Hyposialylation of Integrins Stimulates the Activity of Myeloid Fibronectin Receptors

Abstract

Despite numerous reports suggesting that β1 integrin receptors undergo differential glycosylation, the potential role of N-linked carbohydrates in modulating integrin function has been largely ignored. In the present study, we find that β1 integrins are differentially glycosylated during phorbol ester (PMA)-stimulated differentiation of myeloid cells along the monocyte/macrophage lineage. PMA treatment of two myeloid cell lines, U937 and THP-1, induces a down-regulation in expression of the ST6Gal I sialyltransferase. Correspondingly, the β1 integrin subunit becomes hyposialylated, suggesting that the β1 integrin is a substrate for this enzyme. The expression of hyposialylated β1 integrin isoforms is temporally correlated with enhanced binding of myeloid cells to fibronectin, and, importantly, fibronectin binding is inhibited when the Golgi disrupter, brefeldin A, is used to block the expression of the hyposialylated form. Consistent with the observation that cells with hyposialylated integrins are more adhesive to fibronectin, we demonstrate that the enzymatic removal of sialic acid residues from purified α5β1integrins stimulates fibronectin binding by these integrins. These data support the hypothesis that unsialylated β1 integrins are more adhesive to fibronectin, although desialylation of α5 subunits could also contribute to increased fibronectin binding. Collectively our results suggest a novel mechanism for regulation of the β1 integrin family of cell adhesion receptors. Despite numerous reports suggesting that β1 integrin receptors undergo differential glycosylation, the potential role of N-linked carbohydrates in modulating integrin function has been largely ignored. In the present study, we find that β1 integrins are differentially glycosylated during phorbol ester (PMA)-stimulated differentiation of myeloid cells along the monocyte/macrophage lineage. PMA treatment of two myeloid cell lines, U937 and THP-1, induces a down-regulation in expression of the ST6Gal I sialyltransferase. Correspondingly, the β1 integrin subunit becomes hyposialylated, suggesting that the β1 integrin is a substrate for this enzyme. The expression of hyposialylated β1 integrin isoforms is temporally correlated with enhanced binding of myeloid cells to fibronectin, and, importantly, fibronectin binding is inhibited when the Golgi disrupter, brefeldin A, is used to block the expression of the hyposialylated form. Consistent with the observation that cells with hyposialylated integrins are more adhesive to fibronectin, we demonstrate that the enzymatic removal of sialic acid residues from purified α5β1integrins stimulates fibronectin binding by these integrins. These data support the hypothesis that unsialylated β1 integrins are more adhesive to fibronectin, although desialylation of α5 subunits could also contribute to increased fibronectin binding. Collectively our results suggest a novel mechanism for regulation of the β1 integrin family of cell adhesion receptors. phorbol myristate acetate horseradish peroxidase Maackia amurensis phosphate-buffered saline Sambucus nigra N-glycosidase F enzyme-linked immunosorbent assay bovine serum albumin The U937 and THP-1 myeloid cell lines have been widely used to study myeloid differentiation along the monocyte/macrophage lineage. Following treatment with the phorbol ester, phorbol myristate acetate (PMA),1 U937 and THP-1 cells express proteins characteristic of terminally differentiated cells, including components of the respiratory burst oxidase (gp91phox, p67phox, and p47phox), granule proteins (myeloperoxidase and elastase) and cell adhesion receptors (CD11, CD18, and CD49e) (1Tenen D.G. Hromas R. Licht J.D. Zhang D.E. Blood. 1997; 90: 489-519Crossref PubMed Google Scholar, 2Boles B.K. Ritzenthaler J. Birkenmeier T. Roman J. Am. J. Physiol. 