Periostin, a Member of a Novel Family of Vitamin K-dependent Proteins, Is Expressed by Mesenchymal Stromal Cells

The modification of glutamic acid residues to γ-carboxyglutamic acid (Gla) is a post-translational modification catalyzed by the vitamin K-dependent enzyme γ-glutamylcarboxylase. Despite ubiquitous expression of the γ-carboxylation machinery in mammalian tissues, only 12 Gla-containing proteins have so far been identified in humans. Because bone tissue is the second most abundant source of Gla-containing proteins after the liver, we sought to identify Gla proteins secreted by bone marrow-derived mesenchymal stromal cells (MSCs). We used a proteomics approach to screen the secretome of MSCs with a combination of two-dimensional gel electrophoresis and tandem mass spectrometry. The most abundant Gla-containing protein secreted by MSCs was identified as periostin, a previously unrecognized γ-carboxylated protein. In silico amino acid sequence analysis of periostin demonstrated the presence of four consensus γ-carboxylase recognition sites embedded within fasciclin-like protein domains. The carboxylation of periostin was confirmed by immunoprecipitation and purification of the recombinant protein. Carboxylation of periostin could be inhibited by warfarin in MSCs, demonstrating its dependence on the presence of vitamin K. We were able to demonstrate localization of carboxylated periostin to bone nodules formed by MSCs in vitro, suggesting a role in extracellular matrix mineralization. Our data also show that another fasciclin I-like protein, βig-h3, contains Gla. In conclusion, periostin is a member of a novel vitamin K-dependent γ-carboxylated protein family characterized by the presence of fasciclin domains. Furthermore, carboxylated periostin is produced by bone-derived cells of mesenchymal lineage and is abundantly found in mineralized bone nodules in vitro. The modification of glutamic acid residues to γ-carboxyglutamic acid (Gla) is a post-translational modification catalyzed by the vitamin K-dependent enzyme γ-glutamylcarboxylase. Despite ubiquitous expression of the γ-carboxylation machinery in mammalian tissues, only 12 Gla-containing proteins have so far been identified in humans. Because bone tissue is the second most abundant source of Gla-containing proteins after the liver, we sought to identify Gla proteins secreted by bone marrow-derived mesenchymal stromal cells (MSCs). We used a proteomics approach to screen the secretome of MSCs with a combination of two-dimensional gel electrophoresis and tandem mass spectrometry. The most abundant Gla-containing protein secreted by MSCs was identified as periostin, a previously unrecognized γ-carboxylated protein. In silico amino acid sequence analysis of periostin demonstrated the presence of four consensus γ-carboxylase recognition sites embedded within fasciclin-like protein domains. The carboxylation of periostin was confirmed by immunoprecipitation and purification of the recombinant protein. Carboxylation of periostin could be inhibited by warfarin in MSCs, demonstrating its dependence on the presence of vitamin K. We were able to demonstrate localization of carboxylated periostin to bone nodules formed by MSCs in vitro, suggesting a role in extracellular matrix mineralization. Our data also show that another fasciclin I-like protein, βig-h3, contains Gla. In conclusion, periostin is a member of a novel vitamin K-dependent γ-carboxylated protein family characterized by the presence of fasciclin domains. Furthermore, carboxylated periostin is produced by bone-derived cells of mesenchymal lineage and is abundantly found in mineralized bone nodules in vitro. Bone marrow stroma contains a population of adherent cells referred to as mesenchymal stromal cells (MSCs), 4The abbreviations used are: MSC, marrow-derived mesenchymal stromal cells; Gla, γ-carboxyglutamic acid; CM, conditioned medium; ECM, extracellular matrix; PN, periostin; OC, osteocalcin; MGP, matrix Gla protein; MS, mass spectrometry; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; HPLC, high pressure liquid chromatography; CRS, carboxylase recognition site(s). which have the capacity to differentiate into many mesenchymal cell lineages, including osteoblasts, chondrocytes, and adipocytes (1Friedenstein A.J. Chailakhyan R.K. Latsinik N.V. Panasyuk A.F. Keiliss-Borok I.V. Transplantation. 1974; 17: 331-340Crossref PubMed Scopus (1126) Google Scholar, 2Clark B.R. Keating A. Ann. N. Y. Acad. Sci. 1995; 770: 70-78Crossref PubMed Scopus (174) Google Scholar, 3Pittenger M.F. Mackay A.M. Beck S.C. Jaiswal R.K. Douglas R. Mosca J.D. Moorman M.A. Simonetti D.W. Craig S. Marshak D.R. Science. 