cranial sutures (fontanelles), and generalized arthritis. In humans, mutation of MT1-MMP causes the multicentric osteolysis and arthritis disease, Winchester syndrome, which recapitulates the dramatic skeletal phenotype of the MT1-MMP-/- mouse, showing the fundamental role of MT1-MMP in postnatal bone development in humans. Because MT1-MMP is essential in ECM remodeling, it is universally accepted that the severe phenotype of MT1-MMP-/- mice results from defective collagen turnover. However, a large body of evidence shows that the cytoplasmic tail of MT1-MMP contains multiple aa residues and motifs that control intracellular signaling pathways and cell functions by proteolysis-independent mechanisms. We generated a mouse with a point mutation of the unique tyrosine in the MT1-MMP cytoplasmic domain (MT1-MMP Y573D), and found that this non-proteolytic domain controls Wnt signaling and bone homeostasis in vivo. In the MT1- MM-/- mouse both the proteolytic and non-proteolytic functions of MT1-MMP are abolished, but the phenotypic effects are universally ascribed only to the lack of proteolytic activity. However, because MT1-MMP is a bifunctional protein, to understand its roles in vivo it is necessary to study its proteolytic activity independently of its non-proteolytic functions. This can be done by mutating the conserved Glu240 (E240A) in the MT1-MMP catalytic domain, a mutation that abrogates the proteolytic activity without affecting the non-proteolytic functions of MT1-MMP. Therefore, we propose to study the roles of MT1-MMP in bone homeostasis by developing the following Specific Aim: To generate and characterize the phenotype of mice with conditional expression of MT1-MMP E240A. We will generate a genetically modified mouse in which expression of proteolytically inactive MT1-MMP E240A can be induced in a time- and tissue-specific manner. We will then analyze the bone phenotype of conditional MMP14 E240A, MT1-MM-/- and MT1-MMP Y573D mice in which the respective mutations are induced in osteoblasts at different stages of skeletal development. Osteoblasts and mesenchymal stem cells from these mice will also be characterized for Wnt signaling and differentiation into the osteogenic lineage. We expect that MT1-MMP E240A mice devoid of MT1-MMP proteolytic activity will show milder osteopenia and dwarfism than MT1-MMP-/- mice, but a bone phenotype opposite to that of MT1-MMP Y573D mice (mild osteopenia and dwarfism vs. increased bone mass, respectively). The results will elucidate the relative contribution of the proteolysis-dependent and independent functions of MT1-MMP to bone homeostasis, and unveil non-proteolytic functions thus far undetected in vivo. The knowledge derived from our study can ultimately provide important information for the design of highly innovative approaches for the treatment of osteoporosis and other bone fragility conditions.