The cell surface hyaluronidase TMEM2 regulates cell adhesion and migration via degradation of hyaluronan at focal adhesion sites

Abstract

The extracellular matrix (ECM) plays an important role in maintaining tissue homeostasis and poses a significant physical barrier to in vivo cell migration. Accordingly, as a means of enhancing tissue invasion, tumor cells use matrix metalloproteinases to degrade ECM proteins. However, the in vivo ECM is comprised not only of proteins but also of a variety of nonprotein components. Hyaluronan (HA), one of the most abundant nonprotein components of the interstitial ECM, forms a gel-like antiadhesive barrier that is impenetrable to particulate matter and cells. Mechanisms by which tumor cells penetrate the HA barrier have not been addressed. Here, we demonstrate that transmembrane protein 2 (TMEM2), the only known transmembrane hyaluronidase, is the predominant mediator of contact-dependent HA degradation and subsequent integrin-mediated cell–substrate adhesion. We show that a variety of tumor cells are able to eliminate substrate-bound HA in a tightly localized pattern corresponding to the distribution of focal adhesions (FAs) and stress fibers. This FA-targeted HA degradation is mediated by TMEM2, which itself is localized at site of FAs. TMEM2 depletion inhibits the ability of tumor cells to attach and migrate in an HA-rich environment. Importantly, TMEM2 directly binds at least two integrins via interaction between extracellular domains. Our findings demonstrate a critical role for TMEM2-mediated HA degradation in the adhesion and migration of cells on HA-rich ECM substrates and provide novel insight into the early phase of FA formation. The extracellular matrix (ECM) plays an important role in maintaining tissue homeostasis and poses a significant physical barrier to in vivo cell migration. Accordingly, as a means of enhancing tissue invasion, tumor cells use matrix metalloproteinases to degrade ECM proteins. However, the in vivo ECM is comprised not only of proteins but also of a variety of nonprotein components. Hyaluronan (HA), one of the most abundant nonprotein components of the interstitial ECM, forms a gel-like antiadhesive barrier that is impenetrable to particulate matter and cells. Mechanisms by which tumor cells penetrate the HA barrier have not been addressed. Here, we demonstrate that transmembrane protein 2 (TMEM2), the only known transmembrane hyaluronidase, is the predominant mediator of contact-dependent HA degradation and subsequent integrin-mediated cell–substrate adhesion. We show that a variety of tumor cells are able to eliminate substrate-bound HA in a tightly localized pattern corresponding to the distribution of focal adhesions (FAs) and stress fibers. This FA-targeted HA degradation is mediated by TMEM2, which itself is localized at site of FAs. TMEM2 depletion inhibits the ability of tumor cells to attach and migrate in an HA-rich environment. Importantly, TMEM2 directly binds at least two integrins via interaction between extracellular domains. Our findings demonstrate a critical role for TMEM2-mediated HA degradation in the adhesion and migration of cells on HA-rich ECM substrates and provide novel insight into the early phase of FA formation. Hyaluronan (HA) is a high–molecular weight polysaccharide belonging to the family of glycosaminoglycans. It is a long unbranched polymer composed of repeating disaccharide units of a N-acetylglucosamine and a glucuronic acid, with a molecular weight reaching as high as 107 Da (1Laurent T.C. Fraser J.R. Hyaluronan.FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2012) Google Scholar). Because of its highly hydrophilic nature, HA has an extremely large hydrodynamic volume in solution and assumes semiflexible secondary and tertiary conformations based on intramolecular and intermolecular hydrogen bonding (2Scott J.E. Heatley F. Hyaluronan forms specific stable tertiary structures in aqueous solution: A 13C NMR study.Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4850-4855Crossref PubMed Scopus (194) Google Scholar, 3Cowman M.K. Schmidt T.A. Raghavan P. Stecco A. Viscoelastic properties of hyaluronan in physiological conditions.F1000Res. 2015; 4: 622Crossref PubMed Scopus (114) Google Scholar). Furthermore, HA associates with a variety of matrix proteins and proteoglycans, such as aggrecan-type chondroitin sulfate proteoglycans and link proteins (4Day A.J. Prestwich G.D. Hyaluronan-binding proteins: Tying up the giant.J. Biol. Chem. 2002; 277: 4585-4588Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar), to form higher-order complexes of extracellular matrix (ECM) molecules. Because of these unique properties, HA exerts a profound influence on the biomechanical properties of the ECM. Most notably, HA forms a gel-like meshwork that is permeable to small molecules but impermeable to particulate matter, including cells (5Bard J.B. McBride W.H. Ross A.R. Morphology of hyaluronidase-sensitive cell coats as seen in the SEM after freeze-drying.J. Cell Sci. 1983; 62: 371-383Crossref PubMed Google Scholar, 6McBride W.H. Bard J.B. Hyaluronidase-sensitive halos around adherent cells. Their role in blocking lymphocyte-mediated cytolysis.J. Exp. Med. 1979; 149: 507-515Crossref PubMed Scopus (149) Google Scholar, 7Goldberg R.L. Toole B.P. Pericellular coat of chick embryo chondrocytes: Structural role of hyaluronate.J. Cell Biol. 1984; 99: 2114-2122Crossref PubMed Scopus (54) Google Scholar). While HA coated on a glass or plastic substrate can mediate weak attachment of cells that express HA receptors, such as CD44, high levels of HA in the extracellular and pericellular space act as a repulsive barrier to cell adhesion and migration. This antiadhesive effect is primarily because of steric exclusion by the thick, impermeable, and gel-like HA matrix, which is impenetrable to particles and cells, preventing the engagement of cell surface adhesion receptors with their ECM ligands (8Toole B.P. Hyaluronan in morphogenesis.Semin. Cell Dev. Biol. 2001; 12: 79-87Crossref PubMed Scopus (414) Google Scholar, 9Knudson W. The role of CD44 as a cell surface hyaluronan receptor during tumor invasion of connective tissue.Front. Biosci. 1998; 3: d604-d615Crossref PubMed Scopus (47) Google Scholar, 10Zimmerman E. Geiger B. Addadi L. Initial stages of cell-matrix adhesion can be mediated and modulated by cell-surface hyaluronan.Biophys. J. 2002; 82: 1848-1857Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). HA is a major factor in defining the biophysical and biological properties of the tumor microenvironment. HA accumulation is especially prominent in tumors that exhibit desmoplastic reactivity (11Voutouri C. Stylianopoulos T. Accumulation of mechanical forces in tumors is related to hyaluronan content and tissue stiffness.PLoS One. 2018; 13e0193801Crossref PubMed Scopus (22) Google Scholar, 12Provenzano P.P. Hingorani S.R. Hyaluronan, fluid pressure, and stromal resistance in pancreas cancer.Br. J. Cancer. 2013; 108: 1-8Crossref PubMed Scopus (187) Google Scholar). It has been shown that HA deposited in the stroma of adenocarcinomas is produced predominantly by stromal cells, rather than by neoplastic tumor cells (13Stern R. Hyaluronan metabolism: A major paradox in cancer biology.Pathol. Biol. (Paris). 2005; 53: 372-382Crossref PubMed Scopus (135) Google Scholar, 14Lipponen P. Aaltomaa S. Tammi R. Tammi M. Agren U. Kosma V.M. High stromal hyaluronan level is associated with poor differentiation and metastasis in prostate cancer.Eur. J. Cancer. 2001; 37: 849-856Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 15Aaltomaa S. Lipponen P. Tammi R. Tammi M. Viitanen J. Kankkunen J.P. Kosma V.M. Strong stromal hyaluronan expression is associated with PSA recurrence in local prostate cancer.Urol. Int. 2002; 69: 266-272Crossref PubMed Scopus (65) Google Scholar, 16Tammi R.H. Kultti A. Kosma V.M. Pirinen R. Auvinen P. Tammi M.I. Hyaluronan in human tumors: Pathobiological and prognostic messages from cell-associated and stromal hyaluronan.Semin. Cancer Biol. 2008; 18: 288-295Crossref PubMed Scopus (206) Google Scholar), whereas hyaluronidase activities are associated with the neoplastic cells (17Lokeshwar V.B. Rubinowicz D. Schroeder G.L. Forgacs E. Minna J.D. Block N.L. Nadji M. Lokeshwar B.L. Stromal and epithelial expression of tumor markers hyaluronic acid and hyaluronidase in prostate Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). on the antiadhesive properties of is to that tumor cells degradation of extracellular HA as a means of enhancing adhesion and a that tumor and The hyaluronidase of tumor cells is to their matrix ability to degrade protein components of the ECM as an means of the in a that is for tumor and the on HA degradation have on the family proteins (13Stern R. Hyaluronan metabolism: A major paradox in cancer biology.Pathol. Biol. (Paris). 