1999; 284: 143-147Crossref PubMed Scopus (18139) Google Scholar). To date, no single marker that is able to identify and localize MSCs in vivo has been described. MSCs are therefore identified in vitro based on their abilities to differentiate into mesenchymal lineage cells and express specific membrane-bound surface antigens that distinguish them from hematopoietic, endothelial, and fibroblastic cells (4Deans R.J. Moseley A.B. Exp. Hematol. 2000; 28: 875-884Abstract Full Text Full Text PDF PubMed Scopus (1319) Google Scholar, 5Horwitz E.M. Le Blanc K. Dominici M. Mueller I. Slaper-Cortenbach I. Marini F.C. Deans R.J. Krause D.S. Keating A. Cytotherapy. 2005; 7: 393-395Abstract Full Text Full Text PDF PubMed Scopus (1555) Google Scholar). It is widely accepted that one of the main biological functions of marrow MSCs is to provide the microenvironment necessary for support of hematopoiesis, but their role in development, maintenance, and repair of bone is also well established (6Prockop D.J. Science. 1997; 276: 71-74Crossref PubMed Scopus (4157) Google Scholar, 7Prockop D.J. Gregory C.A. Spees J.L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11917-11923Crossref PubMed Scopus (371) Google Scholar, 8Bruder S.P. Fink D.J. Caplan A.I. J. Cell Biochem. 1994; 56: 283-294Crossref PubMed Scopus (770) Google Scholar). It is also known that a subset of proteins post-translationally modified in a vitamin K-dependent manner with γ-carboxylated glutamic acid (Gla), such as osteocalcin (OC) and matrix Gla protein (MGP), play a pivotal role in normal bone development and repair (9Cranenburg E.C. Schurgers L.J. Vermeer C. Thromb. Haemost. 2007; 98: 120-125Crossref PubMed Scopus (190) Google Scholar). OC is the most abundant noncollagenous protein in bone, suggesting an important role in bone homeostasis. The physiologic role of the Gla-containing proteins in mineralized tissues is still a matter of debate. MGP is recognized as a potent inhibitor of the calcification of soft tissues, whereas OC is involved in the homeostatic regulation of bone mass (10Luo G. Ducy P. McKee M.D. Pinero G.J. Loyer E. Behringer R.R. Karsenty G. Nature. 1997; 386: 78-81Crossref PubMed Scopus (1764) Google Scholar, 11Ducy P. Desbois C. Boyce B. Pinero G. Story B. Dunstan C. Smith E. Bonadio J. Goldstein S. Gundberg C. Bradley A. Karsenty G. Nature. 1996; 382: 448-452Crossref PubMed Scopus (1405) Google Scholar). There is, however, little doubt that γ-carboxylation of these proteins and possibly others is essential for normal bone development, as is exemplified by the well described congenital bone defects observed in warfarin embryopathy (warfarin being a vitamin K antagonist blocking γ-carboxylation of proteins) (12Hall J.G. Pauli R.M. Wilson K.M. Am. J. Med. 1980; 68: 122-140Abstract Full Text PDF PubMed Scopus (766) Google Scholar). Since MSCs give rise to all nonhematopoietic cell lineages in the bone (stromal fibroblasts, osteocytes, chondrocytes, and adipocytes) and are also known to support hematopoiesis, we sought to identify which Gla-containing proteins are produced by these cells. To identify the Gla-containing proteins secreted by MSCs, we used a proteomic approach, which permitted an unbiased screening of the entire secretome of MSCs. Two-dimensional gel electrophoresis and tandem mass spectrometry (MS/MS) analysis unambiguously identified periostin (PN) as the most abundant Gla-containing protein secreted by MSCs. This was a surprising and unexpected finding, since periostin is not known as a Gla-containing protein. We further show that βig-h3, another fasciclin I-like protein highly homologous to periostin, is also carboxylated. Thus, periostin is a member of a new family of Gla-containing proteins characterized by tandem repeat fasciclin-like with carboxylase recognition sites embedded in and Cell used were as periostin also with and by The were from and used were from cells and cells were from the and from were as previously described N. J. S. J. 2005; PubMed Scopus Google Scholar). cells were in modified with and to for to a population being by cell the and was as described N. J. S. J. 2005; PubMed Scopus Google Scholar). was to the of γ-carboxylation was To the cells were in modified with and warfarin and MSCs from were from the for were and for The cells were and to the MSCs cells were on a tissue and to to in the presence of vitamin K. The cells were with and modified was was after being on an the were with with and in was the two-dimensional Two-dimensional and of protein in of with were in were in the and for for The was in and a was as by the of was were with with and with The was for with and and were on and with was the of the one of the was in acid and with by modified was on for and of gel and were and the were The were from the gel and to on and was an of was a and for with was a The was acid to acid in The used for mass spectrometry was a analysis was on the most from with for The used was an from to mass data were for from to a and with and were with of from