In the mature gut, laminin-5 is expressed at the basal aspect of the differentiating epithelial cells. In vitro, we show that three more or less differentiated human colonic cancer HT29 cell lines produce and deposit laminin-5; they predominantly synthesize and secrete the 440 kDa form of laminin-5 that comprises the unprocessed 155 kDa gamma2 chain, as determined by immunoprecipitation analysis. In contrast, the highly differentiated colon carcinoma Caco-2 cells produce almost no laminin-5. Using anti-integrin antibodies, we show that adhesion of the two colonic cancer cell lines to laminin-5 is mediated by multiple integrin receptors including those for alpha3beta1, alpha6beta1 and alpha6beta4 integrins like in other cell types. In addition, the implication of integrin alpha2beta1 in this adhesion process is demonstrated for the first time. This has been shown by cell adhesion inhibition experiments, solid phase assays and confocal analysis. Together with previous in situ observations, these data provide a baseline knowledge for the understanding of the regulation of laminin-5 in normal and pathological intestine.

REFERENCES

Aberdam
D.
,
Galliano
M. F.
,
Mattei
M. G.
,
Pisani-Spadafora
A.
,
Ortonne
J. P.
,
Meneguzzi
G.
(
1994
).
Assignment of mouse nicein genes to chromosomes 1 and 18.
Mamm. Genome
5
,
229
233
Aberdam
D.
,
Galliano
M. F.
,
Vailly
J.
,
Pulkkinen
L.
,
Bonifas
J.
,
Christiano
A. M.
,
Tryggvason
K.
,
Uitto
J.
,
Epstein
E. H.
Jr.
,
Ortonne
J. P.
(
1994
).
Herlitz's junctional epidermolysis bullosa is linked to mutations in the gene (LAMC2) for the gamma 2 subunit of nicein/kalinin (LAMININ-5).
Nature Genet
6
,
299
304
Basson
M. D.
,
Modlin
I. M.
,
Madri
J. A.
(
1992
).
Human enterocyte (Caco-2) migration is modulated in vitro by extracellular matrix composition and epidermal growth factor.
J. Clin. Invest
90
,
15
23
Basson
M. D.
,
Turowski
G.
,
Emenaker
N. J.
(
1996
).
Regulation of human (Caco-2) intestinal epithelial cell differentiation by extracellular matrix proteins.
Exp. Cell Res
225
,
301
305
Brown
T. A.
,
Gil
S. G.
,
Sybert
V. P.
,
Lestringant
G. G.
,
Tadini
G.
,
Caputo
R.
,
Carter
W. G.
(
1996
).
Defective integrin alpha 6 beta 4 expression in the skin of patients with junctional epidermolysis bullosa and pyloric atresia.
J. Invest. Dermatol
107
,
384
391
Calderwood
D. A.
,
Tuckwell
D. S.
,
Eble
J.
,
Kuhn
K.
,
Humphries
M. J.
(
1997
).
The integrin1 A-domain is a ligand binding site for collagens and laminin.
J. Biol. Chem
272
,
12311
12317
Carter
W. G.
,
Ryan
M. C.
,
Gahr
P. J.
(
1991
).
Epiligrin, a new cell adhesion ligand for integrin3 1 in epithelial basement membranes.
Cell
65
,
599
610
Champliaud
M. F.
,
Lunstrum
G. P.
,
Rousselle
P.
,
Nishiyama
T.
,
Keene
D. R.
,
Burgeson
R. E.
(
1996
).
Human amnion contains a novel laminin variant, laminin 7, which like laminin 6, covalently associates with laminin 5 to promote stable epithelial-stromal attachment.
J. Cell Biol
132
,
1189
1198
Chan
B. M. C.
,
Hemler
M. E.
(
1993
).
Multiple functional forms of the integrin VLA-2 can be derived from a single(2) cDNA clone—Interconversion of forms induced by an anti- (1) antibody.
J. Cell Biol
120
,
537
543
Chantret
I.
,
Barbat
A.
,
Dussaulx
E.
,
Brattain
M. G.
,
Zweibaum
A.
(
1988
).
Epithelial polarity, villin expression, and enterocytic differentiation of cultured human colon carcinoma cells: a survey of twenty cell lines.
