Molecular Cloning of POEM

Cell adhesion molecules are involved in a number of biological functions, such as cell survival, cell differentiation, tissue repair, and development. A novel molecule, POEM (preosteoblast epidermal growth factor-like repeat protein with meprin, A5 protein, and receptor protein-tyrosine phosphatase μ domain), was isolated by reverse transcription-polymerase chain reaction using a set of degenerate primers designed after other known epidermal growth factor (EGF)-like motifs. From its structure, POEM was suggested to be a novel adhesion molecule with five EGF-like domains, an Arg-Gly-Asp (RGD) cell binding motif, and a meprin, A5 protein, and receptor protein-tyrosine phosphatase μ (MAM) domain. By in situhybridization using embryonic day 16.5 (E16.5) mouse embryos, strong expression of POEM mRNA was observed in developing kidney renal tubules, parathyroid and thyroid glands, developing bone, tooth germ, and endocrine organs of the brain. The inner ear, skeletal muscle, smooth muscle (except for the vascular system), and skin were also positive for POEM expression. Bacterial recombinant POEM protein containing the RGD sequence and MAM domain showed strong cell adhesion, spreading, and survival-promoting activities. By mutating the RGD sequence to RGE, the cell spreading and survival activities were significantly decreased, but the MAM domain was shown to contribute only to cell adhesion and not to cell spreading and survival-promoting activities. The distribution of POEM in several tissues was close to that of α8β1integrin. Therefore, we conducted cell adhesion assays using KA8 cells, a K562 leukemia clone stably expressing α8integrin. Parental K562 cells, which expressed α5β1 integrin, bound to fibronectin but not to POEM. On the other hand, KA8 cells showed strong binding and spreading on both fibronectin and POEM. These results suggest that POEM is a novel ligand for α8β1integrin and that POEM may be involved in the development and function of various tissues, such as kidney, bone, muscles, and endocrine organs. Cell adhesion molecules are involved in a number of biological functions, such as cell survival, cell differentiation, tissue repair, and development. A novel molecule, POEM (preosteoblast epidermal growth factor-like repeat protein with meprin, A5 protein, and receptor protein-tyrosine phosphatase μ domain), was isolated by reverse transcription-polymerase chain reaction using a set of degenerate primers designed after other known epidermal growth factor (EGF)-like motifs. From its structure, POEM was suggested to be a novel adhesion molecule with five EGF-like domains, an Arg-Gly-Asp (RGD) cell binding motif, and a meprin, A5 protein, and receptor protein-tyrosine phosphatase μ (MAM) domain. By in situhybridization using embryonic day 16.5 (E16.5) mouse embryos, strong expression of POEM mRNA was observed in developing kidney renal tubules, parathyroid and thyroid glands, developing bone, tooth germ, and endocrine organs of the brain. The inner ear, skeletal muscle, smooth muscle (except for the vascular system), and skin were also positive for POEM expression. Bacterial recombinant POEM protein containing the RGD sequence and MAM domain showed strong cell adhesion, spreading, and survival-promoting activities. By mutating the RGD sequence to RGE, the cell spreading and survival activities were significantly decreased, but the MAM domain was shown to contribute only to cell adhesion and not to cell spreading and survival-promoting activities. The distribution of POEM in several tissues was close to that of α8β1integrin. Therefore, we conducted cell adhesion assays using KA8 cells, a K562 leukemia clone stably expressing α8integrin. Parental K562 cells, which expressed α5β1 integrin, bound to fibronectin but not to POEM. On the other hand, KA8 cells showed strong binding and spreading on both fibronectin and POEM. These results suggest that POEM is a novel ligand for α8β1integrin and that POEM may be involved in the development and function of various tissues, such as kidney, bone, muscles, and endocrine organs. epidermal growth factor extracellular matrix polymerase chain reaction meprin, A5 protein, and receptor protein-tyrosine