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Myeloid Leukemia Cells Macrophage Differentiation of Human HL-60

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Myeloid Leukemia Cells Macrophage Differentiation of Human HL-60 Interaction Between α5β1 Integrin and Secreted Fibronectin Is Involved in Macrophage Differentiation of Human HL-60 Myeloid Leukemia Cells This information is current as of September 26, 2021. Amale Laouar, Frank R. Collart, Cynthia B. H. Chubb, Bei Xie and Eliezer Huberman J Immunol 1999; 162:407-414; ; http://www.jimmunol.org/content/162/1/407 Downloaded from References This article cites 41 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/162/1/407.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 26, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. a b Interaction Between 5 1 Integrin and Secreted Fibronectin Is Involved in Macrophage Differentiation of Human HL-60 Myeloid Leukemia Cells1 Amale Laouar, Frank R. Collart, Cynthia B. H. Chubb, Bei Xie, and Eliezer Huberman2 a b We examined the role of fibronectin (FN) and FN-binding integrins in macrophage differentiation. Increased FN and 5 1 integrin gene expression was observed in phorbol 12-myristate 13-acetate PMA-treated HL-60 cells and PMA- or macro- phage-CSF-treated blood monocytes before the manifestation of macrophage markers. After treatment of HL-60 cells and monocytes, newly synthesized FN was released and deposited on the dishes. An HL-60 cell variant, HL-525, which is deficient in the protein kinase Cb (PKC-b) and resistant to PMA-induced differentiation, failed to express FN after PMA treatment. b Transfecting HL-525 cells with a PKC- expression plasmid restored PMA-induced FN gene expression and macrophage Downloaded from a b differentiation. Untreated HL-525 cells (which have a high level of the 5 1 integrin) incubated on FN differentiated into macrophages. The percentage of cells having a macrophage phenotype induced by PMA in HL-60 cells, by FN in HL-525 a b cells, or by either PMA or macrophage-CSF in monocytes was reduced in the presence of mAbs to FN and 5 1 integrin. The integrin-signaling nonreceptor tyrosine kinase, p72Syk, was activated in PMA-treated HL-60 and FN-treated HL-525 cells. We suggest that macrophage differentiation involves the activation of PKC-b and expression of extracellular matrix a b http://www.jimmunol.org/ proteins such as FN and the corresponding integrins, 5 1 integrin in particular. The stimulated cells, through the integrins, attach to substrates by binding to the deposited FN. This attachment, in turn, may through integrin signaling activate nonreceptor tyrosine kinases, including p72Syk, and later lead to expression of other genes involved in evoking the macro- phage phenotype. The Journal of Immunology, 1999, 162: 407–414. he extracellular matrix (ECM)3 is an intricate assembly of phosphorylation in hemopoietic cells (11–14). These changes in proteins that includes collagen, laminin, and fibronectin tyrosine phosphorylation are likely caused by the activation of T (FN) (1). Cells interact with these proteins and with each nonreceptor tyrosine kinases, such as p125FAK or p72Syk (9, 11, 14, other via specific receptors located on their surface. A major class 15). Subsequent to such a tyrosine phosphorylation event, there is of these receptors is the integrins, each of which is composed of a rapid induction of immediate-early genes, including transcription by guest on September 26, 2021 two distinct a and b transmembrane glycoprotein subunits that are factors such as c-fos,c-jun, IkB, and MAD-6, as well as cytokines noncovalently linked (2). The ab associations determine the li- such as IL-1b, IL-8, and TNF-a (16). gand-binding specificities of the integrin heterodimers for various Macrophages secrete FN, bind FN, and migrate in response to ECM proteins (3). In some cases, two integrins that share a ligand FN (17–20). In addition, FN provides signals that lead to en- recognize different regions of the ligand molecule, as is true of the hanced TNF-a production, respiratory burst activity, and a b a b 5 1 and 4 1 FN receptors, that bind to the Arg-Gly-Asp-Ser phagocytosis of foreign microorganisms (18, 21–23), and (RGDS) and Leu-Asp-Val (LDV) motif of FN, respectively (4). promote the differentiation of blood monocytes into tissue Alternatively, two distinct integrins can bind to the same region of macrophages (24–26). The process of FN secretion and adhe- the same ligand, such as the a b and a b integrins, which both 5 1 v 3 sion was