Cancer Res
48
,
1936
1942
De Arcangelis
A.
,
Simo
P.
,
Lesuffleur
T.
,
Kedinger
M.
,
Simon-Assmann
P.
(
1994
).
L'expression de la laminine est correlee à la differenciation des cellules cancereuses coliques humaines.
Gastroenterol. Clin. Biol
18
,
630
637
De Arcangelis
A.
,
Neuville
P.
,
Boukamel
R.
,
Lefebvre
O.
,
Kedinger
M.
,
Simon-Assmann
P.
(
1996
).
Inhibition of laminin1-chain expression leads to alteration of basement membrane assembly and cell differentiation.
J. Cell Biol
133
,
417
430
Delwel
G. O.
,
Hogervorst
F.
,
Kuikman
I.
,
Paulsson
M.
,
Timpl
R.
,
Sonnenberg
A.
(
1993
).
Expression and function of the cytoplasmic variants of the integrin-6 subunit in transfected K562 cells—Activation-dependent adhesion and interaction with isoforms of laminin.
J. Biol. Chem
268
,
25865
25875
Delwel
G. O.
,
De Melker
A. A.
,
Hogervorst
F.
,
Jaspars
L. H.
,
Fles
D. L.
,
Kuikman
I.
,
Lindblom
A.
,
Paulsson
M.
,
Timpl
R.
,
Sonnenberg
A.
(
1994
).
Distinct and overlapping ligand specificities of the3A 1 and6A 1 integrins: recognition of laminin isoforms.
Mol. Biol. Cell
5
,
203
215
Diamond
M. S.
,
Springer
T. A.
(
1994
).
The dynamic regulation of integrin adhesiveness.
Curr. Biol
4
,
506
517
Dieckgraefe
B. K.
,
Santoro
S. A.
,
Alpers
D. H.
(
1996
).
Immunolocalization of-integrin subunits and extracellular matrix components during human colonic organogenesis.
Gastroenterology
110
,
58
71
Ebert
E. C.
(
1996
).
Mechanisms of colon cancer binding to substratum and cells.
Dig. Dis. Sci
41
,
1551
1556
Fogh
J.
,
Fogh
J. M.
,
Orfeo
T.
(
1977
).
One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice.
J. Nat. Cancer Inst
59
,
221
226
Fontao
L.
,
Dirrig
S.
,
Owaribe
K.
,
Kedinger
M.
,
Launay
J. F.
(
1997
).
Polarized expression of HD1: relationship with the cytoskeleton in cultured human colonic carcinoma cells.
Exp. Cell Res
231
,
319
327
Galliano
M. F.
,
Aberdam
D.
,
Aguzzi
A.
,
Ortonne
J. P.
,
Meneguzzi
G.
(
1995
).
Cloning and complete primary structure of the mouse laminin3 chain. Distinct expression pattern of the laminin 3A and3B chain isoforms.
J. Biol. Chem
270
,
21820
21826
Giannelli
G.
,
Falk-Marzillier
J.
,
Schiraldi
O.
,
Stetler-Stevenson
W. G.
,
Quaranta
V.
(
1997
).
Induction of cell migration by matrix metalloprotease-2 cleavage of laminin-5.
Science
277
,
225
228
Goulet
O.
,
Kedinger
M.
,
Brousse
N.
,
Cuenod
B.
,
Colomb
V.
,
Patey
N.
,
De Potter
S.
,
Mougenot
J. F.
,
Canioni
D.
,
Cerf-Bensussan
N.
(
1995
).
Intractable diarrhea of infancy with epithelial and basement membrane abnormalities.
J. Pediatr
127
,
212
219
Heath
J. P.
(
1996
).
Epithelial cell migration in the intestine.
Cell. Biol. Int
20
,
139
146
Karecla
P. I.
,
Timpl
R.
,
Watt
F. M.
(
1994
).
Adhesion of human epidermal keratinocytes to laminin.
Cell Adhes. Commun
2
,
309
318
Kedinger
M.
,
Haffen
K.
,
Simon-Assmann
P.
(
1987
).
Intestinal tissue and cell cultures.
Differentiation
36
,
71
85
Kern
A.
,
Eble
J.
,
Golbik
R.
,
Kuhn
K.
(
1993
).
Interaction of type IV collagen with the isolated integrins1 1and2 1.
Eur. J. Biochem
215
,
151
159
Kikkawa
Y.
,
Umeda
M.
,
Miyazaki
K.
(
1994
).
Marked stimulation of cell adhesion and motility by ladsin, a laminin-like scatter factor.
J. Biochem. (Tokyo)
116
,
862
869
Kikkawa
Y.
,
Akaogi
K.
,
Mizushima
H.
,
Yamanaka
N.
,
Umeda
M.
,
Miyazaki
K.
(
1996
).
Stimulation of endothelial cell migration in culture by ladsin, a laminin-5-like cell adhesion protein.
In Vitro Cell Dev. Biol. Anim
32
,
46
52
Leivo
I.
,
Tani
T.
,
Laitinen
L.
,
Bruns
R.
,
Kivilaakso
E.
,
Lehto
V. P.
,
Burgeson
R. E.
,
Virtanen
I.
(
1996
).
Anchoring complex components laminin-5 and type VII collagen in intestine: association with migrating and differentiating enterocytes.
J. Histochem. Cytochem
44
,
1267
1277
Lesuffleur
T.
,
Barbat
A.
,
Dussaulx
E.
,
Zweibaum
A.
(
1990
).
Growth adaptation to methotrexate of HT-29 human colon carcinoma cells is associated with their ability to differentiate into columnar absorptive and mucus-secreting cells.
Cancer Res
50
,
6334
6343
Lesuffleur
T.
,
Kornowski
A.
,
Luccioni
C.
,
Muleris
M.
,
Barbat
A.
,
Beaumatin
J.
,
Dussaulx
E.
,
Dutrillaux
B.
,
Zweibaum
A.
(
1991
).
Adaptation to 5-fluorouracil of the heterogeneous human colon tumor cell line HT-29 results in the selection of cells committed to differentiation.
Int. J. Cancer
49
,
721
730
Lotz
M. M.
,
Korzelius
C. A.
,
Mercurio
A. M.
(
1990
).
Human colon carcinoma cells use multiple receptors to adhere to laminin: involvement of6 4 and2 1 integrins.
Cell Regul
1
,
249
257
Lotz
M. M.
,
Nusrat
A.
,
Madara
J. L.
,
Ezzell
R.
,
Wewer
U. M.
,
Mercurio
A. M.
(
1997
).
Intestinal epithelial restitution. Involvement of specific laminin isoforms and integrin laminin receptors in wound closure of a transformed model epithelium.
Am. J. Pathol
150
,
747
760
Marinkovich
M. P.
,
Lunstrum
G. P.
,
Burgeson
R. E.
(
1992
).
The anchoring filament protein kalinin is synthesized and secreted as a high molecular weight precursor.
J. Biol. Chem
267
,
17900
17906
Marinkovich
M. P.
,
Lunstrum
G. P.
,
Keene
D. R.
,
Burgeson
R. E.
(
1992
).
The dermal-epidermal junction of human skin contains a novel laminin variant.
J. Cell Biol
119
,
695
703
Messent
A. J.
,
Tuckwell
D. S.
,
Knauper
V.
,
Humphries
M. J.
,
Murphy
G.
,
Gavrilovic
J.
(
1998
).
Effects of collagenase-cleavage of type I collagen on2 1 integrin-mediated cell adhesion.
J. Cell Sci
111
,
1127
1135
Miner
J. H.
,
Patton
B. L.
,
Lentz
S. I.
,
Gilbert
D. J.
,
Snider
W. D.
,
Jenkins
N. A.
,
Copeland
N. G.
,
Sanes
J. R.
(
1997
).
The lamininchains: expression, developmental transitions, and chromosomal locations of 1–5, identification of heterotrimeric laminins 8–11 and cloning of a novel3 isoform.
J. Cell Biol
137
,
685
701
Niessen
C. M.
,
Hogervorst
F.
,
Jaspars
L. H.
,
De Melker
A. A.
,
Delwel
G. O.
,
Hulsman
E. H.
,
Kuikman
I.
,
Sonnenberg
A.
