The Wnt/Planar Cell Polarity Protein-tyrosine Kinase-7 (PTK7) Is a Highly Efficient Proteolytic Target of Membrane Type-1 Matrix Metalloproteinase
2010; Elsevier BV; Volume: 285; Issue: 46 Linguagem: Inglês
10.1074/jbc.m110.165159
ISSN1083-351X
AutoresVladislav S. Golubkov, Alexei V. Chekanov, Piotr Cieplak, Alexander E. Aleshin, Andrei V. Chernov, Wenhong Zhu, Ilian Radichev, Danhua Zhang, P. Duc Si Dong, Alex Y. Strongin,
Tópico(s)Cell Adhesion Molecules Research
ResumoPTK7 is an essential component of the Wnt/planar cell polarity (PCP) pathway. We provide evidence that the Wnt/PCP pathway converges with pericellular proteolysis in both normal development and cancer. Here, we demonstrate that membrane type-1 matrix metalloproteinase (MT1-MMP), a key proinvasive proteinase, functions as a principal sheddase of PTK7. MT1-MMP directly cleaves the exposed PKP621↓LI sequence of the seventh Ig-like domain of the full-length membrane PTK7 and generates, as a result, an N-terminal, soluble PTK7 fragment (sPTK7). The enforced expression of membrane PTK7 in cancer cells leads to the actin cytoskeleton reorganization and the inhibition of cell invasion. MT1-MMP silencing and the analysis of the uncleavable L622D PTK7 mutant confirm the significance of MT1-MMP proteolysis of PTK7 in cell functions. Our data also demonstrate that a fine balance between the metalloproteinase activity and PTK7 levels is required for normal development of zebrafish (Danio rerio). Aberration of this balance by the proteinase inhibition or PTK7 silencing results in the PCP-dependent convergent extension defects in the zebrafish. Overall, our data suggest that the MT1-MMP-PTK7 axis plays an important role in both cancer cell invasion and normal embryogenesis in vertebrates. Further insight into these novel mechanisms may promote understanding of directional cell motility and lead to the identification of therapeutics to treat PCP-related developmental disorders and malignancy. PTK7 is an essential component of the Wnt/planar cell polarity (PCP) pathway. We provide evidence that the Wnt/PCP pathway converges with pericellular proteolysis in both normal development and cancer. Here, we demonstrate that membrane type-1 matrix metalloproteinase (MT1-MMP), a key proinvasive proteinase, functions as a principal sheddase of PTK7. MT1-MMP directly cleaves the exposed PKP621↓LI sequence of the seventh Ig-like domain of the full-length membrane PTK7 and generates, as a result, an N-terminal, soluble PTK7 fragment (sPTK7). The enforced expression of membrane PTK7 in cancer cells leads to the actin cytoskeleton reorganization and the inhibition of cell invasion. MT1-MMP silencing and the analysis of the uncleavable L622D PTK7 mutant confirm the significance of MT1-MMP proteolysis of PTK7 in cell functions. Our data also demonstrate that a fine balance between the metalloproteinase activity and PTK7 levels is required for normal development of zebrafish (Danio rerio). Aberration of this balance by the proteinase inhibition or PTK7 silencing results in the PCP-dependent convergent extension defects in the zebrafish. Overall, our data suggest that the MT1-MMP-PTK7 axis plays an important role in both cancer cell invasion and normal embryogenesis in vertebrates. Further insight into these novel mechanisms may promote understanding of directional cell motility and lead to the identification of therapeutics to treat PCP-related developmental disorders and malignancy. IntroductionSecreted Wnt glycoproteins regulate β-catenin-dependent (canonical) and β-catenin-independent (non-canonical) signaling pathways (1Gao C. Chen Y.G. Cell. Signal. 