Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 May 1;108(5):1925–1933. doi: 10.1083/jcb.108.5.1925

Phorbol ester modulation of integrin-mediated cell adhesion: a postreceptor event

PMCID: PMC2115550  PMID: 2785521

Abstract

Chinese hamster ovary (CHO) suspension culture cells adhere readily to substrata coated with extracellular matrix proteins such as fibronectin, vitronectin, or laminin. In the case of fibronectin, it is known that adhesion is mediated by an integrin-type, cell surface fibronectin receptor (FnR). We demonstrate here that treatment of CHO cells with submicromolar concentrations of phorbol ester produces a remarkable increase in the ability of these cells to adhere to fibronectin. Both the rate of adhesion and the efficiency of adhesion are enhanced about four- to fivefold. Further, phorbol ester treatment renders the fibronectin-mediated adhesion process less sensitive to inhibitors, including GRGDSP peptide and PB1, a monoclonal anti-FnR antibody. By contrast, nonspecific adhesion processes, for example cell attachment to substrata coated with polylysine or concanavalin A, are not affected by phorbol ester treatment. Thus integrin-mediated adhesion is modulated by phorbol esters, but nonspecific adhesion is not. Neither the number of cell surface FnRs nor the receptor affinity, as measured by 125I-fibronectin and 125I-anti-FnR antibody binding, is altered by phorbol ester treatment. Thus, the effect of phorbol ester on cell adhesion seems to occur at a step subsequent to initial ligand- receptor binding events. Since phorbol ester is a potent activator of protein kinase C, we examined phosphorylation patterns in control and phorbol-treated cells. In immunoprecipitates of lysates from suspension culture cells, there was no evidence of phorbol ester-stimulated phosphorylation of FnR or of talin, a protein thought to interact with FnR. These results suggest that phorbol ester effects on fibronectin- dependent adhesion are not due to phosphorylation of the FnR itself but rather may be due to postreceptor events, possibly the phosphorylation of cytoskeletal proteins involved in integrin-mediated adhesion.

Full Text

The Full Text of this article is available as a PDF (1.4 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akiyama S. K., Yamada K. M. Synthetic peptides competitively inhibit both direct binding to fibroblasts and functional biological assays for the purified cell-binding domain of fibronectin. J Biol Chem. 1985 Sep 5;260(19):10402–10405. [PubMed] [Google Scholar]
  2. Akiyama S. K., Yamada K. M. The interaction of plasma fibronectin with fibroblastic cells in suspension. J Biol Chem. 1985 Apr 10;260(7):4492–4500. [PubMed] [Google Scholar]
  3. Argraves W. S., Suzuki S., Arai H., Thompson K., Pierschbacher M. D., Ruoslahti E. Amino acid sequence of the human fibronectin receptor. J Cell Biol. 1987 Sep;105(3):1183–1190. doi: 10.1083/jcb.105.3.1183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ashendel C. L. The phorbol ester receptor: a phospholipid-regulated protein kinase. Biochim Biophys Acta. 1985 Sep 9;822(2):219–242. doi: 10.1016/0304-4157(85)90009-7. [DOI] [PubMed] [Google Scholar]
  5. Bernal S. D., Chen L. B. Induction of cytoskeleton-associated proteins during differentiation of human myeloid leukemic cell lines. Cancer Res. 1982 Dec;42(12):5106–5116. [PubMed] [Google Scholar]
  6. Brown P. J., Juliano R. L. Association between fibronectin receptor and the substratum: spare receptors for cell adhesion. Exp Cell Res. 1987 Aug;171(2):376–388. doi: 10.1016/0014-4827(87)90170-4. [DOI] [PubMed] [Google Scholar]
  7. Brown P. J., Juliano R. L. Expression and function of a putative cell surface receptor for fibronectin in hamster and human cell lines. J Cell Biol. 1986 Oct;103(4):1595–1603. doi: 10.1083/jcb.103.4.1595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brown P. J., Juliano R. L. Monoclonal antibodies to distinctive epitopes on the alpha and beta subunits of the fibronectin receptor. Exp Cell Res. 1988 Aug;177(2):303–318. doi: 10.1016/0014-4827(88)90464-8. [DOI] [PubMed] [Google Scholar]
