Extracellular matrix (ECM) deposition and organization is maintained by transmembrane signaling and integrins play major roles. We now show that a second transmembrane component, syndecan-2 heparan sulfate proteoglycan, is pivotal in matrix assembly. Chinese Hamster Ovary (CHO) cells were stably transfected with full length (S2) or truncated syndecan-2 lacking the C-terminal 14 amino acids of the cytoplasmic domain (S2deltaS). No differences in the amount of matrix assembly were noted with S2 cells, but those expressing S2deltaS could not assemble laminin or fibronectin into a fibrillar matrix. The loss of matrix formation was not caused by a failure to synthesize or externalize ECM components as determined by metabolic labeling or due to differences in surface expression of alpha5 or beta1 integrin. The matrix assembly defect was at the cell surface, since S2deltaS cells also lost the ability to rearrange laminin or fibronectin substrates into fibrils and to bind exogenous fibronectin. Transfection of activated alphaIIbalphaLdeltabeta3 integrin into alpha(5)-deficient CHO B2 cells resulted in reestablishment of the previously lost fibronectin matrix. However, cotransfection of this cell line with S2deltaS could override the presence of activated integrins. These results suggest a regulatory role for syndecan-2 in matrix assembly, along with previously suggested roles for activated integrins.

REFERENCES

Akiyama
S. K.
,
Yamada
S. S.
,
Chen
W. T.
,
Yamada
K. M.
(
1989
).
Analysis of fibronectin receptor function with monoclonal antibodies: Roles in cell adhesion, migration, matrix assembly, and cytoskeletal organization.
J. Cell Biol
109
,
863
875
Ali
I. U.
,
Hynes
R. O.
(
1977
).
Effects of cytochalasin B and colchicine on attachment of a major surface protein of fibroblasts.
Biochim. Biophys. Acta
471
,
16
24
Aota
S.
,
Nomizu
M.
,
Yamada
K. M.
(
1994
).
The short amino acid sequence Pro-His-Ser-Arg-Asn in human fibronectin enhances cell-adhesive function.
J. Biol. Chem
269
,
24756
24761
Baciu
P. C.
,
Goetinck
P. F.
(
1995
).
Protein kinase C regulates the recruitment of syndecan-4 into focal contacts.
Mol. Biol. Cell
6
,
1503
1513
Barry
E. L. R.
,
Mosher
D. F.
(
1989
).
Factor XIIIa-mediated cross-linking of fibronectin in fibroblast cell layers.
J. Biol. Chem
264
,
4179
4185
Bernfield
M.
,
Kokenyesi
R.
,
Kato
M.
,
Hinkes
M. T.
,
Spring
J.
,
Gallo
R. L.
,
Lose
E. J.
(
1992
).
Biology of the syndecans.
Annu. Rev. Cell Biol
8
,
365
393
Brown
P. J.
,
Juliano
R. L.
(
1988
).
Monoclonal antibodies to distinctive epitopes on theand subunits of the fibronectin receptor.
Exp. Cell Res
177
,
303
318
Bultmann
H.
,
Santas
A. J.
,
Pesciotta Peters
D. M.
) (
1998
).
Fibronectin fibrillogenesis involves the Heparin II binding domain of fibronectin.
J. Biol. Chem
273
,
2601
2609
Carey
D. J.
(
1997
).
Syndecans: multifunctional cell-surface co-receptors.
Biochem. J
327
,
1
16
Chen
H.
,
Mosher
D. F.
(
1996
).
Formation of sodium dodecyl sulfate-stable fibronectin multimers. Failure to detect products of thiol-disulfide exchange in cyanogen bromide or limited acid digests of stabilized matrix fibronectin.
J. Biol. Chem
271
,
9084
9089
Christopher
R. A.
,
Kowalczyk
A. P.
,
McKeown-Longo
P. J.
) (
1997
).
Localization of fibronectin matrix assembly sites on fibroblasts and endothelial cells.
J. Cell Sci
110
,
569
581
Chung
C. Y.
,
Erickson
H. P.
(
1997
).
Glycosaminoglycans modulate fibronectin matrix assembly and are essential for matrix incorporation of tenascin-C.
J. Cell Sci
110
,
1413
1419
David
G.
,
Bai
X. M.
,
Van der Schueren
B.
,
Cassiman
J.-J.
,
Van den Berghe
H.
(
1992
).
Developmental changes in heparan sulfate expression: In situ detection with mAbs.