2000; 278: L703-L712PubMed Google Scholar). Consistent with the expression of these proteins, PMA-treated cells acquire functions characteristic of mature phagocytes, including respiratory burst activity, phagocytosis, and increased adhesiveness to endothelial cells as well as to extracellular matrix components such as fibronectin. In vivo, the increased adhesiveness of monocyte/macrophage cells contributes to the extravasation of cells from the vasculature and may further tether cells at sites of inflammation within the tissues. The PMA-induced adhesion of myeloid cells to extracellular matrix components is mediated by the integrin family of cell adhesion receptors. Integrins are heterodimeric glycoproteins composed of one α and one β subunit (3Shimizu Y. Rose D.M. Ginsberg M.H. Adv. Immunol. 1999; 72: 325-380Crossref PubMed Google Scholar, 4Aplin A.E. Howe A. Alahari S.K. Juliano R.L. Pharmacol. Rev. 1998; 50: 197-263PubMed Google Scholar). The specificity of integrin heterodimers is dictated by the pairing of various α and β subunits; for example, the α5β1 integrin (VLA5) associates with fibronectin, whereas the α2β1 integrin (VLA2) binds to either collagen or laminin. The binding of integrins to ligand, followed by receptor clustering, initiates signal transduction events that ultimately regulate many fundamental cellular processes, including initiation of gene transcription, cell survival, cell motility/invasiveness, and cytoskeletal reorganization. The mechanisms that underlie the PMA-induced adhesion of differentiated myeloid cells to fibronectin have not been well-defined. Several groups have reported that sustained PMA treatment of U937 cells stimulates both the transcription and cell surface expression of the α5 and β1 integrin subunits (2Boles B.K. Ritzenthaler J. Birkenmeier T. Roman J. Am. J. Physiol. 2000; 278: L703-L712PubMed Google Scholar, 5Bellon T. Lopez-Rodriguez C. Rubio M.A. Jochems G. Bernabeu C. Corbi A.L. Eur. J. Immunol. 1994; 24: 41-47Crossref PubMed Scopus (40) Google Scholar, 6Ferreira O.C., Jr. Valinsky J.E. Sheridan K. Wayner E.A. Bianco C. Garcia-Pardo A. Exp. Cell Res. 1991; 193: 20-26Crossref PubMed Scopus (51) Google Scholar, 7Laouar A. Collart F.R. Chubb C.B. Xie B. Huberman E. J. Immunol. 1999; 162: 407-414PubMed Google Scholar). However, untreated U937 cells express an abundant amount of the α5β1 integrin heterodimer, and yet these cells do not bind to fibronectin. This finding suggests that these receptors are in an inactive state. Increased expression may therefore contribute to enhanced binding but is unlikely to fully account for the robust cell adhesion that occurs following PMA treatment. Intriguingly, it has been shown that PMA treatment of U937 and THP-1 cells induces the synthesis of a β1 integrin subunit with alteredN-glycosylation (8Van de Water L. Aronson D. Braman V. Cancer Res. 1988; 48: 5730-5737PubMed Google Scholar). Hence, PMA not only increases the expression of β1 integrins but also directs the synthesis of β1 integrins that are structurally different from the β1 integrins expressed by untreated cells. In the present study we provide evidence implicating differentially glycosylated β1 integrins as mediators of the PMA-dependent fibronectin binding of U937 and THP-1 cells. Our studies show that PMA treatment causes a down-regulation in the ST6Gal I sialyltransferase, a trans-Golgi enzyme that adds the negatively charged sugar, α2–6-linked sialic acid, to glycoproteins. In turn, β1 integrins that are synthesized following PMA treatment are hyposialylated and demonstrate enhanced fibronectin-binding capability. Collectively, our results suggest a novel mechanism for the regulation of β1 integrin function during myeloid differentiation. A U937 cell subclone selected for sensitivity to granulocyte macrophage-colony stimulating factor was obtained from Dr. Andrew Kraft (University of Colorado, Denver, CO). THP-1 cells were obtained from the American Type Culture Collection (ATCC). U937 cells were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and gentamicin, whereas THP-1 cells were maintained in RPMI 1640 media with 10% fetal bovine serum and gentamicin. The PMA-resistant cell line, PRU, was derived from U937 cell cultures by treating U937 cells with 100 ng/ml PMA for 36 h and then by selecting cells that did not adhere to the tissue culture dish. The