Stromelysin-1 regulates adipogenesis during mammary gland involution - PubMed (original) (raw)

Stromelysin-1 regulates adipogenesis during mammary gland involution

C M Alexander et al. J Cell Biol. 2001.

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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Figures

Figure 1

Figure 1

Apoptosis and epithelial function during gland involution in transgenic mice. (a) Total RNA was extracted from mammary glands at various time points, separated by agarose gel electrophoresis (10 μg/track), transferred to membranes, and probed with Str1, TIMP-1, -2 (2 mRNA species), TIMP-3, vimentin (Vim; an internal standard related to mesenchymal cell number), or cytokeratin 18 (CK18; an internal standard related to epithelial cell number) cDNAs. The ethidium bromide–stained 28S rRNA band is also shown for comparison. V, virgin; dP, days pregnant; dL, days lactating; di, days after weaning of pups. _(_b) To measure the onset of programmed cell death, DNA was extracted from glands at various stages and analyzed by agarose gel electrophoresis for evidence of laddering. Epithelial cells began to die between 1 and 3 d after weaning of pups. 1L and 8L, 1 and 8 d lactation; 1i–20i: 1 – 20 d involution after removal of pups after 8 d of lactation. (c) DNA samples from key time points, 2 and 3 d after weaning, are shown for two each of TO and control mice. The onset of apoptosis is normal in TO mice. (d) Differentiated epithelial function was assayed by measuring the expression of mRNA for the milk protein WAP. RNA was extracted from glands at various stages of involution (L, lactating; 1i–4i, 1–4 d of involution after removal of pups after 8 d of lactation) from TO and control mice, and from Str1_−/_− and Str1+/+ mice, and was analyzed by hybridizing Northern blots with a WAP cDNA probe. Ethidium bromide–stained gel tracks before membrane transfer are shown for comparison (18S rRNA). No significant differences were observed in either transgenic line.

Figure 7

Figure 7

Mammary involution is divided into two interactive phases for epithelial and stromal remodeling. This scheme summarizes the relative time course of the catabolic events associated with epithelial involution, namely the loss of milk protein synthesis (differentiated function), together with apoptosis and regression of the majority of the epithelial cells, and the anabolic events responsible for reexpanding the fat pad.

Figure 2

Figure 2

Inhibition of MMP activity affects the dynamics of the remodeling involuting mammary gland. H&E-stained sections of wild type (a, c, and e), TO (b, d, and f), Str+/+ (g and i), and Str1_−/_− (h and j) at mammary glands at 8 d lactation and 2 d involution (e and f), 8 d lactation and 4 d involution (c and d), 2 d lactation and 3 d involution (e and f), 8 d lactation and 3 d involution (g, h), and 8 d lactation and 6 d involution (i and j). Glands deficient in MMP activity show increased numbers of hypertrophic adipocytes during mammary gland involution.

Figure 3

Figure 3

Upregulation of Str1 mRNA expression during stromal remodeling of involuting mammary gland. (a) mRNA was extracted from mammary glands at various time points (L, lactating; 1i–10i, 1–10 d of involution after weaning pups), Northern blots were prepared and probed with cDNAs encoding the milk protein WAP, Str1, the basement membrane protein entactin, and the endothelial cell surface adhesion molecule PECAM-1. (b) ECM-enriched extracts of 8 mg of mammary tissue from control and TO mice 1–4 d after weaning (1i–4i) were analyzed by SDS-PAGE; gels were either Coomassie blue stained (CB) or transferred to membranes for incubation with antisera to either entactin or laminin. Molecular weights of proteins are indicated at the left-hand side. Entactin migrates at 150 kD, with specific MMP-derived cleavage fragments migrating at 100 and 110 kD. The ECM-enriched fraction changes dramatically during the involutive phase of mammary gland remodeling (2–4 d after weaning). The collagens (arrowheads) and basement membrane proteins (entactin and laminin) are substantially reduced.

Figure 4

Figure 4

Immunostaining of entactin shows that epithelial basement membranes are retained during involution. Fixed cryosections from normal glands 1–4 d after weaning were stained for entactin. During gland involution (1i–4i), basement membranes around epithelial alveoli stained positively for entactin (brown HRP-linked product; arrowheads). 4 d after weaning (4i), entactin also appears around hypertrophying adipocytes (arrows). Immunostaining experiments using a subsaturating, diluted primary antibody (1:2,000), aimed at ensuring that this assay was quantitative for glands 2–3 d after weaning, did not show the dramatic loss of entactin observed biochemically (Fig. 3). Control sections (incubated with rat antiserum in place of primary antibody) are blank except for methylene blue–stained nuclei (c).

Figure 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.

Figure 6

Figure 6

Inhibition of MMP activity accelerates lipogenesis in 3T3-L1 cultures. Confluent cultures of 3T3-L1 cells were induced to differentiate by adding DM, either in the presence or absence of the hydroxamate inhibitor GM6001, recombinant human TIMP-1 (rhTIMP1), or purified human TIMP-1 (hTIMP1). (a) In the presence of GM6001, nuclear C/EBPβ protein appears just 24 h after DM administration, earlier than control cultures. (b) After 4 d, the accumulation of lipid was assessed by Oil red O staining of tissue culture dishes containing 3T3-L1 cells. (c) To quantify Oil red O staining, the cultures were extracted in propanol and the absorbance at 510 nm was measured. Control cultures (con) were untreated with DM. Results shown are the average of six determinations and the bars indicate standard deviations. Cells treated with DM alone were normalized to one to reveal the fold induction of lipogenesis in the presence of MMP inhibitors. P values were determined using the Student's t test.

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