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Site-specific inductive and inhibitory activities of MMP-2 and MMP-3 orchestrate mammary gland branching morphogenesis.

Wiseman BS, Sternlicht MD, Lund LR, Alexander CM, Mott J, Bissell MJ, Soloway P, Itohara S, Werb Z - J. Cell Biol. (2003)

Bottom Line: Unexpectedly, MMP-2 also represses precocious lateral branching during mid-puberty.In contrast, MMP-3 induces secondary and tertiary lateral branching of ducts during mid-puberty and early pregnancy.Thus, specific MMPs refine the mammary branching pattern by distinct mechanisms during mammary gland branching morphogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, University of California, San Francisco, CA 94143-0452, USA.

ABSTRACT
During puberty, mouse mammary epithelial ducts invade the stromal mammary fat pad in a wave of branching morphogenesis to form a complex ductal tree. Using pharmacologic and genetic approaches, we find that mammary gland branching morphogenesis requires transient matrix metalloproteinase (MMP) activity for invasion and branch point selection. MMP-2, but not MMP-9, facilitates terminal end bud invasion by inhibiting epithelial cell apoptosis at the start of puberty. Unexpectedly, MMP-2 also represses precocious lateral branching during mid-puberty. In contrast, MMP-3 induces secondary and tertiary lateral branching of ducts during mid-puberty and early pregnancy. Nevertheless, the mammary gland is able to develop lactational competence in MMP mutant mice. Thus, specific MMPs refine the mammary branching pattern by distinct mechanisms during mammary gland branching morphogenesis.

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MMP-2 −/− mice have reduced ductal invasion and increased lateral branching. (A–D) Whole mounts of mammary glands of an (A and C) MMP-2 +/− and (B and D) MMP-2 −/− mice (A and B) 30 d old and (C and D) 50 d in estrus. Note supernumerary branching in D (inset). Example of ramified branch (arrows) and unramified branch or bud (arrowheads). Bars, 1 mm. (E–H) Sections through TEBs in mammary glands from 30-d-old (E and G) MMP-2 +/− and (F and H) MMP-2 −/− mice. (E and F) Immunohistochemistry for Ln-1. (G and H) TUNEL assay. Apoptotic cells are red. Bars, 25 μm. (I–L) Penetration of mammary ducts of (I) MMP-2 +/− and MMP-2 −/− and (J) MMP-9 +/− and MMP-9 −/− mice, (K) number of branches per millimeter, and (L) number of unramified branches per millimeter at 50 d from primary mammary ducts of MMP-2 +/− and −/− mice. Data are mean ± SEM (I, K, and L) using 8–16 or (H) 4–8 mammary glands per data point. (M and N) Percentage of BrdU (M) or TUNEL (N) positive cells within TEBs of MMP-2 −/− and MMP-2 +/− 30-d-old mice. Data are mean percent per TEB ± SD. ***, P < 0.0005; **, P < 0.005; *, P < 0.05, unpaired, two-tailed t test.
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fig4: MMP-2 −/− mice have reduced ductal invasion and increased lateral branching. (A–D) Whole mounts of mammary glands of an (A and C) MMP-2 +/− and (B and D) MMP-2 −/− mice (A and B) 30 d old and (C and D) 50 d in estrus. Note supernumerary branching in D (inset). Example of ramified branch (arrows) and unramified branch or bud (arrowheads). Bars, 1 mm. (E–H) Sections through TEBs in mammary glands from 30-d-old (E and G) MMP-2 +/− and (F and H) MMP-2 −/− mice. (E and F) Immunohistochemistry for Ln-1. (G and H) TUNEL assay. Apoptotic cells are red. Bars, 25 μm. (I–L) Penetration of mammary ducts of (I) MMP-2 +/− and MMP-2 −/− and (J) MMP-9 +/− and MMP-9 −/− mice, (K) number of branches per millimeter, and (L) number of unramified branches per millimeter at 50 d from primary mammary ducts of MMP-2 +/− and −/− mice. Data are mean ± SEM (I, K, and L) using 8–16 or (H) 4–8 mammary glands per data point. (M and N) Percentage of BrdU (M) or TUNEL (N) positive cells within TEBs of MMP-2 −/− and MMP-2 +/− 30-d-old mice. Data are mean percent per TEB ± SD. ***, P < 0.0005; **, P < 0.005; *, P < 0.05, unpaired, two-tailed t test.