2010; 22: 717-727Crossref PubMed Scopus (528) Google Scholar, 2Schlessinger K. Hall A. Tolwinski N. Genes Dev. 2009; 23: 265-277Crossref PubMed Scopus (275) Google Scholar, 3Simons M. Mlodzik M. Annu. Rev. Genet. 2008; 42: 517-540Crossref PubMed Scopus (428) Google Scholar, 4Katoh M. Oncol. Rep. 2005; 14: 1583-1588PubMed Google Scholar, 5Jessen J.R. Zebrafish. 2009; 6: 21-28Crossref PubMed Scopus (53) Google Scholar, 6Wang Y. Mol. 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As a result, PCP is important for the directed collective cell movements and orchestrates the synchronized cell arrangements within the tissue plane in the course of a plethora of biological processes (2Schlessinger K. Hall A. Tolwinski N. Genes Dev. 2009; 23: 265-277Crossref PubMed Scopus (275) Google Scholar, 4Katoh M. Oncol. Rep. 2005; 14: 1583-1588PubMed Google Scholar, 6Wang Y. Mol. Cancer Ther. 2009; 8: 2103-2109Crossref PubMed Scopus (160) Google Scholar, 7Vladar E.K. Antic D. Axelrod J.D. Cold Spring Harb. Perspect. Biol. 2009; 1: a002964Crossref PubMed Scopus (175) Google Scholar, 8Goodrich L.V. Neuron. 2008; 60: 9-16Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 9Dale R.M. Sisson B.E. Topczewski J. Zebrafish. 2009; 6: 9-14Crossref PubMed Scopus (30) Google Scholar, 10Montcouquiol M. Crenshaw 3rd, E.B. Kelley M.W. Annu. Rev. Neurosci. 2006; 29: 363-386Crossref PubMed Scopus (146) Google Scholar, 11Yin C. Ciruna B. Solnica-Krezel L. Curr. Top. Dev. Biol. 2009; 89: 163-192Crossref PubMed Scopus (56) Google Scholar).The first PCP signaling events occur at a gastrulation stage of embryogenesis to regulate the polarized cell movement and accomplish convergent extension (CE) for the anterior-posterior body axis elongation, neural tube closure, and craniofacial morphogenesis (8Goodrich L.V. Neuron. 2008; 60: 9-16Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 9Dale R.M. Sisson B.E. Topczewski J. Zebrafish. 2009; 6: 9-14Crossref PubMed Scopus (30) Google Scholar, 11Yin C. Ciruna B. Solnica-Krezel L. Curr. Top. Dev. Biol. 2009; 89: 163-192Crossref PubMed Scopus (56) Google Scholar, 12Roszko I. Sawada A. Solnica-Krezel L. Semin. Cell Dev. Biol. 2009; 20: 986-997Crossref PubMed Scopus (176) Google Scholar). CE failure results in the multiple severe developmental defects, including a shortened body axis (dwarfism), defective neural system, and craniofacial abnormalities. Defects in the non-canonical Wnt/PCP pathway are linked to a broad range of diseases, including cancer (3Simons M. Mlodzik M. Annu. Rev. Genet. 2008; 42: 517-540Crossref PubMed Scopus (428) Google Scholar, 5Jessen J.R. Zebrafish. 2009; 6: 21-28Crossref PubMed Scopus (53) Google Scholar). Wnt5a, Wnt5b, and Wnt11, which work through the non-canonical pathway, are often up-regulated in cancer and promote cancer cell motility and invasion (6Wang Y. Mol. Cancer Ther. 2009; 8: 2103-2109Crossref PubMed Scopus (160) Google Scholar, 13Weeraratna A.T. Jiang Y. Hostetter G. Rosenblatt K. Duray P. Bittner M. Trent J.M. Cancer Cell. 2002; 1: 279-288Abstract Full Text Full Text PDF PubMed Scopus (773) Google Scholar). Evidently, an in depth mechanistic understanding of the PCP mechanism and its aberrant regulation in disease is required to control tumor progression and metastasis in a clinically advantageous manner (6Wang Y. Mol. Cancer Ther. 2009; 8: 2103-2109Crossref PubMed Scopus (160) Google Scholar).Human PTK7 pseudokinase (also known as colon carcinoma kinase-4, CCK-4) is required for PCP and CE (14Shnitsar I. Borchers A. Development. 