PMA-containing media was replaced with normal media, and cells were grown for 72 h. This population of cells was then challenged again with 100 ng/ml PMA for 36 h, and nonadherent cells were again collected. The nonadherent cells were diluted and seeded into 96-well dishes to obtain single-cell clones. Clones were subsequently expanded and evaluated for resistance to PMA-induced cell adhesion. U937 cells were treated with or without 50 ng/ml PMA for 15 h. Cells were then lysed in 50 mm Tris-HCl buffer (pH 7.4) containing 1% Triton X-100, 0.5 mm phenylmethylsulfonyl fluoride, 20 μg/ml leupeptin, 4 mm sodium fluoride, and 200 μm sodium pervanadate (“lysis buffer”). Protein concentrations were determined using a modified Bradford assay (Sigma Chemical Co.). One to two hundred micrograms of cell lysate were resolved by reducing SDS-PAGE, and proteins were subsequently transferred to polyvinylidene difluoride membrane. Membranes were blocked for 1–2 h with 3% nonfat dry milk. After blocking, blots were incubated for 2–4 h with a monoclonal antibody specific for the β1 integrin (Transduction Labs) or with a polyclonal antibody specific for ST6Gal I (provided by Dr. Karen Colley). Blots were then washed several times with Tris-buffered saline containing 0.05% Tween-20 (TBST). Following the washes, blots were incubated for 1 h with a horseradish-peroxidase (HRP)-coupled secondary antibody (Amersham Biosciences), then washed again with TBST. Blots were incubated with a luminescent HRP substrate (Amersham Biosciences) for 1 min, and positive signals were detected using the enhanced chemiluminescence method. Cell lysates were boiled in buffer containing 1% SDS to denature cellular proteins. Cell lysates were then diluted 5-fold in PBS, and Triton X-100 was added to a final concentration of 1%. The deglycosylating enzyme, PNGase F (Roche Molecular Biochemicals), was added to a final concentration of 10 units/ml, and lysates were incubated overnight at Cell lysates were treated for h at in the or of 10 (Roche Molecular or lysates were then boiled in buffer and subsequently to hundred micrograms of cell lysate was incubated for h at 4 with 4 of either or 20 of (Sigma Chemical was then and were incubated for an h at 4 with were by and washed times with followed by one with were from the by in The glycoproteins were resolved by SDS-PAGE, then to the β1 Cells were treated with or without 50 ng/ml PMA then were seeded tissue culture dishes that been overnight at 4 with 20 μg/ml fibronectin. Cells were to adhere for 15 h at Following this nonadherent cells were from the dishes by the dishes with cells were for in then for with cells were subsequently in 10% acid, and was used to the amount of in Cells were incubated for 1 h at with a function antibody specific for either the β1 integrin subunit or the or integrin subunits A was as a Following with the antibody concentration 10 50 ng/ml PMA was added to the and cells were seeded Cell adhesion was as Cells were in media containing 50 ng/ml PMA and were then in tissue culture dishes to cell adhesion to the the of cells were from the dishes and seeded tissue culture dishes that been with 20 μg/ml fibronectin. Cells were to adhere for min, and then adhesion was as selected during the adhesion brefeldin A was added to the media at a final concentration of 20 α5β1 integrins were to a final concentration of μg/ml in 50 mm buffer containing mm mm mm and Triton X-100 to hundred of was added to the integrin and were incubated for h at with a an amount of was incubated in buffer for h at in the of Following the the buffer was to by with were to the and the were tissue culture dishes that been with of fibronectin. A final amount of of α5β1 integrin was added to or untreated were also that been with fibronectin, to for binding. integrins were to adhere to or fibronectin for 1 h at The were then washed times with The integrin monoclonal was subsequently added to the and the were incubated for an at The antibody both and β1 integrins and α5β1 from both and PMA-treated U937 and THP-1 cells not this antibody is to in Following the with were washed times with was and were incubated