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Partial reversal of transformed fusiform phenotype by overexpression of calreticulin

Cellular & Molecular Biology Letters volume 12, pages 294–307 (2007)Cite this article

Abstract

Calreticulin, a Ca2+-storage and chaperone protein of the ER, has also been shown to affect cell adhesiveness. To examine the effects of differential expression of calreticulin on cellular adhesiveness, we used L fibroblast cell lines stably expressing either elevated or reduced amounts of full length, ER-targeted calreticulin. Overexpression of calreticulin correlates with an increase in adhesiveness of L fibroblasts such that these transformed cells acquire epithelioid morphology and form an epithelial-cell sheet when crowded. Functionally, the “reversal” of transformed phenotype in L fibroblasts differentially overexpressing calreticulin can be accounted for by changes in levels of expression of N-cadherin and vinculin. Structurally, however, although the form and extent of cell-cell contacts in L fibroblasts overexpressing calreticulin mimicked those in normal epithelia, electron microscopical examination revealed that cell-cell junctions formed by these transformed cells bore only superficial resemblance to those of normal epithelia in culture. Our data imply that overexpression of calreticulin, while partially reverses fusiform transformed phenotype is in itself insufficient to re-establish bona fide zonulae adherens in transformed fibroblasts.

Abbreviations

BSA:

bovine serum albumin

DMEM:

Dulbecco’s modified Eagle’s medium

EDTA:

ethylenediaminetetraacetic acid

EGTA:

glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid

ER:

endoplasmic reticulum

FITC:

fluorescein isothiocyanate

PBS:

phosphate buffered saline

SDS-PAGE:

sodium dodecyl-polyacrylamide gel electrophoresis

References

  1. Gelebart, P., Opas, M. and Michalak, M. Calreticulin, a Ca(2+)-binding chaperone of the endoplasmic reticulum. Int. J. Biochem. Cell Biol. 37 (2005) 260–266.

    Article  CAS  PubMed  Google Scholar 

  2. Bedard, K., Szabo, E., Michalak, M. and Opas, M. Cellular functions of endoplasmic reticulum chaperones calreticulin, calnexin, and ERp57. Int. Rev. Cytol. 245 (2005) 91–121.

    CAS  PubMed  Google Scholar 

  3. Papp, S., Fadel, M.P. and Opas, M. ER-to-cell surface signalling: calreticulin and cell adhesion. J. Appl. Biomed. 2 (2004) 1–14.

    CAS  Google Scholar 

  4. Geiger, B., Volk, T. and Volberg, T. Molecular heterogeneity of adherens junctions. J. Cell Biol. 101 (1985) 1523–1531.

    Google Scholar 

  5. Geiger, B., Volk, T., Volberg, T. and Bendori, R. Molecular interactions in adherens-type contacts. J. Cell Sci. Suppl. 8 (1987) 251–272.

    CAS  PubMed  Google Scholar 

  6. Opas, M., Szewczenko-Pawlikowski, M., Jass, G.K., Mesaeli, N. and Michalak, M. Calreticulin modulates cell adhesiveness via regulation of vinculin expression. J. Cell Biol. 135 (1996) 1913–1923.

    Article  CAS  PubMed  Google Scholar 

  7. Fadel, M.P., Dziak, E., Lo, C.M., Ferrier, J., Mesaeli, N., Michalak, M. and Opas, M. Calreticulin affects focal contact-dependent but not close contactdependent cell-substratum adhesion. J. Biol. Chem. 274 (1999) 15085–15094.

    Article  CAS  PubMed  Google Scholar 

  8. Fadel, M.P., Szewczenko-Pawlikowski, M., Leclerc, P., Dziak, E., Symonds, J.M., Blaschuk, O., Michalak, M. and Opas, M. Calreticulin affects betacatenin associated pathways. J. Biol. Chem. 276 (2001) 27083–27089.

    Article  CAS  PubMed  Google Scholar 

  9. Dedhar, S. Novel functions for calreticulin: Interaction with integrins and modulation of gene expression. Trends Biochem. Sci. 19 (1994) 269–271.

    Article  CAS  PubMed  Google Scholar 

  10. Coppolino, M., Leung-Hagesteijn, C., Dedhar, S. and Wilkins, J. Inducible interaction of integrin α2β1 with calreticulin-Dependence on the activation state of the integrin. J. Biol. Chem. 270 (1995) 23132–23138.

    Article  CAS  PubMed  Google Scholar 

  11. Afshar, N., Black, B.E. and Paschal, B.M. Retrotranslocation of the chaperone calreticulin from the endoplasmic reticulum lumen to the cytosol. Mol. Cell Biol. 25 (2005) 8844–8853.