Mentions: MMP-2 is expressed in the stromal compartment around mammary ducts during branching morphogenesis and is present in an activated form (Fig. 1; Fata et al., 1999). MMP-2 influences branching in the pseudoglandular stage of lung development (Kheradmand et al., 2002), making it a good candidate for regulating mammary branching. Although MMP-2 −/− mice show attenuated tumor growth (Itoh et al., 1997, 1998), the females are able to nurture their young. We compared mammary glands of age- and estrus-matched MMP-2 −/−, MMP-2 +/−, and wild-type mice. Because there was no discernable heterozygous phenotype, we used the MMP-2 +/− mice as controls. There was no difference in the morphology of MMP-2 −/− mammary glands compared with littermate controls at 20 d old, indicating that MMP-2 is not needed before pubertal development. However, we found retarded invasion of the mammary ducts of MMP-2 −/− mice into the stromal fat pad at 30 d (Fig. 4, A, B, and I). This corroborates our in situ data showing that mRNA for the activator of MMP-2, namely MMP-14, is concentrated right at the invasive front of ducts (Fig. 1). This corresponds to the site of highest MMP activity as seen by in situ zymography (unpublished data). This phenotype was most evident in early puberty at 30 d old and the difference was significant relative to the fat pad length. However, the retardation was transient, and by 50 d, the difference was no longer significant (Fig. 4, C, D, and I). Therefore, MMP-2 regulates only the initial events in ductal invasion after puberty.


Site-specific inductive and inhibitory activities of MMP-2 and MMP-3 orchestrate mammary gland branching morphogenesis.

Wiseman BS, Sternlicht MD, Lund LR, Alexander CM, Mott J, Bissell MJ, Soloway P, Itohara S, Werb Z - J. Cell Biol. (2003)

