Stromelysin-1 Is Highly Upregulated during Mammary Gland Involution
The endogenous expression of Str1 mRNA is regulated during mammary gland development. Str1 expression was low in virgin glands, but it was induced during early pregnancy (3 dpc), continued to increase to 9 dpc, and was not detectable after 12 d of pregnancy or during lactation ( a).
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 (more ...)
Expression of a Str1 transgene during late pregnancy induces unscheduled apoptosis in dividing mammary epithelial cells (Boudreau et al. 1995
; Alexander et al. 1996
). Interestingly, the expression of endogenous Str1 mRNA is not detectable during late pregnancy, presumably protecting cells from Str1-induced cell death. Str1 mRNA was highly induced during involution, when the secretory epithelium undergoes apoptosis (Talhouk et al. 1992
; Li et al. 1994
; Lund et al. 1996
), to ~50-fold that typical of pregnant glands.
During mammary development induced by pregnancy, the expression of mRNA for the matrix metalloproteinase inhibitor, TIMP-1, is induced beginning at 6 dpc, continues throughout lactation, and declines sharply on the same day that stromelysin-1 mRNA is maximally induced ( a; Talhouk et al. 1992
). Transgenic huTIMP-1 is expressed in parallel with actin mRNA. TIMP-2 and -3 are expressed throughout mammary development, decreasing during lactation ( a).
TIMP-1 Overexpression and Stromelysin-1 Deficiency Do Not Affect Apoptosis or Loss of Mammary Epithelial Function during Mammary Gland Involution
To determine whether Str1 has a role in inducing epithelial cell apoptosis during mammary gland involution after weaning, we measured DNA laddering in tissue extracts from wild-type, TO transgenic, and Str1
− mice. In wild-type mice, ladders of DNA appeared between 1 and 3 d after weaning ( and ), correlating with the appearance of residual cell bodies in alveolar lumens in histological samples (data not shown; also see Lund et al. 1996
). The peak of cell death was at 3 d, and cell death was significant for 8–10 d. After 10 d, resorption of the epithelium (>90% of epithelial cells) was complete. DNA laddering was also evident 2 and 3 d after the removal of pups ( c) from TO mice, indicating that the onset of epithelial involution was normal. To measure the decline of differentiated epithelial cell function that occurs during weaning, we assayed the expression of the mRNA for the milk protein, whey acidic protein (WAP). Surprisingly, both TO and Str1
− mice showed the wild-type pattern of declining expression between days 2 and 3 postweaning ( d). These data indicate that Str1 and other TIMP-1–inhibitable MMPs do not play a significant role in apoptosis of secretory epithelial cells. Why then is the expression of Str1 so dramatically induced during mammary involution?
Mammary Adipogenesis Is Accelerated in Mice Deficient in MMP Function
We observed a striking alteration in the morphology of MMP-deficient glands during involution (). At first glance, these glands appeared to involute more rapidly because the area occupied by epithelial ducts and alveoli decreased. However, from morphological data, we deduced that this was due to accelerated repopulation of the gland with adipocytes. After weaning of wild-type mice allowed to lactate for 8 d, there is a delay of ~4 d before significant recolonization of the mammary gland with differentiated adipocytes. In TO mice, differentiated hypertrophic adipocytes appeared in greater number than wild type as early as 2 d after weaning (, a and b). By histomorphometry of H&E-stained paraffin sections, mammary glands of TO mice harvested 4 d after weaning contained between 40 and 50% more unilocular adipocytes (, and and ).
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 (more ...)
Altered Adipogenesis and PECAM Expression in Involuting Glands of Str1−/− and TO Mice
Next we altered the time course of mammary involution to determine whether this effect upon adipocyte differentiation was maintained. When pups are removed after 2 instead of 8 d of lactation, complete alveolar development is prevented, the epithelium regresses more rapidly, and cell death is complete by day 4 (Talhouk et al. 1992
). Using this protocol, we found that the process of adipocyte colonization was accelerated in wild-type mice, so that the relative timing of adipocyte expansion and epithelial cell regression is maintained ( e). When TO females were weaned after 2 d of lactation, the differentiation of adipocytes was increased relative to controls ( f).
