Nanomolar concentrations of native fibronectin and its RGDS-containing cell-binding domain have previously been reported to stimulate fibroblast migration in the transmembrane (or ‘Boyden chamber’) assay; in contrast, the gelatin-binding domain (GBD) of fibronectin has consistently been reported to be devoid of migration-stimulating activity in this assay. We have examined the effects of fibronectin and several of its purified functional domains on the migration of human skin fibroblasts in what is presumably a more physiologically relevant assay involving the movement of cells into a 3-D matrix of native type I collagen fibrils. We report that: (a) femtomolar concentrations of GBD stimulate fibroblast migration into such collagen matrices; and (b) fibronectin, as well as peptides containing all other of its functional domains, do not exhibit migration-stimulating activity when tested in the femtomolar to nanomolar concentration range (i.e. 0.1 pg/ml to 1 microgram/ml). The correct assignment of migration-stimulating activity to GBD, rather than to a contaminant, was confirmed by: (a) the use of several fibronectin and GBD purification protocols; (b) the neutralization of GBD migration-stimulating activity by monoclonal antibodies directed against epitopes present in this domain; (c) the time-dependent generation of migration-stimulating activity by the proteolytic degradation of native fibronectin; and (d) obtaining an identical dose-response curve with a genetically engineered GBD peptide. The cryptic migration-stimulating activity of GBD was not affected by the presence of serum or native fibronectin, but was inhibited by TGF-beta 1. Parallel experiments using the transmembrane assay confirmed that GBD was devoid of migration-stimulating activity in this assay when membranes coated with gelatin were used, but revealed that significant stimulation of migration was achieved with membranes coated with native type I collagen. Cells preincubated with GBD for 24 hours whilst growing on plastic tissue culture dishes and then plated onto native collagen matrices in the absence of further GBD also displayed an elevated migration compared to controls. Taken together, these observations suggest that: (a) the interaction of GBD with a putative cell surface receptor (and not the collagen substratum) initiates a persistent alteration in cell phenotype which is manifest by an increase in migratory activity when these cells are cultured on a native collagen substratum; and (b) GBD may play a hitherto unrecognised role in the control of cell migration in response to the local release of proteases during pathological processes, such as tumour invasion and wound repair.

Ali
I. U.
,
Hynes
R. O.
(
1978
).
Effects of LETS glycoprotein on cell motility.
Cell
14
,
439
446
Aznavoorian
S. A.
,
Stracke
M. L.
,
Krutzsch
H.
,
Schiffmann
E.
,
Liotta
L. A.
(
1990
).
Signal transduction for chemotaxis and haptotaxis by matrix molecules in tumour cells.
J. Cell Biol
110
,
1427
1438
Chernousov
M. A.
,
Fogerty
F. J.
,
Koteliansky
V. E.
,
Mosher
D. F.
(
1991
).
Role of the I-9 and III-1 modules of fibronectin in formation of an extracellular fibronectin matrix.
J. Biol. Chem
266
,
10851
10858
Clark
R. A. F.
,
Wikner
N. E.
,
Doherty
D. E.
,
Norris
D. A.
(
1988
).
Cryptic chemotactic activity of fibronectin for human monocytes resides in the 120-kDa fibroblastic cell-binding fragment.
J. Biol. Chem
263
,
12115
12123
Clark
R. A. F.
(
1990
).
Fibronectin matrix deposition and fibronectin receptor expression in healing and normal skin.
J. Invest.Dermatol
90
,
6
–.
Dang
C. V.
,
Bell
W. R.
,
Kaiser
D.
,
Wong
A.
(
1985
).
Disorganization of cultured vascular endothelial cell monolayers by fibrinogen fragment D.
Science
227
,
1487
1490
De Petro
G.
,
Barlati
S.
,
Vartio
T.
,
Vaheri
A.
(
1981
).
Transformation-enhancing activity of gelatin-binding fragments of fibronectin.
Proc. Nat. Acad. Sci. USA
78
,
4965
4969
Ellis
I.
,
Grey
A. M.
,
Schor
A. M.
,
Schor
S. L.
(
1992
).
Antagonostic effects of TGF1 and MSF on fibroblast migration and hyaluronic acid synthesis: Possible implications for dermal wound healing.
J.Cell Sci
102
,
447
456
Ellis
I.
,
Schor
S. L.
(
1995
).
The interdependent modulation of hyaluronan synthesis by TGF1 and extracellular matrix: Consequences for the control of cell migration.