2005; 53: 372-382Crossref PubMed Scopus (135) Google Scholar, R. Their and of PubMed Scopus Google Scholar). are that family proteins can be on the cell surface A. M.I. tumor is a cell-surface receptor for the protein of which Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar, B. C. J. M. B. is a PubMed Scopus Google Scholar), of that are primarily localized and in the of and W. is localized in and cell Full Text Full Text PDF PubMed Scopus Google Scholar, B. a human in cells, a hyaluronidase with a novel of Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, E. F. S. B. M. M. and of and its forms in PubMed Scopus Google Scholar). The for hyaluronidase B. a human in cells, a hyaluronidase with a novel of Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. R. and of human PubMed Scopus Google for is also with that of to a hyaluronidase that on the cell we have in that the transmembrane protein 2 is such a hyaluronidase T. F. C. A of the transmembrane protein 2 is the cell surface Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, F. A link in hyaluronan Biol. PubMed Scopus Google Scholar). Our that TMEM2 is a transmembrane with the hyaluronidase in its large extracellular TMEM2 high–molecular weight HA into as small as with a T. F. C. A of the transmembrane protein 2 is the cell surface Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). properties are with of a hyaluronidase that on the cell to the cell biological of TMEM2 in activities such as cell adhesion and migration. we show that a variety of tumor cells exhibit the ability to eliminate HA in a pattern to the distribution of focal adhesions (FAs) and stress and that FA-targeted HA degradation is mediated predominantly by TMEM2 depletion inhibits adhesion and migration of tumor cells in an HA-rich that are by the in the and of with the of hyaluronidase to the TMEM2 protein itself is at FA in tumor cells to Furthermore, we that TMEM2 directly with and Our findings demonstrate a critical role for TMEM2-mediated HA degradation in the adhesion and migration of cells on HA-rich ECM substrates and in provide novel insight into the early phase of FA formation. cell HA we have an in which cells are on a glass substrate with HA T. F. C. A of the transmembrane protein 2 is the cell surface Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). to as in HA degradation we HA in a variety of tumor cell human human human prostate and that express high levels of TMEM2 to and these tumor cells eliminate HA in a pattern that the distribution of and stress the cells of these cells for a for that the of HA degradation with The pattern of HA degradation the of during the of cell adhesion and the rather than of the degradation of of in cell 107 107 107 107 107 107 not by expression with by shown as from biological in a not by expression with by shown as from biological are or and TMEM2 is the only known cell surface hyaluronidase, TMEM2 to be for the in HA of these tumor cells. that is the we to TMEM2 and hyaluronidase proteins in cells and the of TMEM2 on in HA in levels by to of TMEM2 in HA degradation the of and effect on in HA degradation that TMEM2 is the hyaluronidase primarily for contact-dependent HA that TMEM2 hyaluronidase is in with at of in HA directly the of TMEM2 protein at these we cells TMEM2 to as depletion of human TMEM2 in these cells specific for human TMEM2, we their pattern of in HA cells exhibit a pattern of in HA degradation to that seen with cells of these cells for that exhibit with and of HA these demonstrate that TMEM2 is the not the hyaluronidase for HA degradation and a role for TMEM2 in FA or in the of cell adhesion on While the of integrin-mediated cell–substrate adhesion has been in the role of or antiadhesive ECM components in cell adhesion addressed. HA is to cell adhesion E. Geiger B. Addadi L. Initial stages of