Previously, we demonstrated that proteolytic processing within the globular domain of the alpha3 subunit of laminin-5 (LN5) converts LN5 from a cell motility-inducing factor to a protein complex that can trigger the formation of hemidesmosomes, certain cell-matrix attachment sites found in epithelial cells. We have prepared a monoclonal antibody (12C4) whose epitope is located toward the carboxy terminus of the globular domain of the alpha3 laminin subunit. This epitope is lost from the alpha3 subunit as a consequence of proteolytic processing. Antibody 12C4 stains throughout the matrix of cells that fail to process the alpha3 laminin subunit, but does not recognize the matrix of confluent cultures of MCF-10A cells, which efficiently process their alpha3 laminin chain. In subconfluent populations of MCF-10A cells, 12C4 only stains matrix deposited at the outer edges of cell colonies. In these cells, integrin alpha3beta1 occasionally colocalizes with the staining generated by the 12C4 antibody but alpha6beta4 integrin does not. In wounded MCF-10A cell cultures, the 12C4 antibody stains the extracellular matrix beneath those cells at the very edge of the cellular sheet that moves to cover the wound site. A similar phenomenon is observed in human skin wounds, since we also detect expression of the unprocessed alpha3 laminin subunit at the leading tip of the sheet of epidermal cells that epithelializes skin wounds in vivo. In addition, using alpha3 laminin subunit and integrin function-inhibiting antibodies, we provide evidence that LN5 and its two integrin receptors (alpha6beta4 and alpha3beta1) appear necessary for wound healing to occur in MCF-10A cell culture wounds. We propose a model for healing of wounded epithelial tissues based on these results.

REFERENCES

Baker
S. E.
,
Hopkinson
S. B.
,
Fitchmun
M.
,
Andreason
G. L.
,
Frasier
F.
,
Plopper
G.
,
Quaranta
V.
,
Jones
J. C. R.
(
1996
).
Laminin-5 and hemidesmosomes: role of the alpha 3 chain subunit in hemidesmosome stability and assembly.
J. Cell Sci
109
,
2509
2520
Beattie
G. M.
,
Rubin
J. S.
,
Mally
M. I.
,
Otonkoski
T.
,
Hayek
A.
(
1996
).
Regulation of proliferation and differentiation of human fetal pancreatic islet cells by extracellular matrix, hepatocyte growth factor, and cell-cell contact.
Diabetes
45
,
1223
1228
Blaschke
R. J.
,
Howlett
A. R.
,
Desprez
P. Y.
,
Petersen
O. W.
,
Bissell
M. J.
(
1994
).
Cell differentiation by extracellular matrix components.
Meth. Enzymol
245
,
535
556
Carter
W. G.
,
Kaur
P.
,
Gill
S. G.
,
Gahr
P. J.
,
Wayner
E. A.
(
1990
).
Distinct functions for integrins3 1 in focal adhesions and a6b4/bullous pemphigoid antigen in a new stable anchoring contact (SAC) of keratinocytes: relation to hemidesmosomes.
J. Cell Biol
111
,
3141
3154
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
Cavani
A.
,
Zambruno
G.
,
Marconi
A.
,
Manca
V.
,
Marchetti
M.
,
Giannetti
A.
(
1993
).
Distinctive integrin expression in the newly forming epidermis during wound healing in humans.
J. Invest. Dermatol
101
,
600
604
Champliaud
M. F.
,
Lunstrum
G. P.
,
Rousselle
P.
,
Nishiyama
T.
,
Keene
D. R.
,
Burgeson
R. E.
(
1997
).
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
DiPersio
C. M.
,
Hodivala-Dilke
K. M.
,
Jaenisch
R.
,
Kreidberg
J. A.
,
Hynes
R. O.
(
1997
).
alpha3beta1 Integrin is required for normal development of the epidermal basement membrane.
J. Cell Biol
137
,
729
742
Fuchs
E.
(
1990
).
Epidermal Differentiation: The Bare Essentials.
J. Cell Biol
111
,
2807
2814
Galliano
M.-F.
,
Aberdam
D.
,
Aguzzi
A.
,
Ortonne
J.-P.
,
Meneguzzi
G.
(
1995
).
Cloning and complete primary structure of the mouse laminin3 chain.
J. Biol. Chem
270
,
21820
21826
Garlick
J.
,
Taichman
L.
(
1994
).
Fate of human keratinocytes during reepithelialization in an organotypic culture model.
Lab. Invest
70
,
916
24
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
Goldfinger
L. E.
,
Stack
M. S.
,
Jones
J. C. R.
(
1998
).
Processing of laminin-5 and its functional consequences: role of plasmin and tissue-type plasminogen activator.
J. Cell Biol
141
,
255
265
Gonzales
M.
,
Haan
K.
,
Baker
S. E.
,
Fitchmun
M. I.
,
Todorov
I.
,
Weitzman
S.
,
Jones
J. C. R.
(
1999
).
A cell signal pathway involving laminin-5,3 1 integrin, and mitogen-activated protein kinase can regulate epithelial cell proliferation.
Mol. Bio. Cell
10
,
259
270
Green
K. J.
,
Jones
J. C. R.
(
1996
).
Desmosomes and hemidesmosomes: structure and function of molecular components.
FASEB J
10
,
871
880
Haapasalmi
K.
,
Zhang
K.
,
Tonnesen
M.
,
Olerud
J.
,
Sheppard
D.
,
Salo
T.
,
Kramer
R.
,
Clark
R.
,
Uitto
V.
,
Larjava
H.
(
1996
).
Keratinocytes in human wounds expressed alpha v beta 6 integrin.
J. Invest. Derm
106
,
42
8
Hay
E. D.
(
1993
).
Extracellular matrix alters epithelial differentiation.
Curr. Opin. Cell Biol
5
,
1029
1035
Hodivala-Dilke
K. M.
,
DiPersio
C. M.
,
Kreidberg
J. A.
,
Hynes
R. O.
(
1998
).
Novel roles for alpha3beta1 integrin as a regulator of cytoskeletal assembly and as a trans-dominant inhibitor of integrin receptor function in mouse keratinocytes.
J. Cell Biol
142
,
1357
1369
Jones
J. C. R.
,
Kurpakus
M. A.
,
Cooper
H. M.
,
Quaranta
V.
(
1991
).
A function for the integrin alpha 6 beta 4 in the hemidesmosome.
Cell Reg
2
,
427
38
Jones
J. C. R.
,
Hopkinson
S. B.
,
Goldfinger
L. E.
(
1998
).
Structure and assembly of hemidesmosomes.
BioEssays
20
,
488
494
Kainulainen
T.
,
Hakkinen
L.
,
Hamidi
S.
,
Larjava
K.
,
Kallioinen
M.
,
Petlonen
J.
,
Salo
T.
,
Larjava
H.
,
Oikarinen
A.
(
1998
).
Laminin-5 expression is independent of the injury and the microenvironment during reepithelialization of wounds.
J. Histochem. Cytochem
46
,
353
360
Klatte
D. H.
,
Kurpakus
M. A.
,
Grelling
K. A.
,
Jones
J. C. R.
(
1989
).
Immunochemical characterization of three components of the hemidesmosome and their expression in cultured epithelial cells.
J. Cell Biol
109
,
3377
3390
Kleinman
H. K.
,
Weeks
B. S.
,
Schnaper
H. W.
,
Kibbey
M. C.
,
Yamamura
K.
,
Grant
D. S.
(
1993
).
The laminins: A family of basement membrane glycoproteins important in cell differentiation and tumor metastases.
Vitamins and Hormones
47
,
161
187
Kuhn
K.
(
1994
).
Basement membrane (type IV) collagen.
Matrix Biol
14
,
439
445
Kurpakus
M. A.
,
Quaranta
V.
,
Jones
J. C. R.
(
1991
).