2008; 135: 4015-4024Crossref PubMed Scopus (104) Google Scholar, 15Yen W.W. Williams M. Periasamy A. Conaway M. Burdsal C. Keller R. Lu X. Sutherland A. Development. 2009; 136: 2039-2048Crossref PubMed Scopus (97) Google Scholar, 16Lu X. Borchers A.G. Jolicoeur C. Rayburn H. Baker J.C. Tessier-Lavigne M. Nature. 2004; 430: 93-98Crossref PubMed Scopus (380) Google Scholar). The full-length membrane PTK7 receptor consists of seven extracellular Ig domains, a transmembrane region, and a catalytically inert cytoplasmic tyrosine kinase (PTK) domain (17Jung J.W. Ji A.R. Lee J. Kim U.J. Lee S.T. Biochim. Biophys. Acta. 2002; 1579: 153-163Crossref PubMed Scopus (32) Google Scholar, 18Jung J.W. Shin W.S. Song J. Lee S.T. 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An N-terminal, soluble PTK7 fragment (sPTK7) inhibited angiogenesis in vitro and in vivo in a dominant negative fashion by competing with the full-length PTK7 (22Shin W.S. Maeng Y.S. Jung J.W. Min J.K. Kwon Y.G. Lee S.T. Biochem. Biophys. Res. Commun. 2008; 371: 793-798Crossref PubMed Scopus (65) Google Scholar). The expression of PTK7 is frequently deregulated in cancers (4Katoh M. Oncol. Rep. 2005; 14: 1583-1588PubMed Google Scholar, 23Boudeau J. Miranda-Saavedra D. Barton G.J. Alessi D.R. Trends Cell Biol. 2006; 16: 443-452Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 24Müller-Tidow C. Schwäble J. Steffen B. Tidow N. Brandt B. Becker K. Schulze-Bahr E. Halfter H. Vogt U. Metzger R. Schneider P.M. Büchner T. Brandts C. Berdel W.E. Serve H. Clin. Cancer Res. 2004; 10: 1241-1249Crossref PubMed Scopus (97) Google Scholar, 25Easty D.J. Mitchell P.J. Patel K. Flørenes V.A. Spritz R.A. Bennett D.C. Int. J. Cancer. 1997; 71: 1061-1065Crossref PubMed Scopus (111) Google Scholar).Directional cell locomotion is highly dependent on both well orchestrated actin cytoskeleton dynamics and efficient pericellular proteolysis (26Zhu S. Liu L. Korzh V. Gong Z. Low B.C. Cell. Signal. 2006; 18: 359-372Crossref PubMed Scopus (92) Google Scholar, 27Coyle R.C. Latimer A. Jessen J.R. Exp. Cell Res. 2008; 314: 2150-2162Crossref PubMed Scopus (64) Google Scholar). Proinvasive, promigratory MT1-MMP (MMP-14), a prototypic member of the MMP family, is a major mediator of pericellular proteolytic events in cancer cells (28Barbolina M.V. Stack M.S. Semin. Cell Dev. Biol. 2008; 19: 24-33Crossref PubMed Scopus (117) Google Scholar). MT1-MMP cleaves ECM proteins, initiates activation of soluble MMPs, and controls the functionality of cell adhesion and signaling receptors. MT1-MMP is a prototypic member of a membrane-anchored MMP subfamily and is distinguished from soluble MMPs by a C-terminal transmembrane domain and a cytoplasmic tail (29Egeblad M. Werb Z. Nat. Rev. Cancer. 2002; 2: 161-174Crossref PubMed Scopus (5079) Google Scholar, 30Itoh Y. Seiki M. J. Cell. Physiol. 2006; 206: 1-8Crossref PubMed Scopus (413) Google Scholar, 31Seiki M. Cancer Lett. 2003; 194: 1-11Crossref PubMed Scopus (356) Google Scholar). MT1-MMP is synthesized as a latent zymogen that requires proteolytic processing of the N-terminal inhibitory prodomain (32Roghi C. Jones L. Gratian M. English W.R. Murphy G. FEBS J. 2010; 277: 3158-3175Crossref PubMed Scopus (29) Google Scholar, 33Golubkov V.S. Cieplak P. Chekanov A.V. Ratnikov B.I. Aleshin A.E. Golubkova N.V. Postnova T.I. Radichev I.A. Rozanov D.V. Zhu W. Motamedchaboki K. Strongin A.Y. J. Biol. Chem. 