the data a were with as of as as and and were from and were the on the of protein was with an for was with protein for The were in and for to the proteins and protein on were to and for were with and was by the in and the was by by the of periostin to a in the was a from S. G. C. M. R. R.M. Full Text Full Text PDF PubMed Scopus Google Scholar). cells were with the and were for cells were in the presence of vitamin and conditioned was and as of recombinant periostin was on an acid as by the of was to from cells and for The were and the was with of periostin was of by were into as mineralized nodules of the tissue was as the CM, the was and for with The was to and for and were and into on The has been described K. M. A. A.J. 7: PubMed Scopus Google Scholar). cells were with and with in being with the and were a and were of and and were with and and of of fasciclin which contains was used as the for and as well as for and on sequence the from to in the fasciclin was used as a for the and of periostin were the of M.A. A. R. A. 2000; PubMed Scopus Google Scholar). The with the was for further the and of the periostin were as G. J. PubMed Scopus Google Scholar). of Gla-containing by identify the γ-carboxylated proteins secreted by MSCs, we used a two-dimensional gel electrophoresis approach, which screening the entire We MSCs in the presence of vitamin K to and was were the one of them was to a and by an whereas the was The from the and the gel were and on the gel were and by The carboxylated protein by as well as a analysis and of periostin as a high with in for was and of the identified the identified most of the with This in the of was also found in with identified of the and a of Because of the in the was to and were This for the and The presence of periostin in the of these identified periostin, a previously unrecognized vitamin K-dependent protein, as the γ-carboxylated protein secreted by MSCs. 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MSCs were from and to in The cells were to lineages by with vitamin K. of was and was from one of the are in We observed that of periostin as to differentiate into adipocytes and in was no periostin expression could be the protein secreted periostin carboxylated in all produced by the secreted of the protein. It is known that carboxylated OC the extracellular matrix in Y. Biochem. PubMed Scopus Google Scholar, C. 1995; PubMed Google we that carboxylated periostin produced by be within the matrix and not be in the of cells. To we from and also the mineralized matrix the The proteins from the by were by This that are able to periostin but that the protein is within the in a To the presence of periostin in the mineralized matrix produced by osteoblasts, we analysis of bone by MSCs. we could periostin in the of cells and the nodules data demonstrate of periostin only in the mineralized matrix of bone nodules in vitro The extracellular matrix the nodules not for periostin It also be that cells the are embedded in a extracellular which for periostin within the and The of the observed for periostin on the bone is to the of the to the highly mineralized since within also to with the periostin only the surface of the also within to be of to periostin, we a of periostin based on its with the fasciclin protein, for which a is The is in the four fasciclin are in There is, however, one to the the and were as we the that to the Gla residues on periostin in the of the protein. The is still to the of the protein its modification by the carboxylase The that all four in periostin have a of a as in This is with the of the as by C. J.L. B. PubMed Scopus Google Scholar). the that all four are and with the This is since γ-carboxylation is known to be a post-translational modification as to a In Gla-containing the is to be the of the carboxylase and is to the Gla in to the The γ-carboxylated residues in all known Gla-containing proteins are found within amino of the Gla protein was previously the only Gla protein in which the sequence is not and the that residues of the also be carboxylated J. Full Text PDF PubMed Google Scholar). There is also that glutamic acid residues within the sequence be such as that of which contains a G. J. PubMed Scopus Google Scholar). we identified all glutamic acid residues in the periostin sequence that were within amino of the and of the four CRS, since these for The identified residues are in with their to the The residues are as in the There is a in the and of these glutamic acid residues in the fasciclin domains. Furthermore, no consensus could be found in the sequence of The of carboxylation sites from to in fasciclin and residues are whereas others within the protein the observed in the of the sites not to which residues are for and further be to identify of the glutamic acid residues that could be for carboxylation in of to in