Growth Factors
12
,
211
222
Fava
R. A.
,
McClure
D. B.
(
1987
).
Fibronectin-associated transforming growth factor.
J. Cell. Physiol
131
,
184
189
Fukai
F.
,
Suzuki
H.
,
Suzuki
K.
,
Tsugita
A.
,
Katayama
T.
(
1991
).
Rat plasma fibronectin contains two distinct chemotactic domains for fibroblastic cells.
J. Biol. Chem
246
,
8807
8813
Fukai
F.
,
Iso
T.
,
Sekiguchi
K.
,
Miyatake
N.
,
Tsugita
A.
,
Katayama
T.
(
1993
).
An amino-terminal fibronectin fragment stimulates the differentiation of ST-13 preadipocytes.
Biochemistry
32
,
5746
5751
Fukai
F.
,
Ohtaki
M.
,
Fugii
N.
,
Yajima
H.
,
Ishii
T.
,
Nishizawa
Y.
,
Miyazaki
K.
,
Katayama
T.
(
1995
).
Release of biological activities from quiescent fibronectin by a conformational change and limited proteolysis by matrix metalloproteinases.
Biochemistry
34
,
11453
11459
Gheradi
E.
,
Gray
J.
,
Stoker
M.
,
Perryman
M.
,
Furlong
R.
(
1989
).
Purification of scatter factor, a fibroblast derived basic protein that modulates epithelial interactions and movement.
Proc. Nat. Acad. Sci. USA
86
,
5844
6848
Grinnell
F.
,
Billingham
R. E.
,
Burgess
L.
(
1981
).
Distribution of fibronectin during wound healing in vivo.
J. Invest. Dermatol
76
,
181
189
Homandberg
G. A.
,
Meyers
R.
,
Xie
D. L.
(
1992
).
Fibronectin fragments cause chondrolysis of bovine articular cartilage slices in culture.
J. Biol. Chem
267
,
3597
3604
Humphries
M. J.
,
Kororiya
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 interaction.
J. Cell Sci
97
,
585
592
Ingham
K. C.
,
Brew
S. A.
,
Migliorini
M. M.
(
1989
).
Further localization of the gelatin-binding determinants within fibronectin.
J. Biol. Chem
264
,
16977
16980
Lacovara
J.
,
Cramer
E. B.
,
Quigley
J. P.
(
1984
).
Fibronectin enhancement of directed migration of B16 melanoma cells.
Cancer Res
44
,
1657
1663
Laemmli
U. K.
(
1970
).
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227
,
680
685
Lambert-Vidmar
S.
,
Lottspeich
F.
,
Emod
I.
,
Planchenault
T.
,
Keil-Dlouha
V.
(
1991
).
Latent fibronectin degrading serine proteinase activity in N-terminal heparin binding domain of human plasma fibronectin.
Eur. J. Biochem
201
,
71
77
Lambert-Vidmar
S.
,
Lottspeich
F.
,
Emod
I.
,
Imhoff
J.-M.
,
Keil-Dlouha
V.
(
1991
).
Collagen-binding domain of human plasma fibronectin contains a latent type-IV collagenase.
Eur. J. Biochem
201
,
79
84
Lesot
H.
,
Fausser
J.-L.
,
Akiyama
S. K.
,
Staub
A.
,
Black
D.
,
Kubler
M.-D.
,
Ruch
J. V.
(
1992
).
The carboxy-terminal extension of the collagen binding domain of fibronectin mediates interaction with a 165kDa membrane protein involved in odontoblast differentiation.
Differentiation
49
,
109
118
Litvinovich
S. V.
,
Strickland
D. K.
,
Medved
L. V.
,
Ingham
K. C.
(
1991
).
Domain structure and interactions of the type I and type II modules in the gelatin-binding region of fibronectin.
J. Mol. Biol
217
,
563
575
McCarthy
J. B.
,
Hagen
S. T.
,
Furcht
L. T.
(
1986
).
Human fibronectin contains distinct adhesion-and motility-promoting domains for metastatic melanoma cells.
J. Cell Biol
102
,
179
188
Mooradian
D. L.
,
Lucas
R. C.
,
Weatherbee
J. A.
,
Furcht
L. T.
(
1989
).
Transforming growth factor-beta 1 binds to immobilized fibronectin.
J. Cell. Biochem
41
,
189
200
Morla
A.
,
Ruoslahti
E.
(
1992
).
