VLA-5-mediated Adhesion to Fibronectin Accelerates Hemin-stimulated Erythroid Differentiation of K562 Cells through Induction of VLA-4 Expression

Abstract

Fibronectin plays important roles in erythropoiesis through the fibronectin receptors VLA-4 and VLA-5. However, the substantial role of these fibronectin receptors and their functional assignment in erythroid differentiation are not yet fully understood. Here, we investigated the effects of cell adhesion to fibronectin on erythroid differentiation using K562 human erythroid progenitor cells. Erythroid differentiation could be induced in K562 cells in suspension by stimulating with hemin. This hemin-stimulated erythroid differentiation was highly accelerated when cells were induced to adhere to fibronectin by treatment with TNIIIA2, a peptide derived from tenascin-C, which has recently been found to induce β1-integrin activation. Another integrin activator, Mn2+, also accelerated hemin-stimulated erythroid differentiation. Adhesive interaction with fibronectin via VLA-4 as well as VLA-5 was responsible for acceleration of the hemin-stimulated erythroid differentiation in response to TNIIIA2, although K562 cells should have been lacking in VLA-4. Adhesion to fibronectin forced by TNIIIA2 causally induced VLA-4 expression in K562 cells, and this was blocked by the RGD peptide, an antagonist for VLA-5. The resulting adhesive interaction with fibronectin via VLA-4 strongly enhanced the hemin-stimulated activation of p38 mitogen-activated protein kinase, which was shown to serve as a signaling molecule crucial for erythroid differentiation. Suppression of VLA-4 expression by RNA interference abrogated acceleration of hemin-stimulated erythroid differentiation in response to TNIIIA2. Thus, VLA-4 and VLA-5 may contribute to erythropoiesis at different stages of erythroid differentiation. Fibronectin plays important roles in erythropoiesis through the fibronectin receptors VLA-4 and VLA-5. However, the substantial role of these fibronectin receptors and their functional assignment in erythroid differentiation are not yet fully understood. Here, we investigated the effects of cell adhesion to fibronectin on erythroid differentiation using K562 human erythroid progenitor cells. Erythroid differentiation could be induced in K562 cells in suspension by stimulating with hemin. This hemin-stimulated erythroid differentiation was highly accelerated when cells were induced to adhere to fibronectin by treatment with TNIIIA2, a peptide derived from tenascin-C, which has recently been found to induce β1-integrin activation. Another integrin activator, Mn2+, also accelerated hemin-stimulated erythroid differentiation. Adhesive interaction with fibronectin via VLA-4 as well as VLA-5 was responsible for acceleration of the hemin-stimulated erythroid differentiation in response to TNIIIA2, although K562 cells should have been lacking in VLA-4. Adhesion to fibronectin forced by TNIIIA2 causally induced VLA-4 expression in K562 cells, and this was blocked by the RGD peptide, an antagonist for VLA-5. The resulting adhesive interaction with fibronectin via VLA-4 strongly enhanced the hemin-stimulated activation of p38 mitogen-activated protein kinase, which was shown to serve as a signaling molecule crucial for erythroid differentiation. Suppression of VLA-4 expression by RNA interference abrogated acceleration of hemin-stimulated erythroid differentiation in response to TNIIIA2. Thus, VLA-4 and VLA-5 may contribute to erythropoiesis at different stages of erythroid differentiation. Hematopoietic stem and progenitor cells proliferate and differentiate in the bone marrow and fetal liver (1Williams D.A. Pediatr. Res. 1994; 36: 557-560Crossref PubMed Scopus (6) Google Scholar, 2Clark B.R. Gallagher J.T. Dexter T.M. Baillieres Clin. Haematol. 