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Stromelysin-1 regulates adipogenesis during mammary gland involution.

Alexander CM, Selvarajan S, Mudgett J, Werb Z - J. Cell Biol. (2001)

Bottom Line: Cell Biol. 135:1669-1677), we found that during post-lactational involution, mammary glands from transgenic mice that overexpress the tissue inhibitor of metalloproteinases, TIMP-1 (TO), or mice carrying a targeted mutation in Str1 showed accelerated differentiation and hypertrophy of adipocytes, while epithelial apoptosis was unaffected.These data suggest that matrix metalloproteinases (MMPs) do not induce unscheduled epithelial cell death after weaning, but instead alter the stromal microenvironment.We conclude that MMPs, especially Str1, determine the rate of adipocyte differentiation during involutive mammary gland remodeling.

View Article: PubMed Central - PubMed

Affiliation: McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin 53706-1599, USA. alexander@oncology.wisc.edu

ABSTRACT
The matrix metalloproteinase MMP-3/stromelysin-1 (Str1) is highly expressed during mammary gland involution induced by weaning. During involution, programmed cell death of the secretory epithelium takes place concomitant with the repopulation of the mammary fat pad with adipocytes. In this study, we have used a genetic approach to determine the role of Str1 during mammary involution. Although Str1 has been shown to induce unscheduled apoptosis when expressed ectopically during late pregnancy (Alexander, C.M., E.W. Howard, M.J. Bissell, and Z. Werb. 1996. J. Cell Biol. 135:1669-1677), we found that during post-lactational involution, mammary glands from transgenic mice that overexpress the tissue inhibitor of metalloproteinases, TIMP-1 (TO), or mice carrying a targeted mutation in Str1 showed accelerated differentiation and hypertrophy of adipocytes, while epithelial apoptosis was unaffected. These data suggest that matrix metalloproteinases (MMPs) do not induce unscheduled epithelial cell death after weaning, but instead alter the stromal microenvironment. We used adipogenic 3T3-L1 cells as a cell culture model to test the function of MMPs during adipocyte differentiation. Fibroblastic 3T3-L1 progenitor cells expressed very low levels of MMPs or TIMPs. The transcription of a number of MMP and TIMP mRNAs [Str1, MT1-MMP, (MMP-14) collagenase-3 (MMP-13), gelatinase A (MMP-2), and TIMP-1, -2 and -3] was induced in committed preadipocytes, but only differentiated adipocytes expressed an activated MMP, gelatinase A. The addition of MMP inhibitors (GM 6001 and TIMP-1) dramatically accelerated the accumulation of lipid during differentiation. We conclude that MMPs, especially Str1, determine the rate of adipocyte differentiation during involutive mammary gland remodeling.

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Expression of MMPs and TIMPs during adipocyte differentiation. (a) RNA was extracted from cultures of 3T3-L1 cells at various stages of differentiation. pre, subconfluent fibroblastic precursor; com, confluent, committed stage at day 0 of differentiation; 2–8 dD, 2–8 d differentiation after administration of DM. RNA was separated by agarose gel electrophoresis and transferred to membranes for analysis of expression of specific MMP and inhibitor mRNAs. Autoradiograms were scanned and the relative expression of specific mRNAs is a ratio of EtBr-stained 18S rRNA was quantified. (b) Enzymes secreted by adipocytes into supernatant media were analyzed by gelatin zymography. Gelatinase A, identified by comparison with mouse enzyme standards, was progressively upregulated and activated (*) as 3T3-L1 cells differentiated.
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Figure 5: Expression of MMPs and TIMPs during adipocyte differentiation. (a) RNA was extracted from cultures of 3T3-L1 cells at various stages of differentiation. pre, subconfluent fibroblastic precursor; com, confluent, committed stage at day 0 of differentiation; 2–8 dD, 2–8 d differentiation after administration of DM. RNA was separated by agarose gel electrophoresis and transferred to membranes for analysis of expression of specific MMP and inhibitor mRNAs. Autoradiograms were scanned and the relative expression of specific mRNAs is a ratio of EtBr-stained 18S rRNA was quantified. (b) Enzymes secreted by adipocytes into supernatant media were analyzed by gelatin zymography. Gelatinase A, identified by comparison with mouse enzyme standards, was progressively upregulated and activated (*) as 3T3-L1 cells differentiated.

Mentions: We first determined the expression of MMPs and TIMPs in 3T3-L1 cells. Str1 expression was developmentally regulated in differentiating 3T3-L1 cells. Str1 mRNA was highly induced in confluent, committed preadipocytes, and expression continued in differentiating cultures (Fig. 5 a). We used the expression of two transcription factors that are expressed by differentiated adipocytes (peroxisome proliferator-activated receptor-γ (pPARγ) mRNA, a nuclear hormone receptor, and C/EBPβ) to monitor the differentiation reaction. Thus, C/EBPβ was highly induced after 2 d of treatment with DM (Fig. 6 a), and pPARγ after 4 d (Fig. 5 a) in parallel with lipid accumulation (Fig. 6b and Fig. c). Since Str1 can activate other MMPs, leading to a cascade of MMP-dependent proteolysis, we determined the expression of other MMPs. mRNA for collagenase-3 (MMP-13) was induced in parallel with Str1. mRNA for the cell-surface bound MT1-MMP (MMP-14), was also induced in committed cells, and increased during differentiation. mRNA for matrilysin (MMP-7) and collagenase (MMP-1) were not detected (data not shown).


