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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 inhibition disrupts mammary gland branching morphogenesis. (A and B) Mammary gland whole mounts from 6.5-wk-old mice treated daily with (A) vehicle or (B) GM6001 from 3.5 wk old. Note the extra ductal budding in the GM6001 treated mammary glands (inset). LN, lymph node; arrowheads, TEBs. Bar, 1 mm. (C–E) Mammary gland whole mounts from (C) nontransgenic control mice and (D) mice hemizygous and (E) homozygous for the β-actin human TIMP-1 transgene at 35 d old. n, nipple. Bar, 1 mm. (F and G) Primary mammary organoids grown for 7 d in the presence of EGF in collagen gels from (F) nontransgenic control mice or (G) huTIMP-1 transgenic mice. (H and I) Penetration of mammary ducts into fat pad of control mice, mice continually treated with GM6001 until sacrifice and mice that were treated with GM6001 from 3.5- to 6.5-wk-old using 8–12 mammary glands per data point (t test compared vehicle-control with mice continually treated with GM6001; H) and (I) nontransgenic control, Tg/+ and Tg/Tg hu-TIMP-1 transgenic mice using 4–12 mammary glands per data point. Data are mean ± SEM. t test compared Tg/+ with Tg/Tg. (J) The percentage of branched organoids in response to 7-d treatment with EGF. Organoids were derived from hu-TIMP-1 transgenic mice, nontransgenic controls, or wild-type mice and grown in absence or presence of GM6001 or recombinant TIMP-1 as indicated. In all panels: ***, P < 0.0005; **, P < 0.005, unpaired, two-tailed t test.
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fig2: MMP inhibition disrupts mammary gland branching morphogenesis. (A and B) Mammary gland whole mounts from 6.5-wk-old mice treated daily with (A) vehicle or (B) GM6001 from 3.5 wk old. Note the extra ductal budding in the GM6001 treated mammary glands (inset). LN, lymph node; arrowheads, TEBs. Bar, 1 mm. (C–E) Mammary gland whole mounts from (C) nontransgenic control mice and (D) mice hemizygous and (E) homozygous for the β-actin human TIMP-1 transgene at 35 d old. n, nipple. Bar, 1 mm. (F and G) Primary mammary organoids grown for 7 d in the presence of EGF in collagen gels from (F) nontransgenic control mice or (G) huTIMP-1 transgenic mice. (H and I) Penetration of mammary ducts into fat pad of control mice, mice continually treated with GM6001 until sacrifice and mice that were treated with GM6001 from 3.5- to 6.5-wk-old using 8–12 mammary glands per data point (t test compared vehicle-control with mice continually treated with GM6001; H) and (I) nontransgenic control, Tg/+ and Tg/Tg hu-TIMP-1 transgenic mice using 4–12 mammary glands per data point. Data are mean ± SEM. t test compared Tg/+ with Tg/Tg. (J) The percentage of branched organoids in response to 7-d treatment with EGF. Organoids were derived from hu-TIMP-1 transgenic mice, nontransgenic controls, or wild-type mice and grown in absence or presence of GM6001 or recombinant TIMP-1 as indicated. In all panels: ***, P < 0.0005; **, P < 0.005, unpaired, two-tailed t test.

Mentions: To determine if MMP activity plays a role in branching morphogenesis during pubertal mammary development, we compared mammary glands from mice treated from 3.5 wk old with a broad spectrum MMP inhibitor to controls treated with the vehicle. GM6001 works well in inhibiting MMP function in vivo (Alexander et al., 1996; Kheradmand et al., 2002), and inhibits all MMPs tested with Ki values of <100 nM (Grobelny et al., 1992; Gijbels et al., 1994). Ducts of mice treated with GM6001 showed much less invasion than controls (Fig. 2, A, B, and H), and stopped about where they were at the beginning of the treatment (not depicted). An unexpected observation in the GM6001-treated mice was that the mammary ducts displayed supernumerary budding along the primary ducts compared with the controls (Fig. 2 B, inset). These buds appeared to be lateral branches that were initiated, but failed to elongate and invade into the mammary fat pad.