1992; 5: 619-652Abstract Full Text PDF PubMed Scopus (85) Google Scholar, 3Coulombel L. Vuillet M.H. Leroy C. Tchernia G. Blood. 1988; 71: 329-334Crossref PubMed Google Scholar, 4Murti K.G. Brown P.S. Kumagai M. Campana D. Exp. Cell Res. 1996; 226: 47-58Crossref PubMed Scopus (40) Google Scholar, 5Williams D.A. Rios M. Stephens C. Patel V.P. Nature. 1991; 352: 438-441Crossref PubMed Scopus (450) Google Scholar, 6Wilson A. Trumpp A. Nat. Rev. Immunol. 2006; 6: 93-106Crossref PubMed Scopus (1074) Google Scholar). Stromal cells of the bone marrow and fetal liver form a hematopoietic microenvironment called a “niche.” This microenvironment niche plays a crucial role in the regulation of the proliferation and differentiation of hematopoietic stem and progenitor cells. Besides humoral factors that include hematopoietic growth factors, adhesive interaction of hematopoietic stem and progenitor cells with stromal cells and/or the extracellular matrix (ECM) 2The abbreviations used are: ECMextracellular matrixFNfibronectinFNIIIFN type III-likeTNtenascinMAPmitogen-activated proteinAbantibodyERKextracellular signal-regulated kinaseJNKc-Jun N-terminal kinasemAbmonoclonal antibodyMEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinaseo-dianisidine3,3-dimethoxybenzidineFITCfluorescein isothiocyanatesiRNAsmall interfering RNA. in the hematopoietic microenvironment is indispensable for hematopoietic development (1Williams D.A. Pediatr. Res. 1994; 36: 557-560Crossref PubMed Scopus (6) Google Scholar, 2Clark B.R. Gallagher J.T. Dexter T.M. Baillieres Clin. Haematol. 1992; 5: 619-652Abstract Full Text PDF PubMed Scopus (85) Google Scholar, 3Coulombel L. Vuillet M.H. Leroy C. Tchernia G. Blood. 1988; 71: 329-334Crossref PubMed Google Scholar, 4Murti K.G. Brown P.S. Kumagai M. Campana D. Exp. Cell Res. 1996; 226: 47-58Crossref PubMed Scopus (40) Google Scholar, 5Williams D.A. Rios M. Stephens C. Patel V.P. Nature. 1991; 352: 438-441Crossref PubMed Scopus (450) Google Scholar, 6Wilson A. Trumpp A. Nat. Rev. Immunol. 2006; 6: 93-106Crossref PubMed Scopus (1074) Google Scholar). The ECM in the hematopoietic microenvironment is composed of various macromolecules, such as fibronectin (FN), collagens, laminins, and proteoglycans. Among them, FN is one of the most important parts of the microenvironment niche (7Patel V.P. Lodish H.F. J. Cell Biol. 1987; 105: 3105-3118Crossref PubMed Scopus (128) Google Scholar, 8Vuillet-Gaugler M.H. Breton-Gorius J. Vainchenker W. Guichard J. Leroy C. Tchernia G. Coulombel L. Blood. 1990; 75: 865-873Crossref PubMed Google Scholar, 9Tada T. Widayati D.T. Fukuta K. Anat. Histol. Embryol. 2006; 35: 235-240Crossref PubMed Scopus (23) Google Scholar, 10Weinstein R. Riordan M.A. Wenc K. Kreczko S. Zhou M. Dainiak N. Blood. 1989; 73: 111-116Crossref PubMed Google Scholar, 11Eshghi S. Vogelezang M.G. Hynes R.O. Griffith L.G. Lodish H.F. J. Cell Biol. 2007; 177: 871-880Crossref PubMed Scopus (78) Google Scholar). Also, in erythropoiesis, the importance of the adhesion of erythroid progenitors to FN via the FN receptors VLA-4 and VLA-5 has been reported (11Eshghi S. Vogelezang M.G. Hynes R.O. Griffith L.G. Lodish H.F. J. Cell Biol. 2007; 177: 871-880Crossref PubMed Scopus (78) Google Scholar, 12Kapur R. Cooper R. Zhang L. Williams D.A. Blood. 2001; 97: 1975-1981Crossref PubMed Scopus (78) Google Scholar, 13Papayannopoulou T. Priestley G.V. Nakamoto B. Blood. 1998; 91: 2231-2239Crossref PubMed Google Scholar, 14van der Loo J.C. Xiao X. McMillin D. Hashino K. Kato I. Williams D.A. J. Clin. Invest. 1998; 102: 1051-1061Crossref PubMed Scopus (135) Google Scholar, 15Yanai N. Sekine C. Yagita H. Obinata M. Blood. 1994; 83: 2844-2850Crossref PubMed Google Scholar, 16Hamamura K. Matsuda H. Takeuchi Y. Habu S. Yagita H. Okumura K. Blood. 