for 1 h at were washed times and then incubated for at with the HRP The amount of integrin to was by using a of for specific binding were obtained by the binding from the fibronectin binding. U937 or THP-1 myeloid cells were incubated for 15 h in the of 50 ng/ml a treatment that is to the differentiation of myeloid cells along the monocyte/macrophage lineage. Following this PMA-treated and untreated cells were and lysates were subsequently resolved by β1 integrins were detected by shown in the mature β1 integrins from PMA-treated U937 and THP-1 cells more during β1 integrins from untreated U937 and THP-1 cells. The of the β1 integrin was by PMA treatment. the in the of mature β1 integrins was to lysates were treated with PNGase an enzyme that N-linked carbohydrates from glycoproteins. of PNGase lysates that β1 integrins from and PMA-treated cells that the differential of glycosylated integrins was to an in the at the of the in that the glycosylated β1 an J. C. J. J. PubMed Google Scholar, D. J. 1994; PubMed Google did not differential in to One of the of myeloid differentiation is increased cell adhesiveness to a of extracellular matrix the expression of an was with myeloid we evaluated the of PMA the of β1 integrins from the cell line, a U937 subclone that not increased cell adhesiveness in to PMA In to U937 and THP-1 cells, PMA did not of mature β1 integrins from cells studies of in cell have that β1 integrins undergo in the of sialic K. K. T. A. Cancer Res. 1997; Google Scholar, L. A. 1994; PubMed Scopus Google Scholar, J.E. J. Immunol. Google Scholar, J. PubMed Google Scholar). PMA a in β1 integrin we used a cell lysates containing an amount of β1 integrin were incubated with a followed by the of to were by and resolved by The was then detected by were a that α2–6-linked sialic and a that binds to sialic shown in from untreated U937 and THP-1 cells were by suggesting that the β1 integrins of myeloid cells α2–6-linked sialic acid However, to β1 integrins from the lysates of PMA-treated U937 and THP-1 cells. These data suggest that PMA treatment of U937 and THP-1 cells the expression of β1 integrins that in sialic U937 and THP-1 cells, PMA-resistant cells did not express hyposialylated β1 integrins following PMA treatment with data suggesting that the β1 integrins of cells do not undergo in to PMA In to the differential of β1 integrins by the the to β1 integrins from of the cell lysates The that the more of β1 integrins from PMA-treated U937 and THP-1 cells from a PMA-induced in sialic further that the PMA-induced in glycosylation, we the of β1 integrins from PMA-treated cells with that of β1 integrins from cell lysates that been by the enzyme, C. (Roche Molecular cell lysates were incubated for h at in the or of Following this the lysates were resolved by then was for the β1 shown in the of cell β1 integrins that been was to that of the hyposialylated β1 integrins expressed by PMA-treated cells. that PMA treatment of U937 and THP-1 cells the expression of β1 integrins that were in sialic we that PMA a down-regulation in the enzyme that directs the ST6Gal 1 sialyltransferase. we used to the of ST6Gal 1 in treated and untreated cell shown in PMA a down-regulation of ST6Gal 1 in U937 and THP-1 cell lines but not in the PMA-resistant cell were also for as a the expression of a hyposialylated was correlated with an in integrin we cell adhesion to fibronectin. THP-1, and cells were incubated in the or of PMA for 15 h and were then for adhesiveness to fibronectin using a assay U937 and THP-1 enhanced binding to fibronectin following PMA whereas the binding of cells was by PMA The PMA-dependent binding of U937 cells to fibronectin from in the of binding was inhibited by that block the function of β1 but not or integrins the expression of a hyposialylated β1 integrin was temporally correlated with PMA-induced cell binding to fibronectin. this we a for cell adhesion to fibronectin. cells were treated with PMA and then in tissue culture dishes for Cells were subsequently seeded tissue culture and adhesion was as PMA the binding of cells to fibronectin However, this cell adhesiveness to and h and then again from to 15 h. that the expression of a hyposialylated integrin with the more of cell the that of integrins the and Golgi several in cell J. J. J. PubMed Google Scholar, S.K. J. PubMed Google Scholar, J. PubMed Scopus Google Scholar, E. E.A. J. Physiol. 