J. Cell Biol
119
,
961
975
David
G.
,
Van der Schueren
B.
,
Marynen
P.
,
Cassiman
J.-J.
,
Van den Berghe
H.
(
1992
).
Molecular cloning of amphiglycan, a novel integral membrane heparan sulfate proteoglycan expressed by epithelial and fibroblastic cells.
J. Cell Biol
118
,
961
969
David
G.
,
Bai
X. M.
,
Van der Schueren
B.
,
Marynen
P.
,
Cassiman
J.-J.
,
Van den Berghe
H.
(
1993
).
Spatial and temporal changes in the expression of fibroglycan (syndecan-2) during mouse embryonic development.
Development
119
,
841
854
Dzamba
B. J.
,
Bultmann
H.
,
Akiyama
S. K.
,
Peters
D. M.
(
1994
).
Substrate-specific binding of the amino terminus of fibronectin to an integrin complex in focal adhesions.
J. Biol. Chem
269
,
19646
19652
Fath
K. R.
,
Edgell
C.-J.
,
Burridge
K.
(
1989
).
The distribution ofdistinct integrins in focal contacts is determined by the substratum composition.
J. Cell Sci
92
,
67
75
Fogerty
F. J.
,
Akiyama
S. K.
,
Yamada
K. M.
,
Mosher
D. F.
(
1990
).
Inhibition of binding of fibronectin to matrix asssembly sites by anti-integrin (5 1) antibodies.
J. Cell Biol
111
,
699
708
Furtado
G. C.
,
Cao
Y.
,
Joiner
K. A.
(
1992
).
Laminin on Toxoplasma gondii mediates parasite binding to the beta 1 integrin receptor alpha 6 beta 1 on human foreskin fibroblasts and Chinese hamster ovary cells.
Infect. Immun
60
,
4925
4931
Giancotti
F.G.
,
Ruoslahti
E.
(
1990
).
Elevated levels of the5 1 fibronectin receptor suppress the transformed phenotype of Chinese hamster ovary cells.
Cell
60
,
849
859
Grinnell
F.
(
1986
).
Focal adhesion sites and the removal of substratum-bound fibronectin.
J. Cell Biol
103
,
2697
2706
Hocking
D. C.
,
Sottile
J.
,
McKeown-Longo
P. J.
) (
1994
).
Fibronectin's III-1 module contains a conformation-dependent binding site for the amino-terminal region of fibronectin.
J. Biol. Chem
269
,
19183
19187
Itano
N.
,
Oguri
K.
,
Nagayasu
Y.
,
Kusano
Y.
,
Nakanishi
H.
,
David
G.
,
Okayama
M.
(
1996
).
Phosphorylation of a membrane-intercalated proteoglycan, syndecan-2, expressed in a stroma-inducing clone from a Lewis mouse sarcoma.
Biochem. J
315
,
925
930
Kim
C. W.
,
Goldberger
O. A.
,
Gallo
R. L.
,
Bernfield
M.
(
1994
).
Members of the syndecan family of heparan sulfate proteoglycans are expressed in distinct cell-, tissue-, and development-specific patterns.
Mol. Biol. Cell
5
,
797
805
Koff
A.
,
Cross
F.
,
Fisher
A.
,
Schumacher
J.
,
Leguellec
K.
,
Philippe
M.
,
Roberts
J. M.
(
1991
).
Human cyclin E, a new cyclin that interacts with two members of the CDC2 gene family.
Cell
66
,
1217
1228
Koff
A.
,
Giordano
A.
,
Desai
D.
,
Yamashita
K.
,
Harper
J. W.
,
Elledge
S.
,
Nishimoto
T.
,
Morgan
D. O.
,
Franza
B. R.
,
Roberts.
J. M.
(
1992
).
Formation and activation of a cyclin E-cdk2 complex during the G1phase of the human cell cycle.
Science
257
,
1689
1694
Kojima
T.
,
Shworak
N. W.
,
Rosenberg
R. D.
(
1992
).
Molecular cloning and expression of two distinct cDNA-encoding heparan sulfate proteoglycan core proteins from a rat endothelial cell line.
J. Biol. Chem
267
,
4870
4877
LeBaron
R. G.
,
Esko
J. D.
,
Woods
A.
,
Johansson
S.
,
Höök
M.
(
1988
).
Adhesion of glycosaminoglycan-deficient Chinese hamster ovary cell mutants to fibronectin substrata.