    Article  CAS  PubMed  Google Scholar 

  12. Shaffer, K.L., Sharma, A., Snapp, E.L. and Hegde, R.S. Regulation of protein compartmentalization expands the diversity of protein function. Dev. Cell 9 (2005) 545–554.

    Article  CAS  PubMed  Google Scholar 

  13. Michalak, M., Baksh, S. and Opas, M. Identification and immunolocalization of calreticulin in pancreatic cells: no evidence for “calciosomes”. Exp. Cell Res. 197 (1991) 91–99.

    Article  CAS  PubMed  Google Scholar 

  14. Fliegel, L., Burns, K., Opas, M. and Michalak, M. The high-affinity calcium binding protein of sarcoplasmic reticulum. Tissue distribution, and homology with calregulin. Biochim. Biophys. Acta 982 (1989) 1–8.

    Article  CAS  PubMed  Google Scholar 

  15. Opas, M., Dziak, E., Fliegel, L. and Michalak, M. Regulation of expression and intracellular distribution of calreticulin, a major calcium binding protein of nonmuscle cells. J. Cell. Physiol. 149 (1991) 160–171.

    Article  CAS  PubMed  Google Scholar 

  16. Szabo, E., Papp, S. and Opas, M. Calreticulin and cellular adhesion/migration-specific signalling pathways. J. Appl. Biomed. 4 (2006) 45–52.

    CAS  Google Scholar 

  17. Burns, K., Duggan, B., Atkinson, E.A., Famulski, K.S., Nemer, M., Bleackley, R.C. and Michalak, M. Modulation of gene expression by calreticulin binding to the glucocorticoid receptor. Nature 367 (1994) 476–480.

    Article  CAS  PubMed  Google Scholar 

  18. Burridge, K. and Chrzanowska-Wodnicka, M. Focal adhesions, contractility, and signaling. Annu. Rev. Cell Dev. Biol. 12 (1996) 463–518.

    Article  CAS  PubMed  Google Scholar 

  19. Gumbiner, B.M. Regulation of cadherin adhesive activity. J. Cell Biol. 148 (2000) 399–403.

    Article  CAS  PubMed  Google Scholar 

  20. Milner, R.E., Baksh, S., Shemanko, C., Carpenter, M.R., Smillie, L., Vance, J.E., Opas, M. and Michalak, M. Calreticulin, and not calsequestrin, is the major calcium binding protein of smooth muscle sarcoplasmic reticulum and liver endoplasmic reticulum. J. Biol. Chem. 266 (1991) 7155–7165.

    CAS  PubMed  Google Scholar 

  21. Geiger, B., Volberg, T., Ginsberg, D., Bitzur, S., Sabanay, I. and Hynes, R.O. Broad spectrum pan-cadherin antibodies, reactive with the C-terminal 24 amino acid residues of N-cadherin. J. Cell Sci. 97 (1990) 607–614.

    CAS  PubMed  Google Scholar 

  22. Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 (1970) 680–685.

    Article  CAS  PubMed  Google Scholar 

  23. Texteira, C.C., Hatori, M., Leboy, P.S., Pacifici, M. and Shapiro, I.M. A rapid and ultrasensitive method for measurement of DNA, calcium and protein content, and alkaline phosphatase activity of chondrocyte cultures. Calcif. Tissue Int. 56 (1995) 252–265.

    Article  Google Scholar 

  24. Rohrschneider, L. and Reynolds, S. Regulation of cellular morphology by the Rous sarcoma virus src gene: analysis of fusiform mutants. Mol. Cell. Biol. 5 (1985) 3097–3107.

    CAS  PubMed  Google Scholar 

  25. Papkoff, J. Regulation of complexed and free catenin pools by distinct mechanisms. Differential effects of Wnt-1 and v-Src. J. Biol. Chem. 272 (1997) 4536–4543.

    CAS  PubMed  Google Scholar 

  26. Kowalczyk, A.P., Palka, H.L., Luu, H.H., Nilles, L.A., Anderson, J.E., Wheelock, M.J. and Green, K.J. Posttranslational regulation of plakoglobin expression. Influence of the desmosomal cadherins on plakoglobin metabolic stability. J. Biol. Chem. 269 (1994) 31214–31223.

    CAS  PubMed  Google Scholar 

  27. Takeichi, M. Cadherins: A molecular family important in selective cell-cell adhesion. Annu. Rev. Biochem. 59 (1990) 237–252.