Histomorphometric analysis of involuting glands from Str1−/− mice at 3 d ( and ) and 6 d ( and ) after weaning revealed changes of adipocyte colonization that resembled those seen in TO mice. Thus, the number of adipocytes/microscopic field and the area that they occupied increased by 30% 6 d after weaning Str1−/− mice, and by 40% 4 d after weaning TO mice (). Note that glands are scored when adipocyte hypertrophy is maximal, and this is different depending upon the mouse strain (TO mice and Str1−/− mice are on CD1 and 129 strain backgrounds, respectively). These data indicate that TIMP-1–sensitive MMPs, including Str1, regulate mammary adipogenesis.
Str1 Upregulation Parallels Mammary Gland Adipogenesis, Angiogenesis, and Remodeling of Stromal Matrix during Involution
Why does the absence of Str1 affect the rate of adipocyte differentiation? Previous studies using in situ hybridization have shown that Str1 mRNA is expressed by fibroblastic cells, some of which may be preadipocytes (Lund et al. 1996
). Str1 protein is frequently associated with blood vessels (Talhouk et al. 1992
). Detailed analysis of the time course of expression of Str1
mRNA, compared with other markers of cell function (such as WAP mRNA expression) showed that Str1
was induced only after the loss of differentiated epithelial cell function, and after the majority of epithelial cell death. Notably, Str1
was induced in parallel with markers usually associated with active remodeling and morphogenesis ().
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 (more ...)
Repopulation of the mammary gland by differentiated adipocytes requires replacement of the interstitial ECM around the fibroblast-like preadipocytes, which is rich in fibrillar collagens and fibronectin (data not shown), by the basement membranes that surround differentiated adipocytes (Smas and Sul 1995
). Concomitantly, the vasculature of the fat pad is remodeled so that there is a dense weave of capillaries in intimate contact with adipocytes (Crandall et al. 1997
). mRNA for nidogen-1/entactin, an ECM molecule that is a prominent component of adipocyte basement membranes (see ), was induced at 3 d, at the same time as Str1 (, a and b). We observed that the expression of PECAM-1 mRNA, a cell adhesion molecule specific to endothelial cells, was induced at 3 d, and peaked at 6 d after weaning. Interestingly, PECAM-1 mRNA was expressed at a higher level in involuting glands from Str1
− mice (). These data lead us to conclude that the timing of induction of Str1 mRNA is consistent with its expression during angiogenesis and remodeling by the stromal compartment.
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 (more ...)
Mammary Gland Involution Is Characterized by a Biosynthetic Phase
We next verified that there was a switch in ECM at this time in involution. The activation of a biosynthetic stromal compartment was reflected in changes in the ECM of involuting glands. We observed dramatic changes in the protein profiles by SDS-PAGE analysis of ECM-enriched extracts in response to weaning. At 1 d after weaning, the mammary gland extracts contained collagens ( b, arrowheads), entactin, and laminin. These protein profiles resembled extracts from pregnant and lactating glands (Alexander et al. 1996
; and data not shown). Coincident with the induction of epithelial apoptosis at 2 d after weaning, most basement membrane proteins, including basement membranes entactin and laminin, disappeared from the ECM-enriched fraction ( b). Immunoreactive entactin and laminin began to reappear after 3 d of involution ( b), when their mRNA transcripts were upregulated ( a, and data not shown) and were present in high amounts after 4 d.
To better define the cellular events that lead to these striking changes, we stained sections of involuting gland with antibodies to basement membrane proteins. Surprisingly, 2 and 3 d after weaning, the amount of entactin in basement membranes around epithelial alveoli appeared similar to that during lactation ( and ). We conclude that during the initiation phase of apoptosis in involuting gland, ECM proteins normally SDS soluble become insoluble, leaving their antigenicity unaffected and the morphology of the basement membranes (at the light microscopic level) unchanged. We suggest that these biochemical changes are the result of extracellular cross linking by tissue transglutaminase, an enzyme known to be induced during apoptosis of other cell types (see Discussion). The increase of entactin protein measured biochemically by SDS-PAGE analysis of glands 4 d after weaning () paralleled the increase of entactin observed by immunostaining around hypertrophying adipocytes in sections of similar glands (, ). During the biosynthetic phase (arrows), entactin protein localized not only to the regressing, insoluble epithelial basement membranes, but to the assembling, soluble basement membranes that surround hypertrophying adipocytes.
