The interplay of matrix metalloproteinases, morphogens and growth factors is necessary for branching of mammary epithelial cells
Marina Simian1,
Yohei Hirai1,*,
Marc Navre2,
Zena Werb3,
Andre Lochter1,
and
Mina J. Bissell1,
1
Life Sciences Division, Lawrence Berkeley National Laboratory, University of
California, 1 Cyclotron Road, Berkeley, CA 94720, USA
2
Affymax Research Institute, Santa Clara, CA 95051, USA
3
Department of Anatomy, Box 0750, University of California, San Francisco, CA
94143, USA
*
Present address: Biomedical and Research Development Department, Sumitomo
Electric Industries, Yokohama 244, Japan
Present address: Department for Basic Research, Center for Clinical and Basic
Research, Ballerup Byvej 222, DK-2750 Ballerup, Denmark
Author for correspondence (e-mail:
mjbissell{at}lbl.gov
)
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Fig. 2. Growth factors induce branching morphogenesis in mammary organoids. Primary
mammary organoids were embedded in collagen I gels and treated for 4 to 6 days
with EGF, KGF, bFGF or HGF at a concentration of 50 ng/ml. (A) Quantification
of the percentage of branching organoids for each of the treatments. The
means±s.d. of three independent experiments are shown. The differences
between the control group (no GF) and the growth factor-treated groups was
statistically significant (**P 0.01). (B) Appearance of
control and growth factor-treated primary mammary organoids. (C) Cross
sections of organoids showing the existence of a lumen at the day of
preparation (day 0) and after 4 days of treatment with KGF. Scale bars: 200
µm in B; 100 µm in C.
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Fig. 3. Growth factors increase the level but not the activation status of
proteinases secreted by primary mammary organoids. Mammary organoids were
treated for 4 to 6 days in collagen I gels with no growth factor (No GF) or
with EGF, HGF, bFGF or KGF. Conditioned medium was collected, concentrated and
run on substrate zymograms. (A) Gelatin zymogram. Latent gelatinase B (LGB),
active and latent gelatinase A (AGA, LGA) and a high molecular weight
gelatinase (HG) were detected. (B) Casein zymogram. A band corresponding to
the molecular weight of latent stromelysin-1 (LStr1) was detected. (C)
Plasminogen casein zymogram. A band corresponding to the molecular weight of
uPA was detected. There were no differences in the activation status of the
gelatinases, caseinase or plasminogen activator between growth factor treated
organoids and untreated controls.
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Fig. 4. Proteinase inhibitors suppress branching morphogenesis induced by growth
factors. (A, part a) Quantification of the percentage of branching organoids
treated with (i) KGF or (ii) EGF in the presence of protease inhibitors E64,
pepstatin, leupeptin, aprotinin, GM6001 and GM1210. Only the MMP inhibitor
GM6001 had a statistically significant inhibitory effect on branching
morphogenesis of both basal (*P0.05) and growth
factor-treated organoids (***P0.001). The
mean±s.d. of six samples in two independent experiments are shown. (A,
part b) Primary organoids after 6 days of treatment with KGF and EGF with or
without GM6001. Note the absence of branches in the organoids treated with the
MMP inhibitor. Similar results were obtained with bFGF and HGF (not shown).
(A, part c) Quantification of branching and (A, part d) appearance of the
organoids treated with TIMP1 and TIMP2 in the presence of KGF. The
mean±s.d. of six (TIMP1) and five (TIMP2) samples in two independent
experiments are shown; (***P0.001). (A, part e)
Quantification of the effect of proteinase inhibitors on branching
morphogenesis induced by co-culturing the organoids with primary fibroblasts
from virgin animals (Fv). Only GM6001 had a statistically significant
inhibitory effect (**P0.01). The mean±s.d. of
six samples in two independent experiments are shown. (B) GM6001 inhibits
increase in cell number in organoids (B, part a), but does not alter
apoptosis. Primary organoids were embedded in collagen I gels and treated for
6 days with KGF in the presence and absence of GM6001. (B, part a) Cell number
was measured using Alamar Blue. The mean±s.d. of six samples in two
independent experiments are shown. (B, part b) Whole organoids (3D) and cross
sections are shown. Apoptotic nuclei were stained in green (FITC). Cross
sections were also stained for  -smooth muscle actin-positive cells
(red). There were no statistical differences in the number of apoptotic cells
in control and treated groups. DAPI nuclear counterstain is in blue. Scale
bars: 400 µm in A; 200 µm in B.
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Fig. 5. Recombinant stromelysin-1 induces branching morphogenesis of primary
mammary organoids. (A) Dose response to rStr1. Mean±s.d. of three
independent experiments is shown. (B) GM6001 inhibits rStr1-induced branching.
rStr1 (1µg/ml) induced-branching morphogenesis compared with the control
(**P<0.01). Control samples were treated with trypsin
and soy bean trypsin inhibitor (SBTI), GM6001,
(***P0.001). The means±s.d. of three
independent experiments are shown. To calculate the branching index, control
values were equated to 1. (C) Appearance of organoids in collagen I gels after
6 days with or without Str1 and GM6001 treatments. (D) Gelatin zymogram of
conditioned media of organoids treated with buffer (Control, C), or different
concentration of rStr1, with or without GM6001 (GM6) or trypsin+SBTI (T/S).
Note the activation of gelatinase B in the presence of rStr1. This activation
is inhibited in the presence of GM6001, as expected. Treatment with
trypsin+SBTI (T/S) did not activate gelatinase B. Scale bars: 200 µm in
C.
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Fig. 8. GM6001 inhibits epimorphin-induced branching morphogenesis in a mammary
epithelial cell line. (A) Preclustered SCp2 cells were treated for 6 days in
collagen I gels with EGF, EGF+epimorphin (EPM) with or without GM6001. GM6001
inhibited branching induced by epimorphin. This inhibition was statistically
significant (***P0.001). Other proteinase inhibitors
such as leupeptin, aprotinin, pepstatin and E64 did not show any inhibition of
branching (not shown). The mean±s.d. of three independent experiments
is shown. (B) Appearance of SCp2 cell clusters treated with EGF, EGF+EPM and
EGF+EPM+GM6001. (C) Epimorphin upregulates gelatinases A and B and Str1 in
SCp2 cells. SCp2 cells were treated for 48 hours with epimorphin or PBS as a
control. Conditioned medium was collected, concentrated and separated on
substrate gels. (D) Epimorphin regulates Str1 mRNA levels. RT-PCR analysis of
Str1 expression in SCp2 cells treated with PBS or EPM for 48 hours. Twenty,
23, 25, 30 and 35 cycles of amplification were performed for detection of Str1
and actin. While the level of actin was not different statistically in the PBS
and epimorphin-treated cultures (slightly higher in PBS in this experiment),
Str1 levels were clearly higher in epimorphin-treated cultures. Scale bars:
200 µm in A.
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Fig. 9. GM6001 does not inhibit SCp2 cell proliferation. SCp2 cells were embedded
in collagen I gels and treated for six days with no growth factor, or with
EGF, KGF, HGF, bFGF in the presence and absence of GM6001, GM1210 and
TGFß1. Quantification of cell number was carried out using Alamar Blue.
The mean±s.d. of three independent experiments is shown. The data are
normalized with values obtained for the control set to 1. Only TGFß1
significantly reduced the cell number (***P0.001).
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