    Article  CAS  PubMed  Google Scholar 

  28. Gumbiner, B.M. and McCrea, P.D. Catenins as mediators of the cytoplasmic functions of cadherins. J. Cell Sci. 106 Suppl. 17 (1993) 155–158.

    CAS  Google Scholar 

  29. Aberle, H., Schwartz, H. and Kemler, R. Cadherin-catenin complex: Protein interactions and their implications for cadherin function. J. Cell. Biochem. 61 (1996) 514–523.

    Article  CAS  PubMed  Google Scholar 

  30. Eaton, S. and Cohen, S. Wnt signal transduction: More than one way to skin a (β-)cat? Trends Cell Biol. 6 (1996) 287–290.

    Article  CAS  PubMed  Google Scholar 

  31. Hirano, S., Nose, A., Hatta, K., Kawakami, A. and Takeichi, M. Calciumdependent cell-cell adhesion molecules (dycadherins): Subclass specificities and possible involvement of actin bundles. J. Cell Biol. 105 (1987) 2501–2510.

    Article  CAS  PubMed  Google Scholar 

  32. Itoh, M., Yonemura, S., Nagafuchi, A. and Tsukita, S. A 220-kD undercoatconstitutive protein: Its specific localization at cadherin-based cell-cell adhesion sites. J. Cell Biol. 115 (1991) 1449–1462.

    Article  CAS  PubMed  Google Scholar 

  33. Matsuyoshi, N., Hamaguchi, M., Taniguchi, S., Nagafuchi, A., Tsukita, S. and Takeichi, M. Cadherin-mediated cell-cell adhesion is perturbed by v-src tyrosine phosphorylation in metastatic fibroblasts. J. Cell Biol. 118 (1992) 703–714.

    Article  CAS  PubMed  Google Scholar 

  34. Knudsen, K.A., Soler, A.P., Johnson, K.R. and Wheelock, M.J. Interaction of α-actinin with the cadherin/catenin cell-cell adhesion complex via α-catenin. J. Cell Biol. 130 (1995) 67–77.

    Article  CAS  PubMed  Google Scholar 

  35. Yonemura, S., Itoh, M., Nagafuchi, A. and Tsukita, S. Cell-to-cell adherens junction formation and actin filament organization: Similarities and differences between non-polarized fibroblasts and polarized epithelial cells. J. Cell Sci. 108 (1995) 127–142.

    CAS  PubMed  Google Scholar 

  36. Matsuyoshi, N. and Imamura, S. Multiple cadherins are expressed in human fibroblasts. Biochemical and Biophysical Research Communications 235 (1997) 355–358.

    Article  CAS  PubMed  Google Scholar 

  37. Angres, B., Barth, A. and Nelson, W.J. Mechanism for transition from initial to stable cell-cell adhesion: Kinetic analysis of E-cadherin-mediated adhesion using a quantitative adhesion assay. J. Cell Biol. 134 (1996) 549–557.

    Article  CAS  PubMed  Google Scholar 

  38. Hazan, R.B., Kang, L., Roe, S., Borgen, P.I. and Rimm, D.L. Vinculin is associated with the E-cadherin adhesion complex. J. Biol. Chem. 272 (1997) 32448–32453.

    Article  CAS  PubMed  Google Scholar 

  39. Hazan, R.B. and Norton, L. The epidermal growth factor receptor modulates the interaction of E-cadherin with the actin cytoskeleton. J. Biol. Chem. 273 (1998) 9078–9084.

    Article  CAS  PubMed  Google Scholar 

  40. Provost, E. and Rimm, D.L. Controversies at the cytoplasmic face of the cadherin-based adhesion complex. Curr. Opin. Cell Biol. 11 (1999) 567–572.

    Article  CAS  PubMed  Google Scholar 

  41. Pokutta, S. and Weis, W.I. The cytoplasmic face of cell contact sites. Curr. Opin. Struct. Biol. 12 (2002) 255–262.

    Article  CAS  PubMed  Google Scholar 

  42. Paradies, N.E. and Grunwald, G.B. Purification and characterization of NCAD90, a soluble endogenous form of N-cadherin, which is generated by proteolysis during retinal development and retains adhesive and neuritepromoting function. J. Neurosci. Res. 36 (1993) 33–45.

    Article  CAS  PubMed  Google Scholar 

  43. Lagunowich, L.A. and Grunwald, G.B. Tissue and age-specificity of posttranslational modifications of N-cadherin during chick embryo development. Differentiation 47 (1991) 19–27.