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 inhibition disrupts mammary gland branching morphogenesis. (A and B) Mammary gland whole mounts from 6.5-wk-old mice treated daily with (A) vehicle or (B) GM6001 from 3.5 wk old. Note the extra ductal budding in the GM6001 treated mammary glands (inset). LN, lymph node; arrowheads, TEBs. Bar, 1 mm. (C–E) Mammary gland whole mounts from (C) nontransgenic control mice and (D) mice hemizygous and (E) homozygous for the β-actin human TIMP-1 transgene at 35 d old. n, nipple. Bar, 1 mm. (F and G) Primary mammary organoids grown for 7 d in the presence of EGF in collagen gels from (F) nontransgenic control mice or (G) huTIMP-1 transgenic mice. (H and I) Penetration of mammary ducts into fat pad of control mice, mice continually treated with GM6001 until sacrifice and mice that were treated with GM6001 from 3.5- to 6.5-wk-old using 8–12 mammary glands per data point (t test compared vehicle-control with mice continually treated with GM6001; H) and (I) nontransgenic control, Tg/+ and Tg/Tg hu-TIMP-1 transgenic mice using 4–12 mammary glands per data point. Data are mean ± SEM. t test compared Tg/+ with Tg/Tg. (J) The percentage of branched organoids in response to 7-d treatment with EGF. Organoids were derived from hu-TIMP-1 transgenic mice, nontransgenic controls, or wild-type mice and grown in absence or presence of GM6001 or recombinant TIMP-1 as indicated. In all panels: ***, P < 0.0005; **, P < 0.005, unpaired, two-tailed t test.
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fig2: MMP inhibition disrupts mammary gland branching morphogenesis. (A and B) Mammary gland whole mounts from 6.5-wk-old mice treated daily with (A) vehicle or (B) GM6001 from 3.5 wk old. Note the extra ductal budding in the GM6001 treated mammary glands (inset). LN, lymph node; arrowheads, TEBs. Bar, 1 mm. (C–E) Mammary gland whole mounts from (C) nontransgenic control mice and (D) mice hemizygous and (E) homozygous for the β-actin human TIMP-1 transgene at 35 d old. n, nipple. Bar, 1 mm. (F and G) Primary mammary organoids grown for 7 d in the presence of EGF in collagen gels from (F) nontransgenic control mice or (G) huTIMP-1 transgenic mice. (H and I) Penetration of mammary ducts into fat pad of control mice, mice continually treated with GM6001 until sacrifice and mice that were treated with GM6001 from 3.5- to 6.5-wk-old using 8–12 mammary glands per data point (t test compared vehicle-control with mice continually treated with GM6001; H) and (I) nontransgenic control, Tg/+ and Tg/Tg hu-TIMP-1 transgenic mice using 4–12 mammary glands per data point. Data are mean ± SEM. t test compared Tg/+ with Tg/Tg. (J) The percentage of branched organoids in response to 7-d treatment with EGF. Organoids were derived from hu-TIMP-1 transgenic mice, nontransgenic controls, or wild-type mice and grown in absence or presence of GM6001 or recombinant TIMP-1 as indicated. In all panels: ***, P < 0.0005; **, P < 0.005, unpaired, two-tailed t test.
Mentions: To determine if MMP activity plays a role in branching morphogenesis during pubertal mammary development, we compared mammary glands from mice treated from 3.5 wk old with a broad spectrum MMP inhibitor to controls treated with the vehicle. GM6001 works well in inhibiting MMP function in vivo (Alexander et al., 1996; Kheradmand et al., 2002), and inhibits all MMPs tested with Ki values of <100 nM (Grobelny et al., 1992; Gijbels et al., 1994). Ducts of mice treated with GM6001 showed much less invasion than controls (Fig. 2, A, B, and H), and stopped about where they were at the beginning of the treatment (not depicted). An unexpected observation in the GM6001-treated mice was that the mammary ducts displayed supernumerary budding along the primary ducts compared with the controls (Fig. 2 B, inset). These buds appeared to be lateral branches that were initiated, but failed to elongate and invade into the mammary fat pad.

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