cell-matrix adhesion can be mediated and modulated by cell-surface hyaluronan.Biophys. J. 2002; 82: 1848-1857Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), has been that HA to be or to the of engagement between integrins and their matrix ligands M. D. Geiger B. Addadi L. and of in cell molecular to focal adhesion PubMed Scopus Google Scholar, R. M. Addadi L. Geiger B. of cell-matrix adhesion PubMed Scopus Google Scholar, B. and of matrix Biol. PubMed Scopus Google Scholar). However, molecular by which HA is by cells have been We that TMEM2 is the hyaluronidase that HA during the of cell adhesion to ECM we the adhesion of cells to substrates of and HA of molecular and for with or TMEM2 cells with to a substrate and a substrate a and However, adhesion of cells to the substrate is adhesion of these cells to the substrate is not and that TMEM2 expression is for adhesion to We the effect of the molecular of HA on cell adhesion. the TMEM2 and cells attach that weight HA has antiadhesive effect that the expression of TMEM2 is not to adhesion TMEM2 expression a significant for cell adhesion to substrates HA of molecular the TMEM2 depletion in than a in the of cells in with the the effect of TMEM2 depletion is than that on the but the of cells is by TMEM2 depletion of TMEM2 depletion on cell adhesion are in the cells by the expression of TMEM2, which not the This the of the we the effect of TMEM2 depletion on cell migration in a to a that an we the by of not the in the for the Cell migration into of the substrate is not between and cells in also in the migration into on the and substrates is in cells with cells and in also and in in the of cell expression of TMEM2 in human cells their ability to migrate into on the high–molecular weight HA substrates in the of TMEM2 and the of the these demonstrate that TMEM2 plays a critical role in cell adhesion and migration on HA a role in cell migration on substrates M. Mechanisms of cell Cell Biol. PubMed Scopus Google Scholar). We the to the of in HA degradation and FA by cells. cells exhibit in HA with cells at the of a for in these cells of these HA degradation cells, in of HA degradation are and the of is with cells for and of FA and stress in cells at the and We also in and cells on substrates of HA the and cells exhibit FA with significant in FA and the of and in the are in the distribution and of between and cells in of the of these cells on the and substrates exhibit an distribution of FA from to in the of in cells have than in The FA in cells is than that seen in cells on and substrates The of TMEM2 and the of the human TMEM2 are by the of FA by expression of TMEM2 in these that TMEM2 plays a role in FA on of TMEM2-mediated HA degradation at FA that TMEM2 with FA TMEM2 is a transmembrane we the that TMEM2 with proteins via its However, human cells to specific of TMEM2 with major including and focal adhesion directly the is for TMEM2-mediated in HA degradation to we an expression for TMEM2 that the the transmembrane and cells to as shown in cells exhibit a pattern of in HA degradation that is from that seen with cells Furthermore, in and cells, are localized at the of in HA degradation these demonstrate that the of TMEM2 is not for and in HA the of TMEM2 proteins to FA to be mediated by between their have been shown to with a variety of proteins via between between integrins and Cell Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, J. S. into the of The role of the extracellular Cell PubMed Scopus Google Scholar, R.L. A.R. B. extracellular between and protein Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. and with J. Cell Biol. PubMed Scopus Google Scholar, as Full Text PDF PubMed Scopus Google Scholar). We two to TMEM2 with a in cells. of TMEM2 and on the cell surface by the of cell surface proteins. cells with the and from cells with of with TMEM2 by with and shown in is with TMEM2, that TMEM2 and are in or in on the cell to TMEM2 and directly via their we produced TMEM2 in cells and its to human in a shown in is with with TMEM2 interaction between the of TMEM2 and to TMEM2 can also with we TMEM2 to the major in the of the between TMEM2 and the these demonstrate that TMEM2 binds directly to integrins via between the with the that