2010; 285: 27726-27736Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Once activated, MT1-MMP can be inhibited by its physiological inhibitors, tissue inhibitors of metalloproteinases-2, -3, and -4 (TIMP-2, -3, and -4). In contrast, TIMP-1 is a poor inhibitor of MT1-MMP (34Will H. Atkinson S.J. Butler G.S. Smith B. Murphy G. J. Biol. Chem. 1996; 271: 17119-17123Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar, 35Brew K. Nagase H. Biochim. Biophys. Acta. 2010; 1803: 55-71Crossref PubMed Scopus (875) Google Scholar).MT1-MMP, as opposed to the soluble MMPs, is ideally positioned to regulate pericellular proteolysis and the functionality of cell receptors (36Kessenbrock K. Plaks V. Werb Z. Cell. 2010; 141: 52-67Abstract Full Text Full Text PDF PubMed Scopus (3470) Google Scholar). In migrating cells, MT1-MMP accumulates predominantly at the leading and trailing edges and, as a result, contributes most efficiently to cell locomotion (37Bravo-Cordero J.J. Marrero-Diaz R. Megías D. Genís L. García-Grande A. García M.A. Arroyo A.G. Montoya M.C. EMBO J. 2007; 26: 1499-1510Crossref PubMed Scopus (192) Google Scholar, 38Wolf K. Friedl P. Clin. Exp. Metastasis. 2009; 26: 289-298Crossref PubMed Scopus (167) Google Scholar, 39Wolf K. Wu Y.I. Liu Y. Geiger J. Tam E. Overall C. Stack M.S. Friedl P. Nat. Cell Biol. 2007; 9: 893-904Crossref PubMed Scopus (753) Google Scholar).Knock-out of MT1-MMP has the most significant phenotype among MMP gene knock-out mice; MT1-MMP knock-out mice are dwarfs and die at adulthood (40Holmbeck K. Bianco P. Caterina J. Yamada S. Kromer M. Kuznetsov S.A. Mankani M. Robey P.G. Poole A.R. Pidoux I. Ward J.M. Birkedal-Hansen H. Cell. 1999; 99: 81-92Abstract Full Text Full Text PDF PubMed Scopus (1098) Google Scholar, 41Holmbeck K. Bianco P. Yamada S. Birkedal-Hansen H. J. Cell. Physiol. 2004; 200: 11-19Crossref PubMed Scopus (147) Google Scholar). Likewise, a loss of the structurally similar primordial At2-MMP induces dwarfism in Arabidopsis plants (42Golldack D. Popova O.V. Dietz K.J. J. Biol. Chem. 2002; 277: 5541-5547Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar).Recent studies link MT1-MMP to the non-canonical Wnt/PCP pathway in embryogenesis and cancer (27Coyle R.C. Latimer A. Jessen J.R. Exp. Cell Res. 2008; 314: 2150-2162Crossref PubMed Scopus (64) Google Scholar, 43Cantrell V.A. Jessen J.R. Cancer Lett. 2010; 287: 54-61Crossref PubMed Scopus (42) Google Scholar). Both transcriptional silencing and enforced overexpression of MT1-MMP negatively impacted CE and craniofacial morphogenesis in zebrafish, suggesting that a stringent control of MT1-MMP activity is essential in normal development (27Coyle R.C. Latimer A. Jessen J.R. Exp. Cell Res. 2008; 314: 2150-2162Crossref PubMed Scopus (64) Google Scholar, 44Paudyal A. Damrau C. Patterson V.L. Ermakov A. Formstone C. Lalanne Z. Wells S. Lu X. Norris D.P. Dean C.H. Henderson D.J. Murdoch J.N. BMC Dev. Biol. 2010; 10: 87Crossref PubMed Scopus (69) Google Scholar, 45Zhang J. Bai S. Zhang X. Nagase H. Sarras Jr., M.P. Matrix Biol. 2003; 22: 279-293Crossref PubMed Scopus (34) Google Scholar). The molecular mechanisms involved in the MT1-MMP-dependent regulation of the non-canonical Wnt/PCP signaling pathway, however, remain elusive. Intriguingly, co-expression data of 19,777 human and 21,036 mouse genes from the COEXPRESdb database indicate that MT1-MMP and PTK7 are closely co-expressed.Here, we provide evidence that the full-length membrane PTK7 affects actin cytoskeleton and inhibits cancer