View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by PubMed Central Stromelysin-1 Regulates Adipogenesis during Mammary Gland Involution Caroline M. Alexander,* Sushma Selvarajan,‡ John Mudgett,ʈ and Zena Werb§ *McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin 53706-1599; ‡Department of Pharmaceutical Chemistry and §Department of Anatomy, University of California San Francisco, San Francisco, California 94143-0452; and ʈDepartment of Immunology, Merck Research Laboratory, Rahway, New Jersey 07065 Abstract. The matrix metalloproteinase MMP-3/stro- lial cell death after weaning, but instead alter the stro- melysin-1 (Str1) is highly expressed during mammary mal microenvironment. We used adipogenic 3T3-L1 gland involution induced by weaning. During involu- cells as a cell culture model to test the function of tion, programmed cell death of the secretory epithe- MMPs during adipocyte differentiation. Fibroblastic lium takes place concomitant with the repopulation of 3T3-L1 progenitor cells expressed very low levels of the mammary fat pad with adipocytes. In this study, we MMPs or TIMPs. The transcription of a number of have used a genetic approach to determine the role of MMP and TIMP mRNAs [Str1, MT1-MMP, (MMP-14) Str1 during mammary involution. Although Str1 has collagenase-3 (MMP-13), gelatinase A (MMP-2), and been shown to induce unscheduled apoptosis when ex- TIMP-1, -2 and -3] was induced in committed preadipo- pressed ectopically during late pregnancy (Alexander, cytes, but only differentiated adipocytes expressed an C.M., E.W. Howard, M.J. Bissell, and Z. Werb. 1996. J. activated MMP, gelatinase A. The addition of MMP in- Cell Biol. 135:1669–1677), we found that during post- hibitors (GM 6001 and TIMP-1) dramatically acceler- lactational involution, mammary glands from trans- ated the accumulation of lipid during differentiation. genic mice that overexpress the tissue inhibitor of me- We conclude that MMPs, especially Str1, determine the talloproteinases, TIMP-1 (TO), or mice carrying a targeted rate of adipocyte differentiation during involutive mutation in Str1 showed accelerated differentiation and mammary gland remodeling. hypertrophy of adipocytes, while epithelial apoptosis was unaffected. These data suggest that matrix metallo- Key words: transgenic mouse • mammary involution proteinases (MMPs) do not induce unscheduled epithe- • MMP-3 • TIMP-1 • 3T3-L1 adipocytes Introduction When pups are removed from a lactating mouse, the mam- or by altering cell cycle progression (Boudreau et al., 1995, mary glands involute, losing the expression of the differen- 1996; Wang et al., 1994; Wiesen and Werb, 2000). When tiated gene products associated with milk production and stromelysin-1 (Str1) is misexpressed in mammary gland by resulting in the loss of at least 90% of the lactating mam- transgenic means, mice show precocious growth of virgin mary epithelial cell number. Using this model of induced gland to a mid-pregnant equivalent (Sympson et al., 1994), apoptosis in adult mice, we can determine physiologically stromal alteration, and unscheduled apoptosis during relevant factors that control epithelial cell death. Mam- pregnancy (Boudreau et al., 1995; Alexander et al., 1996; mary gland involution is a two-phase process. The first Thomasset et al., 1998) and tumor formation (Sternlicht et phase is characterized by the onset of epithelial apoptosis, al., 1999). Therefore, we hypothesize that the response of which is p53 dependent (Jerry et al., 1998), and the second mammary epithelial cells to ectopic Str1 reflects normal phase by upregulation of proteinases, repopulation of the roles for this enzyme. mammary stroma by adipocytes, and completion of epi- The prototype MMP, collagenase, was isolated as a col- thelial programmed cell death in a p53-independent man- lagenolytic activity specifically induced during involution ner (Li et al., 1996; Lund et al., 1996). of the tadpole tail (Gross, 1966). Since the description of In cultured mammary epithelial cells, epithelial apopto- this activity, other MMPs have been shown to be induced sis is induced by expression of matrix metalloproteinases during physiological remodeling reactions and to cleave (MMPs),1 by the inhibition of integrin-mediated adhesion, structural proteins important to maintaining basement Address correspondence to Caroline M.Alexander, McArdle Laboratory 1Abbreviations used in this paper: dpc, days post coitum; ECM, extracellu- for Cancer Research, University of Wisconsin Medical School, 1400 Uni- lar matrix; H&E, hematoxylin and eosin; MMP, matrix metalloproteinase; versity Avenue, Madison, WI 53706-1599. Tel.: (608) 265 5182. Fax: (608) Str1, stromelysin-1/MMP-3; TIMP, tissue inhibitor of metalloproteinases; 262 2824. E-mail: [email protected] TO, TIMP overexpressing transgenic mouse; WAP, whey acidic protein. The Rockefeller University Press, 0021-9525/2001/02/693/11 $5.00 The Journal