1996; 87: 2513-2517Crossref PubMed Google Scholar). However, the substantial role of these FN receptors and their functional assignment in erythroid differentiation are not yet fully understood. extracellular matrix fibronectin FN type III-like tenascin mitogen-activated protein antibody extracellular signal-regulated kinase c-Jun N-terminal kinase monoclonal antibody mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 3,3-dimethoxybenzidine fluorescein isothiocyanate small interfering RNA. We previously found that FN, which provides scaffolding for the adhesion of various cell types, has an alternative functional site opposing cell adhesion (17Fukai F. Takahashi H. Habu Y. Kubushiro N. Katayama T. Biochem. Biophys. Res. Commun. 1996; 220: 394-398Crossref PubMed Scopus (33) Google Scholar). A 22-mer peptide derived from the 14th FN type III-like (FNIII) repeat of the FN molecule, termed FNIII14, strongly suppresses cell adhesion to FN by inhibiting the activation of β1-integrins including VLA-4 and VLA-5 (18Fukai F. Hasebe S. Ueki M. Mutoh M. Ohgi C. Takahashi H. Takeda K. Katayama T. J. Biochem. 1997; 121: 189-192PubMed Google Scholar, 19Kamiya S. Kato R. Wakabayashi M. Tohyama T. Enami I. Ueki M. Yajima H. Ishii T. Nakamura H. Katayama T. Takagi J. Fukai F. Biochemistry. 2002; 41: 3270-3277Crossref PubMed Scopus (37) Google Scholar). Conversely, we have recently found that tenascin (TN)-C, which is an anti-adhesive ECM protein (20Erickson H.P. Curr. Opin. Cell Biol. 1993; 5: 869-876Crossref PubMed Scopus (360) Google Scholar, 21Fischer D. Brown-Lüdi M. Schulthess T. Chiquet-Ehrismann R. J. Cell Sci. 1997; 110: 1513-1522Crossref PubMed Google Scholar), has a functional site for stimulating cell adhesion to FN (22Saito Y. Imazeki H. Miura S. Yoshimura T. Okutsu H. Harada Y. Ohwaki T. Nagao O. Kamiya S. Hayashi R. Kodama H. Handa H. Yoshida T. Fukai F. J. Biol. Chem. 2007; 282: 34929-34937Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). A 22-mer peptide derived from the FNIII repeat A2 in the TN-C molecule, termed TNIIIA2, can induce the conformational change necessary for functional activation of FN receptors through binding with syndecan-4 (22Saito Y. Imazeki H. Miura S. Yoshimura T. Okutsu H. Harada Y. Ohwaki T. Nagao O. Kamiya S. Hayashi R. Kodama H. Handa H. Yoshida T. Fukai F. J. Biol. Chem. 2007; 282: 34929-34937Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 23Saito Y. Shiota Y. Nishisaka M. Owaki T. Shimamura M. Fukai F. Biol. Pharm. Bull. 2008; 31: 1003-1007Crossref PubMed Scopus (23) Google Scholar). The active sites of FNIII14 and TNIIIA2 appear to be cryptic in the molecular structures of FN and TN-C but are exposed by conformational change through interaction with other ECM molecules or by processing with matrix metalloproteinase-2 (22Saito Y. Imazeki H. Miura S. Yoshimura T. Okutsu H. Harada Y. Ohwaki T. Nagao O. Kamiya S. Hayashi R. Kodama H. Handa H. Yoshida T. Fukai F. J. Biol. Chem. 2007; 282: 34929-34937Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 24Watanabe K. Takahashi H. Habu Y. Kamiya-Kubushiro N. Kamiya S. Nakamura H. Yajima H. Ishii T. Katayama T. Miyazaki K. Fukai F. Biochemistry. 2000; 39: 7138-7144Crossref PubMed Scopus (48) Google Scholar). Thus, these functional sites found in FN and TN-C molecules, which act in opposition to their parental ECM proteins, may act as a negative feedback loop for preventing excessive cellular responses to these ECM proteins in biological processes with ECM rearrangement. In any case, FNIII14 and TNIIIA2 enable us to control, either negatively or positively, the adhesion of various cell types to FN. Various hematopoietic progenitor cell lines have been used in in vitro studies of hematopoietic differentiation. However, most hematopoietic progenitor cell lines are nonadherent, because their cell surface β1-integrins, including FN receptors, have impaired ligand-binding activity (25Davis G.E. Camarillo C.W. J. Immunol. 