  9. Brown P. J., Juliano R. L. Selective inhibition of fibronectin-mediated cell adhesion by monoclonal antibodies to a cell-surface glycoprotein. Science. 1985 Jun 21;228(4706):1448–1451. doi: 10.1126/science.4012302. [DOI] [PubMed] [Google Scholar]
  10. Brown P. J. Phorbol ester stimulation of fibronectin-mediated cell adhesion. Biochem Biophys Res Commun. 1988 Sep 15;155(2):603–607. doi: 10.1016/s0006-291x(88)80537-0. [DOI] [PubMed] [Google Scholar]
  11. Buck C. A., Horwitz A. F. Cell surface receptors for extracellular matrix molecules. Annu Rev Cell Biol. 1987;3:179–205. doi: 10.1146/annurev.cb.03.110187.001143. [DOI] [PubMed] [Google Scholar]
  12. Burn P., Kupfer A., Singer S. J. Dynamic membrane-cytoskeletal interactions: specific association of integrin and talin arises in vivo after phorbol ester treatment of peripheral blood lymphocytes. Proc Natl Acad Sci U S A. 1988 Jan;85(2):497–501. doi: 10.1073/pnas.85.2.497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chen W. T., Hasegawa E., Hasegawa T., Weinstock C., Yamada K. M. Development of cell surface linkage complexes in cultured fibroblasts. J Cell Biol. 1985 Apr;100(4):1103–1114. doi: 10.1083/jcb.100.4.1103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Cheung E., Brown P. J., Juliano R. L. Altered type I protein kinase in adhesion defective CHO cell variants. J Cell Physiol. 1987 Jan;130(1):118–124. doi: 10.1002/jcp.1041300117. [DOI] [PubMed] [Google Scholar]
  15. Cheung E., Juliano R. L. cAMP-induced phenotypic reversion of adhesion, aggregation, and endocytosis in adhesion-defective CHO cell variants. J Cell Physiol. 1985 Aug;124(2):337–343. doi: 10.1002/jcp.1041240225. [DOI] [PubMed] [Google Scholar]
  16. Culp L. A., Laterra J., Lark M. W., Beyth R. J., Tobey S. L. Heparan sulphate proteoglycan as mediator of some adhesive responses and cytoskeletal reorganization of cells on fibronectin matrices: independent versus cooperative functions. Ciba Found Symp. 1986;124:158–183. doi: 10.1002/9780470513385.ch10. [DOI] [PubMed] [Google Scholar]
  17. Davis G. E., Blaker S. N., Engvall E., Varon S., Manthorpe M., Gage F. H. Human amnion membrane serves as a substratum for growing axons in vitro and in vivo. Science. 1987 May 29;236(4805):1106–1109. doi: 10.1126/science.3576223. [DOI] [PubMed] [Google Scholar]
  18. Graf J., Iwamoto Y., Sasaki M., Martin G. R., Kleinman H. K., Robey F. A., Yamada Y. Identification of an amino acid sequence in laminin mediating cell attachment, chemotaxis, and receptor binding. Cell. 1987 Mar 27;48(6):989–996. doi: 10.1016/0092-8674(87)90707-0. [DOI] [PubMed] [Google Scholar]
  19. Harper P. A., Juliano R. L. Fibronectin-independent adhesion of fibroblasts to the extracellular matrix: mediation by a high molecular weight membrane glycoprotein. J Cell Biol. 1981 Dec;91(3 Pt 1):647–653. doi: 10.1083/jcb.91.3.647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Harper P. A., Juliano R. L. Two distinct mechanisms of fibroblast adhesion. Nature. 1981 Mar 12;290(5802):136–138. doi: 10.1038/290136a0. [DOI] [PubMed] [Google Scholar]
  21. Hinek A., Wrenn D. S., Mecham R. P., Barondes S. H. The elastin receptor: a galactoside-binding protein. Science. 1988 Mar 25;239(4847):1539–1541. doi: 10.1126/science.2832941. [DOI] [PubMed] [Google Scholar]
  22. Hirst R., Horwitz A., Buck C., Rohrschneider L. Phosphorylation of the fibronectin receptor complex in cells transformed by oncogenes that encode tyrosine kinases. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6470–6474. doi: 10.1073/pnas.83.17.6470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Horwitz A., Duggan K., Buck C., Beckerle M. C., Burridge K. Interaction of plasma membrane fibronectin receptor with talin--a transmembrane linkage. Nature. 1986 Apr 10;320(6062):531–533. doi: 10.1038/320531a0. [DOI] [PubMed] [Google Scholar]
  24. Horwitz A., Duggan K., Greggs R., Decker C., Buck C. The cell substrate attachment (CSAT) antigen has properties of a receptor for laminin and fibronectin. J Cell Biol. 1985 Dec;101(6):2134–2144. doi: 10.1083/jcb.101.6.2134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hunter T. A thousand and one protein kinases. Cell. 1987 Sep 11;50(6):823–829. doi: 10.1016/0092-8674(87)90509-5. [DOI] [PubMed] [Google Scholar]