1999; PubMed Scopus Google Scholar). the expression of a hyposialylated integrin was temporally correlated with the of cell we a of β1 integrins expressed at various following PMA treatment 4 h, was in glycosylation, as by a of in β1 integrin h following PMA the more hyposialylated was although integrin was of the β1 integrin to hyposialylated at following PMA treatment and expression of the hyposialylated was sustained at 15 h These data suggest that the expression of a hyposialylated integrin 4 and h following PMA treatment and a by h. the expression of the hyposialylated is temporally correlated with the of PMA-dependent cell adhesion to fibronectin. further the hypothesis that the expression of a hyposialylated β1 integrin is with integrin we treated cells with brefeldin A, a that the Golgi and then cell adhesion that brefeldin A treatment block the expression of the hyposialylated β1 integrin and the but not the of cell adhesion to fibronectin. we PMA-dependent cell binding to fibronectin at two following brefeldin A we cells for h with 20 μg/ml brefeldin A. PMA was then added to the brefeldin media, and cells were incubated with both PMA and brefeldin A for an shown in brefeldin A cell adhesion to fibronectin following a treatment with These data suggest that PMA-induced cell adhesion at this not the synthesis of a integrin In these data that the amount of β1 integrin that occurs within the brefeldin A treatment not cell adhesion to fibronectin. the of brefeldin A cell adhesion at h following PMA treatment. A was added at h following the initiation of PMA a when binding is and then cell adhesion was h brefeldin A inhibited the enhanced cell adhesion that occurs at h following PMA treatment. These data suggest that the expression of a integrin the hyposialylated β1 integrin was for the of PMA-induced cell studies have shown that the enzymatic removal of sialic from the cell surface cell adhesion to extracellular matrix Cell 2000; PubMed Scopus (40) Google Scholar, J. 1994; PubMed Google Scholar). However, cell surface receptors are to by the enzyme, it is not to desialylation β1 integrin function the hypothesis that unsialylated binding to fibronectin, we a novel to purified β1 integrins and then fibronectin binding using a modified assay purified α5β1 integrins were to sialic acid were treated with or without and were then added to dishes and to adhere for 1 h at integrins were detected by using the integrin shown in α5β1 integrins that been enhanced to fibronectin, to α5β1 integrins with α2–6-linked sialic acid These data that unsialylated β1 integrins are more adhesive to fibronectin, although desialylation of α5 subunits may also contribute to increased binding. the results from binding with purified integrins suggest that sialic acid residues a role in the that sialic acid were from β1 integrins by the enzyme, were to assay for in shown in β1 integrins a more untreated β1 with the of sialic acid In a was in the amount of β1 integrins by the further that treatment was in sialic acid In the present study we have the mechanisms that underlie the PMA-induced adhesion of myeloid cells to fibronectin. In we have shown that PMA treatment has a cell PMA induces a in followed by a at h, and then a of enhanced adhesion that by h, and is sustained for at 15 h. Our results suggest that this of enhanced cell adhesion is to the PMA-dependent expression of a β1 integrin that is in the negatively charged sugar, sialic The expression of this hyposialylated β1 integrin is correlated temporally with the of cell adhesion. of the hyposialylated at 4 and h following PMA is expressed with h, and is sustained for at 15 h. the of PMA-dependent cell adhesion is inhibited when the Golgi disrupter, brefeldin A, is used to block the expression of the hyposialylated form. In to the the of PMA-dependent fibronectin binding not to the expression of