Modification of integrin-mediated cell attachment to substrata by serine proteinases in the presence and absence of divalent cations

Kimio Fujii, Gerald Sylvan Lazarus, Norman M. Schechter

Research output: Contribution to journalArticle

Abstract

The sensitivity to serine proteinases of cellular proteins involved in cell-matrix adhesion was investigated using C32 melanoma cells. Cells dissociated from monolayers by the metal chelator ethylenediaminetetraacetic acid were incubated with proteolytic enzymes, and then attachment was quantified by standard cell adhesion assays. The effect of proteinases was found to depend on the presence of Ca2+ in the incubations. Incubation with 100 nM trypsin or chymotrypsin for 1-2 h (37°C) in the absence of Ca2+ reduced cell attachment to vitronectin (Vn), fibrinogen (Fb), laminin, and fibronectin by approximately 80, 80, 40, and 30%, respectively. Viability studies indicated that such treatment with proteinases was not cytotoxic. Inclusion of 0.1 mM CaCl2 in the incubations prevented the loss in attachment to all substrata. In the case of Kb, proteinase treatment in the presence of Ca2+ had an additional effect; it improved cell attachment to this substratum by about 50%. C32 cells have been shown to express the integrin αvβ3 (Vn receptor) which mediates attachment to Vn and Fb in a GRGDS-sensitive manner. Attachment of C32 cells to Vn and Fb prior to proteinase treatment and after proteinase treatment in the presence of Ca2+ was 90% inhibited by the addition of GRGDS peptide to the attachment assays. These results suggest that the adhesion observed both before and after proteinase treatment was mediated by this integrin. Analysis of the Vn receptor from proteinase-treated cells by immunoblotting of cell extracts and by SDS gel electrophoresis of immunoprecipitated receptor revealed no detectable change in either the αv or β3 subunit that correlated with loss in attachment. Similarly proteinase treatment in the presence of Ca2+ did not produce detectable alterations in the subunits which might correlate with the improved attachment to Fb. Consistent with these results, an enzyme-linked immunoassay to quantify cell surface receptors revealed little difference in the amount of Vn receptor on cells treated with proteinase in the presence or absence of Ca2+. Degradation of the αv subunit was demonstrated, however, at proteinase concentrations higher than those required to affect cell attachment. Thus, treatment of cells with serine proteinases can affect integrin-mediated attachment to matrix proteins in a manner moderated by Ca2+, but the alterations in attachment do not appear to be accompanied by detectable proteolytic modification of the integrin.

Original languageEnglish (US)
Pages (from-to)94-103
Number of pages10
JournalExperimental Cell Research
Volume208
Issue number1
StatePublished - 1993
Externally publishedYes

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Divalent Cations
Serine Proteases
Integrins
Peptide Hydrolases
Integrin alphaVbeta3
Vitronectin
Fibrinogen
glycyl-arginyl-glycyl-aspartyl-serine
Therapeutics
Cell-Matrix Junctions
Cell Surface Receptors
Laminin
Chelating Agents
Cell Extracts
Immunoenzyme Techniques
Fibronectins
Immunoblotting
Cell Adhesion
Edetic Acid
Electrophoresis

ASJC Scopus subject areas

  • Cell Biology

Cite this

Modification of integrin-mediated cell attachment to substrata by serine proteinases in the presence and absence of divalent cations. / Fujii, Kimio; Lazarus, Gerald Sylvan; Schechter, Norman M.

In: Experimental Cell Research, Vol. 208, No. 1, 1993, p. 94-103.