  26. Hynes R. O. Integrins: a family of cell surface receptors. Cell. 1987 Feb 27;48(4):549–554. doi: 10.1016/0092-8674(87)90233-9. [DOI] [PubMed] [Google Scholar]
  27. Juliano R. L. Membrane receptors for extracellular matrix macromolecules: relationship to cell adhesion and tumor metastasis. Biochim Biophys Acta. 1987 Nov 25;907(3):261–278. doi: 10.1016/0304-419x(87)90009-6. [DOI] [PubMed] [Google Scholar]
  28. Järvinen M., Ylänne J., Vartio T., Virtanen I. Tumor promoter and fibronectin induce actin stress fibers and focal adhesion sites in spreading human erythroleukemia (HEL) cells. Eur J Cell Biol. 1987 Oct;44(2):238–246. [PubMed] [Google Scholar]
  29. Kato S., Ben T. L., De Luca L. M. Phorbol esters enhance attachment of NIH/3T3 cells to laminin and type IV collagen substrates. Exp Cell Res. 1988 Nov;179(1):31–41. doi: 10.1016/0014-4827(88)90345-x. [DOI] [PubMed] [Google Scholar]
  30. Kelleher P. J., Juliano R. L. An antibody that inhibits fibronectin-independent adhesion of fibroblasts to extracellular matrix material. J Cell Physiol. 1984 Sep;120(3):329–334. doi: 10.1002/jcp.1041200311. [DOI] [PubMed] [Google Scholar]
  31. Klausner R. D., Harford J., van Renswoude J. Rapid internalization of the transferrin receptor in K562 cells is triggered by ligand binding or treatment with a phorbol ester. Proc Natl Acad Sci U S A. 1984 May;81(10):3005–3009. doi: 10.1073/pnas.81.10.3005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kleinman H. K., Ogle R. C., Cannon F. B., Little C. D., Sweeney T. M., Luckenbill-Edds L. Laminin receptors for neurite formation. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1282–1286. doi: 10.1073/pnas.85.4.1282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  34. Liotta L. A. Tumor invasion and metastases--role of the extracellular matrix: Rhoads Memorial Award lecture. Cancer Res. 1986 Jan;46(1):1–7. [PubMed] [Google Scholar]
  35. Litchfield D. W., Ball E. H. Phosphorylation of the cytoskeletal protein talin by protein kinase C. Biochem Biophys Res Commun. 1986 Feb 13;134(3):1276–1283. doi: 10.1016/0006-291x(86)90388-8. [DOI] [PubMed] [Google Scholar]
  36. McGraw T. E., Dunn K. W., Maxfield F. R. Phorbol ester treatment increases the exocytic rate of the transferrin receptor recycling pathway independent of serine-24 phosphorylation. J Cell Biol. 1988 Apr;106(4):1061–1066. doi: 10.1083/jcb.106.4.1061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature. 1984 Apr 19;308(5961):693–698. doi: 10.1038/308693a0. [DOI] [PubMed] [Google Scholar]
  38. Parise L. V., Phillips D. R. Fibronectin-binding properties of the purified platelet glycoprotein IIb-IIIa complex. J Biol Chem. 1986 Oct 25;261(30):14011–14017. [PubMed] [Google Scholar]
  39. Patel V. P., Lodish H. F. The fibronectin receptor on mammalian erythroid precursor cells: characterization and developmental regulation. J Cell Biol. 1986 Feb;102(2):449–456. doi: 10.1083/jcb.102.2.449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Pierschbacher M., Hayman E. G., Ruoslahti E. Synthetic peptide with cell attachment activity of fibronectin. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1224–1227. doi: 10.1073/pnas.80.5.1224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Pytela R., Pierschbacher M. D., Ruoslahti E. A 125/115-kDa cell surface receptor specific for vitronectin interacts with the arginine-glycine-aspartic acid adhesion sequence derived from fibronectin. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5766–5770. doi: 10.1073/pnas.82.17.5766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Pytela R., Pierschbacher M. D., Ruoslahti E. Identification and isolation of a 140 kd cell surface glycoprotein with properties expected of a fibronectin receptor. Cell. 1985 Jan;40(1):191–198. doi: 10.1016/0092-8674(85)90322-8. [DOI] [PubMed] [Google Scholar]