cell invasion. Our results demonstrate that MT1-MMP directly cleaves the full-length membrane PTK7, that this cleavage generates the sPTK7 species, and, most importantly, that this proteolytic event is ubiquitous in multiple cell systems. Taken together, our experimental data suggest that the pericellular proteolysis and PCP mechanisms converge in the regulation of directional cell migration and that they work in concert in processes as diverse as embryogenesis and malignancy.DISCUSSIONThe importance of receptor shedding by MT1-MMP is well documented in cancer (56Suenaga N. Mori H. Itoh Y. Seiki M. Oncogene. 2005; 24: 859-868Crossref PubMed Scopus (83) Google Scholar, 66Belkin A.M. Akimov S.S. Zaritskaya L.S. Ratnikov B.I. Deryugina E.I. Strongin A.Y. J. Biol. Chem. 2001; 276: 18415-18422Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 67Deryugina E.I. Ratnikov B.I. Postnova T.I. Rozanov D.V. Strongin A.Y. J. Biol. Chem. 2002; 277: 9749-9756Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 68Deryugina E.I. Ratnikov B.I. Yu Q. Baciu P.C. Rozanov D.V. Strongin A.Y. Traffic. 2004; 5: 627-641Crossref PubMed Scopus (41) Google Scholar, 69Kajita M. Itoh Y. Chiba T. Mori H. Okada A. 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Our subsequent studies confirmed that the full-length membrane PTK7 is most efficiently targeted by MT1-MMP, especially when compared with other receptors, including CD44. Cellular MT1-MMP functions as a principal sheddase of PTK7 and directly cleaves the exposed PKP621↓LI sequence of the seventh Ig-like domain of the full-length membrane PTK7. MT1-MMP proteolysis generates the C-terminal, membrane-tethered (50-kDa) and the N-terminal, soluble (70-kDa) fragments of PTK7. In turn, inactivation of the cleavage site generates the uncleavable PTK7 mutant (L622D) that is resistant to MT1-MMP proteolysis.Because PTK7 is an essential component of the Wnt/PCP pathway, we analyzed the effect of the full-length membrane PTK7 and sPTK7 on the actin cytoskeleton, a downstream target of the Wnt/PCP signaling. The precise molecular mechanism of PTK7 signaling leading to the regulation of the actomyosin contraction is not yet understood; however, either Dishevelled (Dsh) or plexins may be interacting partners of PTK7 (14Shnitsar I. Borchers A. Development. 2008; 135: 4015-4024Crossref PubMed Scopus (104) Google Scholar, 74Whitford K.L. Ghosh A. Neuron. 2001; 32: 1-3Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 75Winberg M.L. Tamagnone L. Bai J. Comoglio P.M. Montell D. Goodman C.S. Neuron. 2001; 32: 53-62Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar).The availability of the cells transfected with the full-length, soluble, and uncleavable L622D PTK7 constructs allowed us to demonstrate that PTK7 affects the downstream events of the Wnt/PCP pathway and that the membrane full-length PTK7 and especially the uncleavable L622D mutant reorganize the actin cytoskeleton, repress the MLC phosphorylation, alter the actomyosin contraction, and inhibit cancer cell invasion. MT1-MMP silencing recapitulates the effects that are observed in the cells with the enforced expression of the L622D PTK7 mutant. Consistently, MT1-MMP proteolysis reverses the effects of the full-length PTK7 on cell functions.The enforced expression of sPTK7 in HT1080 cells, however, significantly up-regulated RhoA activation, the upstream event of the Wnt/PCP pathway, rather than the downstream pMLC and actin reorganization. It is likely that the high preexisting levels of pMLC in