shown to be under the control of protein kinase C recognize the RGDS site of FN (5). (PKC) (15, 27, 28). In addition to being adhesion receptors, it has become clear that The human HL-60 myeloid leukemia cell line is often used as integrins are also signal-transducing receptors (6) that regulate cell growth (7) and apoptosis (8), influence gene expression (9), and a model system to study terminal differentiation in myeloid modulate tumor behavior (10). Several reports have shown that cells (29, 30). Activation of PKC by PMA is well known to binding of adhesive ligands to integrins can induce protein tyrosine cause the induction of macrophage markers, such as adhesion and spreading, in myeloid cells (31–33). It is clear that HL-60 cells and blood monocytes adhere and spread on plastic tissue Center for Mechanistic Biology and Biotechnology, Argonne National Laboratory, culture plates in response to PMA treatment; however, the pre- Argonne, IL 60439 cise mechanism by which signals are transmitted from PMA to Received for publication June 8, 1998. Accepted for publication August 31, 1998. the intracellular machinery that controls cell adhesion and The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance spreading, and thus differentiation, remains largely unknown. with 18 U.S.C. Section 1734 solely to indicate this fact. The present study was initiated to test the hypothesis that 1 This work was supported by the U.S. Department of Energy, Office of Biological PMA-induced macrophage differentiation in HL-60 cells and and Environmental Research, under Contract W-31-109-ENG-38. peripheral blood monocytes may involve interaction between 2 Address correspondence and reprint requests to Dr. Eliezer Huberman, Center for secreted FN and its integrin receptors; validation of this hy- Mechanistic Biology and Biotechnology, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, IL 60439-4833. E-mail address: [email protected] pothesis would allow the delineation of the signaling role that b Syk 3 Abbreviations used in this paper: ECM, extracellular matrix; FN, fibronectin; PKC- and nonreceptor tyrosine kinase p72 play in this RGDS, Arg-Gly-Asp-Ser; PKC, protein kinase C; M-CSF, macrophage-CSF. differentiation. Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 a b 408 INTERACTION BETWEEN 5 1 INTEGRIN AND SECRETED FIBRONECTIN Materials and Methods entiation induction, cells were incubated with the mAb or peptide for 20 Reagents min before as well as during treatment with the inducers. Isotypic controls (IgG1, IgG2a, and IgG2b), were purchased from Sigma Immunofluorescence (St. Louis, MO) as were dialyzed murine mAbs to human fibronectin (FN- The immunostaining procedures were conducted at 4°C by using either 15, IgG1), an a-naphthyl acetate esterase assay kit, RGDS and Gly-Pro- b 96-microwell plates or tissue culture chamber slides (Nunc). The cells were Arg-Pro (GPRP) peptides, and Ficoll-Hypaque. mAbs to the human 1 washed twice with PBSA (PBS containing 1% BSA and 0.1% NaN ) and a a 3 (K20, IgG2a), 4 (HP211, IgG1), and 5 (SAM1, IgG2b) were from Im- FAK incubated for 45 min with the appropriate primary mAb under saturating munotech (Westbrook, ME). The mAb to focal adhesion kinase (p125 ) conditions. The cells were then washed twice with PBSA and incubated for was from Transduction Laboratories (Lexington, KY) and Upstate Bio- Syk an additional 45 min with the secondary Ab CY3. After a wash with PBSA, technology (Lake Placid, NY), and the mAb to p72 was from Wako the slides were mounted with phosphate-buffered Gelvatol (Becton Dick- Chemicals (Richmond, VA). Indocarcyanine-conjugated anti-murine goat inson, Sunnyvale, CA). Fluorescence was examined by using a Vaytek Ig (CY3) was purchased from Jackson ImmunoResearch (West Grove, a b digital confocal microscope. To examine the blocking effects of the protein PA), and the mAb to the human V 3 integrin mAb (23CG, IgG1) was b a kinase inhibitors, cells were incubated with the inhibitors 20 min before as bought from PharMingen (San Diego, CA). The anti-human 4 6 integrin well as during treatment with the inducers. mAb was kindly provided by Dr. S. Kennel of Oak Ridge National Lab- oratory. The macrophage-CSF (M-CSF) was from Biosource International RT-PCR analysis (Camarillo, CA), and plates precoated with mouse laminin, collagen type I, or collagen type IV were from Becton Dickinson (Bedford, MA). Human RNA was purified by centrifugation through a CsCl cushion as previously a b described (36).
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