J. Cell Biol
106
,
945
952
Longley
R. L.
,
Woods
A.
,
Fleetwood
A.
,
Cowling
G. J.
,
Gallagher
J. T.
,
Couchman
J. R.
(
1999
).
Control of morphology, cytoskeleton and migration by syndecan-4.
J. Cell Sci
112
,
3421
3431
McCarthy
K. J.
,
Bynum
K.
,
St. John
P. L.
,
Abrahamson
D. R.
,
Couchman
J. R.
(
1993
).
Basement membrane proteoglycans in glomerular morphogenesis: chondroitin sulfate proteoglycan is temporally and spatially restricted during development.
J. Histochem. Cytochem
41
,
401
414
McDonald
J. A.
,
Quade
B. J.
,
Broekelmann
T. J.
,
LaChance
R.
,
Forsman
K.
,
Hasegawa
E.
,
Akiyama
S.
(
1987
).
Fibronectin's cell adhesive domain and an amino-terminal matrix assembly domain participate in its assembly into fibroblast pericellular matrix.
J. Biol. Chem
262
,
2957
2967
McKeown-Longo
P. J.
,
Mosher
D. F.
(
1984
).
Mechanism of formation of disulfide-bonded multimers of plasma fibronectin in cell layers of cultured human fibroblasts.
J. Biol. Chem
259
,
12210
12215
McKeown-Longo
P. J.
,
Mosher
D. F.
(
1985
).
Interaction of the 70,000-mol-wt amino-terminal fragment of fibronectin with the matrix-assembly receptor of fibroblasts.
J. Cell Biol
100
,
364
374
Miekka
S. I.
,
Ingham
K. C.
,
Menache
D.
(
1982
).
Rapid methods for isolation of human plasma fibronectin.
Thromb. Res
27
,
1
14
Miettinen
H. M.
,
Edwards
S. N.
,
Jalkanen
M.
(
1994
).
Analysis of transport and targeting of syndecan-1: Effect of cytoplasmic tail deletions.
Mol. Biol. Cell
5
,
1325
1339
O'Toole
T. E.
,
Katagiri
Y.
,
Faull
R. J.
,
Peter
K.
,
Tamura
R. N.
,
Quaranta
V.
,
Loftus
J. C.
,
Shattil
S. J.
,
Ginsberg
M. H.
(
1994
).
Integrin cytoplasmic domains mediate inside-out signal transduction.
J. Cell Biol
124
,
1047
1059
Oh
E.-S.
,
Woods
A.
,
Couchman
J. R.
(
1997
).
Syndecan-4 proteoglycan regulates the distribution and activity of protein kinase C.
J. Biol. Chem
272
,
8133
8136
Oh
E.-S.
,
Woods
A.
,
Couchman
J. R.
(
1997
).
Multimerization of the cytoplasmic domain of syndecan-4 is required for its ability to activate protein kinase C.
J. Biol. Chem
272
,
11805
11811
Oh
E.-S.
,
Couchman
J. R.
,
Woods
A.
(
1997
).
Serine phosphorylation of syndecan-2 proteoglycan cytoplasmic domain.
Arch. Biochem. Biophys
344
,
67
74
Ott
V. L.
,
Rapraeger
A.C.
) (
1998
).
Tyrosine phosphorylation of syndecan-1 and —4 cytoplasmic domains in adherent B82 fibroblasts.
J. Biol. Chem
273
,
35291
35298
Pierce
A.
,
Lyon
M.
,
Hampson
I. N.
,
Cowling
G. J.
,
Gallagher
J. T.
(
1992
).
Molecular cloning of the major cell surface heparan sulfate proteoglycan from rat liver.
J. Biol. Chem
267
,
3894
3900
Prasthofer
T.
,
Ek
B.
,
Ekman
P.
,
Owens
R.
,
Höök
M.
,
Johansson
S.
(
1995
).
Protein kinase C phosphorylates two of the four known syndecan cytoplasmic domains in vitro.
Biochem. Mol. Biol. Int
36
,
793
802
Quade
B. J.
,
McDonald
J. A.
(
1988
).
Fibronectin's amino-terminal matrix assembly site is located within the 29-kDa amino-terminal domain containing five type I repeats.
J. Biol. Chem
263
,
19602
19609
Schreiner
C. L.
,
Bauer
J. S.
,
Danilov
Y. N.
,
Hussein
S.
,
Sczekan
M. M.
,
Juliano
R. L.
(
1989
).