1993; 151: 7138-7150PubMed Google Scholar, 26Ni H. Li A. Simonsen N. Wilkins J.A. J. Biol. Chem. 1998; 273: 7981-7987Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Therefore, in order to investigate the role of cell adhesion to FN in hematopoietic differentiation, their FN receptors must be activated. Since TNIIIA2 can induce activation of FN receptors in various hematopoietic progenitor cell lines (22Saito Y. Imazeki H. Miura S. Yoshimura T. Okutsu H. Harada Y. Ohwaki T. Nagao O. Kamiya S. Hayashi R. Kodama H. Handa H. Yoshida T. Fukai F. J. Biol. Chem. 2007; 282: 34929-34937Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), this peptide factor may be useful for investigating the substantial role of cell adhesion to FN in hematopoietic differentiation. Here, we investigate the effects of cell adhesion to FN on erythroid differentiation using TNIIIA2 and Mn2+ as the integrin activator and the human erythroid progenitor cell line K562, which only expresses VLA-5, as the FN receptor (27Elices M.J. Osborn L. Takada Y. Crouse C. Luhowskyj S. Hemler 1990; Full Text PDF PubMed Scopus Google Scholar). a we that hemin-stimulated erythroid differentiation of K562 cells is strongly enhanced when K562 cells are forced to adhere to FN. adhesion to FN via VLA-5, which is induced by TNIIIA2 or Mn2+, of VLA-4 The resulting adhesive interaction with FN via VLA-4 a in the hemin-stimulated of p38 kinase, which is shown to serve as a signaling molecule crucial for erythroid differentiation of K562 cells. such as RGD TNIIIA2 and (22Saito Y. Imazeki H. Miura S. Yoshimura T. Okutsu H. Harada Y. Ohwaki T. Nagao O. Kamiya S. Hayashi R. Kodama H. Handa H. Yoshida T. Fukai F. J. Biol. Chem. 2007; 282: 34929-34937Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), and FNIII14 S. Kato R. Wakabayashi M. Tohyama T. Enami I. Ueki M. Yajima H. Ishii T. Nakamura H. Katayama T. Takagi J. Fukai F. Biochemistry. 2002; 41: 3270-3277Crossref PubMed Scopus (37) Google and S. Kato R. Wakabayashi M. Tohyama T. Enami I. Ueki M. Yajima H. Ishii T. Nakamura H. Katayama T. Takagi J. Fukai F. Biochemistry. 2002; 41: 3270-3277Crossref PubMed Scopus (37) Google were from to and were from Cell monoclonal integrin and were from A the active of integrin J. T. Takada Y. Y. J. Biochem. 1997; 121: PubMed Scopus Google Scholar), was from for and and were from A of was from and 3,3-dimethoxybenzidine were from K562 cells were in fetal and were to induce erythroid differentiation by in fetal with in the or of various factors to be for the cells were and with a and at for to cells N. M. S. H. T. Hayashi N. M. T. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). cells were at in different as cells. The of cells in the was the of the of cells to the of cells. The cell adhesion was as previously (22Saito Y. Imazeki H. Miura S. Yoshimura T. Okutsu H. Harada Y. Ohwaki T. Nagao O. Kamiya S. Hayashi R. Kodama H. Handa H. Yoshida T. Fukai F. J. Biol. Chem. 2007; 282: 34929-34937Abstract Full Text Full Text PDF PubMed Scopus (76) Google with cells in with or Mn2+ FNIII14 or TNIIIA2 or and their were in a with FN in the or of for VLA-4 and VLA-5, as were for at and with The of cells the well was at in different the cells, the cells in the were the of the of cells to the of cells the The activation of β1-integrins on the cell surface was by as previously (22Saito Y. Imazeki H. Miura S. Yoshimura T. Okutsu H. Harada Y. Ohwaki T. Nagao O. Kamiya S. Hayashi R. Kodama H. Handa H. Yoshida T. Fukai F. J. Biol. Chem. 2007; 282: 34929-34937Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), using which an active J. T. Takada Y. Y. J. Biochem. 