  43. Rao N. C., Barsky S. H., Terranova V. P., Liotta L. A. Isolation of a tumor cell laminin receptor. Biochem Biophys Res Commun. 1983 Mar 29;111(3):804–808. doi: 10.1016/0006-291x(83)91370-0. [DOI] [PubMed] [Google Scholar]
  44. Rothlein R., Springer T. A. The requirement for lymphocyte function-associated antigen 1 in homotypic leukocyte adhesion stimulated by phorbol ester. J Exp Med. 1986 May 1;163(5):1132–1149. doi: 10.1084/jem.163.5.1132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Rovera G., Santoli D., Damsky C. Human promyelocytic leukemia cells in culture differentiate into macrophage-like cells when treated with a phorbol diester. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2779–2783. doi: 10.1073/pnas.76.6.2779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Ruoslahti E., Pierschbacher M. D. Arg-Gly-Asp: a versatile cell recognition signal. Cell. 1986 Feb 28;44(4):517–518. doi: 10.1016/0092-8674(86)90259-x. [DOI] [PubMed] [Google Scholar]
  47. Ruoslahti E., Pierschbacher M. D. New perspectives in cell adhesion: RGD and integrins. Science. 1987 Oct 23;238(4826):491–497. doi: 10.1126/science.2821619. [DOI] [PubMed] [Google Scholar]
  48. Ruoslahti E., Suzuki S., Hayman E. G., Ill C. R., Pierschbacher M. D. Purification and characterization of vitronectin. Methods Enzymol. 1987;144:430–437. doi: 10.1016/0076-6879(87)44192-x. [DOI] [PubMed] [Google Scholar]
  49. Schliwa M., Nakamura T., Porter K. R., Euteneuer U. A tumor promoter induces rapid and coordinated reorganization of actin and vinculin in cultured cells. J Cell Biol. 1984 Sep;99(3):1045–1059. doi: 10.1083/jcb.99.3.1045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Schwarz M. A., Juliano R. L. Interaction of fibronectin-coated beads with CHO cells. Exp Cell Res. 1984 Jun;152(2):302–312. doi: 10.1016/0014-4827(84)90632-3. [DOI] [PubMed] [Google Scholar]
  51. Schwarz M. A., Juliano R. L. Surface activation of the cell adhesion fragment of fibronectin. Exp Cell Res. 1984 Aug;153(2):550–555. doi: 10.1016/0014-4827(84)90624-4. [DOI] [PubMed] [Google Scholar]
  52. Shiba Y., Sasaki Y., Kanno Y. 12-O-tetradecanoylphorbol-13-acetate disrupts actin filaments and focal contacts and enhances binding of fibronectin-coated latex beads to 3T3-L1 cells. Exp Cell Res. 1988 Oct;178(2):233–241. doi: 10.1016/0014-4827(88)90394-1. [DOI] [PubMed] [Google Scholar]
  53. Smalheiser N. R., Schwartz N. B. Cranin: a laminin-binding protein of cell membranes. Proc Natl Acad Sci U S A. 1987 Sep;84(18):6457–6461. doi: 10.1073/pnas.84.18.6457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Staunton D. E., Marlin S. D., Stratowa C., Dustin M. L., Springer T. A. Primary structure of ICAM-1 demonstrates interaction between members of the immunoglobulin and integrin supergene families. Cell. 1988 Mar 25;52(6):925–933. doi: 10.1016/0092-8674(88)90434-5. [DOI] [PubMed] [Google Scholar]
  55. Takada Y., Strominger J. L., Hemler M. E. The very late antigen family of heterodimers is part of a superfamily of molecules involved in adhesion and embryogenesis. Proc Natl Acad Sci U S A. 1987 May;84(10):3239–3243. doi: 10.1073/pnas.84.10.3239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Takada Y., Wayner E. A., Carter W. G., Hemler M. E. Extracellular matrix receptors, ECMRII and ECMRI, for collagen and fibronectin correspond to VLA-2 and VLA-3 in the VLA family of heterodimers. J Cell Biochem. 1988 Aug;37(4):385–393. doi: 10.1002/jcb.240370406. [DOI] [PubMed] [Google Scholar]
  57. Wayner E. A., Carter W. G. Identification of multiple cell adhesion receptors for collagen and fibronectin in human fibrosarcoma cells possessing unique alpha and common beta subunits. J Cell Biol. 1987 Oct;105(4):1873–1884. doi: 10.1083/jcb.105.4.1873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wright S. D., Reddy P. A., Jong M. T., Erickson B. W. C3bi receptor (complement receptor type 3) recognizes a region of complement protein C3 containing the sequence Arg-Gly-Asp. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1965–1968. doi: 10.1073/pnas.84.7.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES