Mammary gland form and function are regulated by interactions between epithelium and extracellular matrix. Major glycoprotein components of extracellular matrix have been identified that give survival, proliferation and differentiation signals to mammary epithelial cells. We provide evidence that proteolytic fragments of the extracellular matrix glycoprotein, fibronectin, suppress growth and can promote apoptosis of mouse mammary epithelial cells. During mammary gland involution, total fibronectin and fibronectin fragment levels are increased. The peak levels of fibronectin protein and fragments are observed 4–6 days post-weaning, coincident with the peak in epithelial cell death. Using a model for hormone withdrawal-induced death of mammary epithelium, elevated levels of fibronectin proteolytic fragments were associated with apoptosis in TM-6 cells, a tumorigenic mouse mammary epithelial cell line. Treatment of TM-6 cells with exogenous fibronectin fragments (FN120) reduced cell number, and induced apoptosis and matrix degrading protease activity. Inhibition of matrix protease activity rescued TM-6 cell viability, indicating that FN120-induced cell loss is mediated through matrix protease activity. In a three-dimensional model for mammary gland development, FN120 reduced alveolar-like and promoted ductal-like development by a matrix protease-dependent mechanism. These data suggest that during post-lactational involution, fibronectin fragments may contribute to epithelial cell loss and dissolution of mammary alveoli by inducing matrix degrading proteinases.

REFERENCES

Alexander
C. M.
,
Howard
E. W.
,
Bissell
M. J.
,
Werb
Z.
(
1996
).
Rescue of mammary epithelial cell apoptosis and entactin degradation by a tissue inhibitor of metalloproteinases-1 transgene.
J. Cell Biol
135
,
1669
1677
Bissell
M. J.
,
Barcellos-Hoff
M. H.
(
1987
).
The influence of extracellular matrix on gene expression: Is structure the message?.
J. Cell Sci
8
,
327
343
Blum
J. L.
,
Zeigler
M. E.
,
Wicha
M. S.
(
1989
).
Regulation of mammary differentiation by the extracellular matrix.
Environ. Health Perspect
80
,
71
83
Boudreau
N.
,
Sympson
C. J.
,
Werb
Z.
,
Bissell
M. J.
(
1995
).
Suppression of ICE and apoptosis in mammary epithelial cells by extracellular matrix.
Science
267
,
891
893
Buckley
C. D.
,
Pilling
D.
,
Henriquez
N. V.
,
Parsonage
G.
,
Threlfall
K.
,
Scheel-Toellner
D.
,
Simmons
D. L.
,
Akbar
A.N.
,
Lord
J. M.
,
Salmon
M.
(
1999
).
RGD peptides induce apoptosis by direct caspase-3 activation.
Nature
397
,
534
539
Chammas
R.
,
Taverna
D.
,
Cella
N.
,
Santos
C.
,
Hynes
N. E.
(
1994
).
Laminin and tenascin assembly and expression regulate HC11 mouse mammary cell differentiation.
J. Cell Sci
107
,
1031
1040
Christensen
L.
(
1992
).
Fibronectin, laminin, and tetranectin in human breast cancer with special attention to extracellular matrix.
APMIS Supplementum
26
,
6
39
Cox
C.
(
1990
).
Fieller's Theorem: the Likelihood and the Delta Method.
Biometrics
46
,
709
718
Cunha
G.
(
1994
).
Role of mesenchymal-epithelial interactions in normal and abnormal development of the mammary gland and prostate.
Cancer
74
,
1030
1044
Damsky
D. C.
,
Tremble
P.
,
Werb
Z.
(
1992
).
Signal transduction via the fibronectin receptor: do integrins regulate matrix remodeling?.
Matrix. Suppl
1
,
184
191
Darcy
K. M.
,
Shoemaker
S. F.
,
Lee
P. H.
,
Vaughan
M. M.
,
Black
J. D.
,
Ip
M. M.
(
1995
).
Prolactin and epidermal growth factor regulation of the proliferation, morphogenesis, and functional differentiation of normal rat mammary epithelial cells in three dimensional primary culture.
J. Cell Physiol
163
,
346
364
Dickson
S. R.
,
Warburton
M. J.
(
1992
).
Enhanced synthesis of gelatinase and stromelysin by myoepithelial cells during involution of the rat mammary gland.
J. Histochem. Cytochem
40
,
697
703
Eyden
B. P.
,
Watson
R. J.
,
Harris
M.
,
Howell
A.
(
1986
).
Intralobular stromal fibroblasts in the resting human mammary gland: Ultrastructural properties and intercellular relationships.
J. Submicrosc. Cytol. Pathol
18
,
397
408
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
Haslam
S. Z.
(
1991
).
Stromal-epithelial interactions in normal and neoplastic mammary gland.
