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Development 140 (2): 343-352

Transmembrane/cytoplasmic, rather than catalytic, domains of Mmp14 signal to MAPK activation and mammary branching morphogenesis via binding to integrin β1

Hidetoshi Mori1,*, Alvin T. Lo1, Jamie L. Inman1, Jordi Alcaraz1,2, Cyrus M. Ghajar1, Joni D. Mott1, Celeste M. Nelson1,3, Connie S. Chen1, Hui Zhang1, Jamie L. Bascom1, Motoharu Seiki4, and Mina J. Bissell1,*

1 Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
2 Unitat de Biofísica i Bioenginyeria, Universitat de Barcelona, Barcelona 08036, Spain.
3 Chemical and Biological Engineering and Molecular Biology, Princeton University, Princeton, NJ 08544, USA.
4 Institute of Medical Science, University of Tokyo, Tokyo, Japan.


Figure 1
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Fig. 1. Mmp14 expression peaks during puberty, and is highly elevated at the invading front of mammary gland (MG) end buds. (A) Quantitative RT-PCR analysis of Mmp14 expression during development of the mouse MG from virgin (V; 2-3, 6-8 and 9-14 weeks after birth), early pregnancy (Early-preg; day 4), mid-pregnancy (Mid-preg; days 8-12), late pregnancy (Late-Preg; days 16-18) and lactating (days 1-10) mice, normalized to 18S rRNA. Data are mean±s.e.m. **P<0.01 when compared with V (2-3w). (B) Mmp14 promoter activity in MGs from Mmp14 (+/lacZ) mice. Images are of glands of 5-week-old mice. β-Gal staining of a whole-mount MG isolated from a virgin transgenic heterozygote mouse bearing the lacZ gene under the control of the endogenous Mmp14 promoter (Yana et al., 2007Go) indicates that Mmp14 promoter activity is high in mammary epithelial cells. (i) β-Gal-stained MG from Mmp14 (+/+) mouse as negative control. (ii) β-gal stained MG from Mmp14 (+/lacZ) mouse. Scale bar: 6.25 mm. (iii) β-Gal-stained mammary end buds in Mmp14 (+/lacZ) mouse showed intense promoter activity at the tip of the bud. (iv) β-Gal and Eosin stain of a MG tissue section from a 5-week-old Mmp14 (+/lacZ) transgenic mouse. Scale bars: 200 μm.

 

Figure 2
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Fig. 2. Mmp14 catalytic activity is required for invasion/branching in dense but not in sparse collagen gels. (A) A 3D organotypic culture model of mammary epithelial cell invasion/branching. Mammary organoids from Balb/c mice or clusters of EpH4 cells were induced to branch by addition of 9 nM FGF2 for 5 days in either 3 mg/ml or 1 mg/ml collagen 1 (CL-1). (B) CL-1 (3 mg/ml). (i,iii) Vehicle control (ctrl; DMSO), (ii,iv) MMP inhibitor GM6001 (40 μM) or pre-treatment with (v) control shRNA or (vi) Mmp14-shRNA. (i,ii) Bright-field image of primary organoids in 3 mg/ml CL-1 gel. (iii-vi) Live dye (Calcein AM) stained EpH4 cell aggregates in 3 mg/ml CL-1 gel. (vii) Invasion/branching of EpH4 cells was scored as positive when displaying three or more branches with lengths of at least half the diameter of the central cell cluster (as described in the Materials and methods). Percentages of cell invasion of control (DMSO treated; red bar) EpH4 cells versus EpH4 treated with GM6001 or infected with control (ctrl_shRNA; green bar) and shMmp14-containing lentivirus (Mmp14_shRNA). Two-hundred colonies were analyzed for each condition in three separate experiments. Data are mean±s.e.m. ***P<0.001 compared with control. (C) Invasion/branching of MECs in sparse CL-1 gels (1 mg/ml) in the presence of (i,iii) vehicle control (ctrl; DMSO), (ii,iv) MMP inhibitor GM6001 (40 μM) or pre-treated with (v) control- or (vi) Mmp14-shRNA. (i,ii) Bright-field image of primary organoids in CL-1 gel. (iii-vi) Live dye (Calcein AM) stained EpH4 cell aggregates in CL-1 gel. (vii) Percentages of cells invading control EpH4 cells (DMSO treated; red bar), EpH4 cells treated with GM6001 (blue bar), EpH4 cells infected with control shRNA (ctrl, green bar) or EpH4 cells infected with Mmp14 shRNA-containing lentivirus. Two-hundred colonies were analyzed for each condition over three separate experiments. Data are mean±s.e.m. ***P<0.001 when compared with control. Scale bars: 200 μm.