Surface relocation of alpha6 beta4 integrins and assembly of hemidesmosomes in an in vitro model of wound healing.
J. Cell Biol
115
,
1737
1750
Laemmli
U. K.
(
1970
).
Cleavage of structural proteins during assembly of the head of bacteriophage T4.
Nature
277
,
680
685
Lampe
P. D.
,
Nguyen
B. P.
,
Gil
S.
,
Usui
M.
,
Olerud
J.
,
Takada
Y.
,
Carter
W. G.
(
1998
).
Cellular interaction of integrin3 1 with laminin 5 promotes gap junctional communication.
J. Cell Biol
143
,
1735
1748
Langhofer
M.
,
Hopkinson
S. B.
,
Jones
J. C. R.
(
1993
).
The matrix secreted by 804G cells contains laminin-related components that participate in hemidesmosome assembly in vitro.
J. Cell Sci
105
,
753
–.
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.
,
Keene
D. R.
,
Rimberg
C. S.
,
Burgeson
R. E.
(
1993
).
Cellular origin of the dermal-epidermal basement membrane.
Dev. Dyn
197
,
255
267
Marinkovich
M. P.
,
Verrando
P.
,
Keene
D. R.
,
Meneguzzi
G.
,
Lunstrum
G. P.
,
Ortonne
J. P.
,
Burgeson
R. E.
(
1993
).
Basement membrane proteins kalinin and nicein are structurally and immunologically identical.
Lab. Invest
69
,
295
299
Martin
P.
(
1997
).
Wound healing—Aiming for perfect skin regeneration.
Science
276
,
75
81
Masunaga
T.
,
Shimizu
H.
,
Ishiko
A.
,
Tomita
Y.
,
Aberdam
D.
,
Ortonne
J. P.
,
Nishikawa
T.
(
1996
).
Localization of laminin-5 in the epidermal basement membrane.
J. Histochem. Cytochem
44
,
1223
1230
Matsui
C.
,
Nelson
C. F.
,
Hernandez
G. T.
,
Herron
G. S.
,
Bauer
E. A.
,
Hoeffler
W. K.
(
1995
).
Gamma 2 chain of laminin-5 is recognized by monoclonal antibody GB3.
J. Invest. Derm
105
,
648
652
Niessen
C. M.
,
Hogervorst
F.
,
Jaspars
L. H.
,
De Melker
A. A.
,
Delwel
G. O.
,
Hulsman
E. H. M.
,
Kuikman
I.
,
Sonnenberg
A.
(
1994
).
The6 4 integrin is a receptor of both laminin and kalinin.
Exp. Cell Res
211
,
360
367
Oda
D.
,
Bigler
L.
,
Lee
P.
,
Blanton
R.
(
1996
).
HPV immortalization of human oral epithelial cells: a model for carcinogenesis.
Exp. Cell Res
226
,
164
169
O'Toole
E. A.
,
Marinkovich
M. P.
,
Hoeffler
W. K.
,
Furthmayr
H.
,
Woodley
D. T.
(
1997
).
Laminin-5 inhibits human keratinocyte migration.
Exp. Cell Res
233
,
330
339
Owaribe
K.
,
Nishizawa
Y.
,
Franke
W. W.
(
1991
).
Isolation and Characterization of Hemidesmosomes from Bovine Corneal Epithelial Cells.
Exp. Cell Res
192
,
622
630
Paladini
R. D.
,
Takahashi
K.
,
Bravo
N. S.
,
Coulombe
P. A.
(
1996
).
Onset of reepithelialization after skin injury correlates with a reorganization of keratin filaments in wound edge keratinocytes: defining a potential role for keratin 16.
J. Cell Biol
132
,
381
397
Rabinovitz
I.
,
Mercurio
A. M.
(
1997
).
The integrin alpha 6 beta 4 functions in carcinoma cell migration on laminin-1 by mediating the formation and stabilization of actin-containing motility structures.