    Article  CAS  PubMed  Google Scholar 

  44. Lee, M.M., Fink, B.D. and Grunwald, G.B. Evidence that tyrosine phosphorylation regulates N-cadherin turnover during retinal development. Dev. Genet. 20 (1997) 224–234.

    Article  CAS  PubMed  Google Scholar 

  45. Navarro, P., Caveda, L., Breviario, F., Mândoteanu, I., Lampugnani, M.G. and Dejana, E. Catenin-dependent and-independent functions of vascular endothelial cadherin. J. Biol. Chem. 270 (1995) 30965–30972.

    Article  CAS  PubMed  Google Scholar 

  46. Kreft, B., Berndorff, D., Böttinger, A., Finnemann, S., Wedlich, D., Hortsch, M., Tauber, R. and Gessner, R. LI-cadherin-mediated cell-cell adhesion does not require cytoplasmic interactions. J. Cell Biol. 136 (1997) 1109–1121.

    Article  CAS  PubMed  Google Scholar 

  47. Volberg, T., Geiger, B., Kam, Z., Pankov, R., Simcha, I., Sabanay, H., Coll, J.-L., Adamson, E. and Ben-Ze’ev, A. Focal adhesion formation by F9 embryonal carcinoma cells after vinculin gene disruption. J. Cell Sci. 108 (1995) 2253–2260.

    CAS  PubMed  Google Scholar 

  48. Rodríguez Fernández, J.L., Geiger, B., Salomon, D. and Ben-Ze’ev, A. Suppression of vinculin expression by antisense transfection confers changes in cell morphology, motility, and anchorage-dependent growth of 3T3 cells. J. Cell Biol. 122 (1993) 1285–1294.

    Article  PubMed  Google Scholar 

  49. Rodríguez Fernández, J.L., Geiger, B., Salomon, D., Sabanay, H., Zöller, M. and Ben-Ze’ev, A. Suppression of tumorigenicity in transformed cells after transfection with vinculin cDNA. J. Cell Biol. 119 (1992) 427–438.

    Article  PubMed  Google Scholar 

  50. Coll, J.L., Ben-Ze’ev, A., Ezzell, R.M., Fernández, J.L.R., Baribault, H., Oshima, R.G. and Adamson, E.D. Targeted disruption of vinculin genes in F9 and embryonic stem cells changes cell morphology, adhesion, and locomotion. Proc. Natl. Acad. Sci. USA 92 (1995) 9161–9165.

    Article  CAS  PubMed  Google Scholar 

  51. Goldmann, W.H., Schindl, M., Cardozo, T.J. and Ezzell, R.M. Motility of vinculin-deficient F9 embryonic carcinoma cells analyzed by video, laser confocal, and reflection interference contrast microscopy. Exp. Cell Res. 221 (1995) 311–319.

    Article  CAS  PubMed  Google Scholar 

  52. Geiger, B., Dutton, A.H., Tokuyasu, K.T. and Singer, S.J. Immunoelectron microscope studies of membrane-microfilament interactions: Distributions of alpha-actinin, tropomyosin, and vinculin in intestinal epithelial brush border and chicken gizzard smooth muscle cells. J. Cell Biol. 91 (1981) 614–628.

    Article  CAS  PubMed  Google Scholar 

  53. Takeichi, M., Atsumi, T., Yoshida, C., Uno, K. and Okada, T.S. Selective adhesion of embryonal carcinoma cells and differentiated cells by Ca2+-dependent sites. Dev. Biol. 87 (1981) 340–350.

    Article  CAS  PubMed  Google Scholar 

  54. Hatta, K., Nose, A., Nagafuchi, A. and Takeichi, M. Cloning and expression of cDNA encoding a neural calcium-dependent cell adhesion molecule: its identity in the cadherin gene family. J. Cell Biol. 106 (1988) 873–881.

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Laboratory Medicine and Pathobiology, University of Toronto, 1 King’s College Circle, Medical Sciences Building, Toronto, Ontario, Canada, M5S 1A8

    Michal Opas

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  1. Michal Opas
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  2. Marc P. Fadel
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Correspondence to Michal Opas.

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Opas, M., Fadel, M.P. Partial reversal of transformed fusiform phenotype by overexpression of calreticulin. Cell Mol Biol Lett 12, 294–307 (2007). https://doi.org/10.2478/s11658-006-0065-8

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  • DOI: https://doi.org/10.2478/s11658-006-0065-8

Key words

  • Calreticulin
  • Adhesion
  • Vinculin
  • N-Cadherin

Cellular & Molecular Biology Letters

ISSN: 1689-1392

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