of Cell Biology, Volume 152, Number 4, February 19, 2001 693–703 http://www.jcb.org/cgi/content/full/152/4/693 693 membrane integrity (Sternlicht and Werb, 1999; Sternlicht moter have been described (Alexander et al., 1996). In brief, these mice ng/ml circulating human TIMP-1 50ف et al., 1999; Vu and Werb, 2000). One such MMP, Str1, is have no gross phenotype and express -ng/ml of tissue lysate from pregnant mammary gland. Mice carry 20ف and expressed and regulated during mammary gland develop- ing this transgene were compared with nontransgenic sibs or CD-1 control ment and involution (Witty et al., 1995). Str1 cleaves many mice (Charles River Laboratories). Mice carrying a targeted null mutation basement membrane proteins (Mayer et al., 1993; Werb, in the Str1 gene (StrlϪ/Ϫ) were made by homologous recombination us- 1997), and can autoactivate pro-Str1 and other pro-MMPs ing the AB2.1 ES cell line from 129 mice (Mudgett et al., 1998), and were to initiate a proteolytic cascade (Nagase, 1997). It is syn- compared with a control line (Str1ϩ/ϩ) on the 129 background. These mice were also grossly normal. To standardize lactation from mouse to thesized and secreted by a subpopulation of stromal cells mouse, litters were adjusted to six pups per dam, and mothers were in mammary gland (Witty et al., 1995). Targets for protein- housed individually before weaning. For time points during pregnancy, 0.5 ase activity are not limited to extracellular matrix (ECM) days post coitum (dpc) is assumed to be noon of the day of observation of molecules. Cell surface molecules such as E-cadherin can the vaginal plug. Lactation is timed from the time of parturition. be cleaved in cultured mammary epithelial cells trans- fected with an inducible Str1 cDNA (Lochter et al., 1997; Histology and Immunochemistry Werb, 1997; Sternlicht and Werb 2000). Other substrates For histologic evaluation, pieces of mammary gland were fixed in 4% include extracellular growth factors and cell-surface mole- paraformaldehyde overnight at 4ЊC, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E) according to standard tech- cules (Werb, 1997). niques. For immunostaining of specific ECM molecules, 5 – 10 mm pieces To test the importance of MMPs in involution in the of fresh mammary gland were infiltrated with 20% sucrose in 1 mM present study, we have used two genetic approaches. We EDTA, 50 mM Tris pH 7.5 (4ЊC) and frozen in OCT for cryosectioning. have investigated mammary gland involution in mice over- Sections (6–10 ␮m) were cut and immediately fixed in 4% freshly pre- expressing the tissue inhibitor of matrix metalloprotein- pared paraformaldehyde for 15 min, blocked in 0.1 M glycine (3 ϫ 1 min in PBS), and then 10% sheep serum in PBS for 30 min at room tempera- ases, human TIMP-1 (TO) (Alexander et al., 1996), and in ture. Anti–entactin antibody (diluted 1:1,000 in 3% bovine serum albumin mice carrying a null mutation in Str1 (Str1Ϫ/Ϫ) (Mudgett in Tris-buffered saline) was incubated on sections overnight at 4ЊC, et al., 1998). Both of these transgenic mice have no overt washed three times in PBS/0.1% Tween for 5 min, and incubated with sec- defects and are able to complete pregnancy and lactate. ondary antibody (HRP-conjugated anti–rat IgG, diluted 1:100 into 20% Carnation skim milk powder in PBS) for 60 min. The wash protocol was Analysis of mammary glands from TO mice shows that the repeated and the sections were counterstained lightly with methylene transgene is expressed and active (Alexander et al., 1996). blue. By combining the information from these two strains, we can identify processes that require Str1 activity (if both Gel Electrophoresis of Mammary Gland Protein Lysates TO and Str1Ϫ/Ϫ transgenic mice share the same pheno- Pieces of mammary gland tissue were homogenized in RIPA buffer (150 type) and those that require TIMP-1–inhibitable MMP ac- mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris-HCl, tivity (if TO but not Str1Ϫ/Ϫ mice show a specific pheno- pH 8.0) at 0.25 mg wet weight/ml. Lysates were spun for 15 min at 4ЊC in a type). We show here that a Str1 deficiency specifically microcentrifuge, and insoluble fractions (ECM-enriched fractions) were accelerates the differentiation of adipocytes during active washed once in RIPA buffer and boiled into sample buffer containing 5% SDS as described previously (Alexander et al., 1996). Extracts equivalent remodeling, and that epithelial cell death is unaffected in to 8 mg wet weight tissue were separated on 6% SDS-PAGE gels, and ei- either transgenic strain. ther stained with Coomassie blue or silver stain, or transferred to Immo- bilon P for immunoblotting of specific ECM constituents. Materials and Methods Assay of Apoptotic DNA Laddering mg) were digested in lysis buffer (0.2 100ف) Materials Fragments of mammary tissue mg/ml proteinase K in 50 mM Tris-HCl, 0.1 M NaCl, 0.1 M EDTA, 1% Ultraspec RNA isolation solution was from Biotecx.