1997; 121: PubMed Scopus Google at for the expression of integrin erythroid differentiation, cell suspension with or TNIIIA2 was in the or of an antagonist for FN for the cells were with with or and with The of cells was by to of cells in the were in and and by using enhanced as previously (22Saito Y. Imazeki H. Miura S. Yoshimura T. Okutsu H. Harada Y. Ohwaki T. Nagao O. Kamiya S. Hayashi R. Kodama H. Handa H. Yoshida T. Fukai F. J. Biol. Chem. 2007; 282: 34929-34937Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). of integrin p38 and the negative were as The were cells by using and the cells were used for K562 cells are of to FN, because of their (22Saito Y. Imazeki H. Miura S. Yoshimura T. Okutsu H. Harada Y. Ohwaki T. Nagao O. Kamiya S. Hayashi R. Kodama H. Handa H. Yoshida T. Fukai F. J. Biol. Chem. 2007; 282: 34929-34937Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, G.E. Camarillo C.W. J. Immunol. 1993; 151: 7138-7150PubMed Google Scholar, 26Ni H. Li A. Simonsen N. Wilkins J.A. J. Biol. Chem. 1998; 273: 7981-7987Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, M.J. Osborn L. Takada Y. Crouse C. Luhowskyj S. Hemler 1990; Full Text PDF PubMed Scopus Google Scholar). these cells were on the FN with Mn2+, an integrin activator (25Davis G.E. Camarillo C.W. J. Immunol. 1993; 151: 7138-7150PubMed Google Scholar, 26Ni H. Li A. Simonsen N. Wilkins J.A. J. Biol. Chem. 1998; 273: 7981-7987Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), the cells to FN FNIII14 S. Kato R. Wakabayashi M. Tohyama T. Enami I. Ueki M. Yajima H. Ishii T. Nakamura H. Katayama T. Takagi J. Fukai F. Biochemistry. 2002; 41: 3270-3277Crossref PubMed Scopus (37) Google this K562 cell adhesion to FN, which has a FNIII14 S. Kato R. Wakabayashi M. Tohyama T. Enami I. Ueki M. Yajima H. Ishii T. Nakamura H. Katayama T. Takagi J. Fukai F. Biochemistry. 2002; 41: 3270-3277Crossref PubMed Scopus (37) Google Scholar), not Conversely, TNIIIA2 induced the adhesion of K562 cells to FN Mn2+ in a in which the active of TNIIIA2 is (22Saito Y. Imazeki H. Miura S. Yoshimura T. Okutsu H. Harada Y. Ohwaki T. Nagao O. Kamiya S. Hayashi R. Kodama H. Handa H. Yoshida T. Fukai F. J. Biol. Chem. 2007; 282: 34929-34937Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), activity negative and of K562 cell adhesion to FN by these were to their effects on the activation of active of which can be by J. T. Takada Y. Y. J. Biochem. 1997; 121: PubMed Scopus Google Scholar), was exposed by cells with TNIIIA2 as well as Mn2+ but by treatment with FNIII14 and adhesion of K562 cells to FN was by the of VLA-5 and RGD peptide but not by the of VLA-4 and peptide most hematopoietic progenitor cell K562 cells can in suspension to the the of adhesion to FN on cellular differentiation of K562 cells, K562 cells were with in the or of TNIIIA2. Erythroid differentiation was by the cells K562 cells were with in the of TNIIIA2, of cells for A and with at not a in the of cells but the of K562 cells not cells the with hemin. erythroid differentiation of K562 cells was with in the of TNIIIA2, in which cells were induced to adhere to the FN in response to TNIIIA2. a a of cells for only and of cells were for A and the most cells to the FN that TNIIIA2 is of the FN receptor for a TNIIIA2 accelerated hemin-stimulated erythroid differentiation in a which activity could not hemin-stimulated differentiation Mn2+, which induced K562 cell adhesion to FN was of hemin-stimulated erythroid differentiation to a TNIIIA2 of cells with TNIIIA2 induced adhesion to FN but not in K562 cells the other FNIII14, which was of forced adhesion of K562 cells induced by Mn2+ or TNIIIA2 on the hemin-stimulated erythroid differentiation of K562 cells in suspension However, accelerated differentiation of K562 cells, which was by in with TNIIIA2, was by the of FNIII14, on This in erythroid differentiation by the of FNIII14 was by a in the of cells to FN not Thus, acceleration of hemin-stimulated erythroid differentiation in response to TNIIIA2 from adhesion of K562 cells to FN. We investigated VLA-5, the only