a new Carboxylation of I-like to the protein fasciclin I. It is characterized by the presence of four fasciclin I-like domains. Because we identified the in periostin within the fasciclin we for the presence of a in the known fasciclin I-like that all of these proteins a sequence within one of their fasciclin domains. We to the presence of Gla in βig-h3, a protein highly homologous to We cells which was and their secretome a as for periostin, by two-dimensional gel electrophoresis and analysis of identified We identified four that were for Gla, and in all four of them we the presence of This that fasciclin I-like proteins a novel family of vitamin γ-carboxylated for identified in the four in in a new We have demonstrated that marrow-derived MSCs periostin and that a subset of secreted protein is γ-carboxylated in a vitamin K-dependent This was not by is known to of There is a of that has the role of periostin in and is an protein expression is and is with bone, and J. A. R. M. A. Cell 2005; PubMed Scopus Google Scholar). It has been to and has been found to cell and Google Scholar, R. S. C. R.M. Cell PubMed Scopus Google Scholar). It is also highly in tissues and and its expression by cells is with a Y. I. 2007; Google Scholar). The periostin demonstrated a role for periostin in the of and J. A. R. M. A. Cell 2005; PubMed Scopus Google Scholar). has been in these that periostin an important role in the to Thromb. 2005; PubMed Scopus Google and these its role as a in the of the the of periostin in the is still has an important role in and J. J. M. M.A. A. J. J.D. 2007; PubMed Scopus Google Scholar, B. R.J. S. Keating Med. 2007; PubMed Scopus Google Scholar). The of periostin in development is to its role in cell and its periostin was described as in S. R. K. E. Biochem. J. PubMed Scopus Google is the of protein being to all in vitro to the functions of periostin have used recombinant periostin of γ-carboxylated residues to its being produced in cells vitamin K. the of periostin γ-carboxylation in vitro as well as in Since we only observed the periostin by we only of periostin as the J. K. J. Cell Biochem. PubMed Scopus Google are also carboxylated. these are produced by the also the and also be carboxylated. on B. 1999; PubMed Google the for a protein to carboxylation as the protein a to the the protein to the its the cells express the γ-carboxylase glutamic acid residues are in the acid to the and vitamin K is in the all of these are the presence of the is necessary and to carboxylation of the protein. we the amino acid sequence of periostin for the presence of the We found a sequence within the fasciclin of periostin that the consensus sequence of known This sequence was in all four fasciclin found within To is the Gla-containing protein identified that contains one all four are in periostin to be we that the four fasciclin are carboxylated the found in periostin fasciclin the which is one of the a fasciclin This to for the in the mammalian fasciclin-like and to the periostin were also found in all of these proteins and we confirmed that one of them was also carboxylated. This that all mammalian proteins fasciclin-like be a new family of γ-carboxylated It is also that in the of are known to in E. 2003; Google Scholar). The of periostin, to its that is produced by MSCs in one that is highly carboxylated and another that is carboxylated. The carboxylation of a glutamic acid an which only to the mass of the protein but has a on its J. Full Text PDF PubMed Google Scholar). It is, however, to the of Gla residues on a protein based on a of its since also its The of observed in periostin, however, from a to a to that periostin is with many Gla residues in a which could biological for highly and carboxylated of This could in vitro. The the with OC and MGP in of which a of the as an carboxylated in of vitamin K (9Cranenburg E.C. Schurgers L.J. Vermeer C. Thromb. Haemost. 2007; 98: 120-125Crossref PubMed Scopus (190) Google Scholar). 12 vitamin K-dependent Gla-containing proteins have so far been unambiguously identified in humans. and be the and to the of the Gla and these proteins be into four (9Cranenburg E.C. Schurgers L.J. Vermeer C. Thromb. Haemost. 2007; 98: 120-125Crossref PubMed Scopus (190) Google Scholar, B. 1999; PubMed Google Scholar, J.D. Proc. Natl. Acad. Sci. U. S. A. 98: PubMed Scopus Google Gla which are involved in and protein and protein Gla-containing proteins found in mineralized tissues, such as bone and and and Gla-containing proteins Gla proteins and and Gla proteins and in the Gla-containing protein to its known with development and into the extracellular to that with OC and This is by that carboxylated periostin with the matrix produced by and is in high on bone nodules in vitro. Gla OC and MGP, from the Gla-containing proteins by the that not the Gla in vitamin K-dependent OC and MGP and Gla MGP from OC and known Gla-containing proteins in that its sequence the is in the secreted protein, and Gla residues are in the and of its J.D. G. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google that the could be in a Gla-containing protein to carboxylation to and a possibly by MGP and and MGP are also Gla-containing protein in that not a their is not with OC and MGP demonstrated their role in the normal and of tissues Y. Biochem. PubMed Scopus Google Scholar, C. 1995; PubMed Google Scholar). MGP is known as one of the most potent of soft tissue (9Cranenburg E.C. Schurgers L.J. Vermeer C. Thromb. Haemost. 2007; 98: 120-125Crossref PubMed Scopus (190) Google Scholar). its of are still being The that is abundantly found in that not for to the to the Gla residues in MGP in the of the protein, with and its R. R. Thromb. Haemost. 2000; PubMed Scopus Google Scholar). on the to with the of to OC also in the protein. The known functions of OC of and of the to It is also a potent to and Gundberg PubMed Scopus Google Scholar). It is to the by periostin and these bone-derived Gla Our data for that periostin an for the mineralized matrix of the recombinant carboxylated of periostin that the carboxylated not the as to the This could be to a in the of the protein, a in the surface of the protein to the carboxylation could also to the periostin, osteocalcin and MGP C. 1995; PubMed Google Scholar, R. J. Full Text PDF PubMed Google is also known as an protein with and cell for many cell The of periostin important the protein. that the of all four were and that are all to This that periostin be the Gla-containing protein characterized to with one the that the of glutamic acid residues the is not the in all four fasciclin domains. We only on the of data are to the of γ-carboxylation in the of the role of fasciclin and the of the specific residues that are in with the of the protein, to the role of Gla in in Gla-containing the Gla residues are within the protein by these Gla residues a which in the proteins and to the in OC, the Gla residues are to and are of a to be involved in the with the of Gundberg PubMed Scopus Google Scholar). In to the carboxylation of periostin by MSCs was also observed in we its expression by MSCs. We also carboxylated periostin was produced by the and of MSCs. of MSCs in vitro of cells and in a cell we were able to from The MSCs as well as all of their of This could be for by the of since others have found to express periostin in vivo C. J. PubMed Scopus Google Scholar). Because of MSCs is in with high but cell the used have of the protein. we were not able to the carboxylated of periostin in the conditioned of osteoblasts, whereas the periostin secreted by normal MSCs and adipocytes was carboxylated. We carboxylated periostin could be found with the matrix of and found to be the periostin was found to be abundantly in bone in were cells are embedded in mineralized extracellular The data demonstrate that bone marrow-derived MSCs are a source of the of periostin in its expression from development to as well as its in bone and A. N. A. M. N. M. J. PubMed Scopus Google Scholar). Because MSCs a specific bone cell in bone development and repair J. Cell Biochem. PubMed Google Scholar, Scopus Google we that periostin is a in these MSCs are also known to be able to and to sites of tissue and the M.F. PubMed Scopus Google Scholar). In J. J. M. M.A. A. J. J.D. 2007; PubMed Scopus Google Scholar, B. R.J. S. Keating Med. 2007; PubMed Scopus Google periostin expression has been to be by mesenchymal cells in the after Thus, is that of these cells are MSCs that have to the and are in tissue the specific role of periostin in the is still a matter of debate. have that periostin show and after J. J. M. M.A. A. J. J.D. 2007; PubMed Scopus Google others have that of periostin after the and B. R.J. S. Keating Med. 2007; PubMed Scopus Google Scholar). In of the be important to the carboxylation of periostin these In all of these however, be to periostin by vitamin K In demonstrate that periostin is a novel γ-carboxylated protein that is highly by MSCs. is known to be an extracellular protein and is also known to with on the cell surface by its fasciclin Google Scholar, Y. I. 2007; Google Scholar). We that of fasciclin are the sites of γ-carboxylation and that modification Since periostin show and J. A. R. M. A. Cell 2005; PubMed Scopus Google Gla residues on periostin also provide that have an important γ-carboxylation of periostin could to is to important and is also in tissues and in cells. the is also in such as after and after It be important to the role of carboxylation of periostin on and since its carboxylation be by vitamin K such as We for and including two-dimensional and data was the and MSCs were by

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