The mechanism of interaction of chondrocytic cells with cartilage-specific type II collagen has been examined using HCS-2/8 human chondrosarcoma cells as a model system. By the criteria of specific collagen secretion and integrin expression profile, HCS-2/8 have a similar differentiated phenotype to normal chondrocytes and are therefore a good model system. HCS-2/8 cells were able to attach and spread on both native and heat-denatured pepsinised type II collagen, and assays using denatured cyanogen bromide fragments apparently localised the major cell binding site to the CB10 fragment. However, when they were used as soluble inhibitors, cyanogen bromide fragments were found to block adhesion to denatured collagen, but had no effect on either attachment or spreading on the native molecule. The inability of cyanogen bromide fragments to reproduce the cell binding site of native collagen demonstrated a strict dependence on collagen conformation. This was also reflected in the receptors that were employed by HCS-2/8 cells for binding to type II collagen: binding to native collagen was mediated by the integrin alpha 2 beta 1 while binding to denatured collagen was mediated by a novel alpha 5 beta 1-fibronectin bridge. The identification of this bridge adds to the mechanisms by which cells can bind to denatured collagens. The previously characterised KDGEA active site peptide from type I collagen was found to be inactive as an inhibitor of type II collagen-mediated adhesion. The implications of these findings for the strategies used to identify adhesive active sites within collagens are discussed. In particular, these data suggest that, unlike other integrin ligands, a synthetic peptide-based approach is not suitable for the identification of collagen active sites.
REFERENCES
Akiyama
S. K.
, Yamada
K. M.
(1987
). Biosynthesis and acquisition of biological activity of the fibronectin receptor.
J. Biol. Chem
262
, 17536
–17542
.Akiyama
S. K.
, Nagata
K.
, Yamada
K. M.
(1990
). Cell surface receptors for extracellular matrix components.
Biochim. Biophys. Acta
1031
, 91
–110
.Aumailley
M.
, Timpl
R.
(1986
). Attachment of cells to basement membrane collagen type VI.
J. Cell Biol
103
, 1569
–1575
.Aumailley
M.
, Mann
K.
, von der Mark
H.
, Timpl
R.
(1989
). Cell attachment properties of collagen type VI and arg-gly-asp dependent binding to its2(VI) and 3(VI) chains.
Exp. Cell Res
181
, 463
–474
.Ayad
S.
, Abedin
M. Z.
, Grundy
S.
, Weiss
J. B.
(1981
). Isolation and characterisation of an unusual collagen from hyaline cartilage and intervertebral disc.
FEBS Lett
123
, 195
–199
.Cardarelli
P. M.
, Yamagata
S.
, Taguchi
I.
, Gorcsan
F.
, Chiang
S. L.
, Lobl
T.
(1992
). The collagen receptor2 1from MG-63 and HT1080 cells, interacts with a cyclic RGD peptide.
J. Biol. Chem
267
, 23159
–23164
.Coller
B. S.
, Beer
J. H.
, Scudder
L. E.
, Steinberg
M. H.
(1989
). Collagen-platelet interactions: evidence for a direct interaction of collagen with platelet GPIa/IIa and an indirect interaction with platelet GPIIa/IIIa mediated by adhesive proteins.
Blood
74
, 182
–192
.Davis
G. E.
(1992
). Affinity of integrins for damaged extracellular matrix:v 3binds to denatured collagen type I through RGD sites.
Biochem. Biophys. Res. Commun
182
, 1025
–1031
.Dedhar
S.
, Gray
V.
(1990
). Isolation of a novel integrin receptor mediating Arg-Gly-Asp-directed cell adhesion to fibronectin and type I collagen from human neuroblastoma cells; association of a novel1-related subunit with v.
J. Cell Biol
110
, 2185
–2193
.Dessau
W.
, Sasse
J.
, Timpl
R.
, von der Mark
K.
(1978
). Roles of fibronectin and collagen types I and II in chondrocytic differentiation in vitro.
Ann. NY Acad. Sci
312
, 404
–405
.Eble
J. A.
, Golbik
R.
, Mann
K.
, Kuhn
K.
(1993
). The1 1integrin recognition site of the basement membrane collagen molecule [1(IV)]2 2(IV).
EMBO J
12
, 4795
–4802
.Enomoto
M.
, Leboy
P. S.
, Menko
A. S.
, Boettiger
D.
(1993
). 1integrins mediate chondrocyte interaction with type I collagen, type II collagen, and fibronectin.
Exp. Cell Res
205
, 276
–285
.Fressinaud
E.
, Girma
J. P.
, Sadler
J. E.
, Baumgartner
H. R.
, Meyer
D.
(1990
). Synthetic RGDS-containing peptides of von Willebrand factor inhibit platelet adhesion to collagen.
Thromb. Haemost
64
, 589
–593
.Gisslow
M. T.
, McBride
B. C.
(1975
). A rapid sensitive collagenase assay.
Anal. Biochem
68
, 70
–78
.Grinnell
F.
(1978
). Cellular adhesiveness and extracellular substrata.
Int. Rev. Cytol
53
, 65
–144
.Grinnell
F.
, Feld
M. K.
(1979
). Initial adhesion of human fibroblasts in serum-free medium: possible role of secreted fibronectin.
Cell
17
, 117
–129
.Grinnell
F.
, Feld
M. K.
(1980
). Spreading of human fibroblasts in serum-free medium: inhibition by dithiothreitol and the effect of cold insoluble globulin (plasma fibronectin).
J. Cell. Physiol
104
, 321
–334
.Guidry
C.
, Miller
E. J.
, Höök
M.
(1990
). A second fibronectin-binding region is present in collagenchains.
J. Biol. Chem
265
, 19230
–19236
. 254–264.Gullberg
D.
, Gehlsen
K. R.
, Turner
D. C.
, Åhlen
K.
, Zijenah
L. S.
, Barnes
M. J.
, Rubin
K.
(1992
). Analysis of1 12 1and3 1integrins in cell-collagen interactions: identification of conformation dependent1 1binding sites in collagen type I.
EMBO J
11
, 3865
–3873
.Hemler
M. E.
(1990
). VLA proteins in the integrin family: structures, functions and their roles on leukocytes.
Annu. Rev. Immunol
8
, 365
–400
.Humphries
M. J.
, Komoriya
A.
, Akiyama
S.K.
, Olden
K.
, Yamada
K.M.
(1987
). Identification of two distinct regions of the type III connecting segment of human plasma fibronectin that promote cell type-specific adhesion.
J. Biol. Chem
262
, 6886
–6892
.Humphries
M. J.
(1990
). The molecular basis and specificity of integrin-ligand interactions.
J. Cell Sci
97
, 585
–592
.Hynes
R. O.
(1987
). Integrins: a family of cell surface receptors.
Cell
48
, 549
–554
.Hynes
R. O.
(1992
). Integrins: Versatility, modulation, and signalling in cell adhesion.
Cell
69
, 11
–25
.Kleinman
H. K.
, McGoodwin
E. B.
, Martin
G. R.
, Klebe
R. J.
, Fietzek
P. P.
, Wooley
D. E.
(1978
). Localization of the binding site for cell attachment in the1(I) chain of collagen.
J. Biol. Chem
253
, 5642
–5646
.Kleinman
H. K.
, Murray
J. C.
, McGoodwin
E. B.
, Martin
G. R.
(1978
). Connective tissue structure: cell binding to collagen.
J. Invest. Derm
71
, 9
–11
.Kramer
R. H.
, Marks
N.
(1989
). Identification of integrin collagen receptors on human melanoma cells.
J. Biol. Chem
264
, 4684
–4688
.Laemmli
U. K.
(1970
). Cleavage of structural protein during the assembly of the head bacteriophage T4.
Nature
227
, 680
–685
.Lebien
T. W.
, Bove
D. R.
, Bradley
J. G.
, Kersey
J. H.
(1982
). Antibody affinity may influence antigenic modulation of the common acute lymphoblastic leukemia antigen in vitro.
J. Immunol
129
, 2287
–2292
.Marshall
J. F.
, Nesbitt
S. A.
, Helfrich
M. H.
, Horton
M. A.
, Polakova
K.
, Hart
I. R.
(1991
). Integrin expression on human melanoma cell lines: Heterogeneity of vitronectin receptor composition and function.
Int. J. Cancer
49
, 924
–931
.Miller
E. J.
, Lunde
L. G.
(1973
). Isolation and characterisation of the cyanogen bromide peptides from the1(II) chain of bovine and human cartilage collagen.
Biochemistry
12
, 3153
–3159
.Miller
E. J.
, Rhodes
R. K.
(1982
). Preparation and characterisation of the different types of collagen.
Meth. Enzymol
82
, 33
–64
.Mollenhauer
J.