FN receptor on K562 cells, to the acceleration of hemin-stimulated erythroid differentiation in response to TNIIIA2. acceleration of hemin-stimulated erythroid differentiation in response to TNIIIA2 was by the of the RGD peptide, a VLA-5 antagonist in which adhesion of K562 cells was also not acceleration of hemin-stimulated erythroid differentiation in response to TNIIIA2 was also with the peptide, a VLA-4 antagonist although K562 cells should have been lacking in VLA-4 (27Elices M.J. Osborn L. Takada Y. Crouse C. Luhowskyj S. Hemler 1990; Full Text PDF PubMed Scopus Google Scholar). We the expression of integrin and their erythroid differentiation of K562 cells by with (27Elices M.J. Osborn L. Takada Y. Crouse C. Luhowskyj S. Hemler 1990; Full Text PDF PubMed Scopus Google Scholar), K562 cells highly integrin and not but integrin in the expression of and and were the hemin-stimulated erythroid differentiation of K562 cells in erythroid differentiation was by adhesive the of TNIIIA2 or for Mn2+ and for and the for Mn2+ and for of the expression was in K562 cells with TNIIIA2 this was not by A and of the expression was when erythroid differentiation was by and TNIIIA2 in the of the RGD peptide In the peptide not of the expression although abrogated acceleration of hemin-stimulated erythroid differentiation in response to TNIIIA2 The integrin was not on K562 cells either or the with and TNIIIA2 not that integrin is as VLA-4 on K562 cell the of VLA-4 expression in the acceleration of hemin-stimulated erythroid differentiation in response to TNIIIA2, K562 cells with or integrin were to for erythroid differentiation. erythroid differentiation was not by with acceleration in response to TNIIIA2 was by with In these cells, of expression was strongly K562 cells with in which of the expression was erythroid differentiation in response to with or TNIIIA2 that of VLA-4 expression is for the acceleration of hemin-stimulated erythroid differentiation of K562 cells. of the kinase such as and are in signaling for erythropoiesis O. K. A. Blood. 2000; PubMed Google Scholar, P.S. Biol. 1997; PubMed Scopus Google Scholar, Y. Takahashi N. K. Blood. 1998; PubMed Google Scholar). the kinase in erythroid differentiation of K562 cells, we investigated of the erythroid differentiation, as by using for in their hemin-stimulated erythroid differentiation was in K562 cells the not In a in p38 was in cells with with TNIIIA2 not the of with in with TNIIIA2 in a in p38 This in p38 was by cells with the peptide the of VLA-4 signaling in the in p38 of integrin expression also blocked the in p38 The RGD peptide also the of p38 to of VLA-4 expression TNIIIA2, Mn2+ the hemin-stimulated of to a TNIIIA2 not The of p38 in hemin-stimulated erythroid differentiation and acceleration was by of K562 cells were with or p38 of p38 was by p38 but by of p38 expression by p38 in of not only acceleration of the hemin-stimulated erythroid differentiation in response to TNIIIA2 but also the differentiation by with not erythroid differentiation of K562 cells by with or TNIIIA2. In with these of K562 cells with a of but not with and of and erythroid differentiation to the In the we found that erythroid differentiation by was enhanced when K562 cells were induced to adhere to FN by VLA-5 with TNIIIA2 Since Mn2+, integrin activator, was also of hemin-stimulated erythroid differentiation and acceleration of hemin-stimulated erythroid differentiation was by of adhesion to FN and is that acceleration of the hemin-stimulated erythroid differentiation is on adhesion of K562 cells to FN. This acceleration of erythroid differentiation was abrogated by not only a VLA-5 antagonist but also a VLA-4 antagonist although K562 cells should have been lacking in VLA-4. This was by that adhesion to FN via VLA-5 causally induced VLA-4 expression in K562 cells In this of VLA-4 