We then sought evidence to verify that the upregulation of Str1 had functional consequences by examining the integrity of one of its substrates in vivo, entactin/nidogen-1. The coinduction of Str1 and entactin during the biosynthetic phase of involution led to a characteristic pattern of entactin fragmentation ( b, Entactin), the result of Str1 cleavage of the 150-kD entactin molecule between the G1 and G2 domains (Alexander et al. 1996
). This proteolysis was almost completely inhibited in parallel samples from TO mice. This result shows that the TIMP-1 transgene was not only highly expressed during involution, but that it was also an effective MMP inhibitor.
MMPs Regulate Adipogenesis in 3T3-L1 Cells in Culture
The results of the genetic experiments in the mice described above suggest that the rate of adipocyte hypertrophy in the mammary gland is enhanced in the absence of Str1. However, adipogenesis could be either a direct or an indirect target of MMPs in vivo. To examine whether there are direct effects of MMPs, we used a model system of adipogenic differentiation, namely cultured 3T3-L1 cells. These fibroblastic cells are not adipogenic in subconfluent cultures. At confluence, the cells become committed preadipocytes. Treatment of confluent cultures with a differentiation-inducing mix (DM; dexamethasone, insulin, and methylisobutylxanthine) induces the expression of proteins associated with mature adipocytes and the accumulation of lipids (Bernlohr et al. 1984
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 ( 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 ( a), and pPARγ after 4 d ( a) in parallel with lipid accumulation ( and ). 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).
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; (more ...)
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), (more ...)
The expression of the proteolytic activity of MMPs is regulated by TIMPs. All four TIMPs were expressed in adipocytes, as they are in vivo in the mammary fat pat of mice during puberty (Fata et al. 1999
). TIMP-1 and -3 were highly induced in committed cells, but showed little expression in differentiated adipocytes. TIMP-4 was expressed specifically by differentiated adipocytes, increasing in parallel with pPARγ. TIMP-2 expression was characteristic of committed and differentiated cells. We conclude that differentiated cells express a higher ratio of mRNAs for MMPs compared with TIMPs than committed cells.
We found that the relative increase in MMP mRNA expression was accompanied by an induction of proteolytic activity specific to the differentiation phase of 3T3-L1 development. Gelatinase A (MMP-2) was the major MMP identified by zymography of enzymes secreted into the media of induced 3T3-L1 cells ( b). MT1-MMP activates gelatinase A in a TIMP-2–dependent fashion (Will et al. 1996
; Holmbeck et al. 1999
; Caterina et al. 2000
; Wang et al. 2000
; Zhou et al. 2000
). We observed significant activation of gelatinase A after 4 d of differentiation, and further induction and activation after 8 d. Thus, as inhibitor expression declined and MT1-MMP expression increased during adipocyte differentiation, this proteinase was activated.
In vivo, we found that ectopic expression of TIMP-1 expression increased the rate of adipocyte differentiation. If this effect is mediated by a direct effect on adipocytes, we would expect that the addition of an MMP inhibitor would increase the rate of adipogenesis during the differentiation of adipocytes in vitro. To test this hypothesis, we added three different MMP inhibitors [a synthetic hydroxamate inhibitor (GM6001; 10 μM), recombinant human TIMP-1 (250 nM), or natural human TIMP-1 purified from transfected BHK cells (250 nM)] to cultures of committed 3T3-L1 cells concomitant with the differentiation-inducing mix. An accelerated rate of differentiation with increased C/EBPβ expression was evident at day one of differentiation ( a). All three inhibitors stimulated lipogenesis by cells 4 d after induction by more than sevenfold ( and ). We conclude that Str1 determines the rate of hypertrophy and lipogenesis in differentiating adipocytes and that, in its absence, differentiation is accelerated.