phosphatase μ phosphate-buffered saline fluorescence-activated cell-sorting maltose-binding protein preosteoblast epidermal growth factor-like repeat protein with meprin, A5 protein, and receptor protein-tyrosine phosphatase μ domain The epidermal growth factor (EGF)1-like repeat structure is present in a number of extracellular matrix (ECM) proteins and cell surface receptors (1Downing A.K. Knott V. Werner J.M. Cardy C.M. Campbell I.D. Handford P.A. Cell. 1996; 85: 597-605Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). Fibrillin (2Handford P.A. Biochim. Biophys. Acta. 2000; 1498: 84-90Crossref PubMed Scopus (70) Google Scholar) and laminin (3Engel J. Biochemistry. 1992; 31: 10643-10651Crossref PubMed Scopus (187) Google Scholar) are typical ECM proteins with multiple EGF-like repeats. Notch and Delta are cell surface molecules that act as a receptor and ligand, respectively, and interact via their EGF-like domains to determine cell fates (4Artavanis-Tsakonas S. Rand M.D. Lake R.J. Science. 1999; 284: 770-776Crossref PubMed Scopus (4874) Google Scholar). Transforming growth factor α (5Massague J. Pandiella A. Annu. Rev. Biochem. 1993; 62: 515-541Crossref PubMed Scopus (600) Google Scholar) and heparin-binding EGF-like growth factor (6Iwamoto R. Mekada E. Cytokine Growth Factor Rev. 2000; 11: 335-344Crossref PubMed Scopus (186) Google Scholar) are generated by cleavage of transmembrane precursors and act through the EGF receptor. Recently, small matrix proteins with several EGF-like repeat structures have been reported. For example, Pref-1 is produced as a membrane protein and controls adipocytic cell differentiation (7Smas C.M. Sul H.S. Cell. 1993; 73: 725-734Abstract Full Text PDF PubMed Scopus (561) Google Scholar). Del-1 is an adhesion molecule with an Arg-Gly-Asp (RGD) sequence and induces angiogenesis (8Penta K. Varner J.A. Liaw L. Hidai C. Schatzman R. Quertermous T. J. Biol. Chem. 1999; 274: 11101-11109Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). DANCE is another secreted molecule with an RGD cell binding motif; it is expressed predominantly in developing arteries and supposedly acts as a cell adhesion molecule in tissue development and repair (9Nakamura T. Ruiz-Lozano P. Lindner V. Yabe D. Taniwaki M. Furukawa Y. Kobuke K. Tashiro K. Lu Z. Andon N.L. Schaub R. Matsumori A. Sasayama S. Chien K.R. Honjo T. J. Biol. Chem. 1999; 274: 22476-22483Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Interactions between cells and ECM play roles in morphogenesis, tissue homeostasis, and remodeling. The ECM presents much information to the cells by working as a multifunctional ligand for cell adhesion receptors (10Adams J.C. Watt F.M. Giger R.J. Urquhart E.R. Gillespie S.K. Levengood D.V. Ginty D.D. Kolodkin A.L. Marchand P. Volkmann M. Bond J.S. Development. 1993; 117: 1183-1198Crossref PubMed Google Scholar, 11Damsky C.H. Moursi A. Zhou Y. Fisher S.J. Globus R.K. Marchand P. Volkmann M. Bond J.S. Kidney Int. 1997; 51: 1427-1433Abstract Full Text PDF PubMed Scopus (27) Google Scholar). Integrins are a large family of heterodimeric cell surface proteins that serve as receptors for various ECM proteins (12Giancotti F.G. Ruoslahti E. Engel J. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3794) Google Scholar, 13Plow E.F. Haas T.A. Zhang L. Loftus J. Smith J.W. Giancotti F.G. Ruoslahti E. Engel J. J. Biol. Chem. 2000; 275: 21785-21788Abstract Full Text Full Text PDF PubMed Scopus (1107) Google Scholar, 14Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8988) Google Scholar). Integrins are involved in tissue repair, development, and immune responses. Integrins also serve important functions in bone development and remodeling. Recently, it was reported that β1 integrin was significantly involved in osteoblastic function (15Zimmerman D. Jin F. Leboy P. Hardy S. Damsky C. Dev. Biol. 2000; 220: 2-15Crossref PubMed Scopus (117) Google Scholar). Expression of dominant negative β1 subunit in osteoblasts significantly reduced the bone-forming activity of osteoblasts. Matrix proteins produced by osteoblastic cells are the major target molecules not only for osteoblasts themselves but also for osteoclastic cells. Therefore, identification of a novel adhesion molecule produced by osteoblastic cells provides a new insight into the biology and development of bone tissue. In this study, we screened a cDNA library derived from preosteoblastic MC3T3-E1 cells and isolated a cDNA clone coding for a novel adhesion protein, POEM. A cell population enriched in osteoblasts was prepared from the calvaria of a 4-week-old C57BL/6 mouse as described previously (16Tezuka K. Takeshita S. Hakeda Y. Kumegawa M. Kikuno R. Hashimoto-Gotoh T. Biochem. Biophys. Res. Commun. 