expression was with the RGD peptide, a VLA-5 antagonist Since acceleration of erythroid differentiation was blocked with the peptide and also by of expression is that the induced VLA-4 plays an indispensable role in the acceleration of erythroid differentiation. VLA-4 was shown to be in a in p38 as Thus, VLA-5 and VLA-4 were shown to contribute to erythropoiesis at different stages of erythroid VLA-5 the of VLA-4 and the resulting VLA-4 the activation of a signaling molecule for erythroid differentiation. studies that activation of p38 and/or but not is for erythroid differentiation induced by O. K. A. Blood. 2000; PubMed Google Scholar), Y. Takahashi N. K. Blood. 1998; PubMed Google Scholar), Cell 2001; Google Scholar), or R. L. S. A. J. Biochem. 2007; PubMed Scopus Google Scholar). In the of p38 and using the of p38 that the of p38 plays a role in the hemin-stimulated erythroid differentiation of K562 cells. induced adhesion of K562 cells to FN with TNIIIA2 or Mn2+ the of p38 and this of p38 was by of expression The that adhesion is responsible for a in p38 However, with TNIIIA2 or Mn2+ was of p38 activation and also cell adhesion to FN is not for erythroid differentiation of K562 cells. adhesion to not induce erythroid differentiation. is for an of the molecular by which adhesion to FN can a activation of p38 in with hemin. to the of integrin expression differentiation of K562 cells, J. Biol. Chem. 1992; Full Text PDF PubMed Google reported that differentiation is by expression of integrin a In to the erythroid differentiation of integrin expression to the of the to adhere to of the signaling via the kinase to sites in the integrin X. Blood. PubMed Google Scholar). of the kinase with a the expression of integrin in cells induced to TN-C is highly by the stromal cells of the hematopoietic or in the microenvironment the hematopoietic cells M. M. A. L. S. A. F. S. G. G. J. 1993; Google Scholar, G. J. R. A. J. 1993; 41: PubMed Scopus (33) Google Scholar, G. S. J. Cell Biol. 1993; PubMed Scopus Google Scholar). a of TN-C that their for the of the of hematopoietic progenitor cells and of hematopoietic cells in bone marrow of are in of M. T. Y. S. M. Blood. 1998; 91: Google Scholar). Thus, TN-C is to a role in to the role of TNIIIA2 in erythroid differentiation. the other have been studies that the ECM proteins, including have active cryptic G.E. M.J. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). sites can be exposed through biological such as conformational change in response to interaction with cells or other ECM and processing with the that are by cells of hematopoietic including hematopoietic stromal cells and cells (22Saito Y. Imazeki H. Miura S. Yoshimura T. Okutsu H. Harada Y. Ohwaki T. Nagao O. Kamiya S. Hayashi R. Kodama H. Handa H. Yoshida T. Fukai F. J. Biol. Chem. 2007; 282: 34929-34937Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 24Watanabe K. Takahashi H. Habu Y. Kamiya-Kubushiro N. Kamiya S. Nakamura H. Yajima H. Ishii T. Katayama T. Miyazaki K. Fukai F. Biochemistry. 2000; 39: 7138-7144Crossref PubMed Scopus (48) Google Scholar, J. 1996; Google Scholar, Cell Biol. 1997; Full Text PDF PubMed Scopus Google Scholar, A. J. H. J. J.A. Blood. PubMed Google Scholar, and Scholar). is important to the is exposed in the microenvironment of the hematopoietic M. J. Takahashi S. H. N. Obinata M. T. Exp. 2006; Full Text Full Text PDF PubMed Scopus Google have TN-C as a molecule stromal erythropoiesis, as by erythroid from fetal liver cells of that of TN-C suppresses the erythroid and the of TN-C in stromal cell the active of the TN-C molecule has not been in their may be that the functional site on the TN-C molecule is in erythroid is also important to acceleration of erythroid differentiation using K562 cells and is for erythroid development in A on the of TNIIIA2 on erythroid is with