interaction forms the for TMEM2 to FA on adhesion have on integrins and their protein ECM in vivo also large of glycosaminoglycans. HA is of because of its biophysical properties, and biological on cell adhesion and migration. The by which cells the of as a means of cell adhesion and we demonstrate that adherent cells degrade HA via the of the cell surface hyaluronidase TMEM2, and that is for cells to cell adhesion and migration on that TMEM2-mediated HA degradation with and that TMEM2 directly associates with that in HA degradation and FA are during the of cell adhesion. While tumor cell degradation of HA has been in tumor R. in cancer Cancer Biol. 2008; 18: PubMed Scopus Google Scholar, V.B. Lokeshwar B.L. Block N.L. of levels of hyaluronidase, a with prostate cancer Google Scholar), has been to the by which tumor cells degrade HA in the Our HA to cell HA activities T. F. C. A of the transmembrane protein 2 is the cell surface Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that tumor cells degrade substrate-bound HA in with FA show that contact-dependent in HA degradation is predominantly mediated by TMEM2, rather than by and This to be with the that TMEM2 is the only known transmembrane the role of TMEM2 in HA we have shown that TMEM2 proteins are with and with with is that a of proteins by J.R. of the focal adhesion a role for in of focal adhesion Cell Biol. PubMed Scopus Google has TMEM2 as one of the proteins in from these demonstrate that cells degrade HA at FA and that localized HA degradation is mediated predominantly by ECM degradation with has also been for the matrix matrix degradation at focal adhesions via by a Cell Biol. PubMed Scopus Google Scholar). matrix TMEM2 can be as an by cells to the ECM to adhesion and migration. of is that TMEM2 binds to via an interaction between the of the two proteins and We also that interaction is not to at least one also with TMEM2 via its The interaction with plays the role in the of TMEM2 to in cells. Furthermore, the that the can in HA degradation in the as TMEM2 that the extracellular interaction is to in HA degradation by are important proteins that with integrins via extracellular a and and between integrins and Cell Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, J. S. into the of The role of the extracellular Cell PubMed Scopus Google Scholar, R.L. A.R. B. extracellular between and protein Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. and with J. Cell Biol. PubMed Scopus Google Scholar, as Full Text PDF PubMed Scopus Google Scholar). we have not the in the interaction because of in of The effect of TMEM2 the of TMEM2 in cell adhesion and migration. the expression of TMEM2 is for cells to form adhesion to substrate high–molecular weight The by which TMEM2 is in FA is of in cells adhesion and migration on a for the role of TMEM2 in integrin-mediated cell based on the in The of the is that TMEM2 HA in the of integrin-mediated engagement and FA and important to be is TMEM2-mediated HA degradation or FA formation. are at TMEM2 has HA or is TMEM2 to The that TMEM2 FA that TMEM2 be a for FA formation. is that the a between these two be to of early adhesions or focal and TMEM2-mediated HA at these adhesion interaction and the of these adhesions into FAs. is that R. M. Addadi L. Geiger B. of cell-matrix adhesion PubMed Scopus Google in that are by early FAs. It is that TMEM2 is the in that the of to early and FAs. the of early cell adhesion and the role of TMEM2 in the to be via use of cell The cell biological of TMEM2 in has to HA is highly in tumor especially in by desmoplastic (11Voutouri C. Stylianopoulos T. Accumulation of mechanical forces in tumors is related to hyaluronan content and tissue stiffness.PLoS One. 2018; 13e0193801Crossref PubMed Scopus (22) Google Scholar, 12Provenzano P.P. Hingorani S.R. Hyaluronan, fluid pressure, and stromal resistance in pancreas cancer.Br. J. Cancer. 2013; 108: 1-8Crossref PubMed Scopus (187) Google Scholar, 16Tammi R.H. Kultti A. Kosma V.M. Pirinen R. Auvinen P. Tammi M.I. Hyaluronan in human tumors: Pathobiological and prognostic messages from cell-associated and stromal hyaluronan.Semin. Cancer Biol. 2008; 18: 288-295Crossref PubMed Scopus (206) Google Scholar, the tumor in hyaluronidase a PubMed Scopus Google Scholar, T. P. A of the tumor PubMed Scopus Google Scholar). tumor HA is produced predominantly by stromal cells, rather than by neoplastic tumor cells (13Stern R. Hyaluronan metabolism: A major paradox in cancer biology.Pathol. Biol. (Paris). 2005; 53: 372-382Crossref PubMed Scopus (135) Google Scholar, 14Lipponen P. Aaltomaa S. Tammi R. Tammi M. Agren U. Kosma V.M. High stromal hyaluronan level is associated with poor differentiation and metastasis in prostate cancer.Eur. J. Cancer. 2001; 37: 849-856Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 15Aaltomaa S. Lipponen P. Tammi R. Tammi M. Viitanen J. Kankkunen J.P. Kosma V.M. Strong stromal hyaluronan expression is associated with PSA recurrence in local prostate cancer.Urol. Int. 2002; 69: 266-272Crossref PubMed Scopus (65) Google Scholar, 16Tammi R.H. Kultti A. Kosma V.M. Pirinen R. Auvinen P. Tammi M.I. Hyaluronan in human tumors: Pathobiological and prognostic messages from cell-associated and stromal hyaluronan.Semin. Cancer Biol. 2008; 18: 288-295Crossref PubMed Scopus (206) Google Scholar), and ECM a for tumor cells P.P. Hingorani S.R. Hyaluronan, fluid pressure, and stromal resistance in pancreas cancer.Br. J. Cancer. 2013; 108: 1-8Crossref PubMed Scopus (187) Google Scholar, Geiger J. E. C. P. pericellular the from to cancer cell Cell Biol. PubMed Scopus Google Scholar). the tissue deposited in The demonstrate that TMEM2 is predominantly in the tumor of and prostate the that tumor cells use TMEM2 to degrade stromal the in a that is for their and is to that TMEM2 is one of the in human cancer expression is highly by a of tumor invasion, that TMEM2 expression with in TMEM2 is a that migration and invasion in PubMed Scopus Google Scholar). High TMEM2 expression is also with of with cancer are in of their of a in which TMEM2 is a in the of tumor expression for TMEM2 with at the as site between the and the site of TMEM2 in by the into T. F. C. A of the transmembrane protein 2 is the cell surface Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). expression for the TMEM2 by of to between and transmembrane expression for the of TMEM2 with by the from in T. F. C. A of the transmembrane protein 2 is the cell surface Biol. Chem. Full Text Full Text PDF PubMed Scopus Google of by The from human TMEM2 and of by by as The of in cells by expression of TMEM2, which not the The cell from prostate prostate cell TMEM2 and its by of the expression by and cell The for and for for of by to from human cells an and by The expression by a with the from TMEM2 and as in a T. F. C. A of the transmembrane protein 2 is the cell surface Biol. Chem. Full Text Full Text PDF PubMed Scopus Google for produced by of into and by of hyaluronidase expression in cells with cells to This as T. F. C. A of the transmembrane protein 2 is the cell surface Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). cells on coated with and for cells with for use in with in for at and with in solution and for at to cell cells with for and with for at by with in and secondary in with coated with only or a of and HA of molecular weight and glass with and with with for at with and with in for at with coated with in acid for 2 at with HA at and with at for cells the coated in at a of cells and for at in a with as Cell adhesion by cells. in biological and by with A to a on coated with and HA as in of to coated into and on the the with cells in of into the and for by the from and into with a migration or the of FA of cells with or as by in a in between two by cells and of by in biological and by with cells for in a coated with with cells with in for at by with and to with at by and with and with secondary with substrate or substrate by on or by with TMEM2 at the with a produced in cells, as T. F. C. A of the transmembrane protein 2 is the cell surface Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). to by for 2 at and the with of of to the and and in at by in and by and with or of as as are the and This The that have of with the content of We for and for the and C. and the F. and the F. A. and the and the F. and the This by of and the The content is the of the and not the of the of

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