(
1994
).
The6 4 integrin is a receptor for both laminin and kalinin.
Exp. Cell Res
211
,
360
367
Niessen
C. M.
,
van Der Raaij-Helmer
L. M. H.
,
Hulsman
E. H. M.
,
van Der Neut
R.
,
Jonkman
M. F.
,
Sonnenberg
A.
(
1996
).
Deficiency of the integrin beta 4 subunit in junctional epidermolysis bullosa with pyloric atresia: consequences for hemidesmosome formation and adhesion properties.
J. Cell Sci
109
,
695
706
Nomizu
M.
,
Kuratomi
Y.
,
Song
S.-Y.
,
Ponce
M. L.
,
Hoffman
M. P.
,
Powell
S. K.
,
Miyoshi
K.
,
Otaka
A.
,
Kleinman
H. K.
,
Yamada
Y.
(
1997
).
Identification of cell binding sequences in mouse laminin gamma-1 chain by systematic peptide screening.
J. Biol. Chem
272
,
32198
32205
Orian-Rousseau
V.
,
Aberdam
D.
,
Fontao
L.
,
Chevalier
L.
,
Meneguzzi
G.
,
Kedinger
M.
,
Simon-Assmann
P.
(
1996
).
Developmental expression of laminin-5 and HD1 in the intestine: epithelial to mesenchymal shift for the laminin gamma-2 chain subunit deposition.
Dev. Dynam
206
,
12
23
Perreault
N.
,
Vachon
P. H.
,
Beaulieu
J. F.
(
1995
).
Appearance and distribution of laminin A chain isoforms and integrin2, 3,6, 1 and4 subunits in the developing human small intestinal mucosa.
Anat. Rec
242
,
242
250
Pfaff
M.
,
Gohring
W.
,
Brown
J. C.
,
Timpl
R.
(
1994
).
Binding of purified collagen receptors (6 1,2 1) and RGD-dependent integrins to laminins and laminin fragments.
Eur. J. Biochem
225
,
975
984
Pinto
M.
,
Robine-Leon
S.
,
Appay
M. D.
,
Kedinger
M.
,
Triadou
N.
,
Dussaulx
E.
,
Lacroix
B.
,
Simon-Assmann
P.
,
Haffen
K.
,
Fogh
J.
, et al. 
(
1983
).
Enterocyte-like differentiation and polarization of the human colon carcinoma cell-line (Caco-2) in culture.
Biol. Cell
47
,
323
330
Pulkkinen
L.
,
Kimonis
V. E.
,
Xu
Y. L.
,
Spanou
E. N.
,
Mclean
W. H. I.
,
Uitto
J.
(
1997
).
Homozygous alpha 6 integrin mutation in junctional epidermolysis bullosa with congenital duodenal atresia.
Hum. Mol. Genet
6
,
669
674
Pyke
C.
,
Romer
J.
,
Kallunki
P.
,
Lund
L. R.
,
Ralfkiaer
E.
,
Dano
K.
,
Tryggvason
K.
(
1994
).
The gamma 2 chain of kalinin/laminin 5 is preferentially expressed in invading malignant cells in human cancers.
Am. J. Pathol
145
,
782
791
Pyke
C.
,
Sale
S.
,
Ralfkiaer
E.
,
Romer
J.
,
Dano
K.
,
Tryggvason
K.
(
1995
).
Laminin-5 is a marker of invading cancer cells in some human carcinomas and is coexpressed with the receptor for urokinase plasminogen activator in budding cancer cells in colon adenocarcinomas.
Cancer Res
55
,
4132
4139
Rousselle
P.
,
Lunstrum
G. P.
,
Keene
D. R.
,
Burgeson
R. E.
(
1991
).
Kalinin—An epithelium-specific basement membrane adhesion molecule that is a component of anchoring filaments.
J. Cell Biol
114
,
567
576
Rousselle
P.
,
Aumailley
M.
(
1994
).
Kalinin is more efficient than laminin in promoting adhesion of primary keratinocytes and some other epithelial cells and has a different requirement for integrin receptors.
J. Cell Biol
125
,
205
214
Ryan
M. C.
,
Tizard
R.