Cancer Treat. Res
53
,
401
420
Hocking
D. C.
,
Sottile
J.
,
McKeown-Longo
P.
(
1998
).
Activation of distinct5 1-mediated signaling pathways by fibronectin's cell adhesion and matrix assembly domains.
J. Cell Biol
141
,
241
253
Hynes
R. O.
,
Yamada
K. M.
(
1982
).
Fibronectins: Multifunctional modular glycoproteins.
J. Cell Biol
95
,
369
377
Ilic
D.
,
Damsky
C. H.
,
Yamamoto
T.
(
1997
).
Focal adhesion kinase: at the crossroads of signal transduction.
J. Cell Sci
110
,
401
407
Kimata
K.
,
Sakakura
T.
,
Inaguma
Y.
,
Kato
M.
,
Nishizuka
Y.
(
1985
).
Participation of two different mesenchymes in the developing mouse mammary gland: Synthesis of basement membrane components by fat pad precursor cells.
J. Embryol. Exp. Morph
89
,
243
257
Kittrell
F. S.
,
Oborn
C.
,
Medina
D.
(
1992
).
Development of mammary Preneoplasias in vivo from mouse mammary epithelial cell lines in vitro.
Cancer Res
52
,
1924
1932
Koukoulis
G. K.
,
Virtanen
I.
,
Korhonen
M.
,
Laitinen
L.
,
Quaranta
V.
,
Gould
V. E.
(
1991
).
Immunohistochemical localization of integrins in the normal, hyperplastic, and neoplastic breast. Correlations with their functions as receptors and cell adhesion molecules.
Am. J. Pathol
139
,
787
799
Li
M. L.
,
Aggeler
J.
,
Farson
D. A.
,
Hatier
C.
,
Hassell
J.
,
Bissell
M. J.
(
1987
).
Influence of a reconstituted basement membrane and its components on casein gene expression and secretion in mouse mammary epithelial cells.
Proc. Natl. Acad. Sci. USA
84
,
136
140
Lund
L. R.
,
Romer
J.
,
Thomasset
N.
,
Solberg
H.
,
Pyke
C.
,
Bissell
M. J.
,
Dano
K.
,
Werb
Z.
(
1996
).
Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and-dependent pathways.
Development
122
,
181
193
McGeehan
G. M.
,
Becherer
J. D.
,
Bast
R. C.
,
Champion
B.
,
Connolly
K. M.
,
Conway
J. G.
,
Furdon
P.
,
Karp
S.
,
Kidao
S.
(
1994
).
Regulation of tumor necrosis factor-alpha processing by a metalloproteinase inhibitor.
Nature
370
,
558
561
Medina
D.
,
Kittrell
F. S.
,
Liu
Y.-J.
,
Schwartz
M.
(
1993
).
Morphological and functional properties of TM preneoplastic mammary outgrowths.
Cancer Res
53
,
663
667
Meredith
J. E.
,
Fazeli
B.
,
Schwartz
M. A.
(
1993
).
The extracellular matrix as a survival factor.
Mol. Cell Biol
4
,
953
961
Merlo
G. R.
,
Cella
N.
,
Hynes
N. E.
(
1997
).
Apoptosis is accompanied by changes in Bcl-2 and Bax expression, induced by loss of attachment, and inhibited by specific extracellular matrix proteins in mammary epithelial cells.
Cell Growth Differ
8
,
251
260
Mohler
W. A.
,
Charlton
C. A.
,
Blau
H. M.
(
1996
).
Spectrophotometric quantitation of tissue culture cell number in any medium.
Biotechniques
21
,
260
262
Monaghan
P.
,
Warburton
M. J.
,
Perusinghe
N.
,
Rudland
P. S.
(
1983
).
Topographical arrangement of basement membrane proteins in lactating rat mammary gland: comparison of the distribution of type IV collagen, laminin, fibronectin, and Thy-1 at the ultrastructural level.
Proc. Natl. Acad. Sci. USA
80
,
3344
3348
Oka
T.
,
Yoshimura
M.
,
Lavandero
S.
,
Wada
K.
,
Ohba
Y.
(
1991
).
Control of growth and differentiation of the mammary gland by growth factors.
J. Dairy Sci
74
,
2788
2800
Ozzello
L.
(
1970
).
Epithelial-stromal junction of normal and dysplastic mammary glands.
Cancer
25
,
586
600
Panayotou
G.
,
End
P.
,
Aumailley
M.
,
Timpl
R.
,
Engel
J.
(
1989
).
Domains of laminin with growth-factor activity.
Cell
56
,
93
101
Pullan
S.
,
Wilson
J.
,
Metcalf
A.
,
Edwards
G. M.
,
Goberdhan
N.
,
Tilly
J.