MMP-2 −/− mice have reduced ductal invasion and increased lateral branching. (A–D) Whole mounts of mammary glands of an (A and C) MMP-2 +/− and (B and D) MMP-2 −/− mice (A and B) 30 d old and (C and D) 50 d in estrus. Note supernumerary branching in D (inset). Example of ramified branch (arrows) and unramified branch or bud (arrowheads). Bars, 1 mm. (E–H) Sections through TEBs in mammary glands from 30-d-old (E and G) MMP-2 +/− and (F and H) MMP-2 −/− mice. (E and F) Immunohistochemistry for Ln-1. (G and H) TUNEL assay. Apoptotic cells are red. Bars, 25 μm. (I–L) Penetration of mammary ducts of (I) MMP-2 +/− and MMP-2 −/− and (J) MMP-9 +/− and MMP-9 −/− mice, (K) number of branches per millimeter, and (L) number of unramified branches per millimeter at 50 d from primary mammary ducts of MMP-2 +/− and −/− mice. Data are mean ± SEM (I, K, and L) using 8–16 or (H) 4–8 mammary glands per data point. (M and N) Percentage of BrdU (M) or TUNEL (N) positive cells within TEBs of MMP-2 −/− and MMP-2 +/− 30-d-old mice. Data are mean percent per TEB ± SD. ***, P < 0.0005; **, P < 0.005; *, P < 0.05, unpaired, two-tailed t test.
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fig4: MMP-2 −/− mice have reduced ductal invasion and increased lateral branching. (A–D) Whole mounts of mammary glands of an (A and C) MMP-2 +/− and (B and D) MMP-2 −/− mice (A and B) 30 d old and (C and D) 50 d in estrus. Note supernumerary branching in D (inset). Example of ramified branch (arrows) and unramified branch or bud (arrowheads). Bars, 1 mm. (E–H) Sections through TEBs in mammary glands from 30-d-old (E and G) MMP-2 +/− and (F and H) MMP-2 −/− mice. (E and F) Immunohistochemistry for Ln-1. (G and H) TUNEL assay. Apoptotic cells are red. Bars, 25 μm. (I–L) Penetration of mammary ducts of (I) MMP-2 +/− and MMP-2 −/− and (J) MMP-9 +/− and MMP-9 −/− mice, (K) number of branches per millimeter, and (L) number of unramified branches per millimeter at 50 d from primary mammary ducts of MMP-2 +/− and −/− mice. Data are mean ± SEM (I, K, and L) using 8–16 or (H) 4–8 mammary glands per data point. (M and N) Percentage of BrdU (M) or TUNEL (N) positive cells within TEBs of MMP-2 −/− and MMP-2 +/− 30-d-old mice. Data are mean percent per TEB ± SD. ***, P < 0.0005; **, P < 0.005; *, P < 0.05, unpaired, two-tailed t test.
Mentions: MMP-2 is expressed in the stromal compartment around mammary ducts during branching morphogenesis and is present in an activated form (Fig. 1; Fata et al., 1999). MMP-2 influences branching in the pseudoglandular stage of lung development (Kheradmand et al., 2002), making it a good candidate for regulating mammary branching. Although MMP-2 −/− mice show attenuated tumor growth (Itoh et al., 1997, 1998), the females are able to nurture their young. We compared mammary glands of age- and estrus-matched MMP-2 −/−, MMP-2 +/−, and wild-type mice. Because there was no discernable heterozygous phenotype, we used the MMP-2 +/− mice as controls. There was no difference in the morphology of MMP-2 −/− mammary glands compared with littermate controls at 20 d old, indicating that MMP-2 is not needed before pubertal development. However, we found retarded invasion of the mammary ducts of MMP-2 −/− mice into the stromal fat pad at 30 d (Fig. 4, A, B, and I). This corroborates our in situ data showing that mRNA for the activator of MMP-2, namely MMP-14, is concentrated right at the invasive front of ducts (Fig. 1). This corresponds to the site of highest MMP activity as seen by in situ zymography (unpublished data). This phenotype was most evident in early puberty at 30 d old and the difference was significant relative to the fat pad length. However, the retardation was transient, and by 50 d, the difference was no longer significant (Fig. 4, C, D, and I). Therefore, MMP-2 regulates only the initial events in ductal invasion after puberty.

Bottom Line: Unexpectedly, MMP-2 also represses precocious lateral branching during mid-puberty.In contrast, MMP-3 induces secondary and tertiary lateral branching of ducts during mid-puberty and early pregnancy.Thus, specific MMPs refine the mammary branching pattern by distinct mechanisms during mammary gland branching morphogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, University of California, San Francisco, CA 94143-0452, USA.

ABSTRACT
During puberty, mouse mammary epithelial ducts invade the stromal mammary fat pad in a wave of branching morphogenesis to form a complex ductal tree. Using pharmacologic and genetic approaches, we find that mammary gland branching morphogenesis requires transient matrix metalloproteinase (MMP) activity for invasion and branch point selection. MMP-2, but not MMP-9, facilitates terminal end bud invasion by inhibiting epithelial cell apoptosis at the start of puberty. Unexpectedly, MMP-2 also represses precocious lateral branching during mid-puberty. In contrast, MMP-3 induces secondary and tertiary lateral branching of ducts during mid-puberty and early pregnancy. Nevertheless, the mammary gland is able to develop lactational competence in MMP mutant mice. Thus, specific MMPs refine the mammary branching pattern by distinct mechanisms during mammary gland branching morphogenesis.

Show MeSH
Related in: MedlinePlus