J. Cell Biol
139
,
1873
1884
Riddelle
K. S.
,
Green
K. J.
,
Jones
J. C. R.
(
1991
).
Formation of hemidesmosomes in vitro by a transformed rat bladder cell line.
J. Cell Biol
112
,
159
168
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
Ryan
M. C.
,
Tizard
R.
,
VanDevanter
D. R.
,
Carter
W. C.
(
1994
).
Cloning of the LamA3 gene encoding the a3 chain of the adhesive ligand epiligrin.
J. Biol. Chem
269
,
22779
22787
Sonnenberg
A.
,
Modderman
P. W.
,
Hogervorst
F.
(
1988
).
Laminin receptor on platelets is the integrin VLA-6.
Nature
336
,
487
489
Sonnenberg
A.
,
Calafat
J.
,
Janssen
H.
,
Daams
H.
,
van der Raaij-Helmer
L. M. H.
,
Falciuoni
R.
,
Kennel
S. J.
,
Aplin
J. D.
,
Baker
J.
,
Loizidou
M.
, et al. 
(
1991
).
Integrin6 4 complex is located inhemidesmosomes, suggesting a major role in epidermal cell-basement membrane adhesion.
J. Cell Biol
113
,
907
917
Stahl
S.
,
Weitzman
S.
,
Jones
J. C. R.
(
1997
).
The role of laminin-5 and its receptors in mammary epithelial cell branching morphogenesis.
J. Cell Sci
110
,
55
63
Timpl
R.
(
1996
).
Macromolecular organization of basement membranes.
Curr. Opin. Cell Biol
8
,
618
624
Timpl
R.
,
Rohde
H.
,
Robey
P. G.
,
Rennard
S. I.
,
Foidart
J.-M.
,
Martin
G. R.
(
1979
).
Laminin—A glycoprotein from basement membrane.
J. Biol. Chem
254
,
9933
9937
Uematsu
J.
,
Nishizawa
Y.
,
Sonnenberg
A.
,
Owaribe
K.
(
1994
).
Demonstration of type II hemidesmosomes in a mammary gland epithelial cell line, BMGE-H.
J. Biochem
115
,
469
476
Uitto
J.
,
Pulkkinen
L.
(
1996
).
Molecular complexity of the cutaneous basement membrane zone.
Mol. Biol. Rep
23
,
35
46
Vailly
J.
,
Verrando
P.
,
Champliaud
M. F.
,
Gerecke
D.
,
Wagman
D. W.
,
Baudoin
C.
,
Aberdam
D.
,
Burgeson
R.
,
Bauer
E.
,
Ortonne
J. P.
(
1994
).
The 100-kDa chain of nicein/kalinin is a laminin B2 chain variant.
Eur. J. Biochem
219
,
209
218
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.
,
Baudoin
C.
,
Miquel
C.
,
Galliano
M. F.
,
Christiano
A. M.
,
Uitto
J.
,
Ortonne
J. P.
,
Meneguzzi
G.
(
1995
).
Cloning of the laminin alpha 3 chain gene (LAMA3) and identification of a homozygous deletion in a patient with Herlitz junctional epidermolysis bullosa.
Genomics
30
,
273
280
Xia
Y.
,
Gill
S. G.
,
Carter
W. G.
(
1996
).
Anchorage mediated by integrin6 4 to laminin 5 (epiligrin) regulates tyrosine phosphorylation of a membrane associated 80-kD protein.
J. Cell Biol
132
,
727
740
Zhang
K.
,
Kramer
R.
(
1996
).
Laminin-5 deposition promotes keratinocyte motility.
Exp. Cell Res
227
,
309
333
Zieske
J. D.
,
Mason
V. S.
,
Wasson
M. E.
,
Meunier
S. F.
,
Nolte
C. J.
,
Fukai
N.
,
Olsen
B. R.
,
Parenteau
N. L.
(
1994
).
Basement membrane assembly and differentiation of cultured corneal cells: importance of culture environment and endothelial cell interaction.
Exp. Cell Res
214
,
621
633
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