1990; 173: PubMed Scopus Google Scholar). a mouse cell was in cells were in KA8 cells, which the integrin subunit K. Biol. Cell. PubMed Scopus Google and K562 cells were by F. of KA8 cells were in containing and K562 cells were in the but the was with and were in a of in A of degenerate primers for polymerase chain reaction was designed on the in structures S. S. T. Dev. 2000; PubMed Scopus Google Scholar). was from the mRNA of MC3T3-E1 cells and by of a reverse The of of for for and for The were into and to by the with an A cDNA library was from the mRNA of MC3T3-E1 cells for by using a cDNA library A cDNA of a that was not in the was and to a MC3T3-E1 for cDNA library in The positive was to in of the as described by the A was with and sequence and a was with The sequence reported in this in the sequence with the number was prepared from kidney, and of 4-week-old In and and were also for The was prepared by the F.M. R. D.D. Smith J.A. K. in Scholar) with were from cells by using a mRNA The were in and to The were with and in by The of the was by with a and were by from mouse POEM cDNA with a The were and and were from and was from These and were in in in a and were for with and for with and In with and was as described previously with a In were with in phosphate-buffered saline for and with The were with with a of for and for in containing A cDNA as shown in from of POEM was by using primers and cDNA to of POEM was also using the and reverse were with and and to the sequence and mouse cDNA of expression by R. of to expression for proteins of POEM and A was into POEM cDNA by using a containing a The sequence of the was a negative was and MC3T3-E1 cells were with the expression by using to the of cells was with In the cells, the cells were with in and cell were a of ECM were from the cell with a small of after the cells were The MC3T3-E1 containing the cells and ECM proteins were with These were by were and with in saline for were with a The membrane was to with cells were with other POEM by using the cells were with and with in The cells were with containing and and with a a of for on cells were with were with a The of POEM cDNA the of POEM protein was into the expression to an expression for maltose-binding protein protein with a the RGD sequence to was by the of with the of POEM and proteins were produced by with The recombinant proteins were by and by after The protein was with a using as a were with of recombinant proteins with and for fibronectin was as a positive and and were as negative were with and for with a of in MC3T3-E1 cells were by in and in the for the were several with of the cells been of the negative cells were and with in and the cell number was as the number of a were in For the cell adhesion using K562 and KA8 cells, were with recombinant a of and as described These cells were a of for and observed a A of mouse integrin sequence to of number was using the and The reverse was conducted as described using cDNA of MC3T3-E1 cells as a The of of for for and for The were on a and with MC3T3-E1 is a cell with preosteoblastic generated a of degenerate primers on the EGF-like repeat structures in various ECM and receptor proteins S. S. T. Dev. 2000; PubMed Scopus Google Scholar). a cDNA of MC3T3-E1 for as a we a sequence was not in the By a MC3T3-E1 cDNA library with cDNA as a we isolated and By and we isolated a cDNA with an of The was to the sequence M. J. Cell Biol. PubMed Scopus Google Scholar). The sequence of this protein an which was to be a sequence J. S. 1997; PubMed Scopus Google but other that serve as transmembrane domains were that is a secreted five EGF-like repeat domains, a an RGD cell adhesion sequence motif, and a MAM domain were Therefore, we this protein