A fibronectin self-assembly site involved in fibronectin matrix assembly: reconstruction in a synthetic peptide.
J. Cell Biol
118
,
421
429
Muir
D.
,
Manthorpe
M.
(
1992
).
Stromelysin generates a fibronectin fragment that inhibits Schwann cell proliferation.
J. Cell Biol
116
,
177
185
Nathan
C.
,
Sporn
M.
(
1991
).
Cytokines in context.
J. Cell Biol
113
,
981
986
O'Reilly
M. S.
,
Holmgren
L.
,
Shing
Y.
,
Chen
C.
,
Rosenthal
R. A.
,
Moses
M.
,
Lanne
W. S.
,
Cao
Y.
,
Sage
E. H.
,
Folkman
J.
(
1994
).
Angiostatin: A novel angiogenesis inhibitor that mediates the supression of metastasis by a Lewis lung carcinoma.
Cell
79
,
315
328
Perris
R.
,
Johansson
S.
(
1987
).
Amphibian neural crest cell migration on purified extracellular matrix components: a chondroitin sulfate proteoglycan inhibits locomotion on fibronectin substrates.
J. Cell Biol
105
,
2511
2521
Pierschbacher
M. D.
,
Ruoslahti
E.
(
1984
).
Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule.
Nature
309
,
30
33
Postlethwaite
A. E.
,
Keski-Oja
J.
,
Balian
G.
,
Kang
A. H.
(
1981
).
Induction of fibroblast chemotaxis by fibronectin. Localization of the chemotactic region to a 140,000-molecular weight non-gelatin-binding fragment.
J. Exp. Med
153
,
494
499
Postlethwaite
A. E.
,
KeskiOja
J.
,
Moses
H. L.
,
Kang
A. H.
(
1987
).
Stimulation of chemotactic migration of human fibroblasts by TGF.
J. Exp. Med
165
,
251
256
Rosen
E. M.
,
Goldberg
I. D.
(
1989
).
Protein factors which regulate cell motility.
In Vitro Cell.Dev. Biol
25
,
1079
1087
Schor
S. L.
(
1980
).
Cell proliferation and migration within three-dimensional collagen gels.
J. Cell Sci
41
,
159
175
Schor
S. L.
(
1994
).
Cytokine control of cell motility: modulation and mediation by the extracellular matrix.
Prog. Growth Factor Res
5
,
223
248
Schor
S. L.
,
Court
J.
(
1979
).
Different mechanisms involved in the attachment of cells to native and denatured collagen.
J. Cell Sci
38
,
267
281
Schor
S. L.
,
Schor
A. M.
,
Bazill
G. W.
(
1981
).
The effects of fibronectin on the migration of human foreskin fibroblasts and syrian hamster melanoma cells into three-dimensional gels of native collagen fibres.
J. Cell Sci
48
,
301
314
Schor
S. L.
,
Schor
A. M.
,
Bazill
G. W.
(
1981
).
The effects of fibronectin on the migration and adhesion of Chinese hamster ovary cells to collagen.
J. Cell Sci
49
,
299
310
Stoker
M.
,
Gherardi
E.
(
1991
).
Regulation of cell movement: the motogenic cytokines.
Biochim. Biophys. Acta
1072
,
81
102
Thiery
J. P.
,
Duband
J. L.
,
Tucker
G. C.
(
1985
).
Cell migration in the vertebrate embryo: Role of cell adhesion and tissue environment in pattern formation.
Annu. Rev. Cell Biol
1
,
91
113
Werb
Z.
,
Tremble
P. M.
,
Behrendsten
O.
,
Cowley
E.
,
Damsky
C. H.
(
1989
).
Signal transduction through fibronectin receptor induces collagenase and stromelysin gene expression.
J. Cell Biol
109
,
877
889
Wysocki
A. B.
,
Grinnell
F.
(
1990
).
Fibronectin profiles in normal and chronic wound fluid.
Lab. Invest
63
,
825
831
Zardi
L.
,
Carnemolla
B.
,
Balza
E.
,
Borsi
L.
,
Castellani
P.
,
Rocco
M.
,
Siri
A.
(
1985
).
Elution of fibronectin proteolytic fragments from a hydroxyapatite chromatography column.
Eur. J. Biochem
146
,
571
579
Zigmond
S. H.
,
Hirsch
J. G.
(
1973
).
Leukocyte locomotion and chemotaxis.
J. Exp. Med
137
,
387
410
This content is only available via PDF.