 

Figure 3
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Fig. 3. MAPK activity and cross-signaling between Mmp14, Erk and Itgb1 are involved in branching morphogenesis. (A) Silencing Mmp14 reduces Erk activity and the level of Itgb1. (i) Immunoblots of phospho-Erk (pErk) in control and Mmp14-silenced EpH4 cells. (ii) Quantification of Erk activity in i. Data are mean±s.e.m. *P<0.05. n=3. (iii) Western blot of Mmp14 and Itgb1 in control and Mmp14-silenced EpH4 cells. LAM A/C was used as the loading control. (iv) MECs from Mmp14 knockout had reduced levels of Itgb1. Immunofluorescence intensity of Itgb1 measured in MG tissues from Mmp14 (+/–, HET) or Mmp14 (–/–, KO) mice (see also supplementary material Fig. S5). Analysis was performed on luminal epithelial cells (LEP) and myoepithelial cells (MEP). Measurement was performed with IMARIS software (Bitplane). At least 50 cells were analyzed per tissue section, n=3 tissue sections. ****P<0.0001. Horizontal lines indicate the mean. (B) Silencing Itgb1 reduced MEC branching, MAPK activity and the Mmp14 levels in sparse CL-1 gels. Branching of (i) control or (ii) Itgb1 shRNA-treated MECs in CL-1 gels of 1 mg/ml. (iii) Silencing Itgb1 reduced Erk phosphorylation. (iv) Quantification of the ratio between pErk and total Erk in Itgb1-shRNA-treated EpH4 cells in sparse CL-1. Data are mean±s.e.m. *P<0.05. (v) Silencing Itgb1 reduced the expression levels of Mmp14 (mean intensity values normalized to Lamin A/C (LAM A/C) calculated via band densitometry from n=3 immunoblots shown below each band). (C) MEK activity is required for cell invasion in CL-1. (i,ii) Control (ctrl; DMSO) and PD98059-treated EpH4 cells in CL-1 gels of 1 mg/ml. (iii) MEK inhibition reduced Mmp14 and Itgb1 levels, as determined by immunoblot (normalized mean intensity values calculated as above are given below each band). Scale bars: 200 μm.

 

Figure 4
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Fig. 4. Co-immunoprecipitation and FRET reveal a direct association between Mmp14 and Itgb1. (A) Co-immunoprecipitation of endogenous Mmp14 and Itgb1. Protein complexes containing Mmp14 were immunoprecipitated from EpH4 cells cultured on a collagen 1-coated dish and probed for Mmp14 (top row) or Itgb1 (bottom row). (B) FRET analysis of monomeric Cypet-tagged MMP14F (FLAG tagged human MMP14) and monomeric Ypet-tagged ITGB1 exogenously expressed in EpH4 MECs. Ypet emission signal was detected as FRET signal when Cypet was excited. Scale bars: 10 μm.

 

Figure 5
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Fig. 5. Assigning functional activity to the non-catalytic domains of MMP14. (A) FLAG-tagged full-length human MMP14 (MMP14F FL) catalytic domain-deleted mutant (MMP14F dCAT) and catalytic/hemopexin domain-deleted mutant (MMP14F dCAT/dPEX). (B) MMP14 overexpression rescued the level of Itgb1 in Mmp14-silenced cells. Expression of MMP14F-FL or other mutant proteins was performed on Mmp14-silenced EpH4 cells at the passage 3 after infection with Mmp14 shRNA containing lentivirus. Samples for cell lysate or branching were used at passage 3 or 4 from silencing Mmp14. Immunoblot analysis of Mmp14 (with an anti-FLAG antibody) and Itgb1 (total and phospho-T788/T789) are indicated. The level of total and phospho-T788/T789 were up-modulated when cells overexpressed MMP14F-FL or MMP14F-dCAT/dPEX. LAM A/C is shown as loading control. Numbers below blots indicate the ratio between Itgb1 (or phospho-T788/T789) and LAM A/C. (C) Full-length MMP14 or MMP14F dCAT/dPEX (i.e. only the transmembrane/cytoplasmic domain) mutant rescued invasion/branching in Mmp14-silenced EpH4 cells in sparse CL-1 gels. (i-iv) Mmp14-silenced EpH4 cells were infected with lentivirus containing (i) control lentivirus (mYpet), and mYpet tagged- (ii) MMP14F-FL, (iii) MMP14F-dCAT and (iv) MMP14F-dCAT/dPEX. Cells were cultured in sparse (1 mg/ml) CL-1 gel. Scale bars: 40 μm.

 

Figure 6
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Fig. 6. Different steps involving Mmp14 and Itgb1 during MEC invasion/branching in a collagen 1 microenvironment. (A) Non-proteolytic activity of MMP14 is involved in mammary epithelial cell sorting in a CL-1 microenvironment (Mori et al., 2009Go). The relationship between Mmp14 expression and MEC sorting. Whereas MECs expressing full-length Mmp14 (FL-Mmp14) or the catalytic domain-deleted mutant (dCAT) sort to the invasive front, the hemopexin domain-deleted mutant (dPEX) or MECs with silenced Mmp14 expression do not. (B) Proteolytic activity of Mmp14 is required for MECs to invade/branch in dense CL-1 (Alcaraz et al., 2011Go). MECs need to degrade collagen 1 to generate a path for invasion/branching in dense collagen. Mmp14 is at the hub of this proteolytic activity for collagen degradation. (C) The association between Mmp14 and Itgb1 during MEC invasion/branching in a sparse CL-1 microenvironment. Whereas MECs do not need MMP activity for invasion/branching in sparse CL-1 gels, Mmp14 itself is required. Specifically, Mmp14 association with Itgb1 is necessary for MEC invasion/branching. Expressing FL-MMP14 or dCAT/dPEX in Mmp14-silenced MECs results in restoration of Itgb1 levels and activity to facilitate branching. Expression of the catalytic domain-deleted mutant (dCAT) was unable to rescue branching and the activation of Itgb1 in sparse CL-1 gels when the catalytic domain is absent. These events (from A to C) suggest that cells use different functions and domains of Mmp14 in a context-dependent manner during branching in collagenous microenvironments.

 


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