POEM (preosteoblast EGF-like repeat protein with MAM The of the five EGF-like and MAM domain of POEM showed with other known as shown in and C. The EGF-like were the in the The MAM domain was and and and the sequence of MAM domains in other The POEM sequence showed to mouse number its number which a an of five EGF-like an RGD motif, and a MAM domain was the sequence and not the domain in POEM. Recently, we a of POEM in the J.C. M.D. R.J. Smith M. P. Zhang L. M. Zhang J. C. S. J. R.J. M. C. M. R. A. D. M. L. A. S. S. S. C. K. K. J. E. K. V. R. M. R. K. Z. P. K. C. F. Z. P. Z. Z. Y. Z. J. Y. Lu F. A.K. D. S. J. A. J. R. C. C. PubMed Scopus Google Scholar) that the with the of mouse POEM. From we that POEM and are molecules to In MC3T3-E1 cells into cells with osteoblastic insight into POEM we to the expression of POEM mRNA in MC3T3-E1 cells, mouse tissues, and mouse POEM mRNA was expressed the of MC3T3-E1 cells the expression as the differentiation Expression of POEM was also in osteoblastic cells and of 4-week-old by not In mouse development, expression of POEM mRNA be as as day of not and that POEM is involved in embryonic development as as in the function of bone and the expression of POEM in developing tissues, we in using of In the expression of POEM mRNA was observed in of the of the the of tubules, and the A and expression was also observed in both the and the the of the of tissue with the of POEM showed positive reaction In POEM expression was also observed in of the kidney not In the parathyroid expression of POEM mRNA was observed in the developing cells of cell in the expression was and tissue of showed of cells into the of structures were the The of POEM mRNA was to the developing cells and cells of the thyroid and A of POEM mRNA was also in the tooth POEM mRNA expression was in the expression was observed in the on the of the and its The of the was POEM expression was also observed in of the inner and in the In the such as the of the the of the bone, and osteoblasts were to the tissues as cells. expression of POEM mRNA was in osteoblasts the bone expression was observed in the cells, developing in both the of the and and the calvaria not In the of the in expression of POEM mRNA was both in osteoblasts on the bone surface and in cells the the of POEM mRNA was also in the cells the of the and In the of cells were cells and POEM mRNA was in of the developing cells to the of cells as in a in the and POEM was expressed in the skeletal the expression was the in the skeletal and the POEM expression was not in the In the POEM was not only in the muscle but also in the and In the muscle, POEM expression was also the in the the and the the muscle In the a of POEM was expressed and to the surface of the POEM expression not be observed in the POEM expression was in the smooth muscle as as in the skeletal In the POEM was expressed in the muscle of both the not and In the expression was observed in both the inner and muscle On the other hand, expression was in the inner muscle of the and the from the to the expression was in the muscle The smooth muscle the skin and the were also positive for POEM smooth muscle of arteries and showed expression of POEM The expression was not in the cells of In the expression of POEM was in the and POEM expression was in the cells of the A and and of the the of the In to expression was observed in the other of the In the were in the of the POEM expression was also observed in the cells in the and in a of the and the expression was in the cells the In of the of the POEM expression was in large cell small cell and their These large cell were as but it was to determine small cell were cells. In the POEM expression was in the in the in the and of the in the and in the of the In the POEM expression was to the cell and their of the In the of POEM was expressed in cell and their and from the to the expression was also in the In the expression of POEM mRNA was in the The expression was observed in the of the and the expression in the In the POEM expression was also observed in the and the expression was in the in the and In the POEM expression was in the cells the of the and In the expression was observed in the cells the developing not In the POEM mRNA