, von der Mark
K.
(1983
). Isolation and characterization of a collagen-binding glycoprotein from chondrocyte membranes.
EMBO J
2
, 45
–50
.Nip
J.
, Shibata
H.
, Loskutoff
D. J.
, Cheresh
D. A.
, Brodt
P.
(1992
). Human melanoma cells derived from lymphatic metastases use integrinv 3to adhere to lymph node vitronectin.
J. Clin. Invest
90
, 1406
–1413
.Piez
K.
(1968
). Molecular weight determination of random coil polypeptides from collagen by molecular sieve chromatography.
Anal. Biochem
26
, 305
–312
.Rubin
K.
, Höök
M.
, Öbrink
B.
, Timpl
R.
(1981
). Substrate adhesion of rat hepatocytes: Mechanism of attachment to collagen substrates.
Cell
24
, 463
–470
.Salter
D. M.
, Hughes
D. E.
, Simpson
R.
, Gardner
D. L.
(1992
). Integrin expression by human articular chondrocytes.
Br. J. Rheum
31
, 231
–234
.Santoro
S. A.
(1986
). Identification of a 160,000 dalton platelet membrane protein that mediates the initial divalent cation-dependent adhesion of platelets to collagen.
Cell
46
, 913
–920
.Schor
S. L.
, Court
J.
(1979
). Different mechanisms in the attachment of cells to native and denatured collagen.
J. Cell Sci
38
, 267
–281
.Staatz
W. D.
, Rajpara
S. M.
, Wayner
E. A.
, Carter
W. G.
, Santoro
S. A.
(1989
). The membrane glycoprotein Ia-IIa (VLA-2) complex mediates the magnesium-dependent adhesion of platelets to collagen.
J. Cell Biol
108
, 1917
–1924
.Staatz
W. D.
, Walsh
J. J.
, Pexton
T.
, Santoro
S. A.
(1990
). The2 1integrin cell surface collagen receptor binds to the1(I)-CB3 peptide of collagen.
J. Biol. Chem
265
, 4778
–4781
.Staatz
W. D.
, Fok
K. F.
, Zutter
M. M.
, Adams
S. P.
, Rodriguez
B. A.
, Santoro
S. A.
(1991
). Identification of a tetrapeptide recognition sequence for the2 1integrin in collagen.
J. Biol. Chem
266
, 7363
–7367
.Takada
Y.
, Wayner
E. A.
, Carter
W. G.
, Hemler
M. E.
(1988
). 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
37
, 385
–393
.Takigawa
M.
, Tajima
K.
, Pan
H.-O.
, Enomoto
M.
, Kinoshita
A.
, Suzuki
F.
, Takano
Y.
, Mori
Y.
(1989
). Establishment of a clonal human chondrosarcoma cell line with cartilage phenotypes.
Cancer Res
49
, 3996
–4002
.Tenchini
M. L.
, Adams
J. C.
, Gilbert
C.
, Steel
J.
, Hudson
D. L.
, Malcovati
M.
, Watt
F. M.
(1993
). Evidence against a major role for integrins in calcium-dependent intercellular adhesion of epidermal keratinocytes.
Cell Adhesion Commun
1
, 55
–66
.Traub
W.
, Piez
K. A.
(1971
). The chemistry and structure of collagen.
Advan. Protein Chem
25
, 243
–352
.Vandenberg
P.
, Kern
A.
, Ries
A.
, Luckenbill-Edds
L.
, Mann
K.
, Kuhn
K.
(1991
). Characterization of a type IV collagen major cell binding site with affinity to the1 1and the2 1integrins.
J. Cell Biol
113
, 1475
–1483
.Verfaille
C.
, McCarthy
J. C.
, McGlave
P. B.
(1992
). Mechanisms underlying abnormal trafficking of malignant progenitors in chronic myelogenous leukemia.
J. Clin. Invest
90
, 1232
–1241
.Wayner
E. A.
, Carter
W. G.
(1987
). Identification of multiple cell adhesion receptors for collagen and fibronectin in human fibrosarcoma cells possessing uniqueand common subunits.
J. Cell Biol
105
, 1873
–1884
.Yamada
K. M.
, Olden
K.
(1978
). Fibronectins- adhesive glycoproteins of cell surface and blood.
Nature
275
, 179
–184
.
This content is only available via PDF.
© 1994 by Company of Biologists
1994
-PolarityCFP.png?versionId=4696)