Isolation and characterization of Chinese hamster ovary cell variantsdeficient in the expression of fibronectin receptor.
J. Cell Biol
109
,
3157
3167
Schwarzbauer
J. E.
(
1991
).
Identification of the fibronectin sequences required for assembly of a fibrillar matrix.
J. Cell Biol
113
,
1463
1473
Sechler
J.
,
Corbett
S. A.
,
Schwarzbauer
J. E.
(
1997
).
Modulatory roles for the integrin activation and the synergy site of fibronectin during matrix assembly.
Mol. Biol. Cell
8
,
2563
2573
Somers
C. E.
,
Mosher
D. F.
(
1993
).
Protein kinase C modulation of fibronectin matrix assembly.
J. Biol. Chem
268
,
22277
22280
Sottile
J.
,
Schwarzbauer
J.
,
Selegue
J.
,
Mosher
D. F.
(
1991
).
Five type I modules of fibronectin form a functional unit that binds to fibroblasts and staphylococcus aureus.
J. Biol. Chem
266
,
12840
12843
Sottile
J.
,
Wiley
S.
(
1994
).
Assembly of amino-terminal fibronectin dimers into the extracellular matrix.
J. Biol. Chem
269
,
17192
17198
Timpl
R.
,
Brown
J. C.
(
1996
).
Supramolecular assembly of basement membranes.
Bioessays
18
,
123
132
Woods
A.
,
Couchman
J. R.
(
1992
).
Protein kinase C involvement in focal adhesion formation.
J. Cell Sci
101
,
277
290
Woods
A.
,
Couchman
J. R.
(
1994
).
Syndecan-4 heparan sulfate proteoglycan is a selectively enriched and widespread focal adhesion component.
Mol. Biol. Cell
5
,
183
192
Woods
A.
,
Johansson
S.
,
Höök
M.
(
1988
).
Fibronectin fibril formation involves cell interactions with two fibronectin domains.
Exp. Cell Res
177
,
272
283
Wu
C.
,
Friedman
R.
,
Chung
A. E.
(
1988
).
Analysis of the assemblyof laminin and the laminin-entactin complex with laminin chain specific monoclonal and polyclonal antibodies.
Biochemistry
27
,
8780
8787
Wu
C.
,
Bauer
J. S.
,
Juliano
R. L.
,
McDonald
J. A.
(
1993
).
The5 1integrin fibronectin receptor, but not the5 cytoplasmic domain, functions in an early and essential step in fibronectin matrix assembly.
J. Biol. Chem
268
,
21883
21888
Wu
C.
,
Chung
A. E.
,
McDonald
J. A.
(
1995
).
A novel role for3 1 integrins in extracellular matrix assembly.
J. Cell Sci
108
,
2511
2523
Wu
C.
,
Keivens
V. M.
,
O'Toole
T. E.
,
McDonald
J. A.
,
Ginsberg
M. H.
(
1995
).
Integrin activation and cytoskeletal interaction are essential for the assembly of a fibronectin matrix.
Cell
83
,
715
724
Wu
C.
,
Fields
A. J.
,
Kapteijn
B. A.
,
McDonald
J. A.
(
1995
).
The role of4 1 integrin in cell motility and fibronectin matrix assembly.
J. Cell Sci
108
,
821
829
Wu
C.
,
Hughes
P. E.
,
Ginsberg
M. H.
,
McDonald
J. A.
(
1996
).
Identification of a new biological function for the integrin alpha v beta 3: initiation of fibronectin matrix assembly.
Cell Adhes. Commun
4
,
149
158
Yatohgo
T.
,
Izumi
M.
,
Kashiwagi
H.
,
Hayashi
M.
(
1988
).
Novel purification of vitronectin from human plasma by heparin affinity chromatography.
Cell Struct. Funct
13
,
281
292
Zhang
Z.
,
Morla
A. O.
,
Vuori
K.
,
Bauer
J. S.
,
Juliano
R. L.
,
Ruoslahti
E.
(
1993
).
The alpha v beta 1 integrin functions as a fibronectin receptor but does not support fibronectin matrix assembly and cell migration on fibronectin.
J. Cell Biol
122
,
235
242
Zhong
C.
,
Kinch
M. S.
,
Burridge
K.
(
1997
).
Rho-stimulated contractility contributes to the fibroblastic phenotype of Ras-transformed epithelial cells.
Mol. Biol. Cell
8
,
2329
2344
This content is only available via PDF.