was to in the and cells, were in POEM was expressed in cells present in the not In expression not be in the POEM expression was in the in the In POEM expression was not observed in the of the EGF-like proteins reported are extracellular we generated an expression for a protein of POEM and and and MC3T3-E1 cells with it for expression For of the of in cells, was on the cell and ECM of cells with the expression protein was in both cell and ECM protein was in that protein was on the cell surface in the of the RGD cell binding not to the a of the POEM protein to be as suggested by and of not expressed in MC3T3-E1 cells, be in the as observed for the cells. In the a of the protein showed a that in cells that was by in MC3T3-E1 cells. determine the cell binding domain of we using cells with expression for proteins protein was on the cell and the cell binding activity was not by the into the RGD the binding activity was significantly reduced by the of the MAM that this domain an important in the of POEM the cell the of the RGD and MAM domain in the cell adhesion and spreading we produced recombinant protein of POEM in E. and it by it as a by on was the of MC3T3-E1 cells to and on protein and fibronectin in a and the of the RGD in this cell adhesion we generated recombinant protein a of with a of the RGD by cell adhesion activity was observed with cell a cell spreading activity with the of the was to in the of MC3T3-E1 cells on the cells on showed an with These results that not only the MAM domain but also the RGD in protein an important in adhesion, spreading, and survival of MC3T3-E1 POEM to be a ligand for the integrin receptor on the The RGD is known as an that integrin subunit was expressed in MC3T3-E1 cells by reverse not the expression is with differentiation osteoblasts Globus R.K. Damsky C.H. J. Cell 1997; PubMed Google Scholar). expression is to that of POEM observed in MC3T3-E1 cells The mRNA expression of POEM in mouse was to that of integrin in E. J. PubMed Scopus Google Scholar). of POEM in kidney and bone tissues was to that of for as binding of phosphatase protein S. Biol. Cell. PubMed Scopus (162) Google Scholar). this we a cell adhesion using KA8 cells, a K562 clone stably with an integrin expression and expressing both α5β1 and K. Biol. Cell. PubMed Scopus Google Scholar). Parental K562 cells, which were as a only KA8 cells to both and fibronectin In K562 cells only to KA8 cells K562 cells to These suggest that POEM is a ligand for integrin, fibronectin is a for both α5β1 as reported previously K. Biol. Cell. PubMed Scopus Google Scholar, S. Biochemistry. PubMed Scopus Google Scholar). the of the integrin a number of biological functions of this molecule were reported. in showed in kidney D. S. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) and of inner cells A. 2000; PubMed Scopus Google Scholar). POEM to be expressed in kidney cells, in the and cells of the expression after and results that POEM is involved in kidney and Expression in the inner is another of POEM. molecules are expressed as of α and The subunit with β1 subunit E. J. PubMed Scopus Google Scholar). and have been shown to be for integrin K. Biol. Cell. PubMed Scopus Google S. Biol. Cell. PubMed Scopus (162) Google Scholar, J.M. D. R. J. Cell PubMed Google Scholar, K. R. C. M. Full Text PDF PubMed Scopus Google Scholar). binding of integrin to showed on the of the protein K. Biol. Cell. PubMed Scopus Google Scholar). S. Biochemistry. PubMed Scopus Google Scholar) generated a integrin phosphatase protein to for of of mouse with the phosphatase protein suggested the of several ligand molecules with from These also developing kidney and bone tissues with phosphatase and that of ligand molecules was to that of in a L. J.S. J.M. J. PubMed Google in not in kidney morphogenesis, the of other ligand molecules for α8β1integrin. the of POEM and the distribution of POEM mRNA are to of the ligand molecule suggested by using and that POEM is a novel ligand molecule for this by using KA8 cells expressing integrin, and we also that K562 cells, which α5β1 fibronectin not to POEM. Therefore, the binding of POEM to other not been POEM to have a binding activity from that of other ligand molecules for From