the highly migratory HT1080 cells make any further increase of these parameters nearly impossible. As a result, the expression of MT1-MMP alone does not cause a noticeable effect on RhoA. In agreement, Rho-ROCK-myosin signaling mediates MT1-MMP-induced cellular aggregation of keratinocytes, but the overexpression of MT1-MMP itself does not result in a readily detectable increase of RhoA activation (76Dangi-Garimella S. Redig A.J. Shields M.A. Siddiqui M.A. Munshi H.G. J. Biol. Chem. 2010; 285: 28363-28372Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). MT1-MMP and Rho-ROCK activity and MLC phosphorylation were also demonstrated to play an important role in podosome formation and cell migration (16Lu X. Borchers A.G. Jolicoeur C. Rayburn H. Baker J.C. Tessier-Lavigne M. Nature. 2004; 430: 93-98Crossref PubMed Scopus (380) Google Scholar, 60Wyckoff J.B. Pinner S.E. Gschmeissner S. Condeelis J.S. Sahai E. Curr. 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Conversely, MT1-MMP silencing affected pMLC more noticeably, especially if compared with RhoA. Evidently, the effects of the multifunctional MT1-MMP on the net levels of pMLC and RhoA in the highly migratory HT1080 cells are more complex (80Lincoln T.M. Circ. Res. 2007; 100: 10-12Crossref PubMed Scopus (40) Google Scholar) than the MT1-MMP/PTK7 interactions alone and involve multiple parameters that are additional and distinct from PTK7.Because of the presence of the link between PTK7 and MT1-MMP that we detected in cancer cells, we investigated the role of the PTK7/MT1-MMP axis in embryogenesis using the zebrafish (D. rerio) developmental model. Our results suggest that zebrafish PTK7 is already expressed as early as 6 h postfertilization and that PTK7 is present at the right time during gastrulation to govern CE in the zebrafish embryo. Both transcriptional silencing of PTK7 and inhibition of MT1-MMP activity, either by small molecule inhibitors or by transcriptional silencing, led to characteristic CE abnormalities, including a short anterior-posterior body axis (dwarfism) and a wide lateral axis in the zebrafish. In agreement, subthreshold dosages of MT1-MMP and PTK7 morpholinos together caused a synergistic effect on the CE phenotype and the developmental abnormalities in zebrafish.Regardless of the opposing effect of PTK7 morpholino and small molecule MMP inhibitors on the levels of the full-length membrane PTK7, these treatments resulted in similar CE defects in the zebrafish embryos. It appears that both in cancer cells and normal embryogenesis, not the level of membrane PTK7 alone but also the well balanced ratio of the full-length PTK7 to sPTK7 plays an important role in regulating the overall effect of this pseudokinase on cell functions. In agreement, according to the findings of others, both overexpression and silencing of MT1-MMP resulted in a similar CE phenotype in zebrafish (27Coyle R.C. Latimer A. Jessen J.R. Exp. Cell Res. 2008; 314: 2150-2162Crossref PubMed Scopus (64) Google Scholar, 45Zhang J. Bai S. Zhang X. Nagase H. Sarras Jr., M.P. Matrix Biol. 2003; 22: 279-293Crossref PubMed Scopus (34) Google Scholar). Taken together, our data suggest that the MT1-MMP/PTK7 axis plays an important role in normal embryogenesis in the course of gastrulation in zebrafish.Our data in zebrafish correlate well with the role PTK7 plays in polarized cell motility and CE during mouse gastrulation (15Yen W.W. Williams M. Periasamy A. Conaway M. Burdsal C. Keller R. Lu X. Sutherland A. Development. 