Stromelysin-1 regulates adipogenesis during mammary gland involution.

Alexander CM, Selvarajan S, Mudgett J, Werb Z - J. Cell Biol. (2001)

Expression of MMPs and TIMPs during adipocyte differentiation. (a) RNA was extracted from cultures of 3T3-L1 cells at various stages of differentiation. pre, subconfluent fibroblastic precursor; com, confluent, committed stage at day 0 of differentiation; 2–8 dD, 2–8 d differentiation after administration of DM. RNA was separated by agarose gel electrophoresis and transferred to membranes for analysis of expression of specific MMP and inhibitor mRNAs. Autoradiograms were scanned and the relative expression of specific mRNAs is a ratio of EtBr-stained 18S rRNA was quantified. (b) Enzymes secreted by adipocytes into supernatant media were analyzed by gelatin zymography. Gelatinase A, identified by comparison with mouse enzyme standards, was progressively upregulated and activated (*) as 3T3-L1 cells differentiated.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195781&req=5

Figure 5: Expression of MMPs and TIMPs during adipocyte differentiation. (a) RNA was extracted from cultures of 3T3-L1 cells at various stages of differentiation. pre, subconfluent fibroblastic precursor; com, confluent, committed stage at day 0 of differentiation; 2–8 dD, 2–8 d differentiation after administration of DM. RNA was separated by agarose gel electrophoresis and transferred to membranes for analysis of expression of specific MMP and inhibitor mRNAs. Autoradiograms were scanned and the relative expression of specific mRNAs is a ratio of EtBr-stained 18S rRNA was quantified. (b) Enzymes secreted by adipocytes into supernatant media were analyzed by gelatin zymography. Gelatinase A, identified by comparison with mouse enzyme standards, was progressively upregulated and activated (*) as 3T3-L1 cells differentiated.
Mentions: We first determined the expression of MMPs and TIMPs in 3T3-L1 cells. Str1 expression was developmentally regulated in differentiating 3T3-L1 cells. Str1 mRNA was highly induced in confluent, committed preadipocytes, and expression continued in differentiating cultures (Fig. 5 a). We used the expression of two transcription factors that are expressed by differentiated adipocytes (peroxisome proliferator-activated receptor-γ (pPARγ) mRNA, a nuclear hormone receptor, and C/EBPβ) to monitor the differentiation reaction. Thus, C/EBPβ was highly induced after 2 d of treatment with DM (Fig. 6 a), and pPARγ after 4 d (Fig. 5 a) in parallel with lipid accumulation (Fig. 6b and Fig. c). Since Str1 can activate other MMPs, leading to a cascade of MMP-dependent proteolysis, we determined the expression of other MMPs. mRNA for collagenase-3 (MMP-13) was induced in parallel with Str1. mRNA for the cell-surface bound MT1-MMP (MMP-14), was also induced in committed cells, and increased during differentiation. mRNA for matrilysin (MMP-7) and collagenase (MMP-1) were not detected (data not shown).

Bottom Line: Cell Biol. 135:1669-1677), we found that during post-lactational involution, mammary glands from transgenic mice that overexpress the tissue inhibitor of metalloproteinases, TIMP-1 (TO), or mice carrying a targeted mutation in Str1 showed accelerated differentiation and hypertrophy of adipocytes, while epithelial apoptosis was unaffected.These data suggest that matrix metalloproteinases (MMPs) do not induce unscheduled epithelial cell death after weaning, but instead alter the stromal microenvironment.We conclude that MMPs, especially Str1, determine the rate of adipocyte differentiation during involutive mammary gland remodeling.

View Article: PubMed Central - PubMed

Affiliation: McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin 53706-1599, USA. alexander@oncology.wisc.edu

ABSTRACT
The matrix metalloproteinase MMP-3/stromelysin-1 (Str1) is highly expressed during mammary gland involution induced by weaning. During involution, programmed cell death of the secretory epithelium takes place concomitant with the repopulation of the mammary fat pad with adipocytes. In this study, we have used a genetic approach to determine the role of Str1 during mammary involution. Although Str1 has been shown to induce unscheduled apoptosis when expressed ectopically during late pregnancy (Alexander, C.M., E.W. Howard, M.J. Bissell, and Z. Werb. 1996. J. Cell Biol. 135:1669-1677), we found that during post-lactational involution, mammary glands from transgenic mice that overexpress the tissue inhibitor of metalloproteinases, TIMP-1 (TO), or mice carrying a targeted mutation in Str1 showed accelerated differentiation and hypertrophy of adipocytes, while epithelial apoptosis was unaffected. These data suggest that matrix metalloproteinases (MMPs) do not induce unscheduled epithelial cell death after weaning, but instead alter the stromal microenvironment. We used adipogenic 3T3-L1 cells as a cell culture model to test the function of MMPs during adipocyte differentiation. Fibroblastic 3T3-L1 progenitor cells expressed very low levels of MMPs or TIMPs. The transcription of a number of MMP and TIMP mRNAs [Str1, MT1-MMP, (MMP-14) collagenase-3 (MMP-13), gelatinase A (MMP-2), and TIMP-1, -2 and -3] was induced in committed preadipocytes, but only differentiated adipocytes expressed an activated MMP, gelatinase A. The addition of MMP inhibitors (GM 6001 and TIMP-1) dramatically accelerated the accumulation of lipid during differentiation. We conclude that MMPs, especially Str1, determine the rate of adipocyte differentiation during involutive mammary gland remodeling.

Show MeSH
Related in: MedlinePlus