,
VanDevanter
D. R.
,
Carter
W. G.
(
1994
).
Cloning of the LamA3 gene encoding the3 chain of the adhesive ligand epiligrin. Expression in wound repair.
J. Biol. Chem
269
,
22779
22787
Saelman
E.
,
Keely
P.
,
Santoro
S.
(
1995
).
Loss of MDCK cell2 1 integrin expression results in reduced cyst formation, failure of hepatocyte growth factor/scatter factor-induced branching morphogenesis, and increased apoptosis.
J. Cell Sci
108
,
3531
3540
Simo
P.
,
Simon-Assmann
P.
,
Bouziges
F.
,
Leberquier
C.
,
Kedinger
M.
,
Ekblom
P.
,
Sorokin
L.
(
1991
).
Changes in the expression of laminin during intestinal development.
Development
112
,
477
487
Simon-Assmann
P.
,
Duclos
B.
,
Orian-Rousseau
V.
,
Arnold
C.
,
Mathelin
C.
,
Engvall
E.
,
Kedinger
M.
(
1994
).
Differential expression of laminin isoforms and6- 4 integrin subunits in the developing human and mouse intestine.
Dev. Dynam
201
,
71
85
Simon-Assmann
P.
,
Leberquier
C.
,
Molto
N.
,
Uezato
T.
,
Bouziges
F.
,
Kedinger
M.
(
1994
).
Adhesive properties and integrin expression profiles of two colonic cancer populations differing by their spreading on laminin.
J. Cell Sci
107
,
577
587
Simon-Assmann
P.
,
Kedinger
M.
,
De Arcangelis
A.
,
Orian-Rousseau
V.
,
Simo
P.
(
1995
).
Extracellular matrix components in intestinal development.
Experientia
51
,
883
900
Tozeren
A.
,
Kleinman
H. K.
,
Wu
S.
,
Mercurio
A. M.
,
Byers
S. W.
(
1994
).
Integrin6 4 mediates dynamic interactions with laminin.
J. Cell Sci
107
,
3153
3163
Tuckwell
D. S.
,
Reid
K. B.
,
Barnes
M. J.
,
Humphries
M. J.
(
1996
).
The A domain of integrin2 binds specifically to a range of collagens but is not in general receptor for the collagenous motif.
Eur. J. Biochem
241
,
732
739
Underwood
P. A.
,
Bennett
F. A.
,
Kirkpatrick
A.
,
Bean
P. A.
,
Moss
B. A.
(
1995
).
Evidence for the location of a binding sequence for the2 1 integrin of endothelial cells, in the1 subunit of laminin.
Biochem. J
309
,
765
771
Vachon
P. H.
,
Beaulieu
J. F.
(
1995
).
Extracellular heterotrimeric laminin promotes differentiation in human enterocytes.
Am. J. Physiol
268
,
857
67
Verrando
P.
,
Hsi
B. L.
,
Yeh
C. J.
,
Pisani
A.
,
Serieys
N.
,
Ortonne
J. P.
(
1987
).
Monoclonal antibody GB3, a new probe for the study of human basement membranes and hemidesmosomes.
Exp. Cell Res
170
,
116
128
Vidal
F.
,
Aberdam
D.
,
Miquel
C.
,
Christiano
A. M.
,
Pulkkinen
L.
,
Uitto
J.
,
Ortonne
J. P.
,
Meneguzzi
G.
(
1995
).
Integrin beta 4 mutations associated with junctional epidermolysis bullosa with pyloric atresia.
Nature Genet
10
,
229
234
Wu
J. E.
,
Santoro
S. A.
(
1994
).
Complex patterns of expression suggest extensive roles for the2 1 integrin in murine development.
Dev. Dynam
199
,
292
314
Zweibaum
A.
,
Pinto
M.
,
Chevalier
G.
,
Dussaulx
E.
,
Triadou
N.
,
Lacroix
B.
,
Haffen
K.
,
Brun
J. L.
,
Rousset
M.
(
1985
).
Enterocytic differentiation of a subpopulation of the human colon tumor cell line HT-29 selected for growth in sugar-free medium and its inhibition by glucose.
J. Cell. Physiol
122
,
21
29
This content is only available via PDF.