2009; 136: 2039-2048Crossref PubMed Scopus (97) Google Scholar). Thus, in embryos mutant for PTK7, the CE is severely affected. Although there is no alternative splicing in the murine PTK7 gene (18Jung J.W. Shin W.S. Song J. Lee S.T. Gene. 2004; 328: 75-84Crossref PubMed Scopus (34) Google Scholar), the presence of the full-length, 140-kDa PTK7 and a PTK7 fragment that is similar to the sPTK7 species was reported in mice (16Lu X. Borchers A.G. Jolicoeur C. Rayburn H. Baker J.C. Tessier-Lavigne M. Nature. 2004; 430: 93-98Crossref PubMed Scopus (380) Google Scholar). Most excitingly, very recently an N-ethyl-N-nitrosourea-induced mutant, named chuzhoi (chz), has been reported in mice (44Paudyal A. Damrau C. Patterson V.L. Ermakov A. Formstone C. Lalanne Z. Wells S. Lu X. Norris D.P. Dean C.H. Henderson D.J. Murdoch J.N. BMC Dev. Biol. 2010; 10: 87Crossref PubMed Scopus (69) Google Scholar). The chz mutation resulted in the insertion of three amino acids (Ala-Asn-Pro) into the junction region between the fifth and the sixth Ig-like domains of PTK7. The Ala-Asn-Pro insertion did not change the membrane PTK7 levels in mice but led to the degradation of the sPTK7 species. The resulting imbalance between membrane PTK7 and sPTK7 led to characteristics consistent with defective CE, including a shortened body axis and multiple defects in heart, lung, and inner ear development. These observations agree very well with our results that we generated in cancer cells and zebrafish.As a result, we believe that a fine balance between the protease and PTK7 is required for normal embryo development. In general, it is now highly likely that the MT1-MMP/PTK7 axis plays an essential role in embryogenesis in the course of gastrulation in vertebrates. Conversely, aberrations of the MT1-MMP/PTK7 axis seem to be the cause of abnormal CE during gastrulation in vertebrates.ConclusionsOverall, we established that the full-length PTK7 down-regulates myosin light chain phosphorylation, actin cytoskeleton organization, and actomyosin contraction (all downstream events in the Wnt/PCP pathway) and that it strongly inhibits cell invasion. PTK7 is a major cleavage target of MT1-MMP in the plasma membrane. MT1-MMP directly cleaves the PKP621↓LI sequence in an exposed region of PTK7, generating the N-terminal, soluble PTK7 ectodomain. The latter forms a complex with the full-length membrane PTK7. MT1-MMP proteolysis reverses the inhibitory action of the full-length PTK on cell locomotion. MT1-MMP silencing and the analysis of the uncleavable L622D PTK7 mutant also confirm the significance of MT1-MMP proteolysis of PTK7 in cell functions. Our novel data suggest that the MT1-MMP/PTK7 axis plays an important role in the regulation of the non-canonical Wnt/PCP pathway and polarized cell motility both in malignancy and vertebrate embryo development. Our results bring us a step closer to the development of selective therapeutics to target the MT1-MMP/PTK7 axis and the Wnt/PCP pathway in a clinically beneficial manner. 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MT1-MMP is a prototypic member of a membrane-anchored MMP subfamily and is distinguished from soluble MMPs by a C-terminal transmembrane domain and a cytoplasmic tail (29Egeblad M. Werb Z. Nat. Rev. Cancer. 2002; 2: 161-174Crossref PubMed Scopus (5079) Google Scholar, 30Itoh Y. Seiki M. J. Cell. Physiol. 