,
Hickman
J. A.
,
Dive
C.
,
Streuli
C. H.
(
1996
).
Requirement of basement membrane for the suppression of programmed cell death in mammary epithelium.
J. Cell Sci
109
,
631
642
Richards
J.
,
Guzman
R.
,
Konrad
M.
,
Yang
J.
,
Nandi
S.
(
1982
).
Growth of mouse mammary gland end buds cultured in a collagen gel matrix.
Exp. Cell Res
141
,
433
443
Ruoslahti
E.
(
1999
).
Fibronectin and its integrin receptors in cancer.
Adv. Cancer Res
76
,
1
20
Sakakura
T.
,
Sakagami
Y.
,
Nishizuka
Y.
(
1982
).
Dual origin of mesenchymal tissues participating in mouse mammary gland embryogenesis.
Dev. Biol
91
,
202
207
Schedin
P. J.
,
Strange
R.
,
Singh
M.
,
Kaeck
M. R.
,
Fontaine
S. C.
,
Thompson
H. J.
(
1995
).
Treatment with chemopreventive agents, difluoromethylornithine and retinyl acetate, results in altered mammary extracellular matrix.
Carcinogenesis
16
,
1787
1794
Schedin
P. J.
,
Thackray
L. B.
,
Malone
P.
,
Fontaine
S. C.
,
Friis
R. R.
,
Strange
R.
(
1996
).
Programmed cell death and mammary neoplasia.
Cancer Treat. Res
83
,
3
22
Strange
R.
,
Li
F.
,
Saurer
S.
,
Burkhardt
A.
,
Friis
R. R.
(
1992
).
Apoptotic cell death and tissue remodeling during mouse mammary gland involution.
Development
115
,
49
58
Sympson
C. J.
,
Talhouk
R. S.
,
Alexander
C. M.
,
Chin
J. R.
,
Clift
S. M.
,
Bissell
M. J.
,
Werb
Z.
(
1994
).
Targeted expression of stromelysin-1 in mammary gland provides evidence for a role of proteinases in branching morphogenesis and the requirement for an intact basement membrane for tissue-specific gene expression.
J. Cell Biol
125
,
681
693
Talhouk
R. S.
,
Bissell
M. J.
,
Werb
Z.
(
1992
).
Coordinated expression of extracellular matrix-degrading proteinases and their inhibitors regulates mammary epithelial function during involution.
J. Cell Biol
118
,
1271
1282
Thompson
H. J.
,
Strange
R.
,
Schedin
P. J.
(
1992
).
Apoptosis in the genesis and prevention of cancer. Cancer Epidemiol.
Biomarkers Prev
1
,
597
602
Vogel
B. E.
,
Tarone
G.
,
Giancotti
F. G.
,
Gailit
J.
,
Ruoslahti
E.
(
1990
).
A novel fibronectin receptor with an unexpected subunit composition (alpha v beta 1).
J. Biol. Chem
265
,
5934
5937
Warburton
M. J.
,
Mitchell
D.
,
Ormerod
E. J.
,
Rudland
P.
(
1982
).
Distribution of myoepithelial cells and basement membrane proteins in the resting, pregnant, lactating, and involuting rat mammary gland.
J. Histochem. Cytochem
30
,
667
676
Watson
R. J.
,
Eyden
B. P.
,
Howell
A.
,
Sellwood
R. A.
(
1988
).
Ultrastructural observations on the basal lamina in the normal human breast.
J. Anat
156
,
1
10
Werb
Z.
,
Tremble
P. M.
,
Behrendtsen
O.
,
Crowley
E.
,
Damsky
C. H.
(
1989
).
Signal transduction through the fibronectin receptor induces collagenase and stromelysin gene expression.
J. Cell Biol
109
,
877
889
Witty
J. P.
,
Wright
J. H.
,
Matrisian
L. M.
(
1995
).
Matrix metalloproteinases are expressed during ductal and alveolar mammary morphogenesis, and misregulation of stromelysin-1 in transgenic mice induces unscheduled alveolar development.
Mol. Biol. Cell
6
,
1287
1303
Yamagata
S.
,
Ito
Y.
,
Tanaka
R.
,
Shimizu
S.
(
1988
).
Gelatinases of metastatic cell lines of murine colonic carcinoma as detected by substrate-gel electrophoresis.
Biochem. Biophys. Res. Commun
151
,
158
162
Zajchowski
D. A.
,
Band
V.
,
Trask
D. K.
,
Kling
D.
,
Connolly
J. L.
,
Sage
R.
(
1990
).
Suppression of tumor-forming ability and related traits in MCF-7 human breast cancer cells by fusion with immortal mammary epithelial cells.
Proc. Natl. Acad. Sci. USA
87
,
2314
2318
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