a integrin brefeldin A when adhesion is at following PMA treatment the hyposialylated integrin is not expressed within the 4 h following PMA treatment is that the in cell adhesiveness is to the PMA-dependent of integrin receptors. has been in cells, that integrins are by events 1994; PubMed Scopus Google Scholar, Cell PubMed Scopus Google Scholar). it has been that PMA may cell adhesion by modulating events that of the Juliano R.L. J. Cell PubMed Scopus Google Scholar, Ginsberg M.H. J. Exp. 1994; PubMed Scopus Google Scholar). further to the mechanisms that underlie the of PMA-dependent myeloid cell adhesion. Our results suggest that the PMA-dependent expression of a hyposialylated β1 integrin is at in to the down-regulation of the ST6Gal I sialyltransferase. of the ST6Gal I is to G. J. PubMed Scopus Google Scholar, A. J. J.D. 1994; PubMed Scopus Google and the of this enzyme in to cell differentiation A. K. Y. K. K. 1998; PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, Res. PubMed Scopus Google Scholar). In our study, two myeloid cell lines that undergo differentiation in to PMA and THP-1 were shown to have of ST6Gal I following PMA treatment. Consistent with these PMA has been shown to ST6Gal I in myeloid cells A. K. Y. K. K. 1998; PubMed Scopus Google Scholar). U937 and THP-1 cells, myeloid cells that have been selected for PMA resistance do not ST6Gal I in to do not express hyposialylated and, do not bind to fibronectin following PMA treatment. Collectively, these results suggest that the β1 integrin is an substrate for ST6Gal I and that integrin function may by the of Several studies support a role for integrin groups in the integrins and S.K. J. PubMed Google reported that the treatment of with blocked cell adhesion to fibronectin. J. 1994; PubMed Google that binding was when N-linked carbohydrates were from cell surface α5β1 integrins. In our study, purified α5β1 integrins were treated with enzyme to the hyposialylated integrins expressed by PMA-treated myeloid cells. α5β1 integrins enhanced binding to fibronectin, with the observation that myeloid cells hyposialylated β1 integrins adhere to fibronectin. The enhanced binding of purified integrins the hypothesis that the of sialic the evidence suggests in cells, of cell surface receptors is correlated with cell adhesion to extracellular matrix J.E. J. Immunol. Google Scholar, Cell 2000; PubMed Scopus (40) Google Scholar, J. Physiol. 1999; PubMed Scopus Google although this has not been J. PubMed Google Scholar). In one study, the enzymatic removal of sialic acid residues from the surface of cells cell adhesion to fibronectin Cell 2000; PubMed Scopus (40) Google Scholar). 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Google Scholar). in β1 integrin are also correlated with in cell or capability. The from have hyposialylated β1 as with β1 integrins expressed by the of L. A. 1994; PubMed Scopus Google Scholar). the β1 integrins of demonstrate enhanced fibronectin, have a different S.K. J. Cell Google Scholar). β1 have been in numerous and cells K. K. T. A. Cancer Res. 1997; Google Scholar, B. A. PubMed Scopus Google Scholar, R. J. PubMed Scopus Google Scholar, A. J. PubMed Scopus Google Scholar, J. Cell Google Scholar, R. Cancer Res. Google Scholar, E. J. 1997; PubMed Scopus Google Scholar). The finding that β1 integrins are expressed by and cell that in cell adhesiveness and suggests that β1 are our results differentially β1 integrin subunits in fibronectin receptor the potential role of differentially glycosylated integrin α subunits has not yet been was reported that the α5 integrin subunits expressed by cells are modified by C. K. K. T. J. PubMed Scopus Google Scholar). 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In of evidence implicating glycosylation, and as a of cell we that in an and novel mechanism for regulation of the β1 integrin Our studies suggest that of a causes the down-regulation of the ST6Gal I sialyltransferase, in turn, to the expression of hyposialylated that bind fibronectin more The expression of hyposialylated β1 integrin a role in the fibronectin-binding of differentiated myeloid cells.