we that POEM is of the important ligand molecules for By and POEM was to several domains, such as five EGF-like and a MAM as as an RGD integrin binding These structures are in secreted by and we that the of the POEM proteins were on the cell surface in the but not in the this was not by the of the RGD cell adhesion the that POEM may interact not only with integrin but also with other cell surface other other family receptor The showed that a molecule the MAM domain from the cell this molecule the MAM domain was in the in cell expressed in cells not is a that the of the MAM domain the structure of POEM protein, results suggest that the MAM domain a for cell surface of POEM as as the RGD in cells. been reported that the MAM domain meprin, A5 protein, and receptor protein-tyrosine cell adhesion activities via MAM domain Z. A. M. Marchand P. Volkmann M. Bond J.S. Full Text Full Text PDF PubMed Scopus Google Scholar, R.J. Urquhart E.R. Gillespie S.K. Levengood D.V. Ginty D.D. Kolodkin A.L. Marchand P. Volkmann M. Bond J.S. Full Text Full Text PDF PubMed Scopus Google Scholar, P. Volkmann M. Bond J.S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, F. M. T. Marchand P. Volkmann M. Bond J.S. Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the that MAM domain is involved in the cell surface binding via and molecules serve as receptor molecules for POEM. MC3T3-E1 cells bound to but K562 and KA8 cells These results also suggest that the binding molecule for the MAM domain of POEM is expressed in a cell The identification of this molecule to the function of POEM. In the developing mouse POEM was expressed in the endocrine organs thyroid and These endocrine organs are to bone and and In the mouse POEM mRNA showed a distribution in and the developing bone, in the tooth germ, and in These also suggest the between POEM and cells a number of ECM For example, and are after M. of Scholar). On the other hand, we that the expression of POEM was as osteoblastic cell differentiation The distribution of POEM expression in the mouse also suggested the of POEM in the of osteoblastic cell POEM an RGD cell adhesion motif, which is known to interact with E.F. Haas T.A. Zhang L. Loftus J. Smith J.W. Giancotti F.G. Ruoslahti E. Engel J. J. Biol. Chem. 2000; 275: 21785-21788Abstract Full Text Full Text PDF PubMed Scopus (1107) Google Scholar, 14Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8988) Google Scholar). In this study, we POEM to be a novel ligand molecule for α8β1integrin. In have been shown to play important roles by cell and bone activity P. S. of Scholar). POEM was expressed in preosteoblastic cells, which not to interact with osteoclastic have been shown to play roles not only in but also in osteoblasts (15Zimmerman D. Jin F. Leboy P. Hardy S. Damsky C. Dev. Biol. 2000; 220: 2-15Crossref PubMed Scopus (117) Google Scholar). in the β1 integrin subunit showed significantly bone-forming Moursi Globus R.K. Damsky C.H. J. Cell 1997; PubMed Google Scholar) also reported that such as and are for and Therefore, POEM may play important roles in osteoblastic function by survival via integrin and POEM is a novel of adhesion molecule with multiple motifs. Recently, a number of novel small EGF-like repeat proteins with RGD cell binding have been reported (8Penta K. Varner J.A. Liaw L. Hidai C. Schatzman R. Quertermous T. J. Biol. Chem. 1999; 274: 11101-11109Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, T. Ruiz-Lozano P. Lindner V. Yabe D. Taniwaki M. Furukawa Y. Kobuke K. Tashiro K. Lu Z. Andon N.L. Schaub R. Matsumori A. Sasayama S. Chien K.R. Honjo T. J. Biol. Chem. 1999; 274: 22476-22483Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, V. A. C. A. Dev. 2000; PubMed Scopus Google Scholar, A. 2000; PubMed Scopus Google Scholar). of molecules a tissue distribution and integrin binding of molecules to a of matrix proteins involved in tissue development and this was R. A. J. D. C. S. J. Cell Biol. Scopus (187) Google Scholar) also reported of a novel integrin ligand The sequence of was with that of POEM. that new into kidney and the development of other tissues expressing L. F. for KA8 and K562 cells, S. for and T. for also R. for the expression and we other of the for for and