Research output: Contribution to journalArticle

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title = "Modification of integrin-mediated cell attachment to substrata by serine proteinases in the presence and absence of divalent cations",
abstract = "The sensitivity to serine proteinases of cellular proteins involved in cell-matrix adhesion was investigated using C32 melanoma cells. Cells dissociated from monolayers by the metal chelator ethylenediaminetetraacetic acid were incubated with proteolytic enzymes, and then attachment was quantified by standard cell adhesion assays. The effect of proteinases was found to depend on the presence of Ca2+ in the incubations. Incubation with 100 nM trypsin or chymotrypsin for 1-2 h (37°C) in the absence of Ca2+ reduced cell attachment to vitronectin (Vn), fibrinogen (Fb), laminin, and fibronectin by approximately 80, 80, 40, and 30{\%}, respectively. Viability studies indicated that such treatment with proteinases was not cytotoxic. Inclusion of 0.1 mM CaCl2 in the incubations prevented the loss in attachment to all substrata. In the case of Kb, proteinase treatment in the presence of Ca2+ had an additional effect; it improved cell attachment to this substratum by about 50{\%}. C32 cells have been shown to express the integrin αvβ3 (Vn receptor) which mediates attachment to Vn and Fb in a GRGDS-sensitive manner. Attachment of C32 cells to Vn and Fb prior to proteinase treatment and after proteinase treatment in the presence of Ca2+ was 90{\%} inhibited by the addition of GRGDS peptide to the attachment assays. These results suggest that the adhesion observed both before and after proteinase treatment was mediated by this integrin. Analysis of the Vn receptor from proteinase-treated cells by immunoblotting of cell extracts and by SDS gel electrophoresis of immunoprecipitated receptor revealed no detectable change in either the αv or β3 subunit that correlated with loss in attachment. Similarly proteinase treatment in the presence of Ca2+ did not produce detectable alterations in the subunits which might correlate with the improved attachment to Fb. Consistent with these results, an enzyme-linked immunoassay to quantify cell surface receptors revealed little difference in the amount of Vn receptor on cells treated with proteinase in the presence or absence of Ca2+. Degradation of the αv subunit was demonstrated, however, at proteinase concentrations higher than those required to affect cell attachment. Thus, treatment of cells with serine proteinases can affect integrin-mediated attachment to matrix proteins in a manner moderated by Ca2+, but the alterations in attachment do not appear to be accompanied by detectable proteolytic modification of the integrin.",
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N2 - The sensitivity to serine proteinases of cellular proteins involved in cell-matrix adhesion was investigated using C32 melanoma cells. Cells dissociated from monolayers by the metal chelator ethylenediaminetetraacetic acid were incubated with proteolytic enzymes, and then attachment was quantified by standard cell adhesion assays. The effect of proteinases was found to depend on the presence of Ca2+ in the incubations. Incubation with 100 nM trypsin or chymotrypsin for 1-2 h (37°C) in the absence of Ca2+ reduced cell attachment to vitronectin (Vn), fibrinogen (Fb), laminin, and fibronectin by approximately 80, 80, 40, and 30%, respectively. Viability studies indicated that such treatment with proteinases was not cytotoxic. Inclusion of 0.1 mM CaCl2 in the incubations prevented the loss in attachment to all substrata. In the case of Kb, proteinase treatment in the presence of Ca2+ had an additional effect; it improved cell attachment to this substratum by about 50%. C32 cells have been shown to express the integrin αvβ3 (Vn receptor) which mediates attachment to Vn and Fb in a GRGDS-sensitive manner. Attachment of C32 cells to Vn and Fb prior to proteinase treatment and after proteinase treatment in the presence of Ca2+ was 90% inhibited by the addition of GRGDS peptide to the attachment assays. These results suggest that the adhesion observed both before and after proteinase treatment was mediated by this integrin. Analysis of the Vn receptor from proteinase-treated cells by immunoblotting of cell extracts and by SDS gel electrophoresis of immunoprecipitated receptor revealed no detectable change in either the αv or β3 subunit that correlated with loss in attachment. Similarly proteinase treatment in the presence of Ca2+ did not produce detectable alterations in the subunits which might correlate with the improved attachment to Fb. Consistent with these results, an enzyme-linked immunoassay to quantify cell surface receptors revealed little difference in the amount of Vn receptor on cells treated with proteinase in the presence or absence of Ca2+. Degradation of the αv subunit was demonstrated, however, at proteinase concentrations higher than those required to affect cell attachment. Thus, treatment of cells with serine proteinases can affect integrin-mediated attachment to matrix proteins in a manner moderated by Ca2+, but the alterations in attachment do not appear to be accompanied by detectable proteolytic modification of the integrin.

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