2006; 206: 1-8Crossref PubMed Scopus (413) Google Scholar, 31Seiki M. Cancer Lett. 2003; 194: 1-11Crossref PubMed Scopus (356) Google Scholar). MT1-MMP is synthesized as a latent zymogen that requires proteolytic processing of the N-terminal inhibitory prodomain (32Roghi C. Jones L. Gratian M. English W.R. Murphy G. FEBS J. 2010; 277: 3158-3175Crossref PubMed Scopus (29) Google Scholar, 33Golubkov V.S. Cieplak P. Chekanov A.V. Ratnikov B.I. Aleshin A.E. Golubkova N.V. Postnova T.I. Radichev I.A. Rozanov D.V. Zhu W. Motamedchaboki K. Strongin A.Y. J. Biol. Chem. 2010; 285: 27726-27736Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Once activated, MT1-MMP can be inhibited by its physiological inhibitors, tissue inhibitors of metalloproteinases-2, -3, and -4 (TIMP-2, -3, and -4). In contrast, TIMP-1 is a poor inhibitor of MT1-MMP (34Will H. Atkinson S.J. Butler G.S. Smith B. Murphy G. J. Biol. Chem. 1996; 271: 17119-17123Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar, 35Brew K. Nagase H. Biochim. Biophys. Acta. 2010; 1803: 55-71Crossref PubMed Scopus (875) Google Scholar).MT1-MMP, as opposed to the soluble MMPs, is ideally positioned to regulate pericellular proteolysis and the functionality of cell receptors (36Kessenbrock K. Plaks V. Werb Z. Cell. 2010; 141: 52-67Abstract Full Text Full Text PDF PubMed Scopus (3470) Google Scholar). In migrating cells, MT1-MMP accumulates predominantly at the leading and trailing edges and, as a result, contributes most efficiently to cell locomotion (37Bravo-Cordero J.J. Marrero-Diaz R. Megías D. Genís L. García-Grande A. García M.A. Arroyo A.G. Montoya M.C. EMBO J. 2007; 26: 1499-1510Crossref PubMed Scopus (192) Google Scholar, 38Wolf K. Friedl P. Clin. Exp. Metastasis. 2009; 26: 289-298Crossref PubMed Scopus (167) Google Scholar, 39Wolf K. Wu Y.I. Liu Y. Geiger J. Tam E. Overall C. Stack M.S. Friedl P. Nat. Cell Biol. 2007; 9: 893-904Crossref PubMed Scopus (753) Google Scholar).Knock-out of MT1-MMP has the most significant phenotype among MMP gene knock-out mice; MT1-MMP knock-out mice are dwarfs and die at adulthood (40Holmbeck K. Bianco P. Caterina J. Yamada S. Kromer M. Kuznetsov S.A. Mankani M. Robey P.G. Poole A.R. Pidoux I. Ward J.M. Birkedal-Hansen H. Cell. 1999; 99: 81-92Abstract Full Text Full Text PDF PubMed Scopus (1098) Google Scholar, 41Holmbeck K. Bianco P. Yamada S. Birkedal-Hansen H. J. Cell. Physiol. 2004; 200: 11-19Crossref PubMed Scopus (147) Google Scholar). Likewise, a loss of the structurally similar primordial At2-MMP induces dwarfism in Arabidopsis plants (42Golldack D. Popova O.V. Dietz K.J. J. 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Nagase H. Sarras Jr., M.P. Matrix Biol. 2003; 22: 279-293Crossref PubMed Scopus (34) Google Scholar). The molecular mechanisms involved in the MT1-MMP-dependent regulation of the non-canonical Wnt/PCP signaling pathway, however, remain elusive. Intriguingly, co-expression data of 19,777 human and 21,036 mouse genes from the COEXPRESdb database indicate that MT1-MMP and PTK7 are closely co-expressed.Here, we provide evidence that the full-length membrane PTK7 affects actin cytoskeleton and inhibits cancer cell invasion. Our results demonstrate that MT1-MMP directly cleaves the full-length membrane PTK7, that this cleavage generates the sPTK7 species, and, most importantly, that this proteolytic event is ubiquitous in multiple cell systems. Taken together, our experimental data suggest that the pericellular proteolysis and PCP mechanisms converge in the regulation of directional cell migration and that they work in concert in processes as diverse as embryogenesis and malignancy.
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