The tumour suppressor IRF-1 is a transcription factor involved in the induction of apoptosis in several in vitro systems. Post-lactational involution of the mammary gland is characterized by extensive apoptosis of the epithelial cells. We have previously shown that signal transducer and activator of transcription (Stat) 3 drives apoptosis and involution in the mouse mammary gland. Since one of the downstream targets of the Stat signalling pathway is IRF-1, we have used IRF-1 knockout mice to address the potential role of this transcription factor in involution. Surprisingly, in the absence of IRF-1 significantly higher numbers of apoptotic cells were found in involuting glands at 48 h compared to control glands. In addition, the alveolar structure in IRF-1 null mammary glands had collapsed whereas in control glands the alveoli remained intact and distended. However, by 72 h control and null glands were morphologically similar suggesting that IRF-1 suppresses apoptosis only during the early, reversible, stage of involution. This suggests a survival role for IRF-1 in mammary epithelia and demonstrates a novel role for IRF-1 in vivo – suppression of premature epithelial apoptosis during mammary gland involution.
The transcription factor interferon regulatory factor (IRF)-1 was originally identified by its ability to bind to the virus-inducible ‘enhancer-like’ elements of the human interferon-β gene and to activate interferon-inducible genes (reviewed by Taniguchi et al., 1997). It is now known to regulate many cellular responses including the suppression of growth (Nguyen et al., 1997; Kirchhoff and Hauser, 1999), susceptibility to oncogenic transformation (Tanaka et al., 1994), and induction of apoptosis by a p53 independent mechanism (Horiuchi et al., 1997; Tamura et al., 1995). Loss of IRF-1 has recently been shown to contribute to c-Ha-ras-induced tumour development and to accelerate and increase tumour development and to alter the tumour spectrum in p53 null mice, thus formally demonstrating its ability to function as a tumour suppressor (Nozawa et al., 1999).
Developmental studies using IRF-1 knockout mice have focused on the role of this factor in regulating the immune system where it has been shown to be required for T cell and NK cell development (Matsuyama et al., 1993; Ogasawara et al., 1998). A function for IRF-1 in epithelial cells has not yet been described. Given the importance of IRF-1 in inducing apoptosis in vitro we have used the IRF-1 knockout mice to examine whether this transcription factor is involved in the regulation of epithelial cell apoptosis in vivo.
Following weaning of the young, the mammary gland undergoes a dramatic remodelling where the epithelial cells are removed by apoptosis and the extracellular matrix is degraded. At the onset of involution there is a specific activation of the signal transducer and activator of transcription, Stat3. We have previously shown that Stat3 is required to drive epithelial apoptosis and involution in the murine mammary gland (Chapman et al., 1999). p53 has also been shown to be increased at the start of involution in some mice (Strange et al., 1992; Jerry et al., 1998). However, its function has remained equivocal as p53 null BALB-C mice displayed delayed involution (Jerry et al., 1998) whereas on an outbred background no difference was seen between p53 wild type and knockout animals (Li et al., 1996). In the absence of Stat3, levels of p53 were increased suggesting that Stat3 may regulate alternative transcription factors and signalling molecules for the induction of apoptosis (Chapman et al., 1999). One putative target of Stat3 is IRF-1 (Yuan et al., 1994) so to establish whether IRF-1 plays a role in regulating epithelial apoptosis we have examined mammary gland involution inIRF-1 null mice.
IRF-1 null mice were kindly provided by Professor Tak Mak (Toronto, Canada). Mice were allowed to lactate for 10 days and then pups were withdrawn to initiate involution. The level of IRF-1 protein present in mammary glands at day 10 of lactation and during involution (days 1–4 and 6) was measured by Western blot (Figure 4). IRF-1 was detected at all time points in wild type mammary glands but densitometric analysis of glands from three independent mice for each time point revealed no significant difference in the average level of IRF-1 over the time course. DNA binding activity was assessed using a consensus IRF-1 binding site. IRF-1 DNA binding was detected in KIM-2 mammary epithelial cells treated for 30 min with IFN-γ (as a positive control) but not following LIF treatment (which activates Stat3) or in extracts from lactating or involuting mammary glands (data not shown) demonstrating that in the mammary gland IRF-1 is not regulated by Stat3. The lack of detectable IRF-1 DNA binding activity during involution could reflect the fact that in the mammary gland IRF-1 does not bind to a consensus DNA binding element but may require a specific DNA sequence (such as a composite element). Alternatively IRF-1 could be part of a multiprotein complex – an IRF association domain has been identified in IRF-1 that may be responsible for such an interaction with other transcription factors (Meraro et al., 1999). Of note, the transcription factor Stat1 has also been shown to regulate apoptosis independently of direct transcriptional activation (Kumar et al., 1997).
Lactating and involuting mammary glands were sectioned and stained with haematoxylin and eosin, Figure 1 shows representative glands from IRF-1 wt (A,C,E,G) and IRF-1 null (B,D,F,H) mice. At day 10 of lactation the glands were composed of alveoli lined by secretory epithelial cells, no phenotypic difference was seen between wild type (Figure 1A) and null (Figure 1B) animals at this stage. No difference was seen earlier during lactation (days 0 and 5) and the mothers were able to raise and feed their pups (data not shown). Twenty-four hours following removal of pups the alveoli of both the wild type (Figure 1C) and null (Figure 1D) glands were expanded due to the accumulation of unsuckled milk. At 48 h many of the alveoli of the control animals remained expanded although a few small areas were apparent where the alveoli had started to collapse (Figure 1E). A low number of apoptotic epithelial cells were seen shed into the alveolar lumina. In contrast, by 48 h the IRF-1 null glands were in a more advanced stage of involution with breakdown of the structure of the glands and collapse of the majority of the alveoli (Figure 1F). By 72 h the control glands had undergone significant involution with the collapse of the alveoli and the reappearance of fat which makes up the majority of tissue in a resting gland (Figure 1G). IRF-1 null glands at this stage had a very similar appearance to control glands with areas of collapsed alveoli surrounded by areas of fat (Figure 1H). Thus IRF-1 appears to regulate the early stages of involution and prevents a premature collapse of the alveoli.
To quantify the degree of alveolar collapse that had occurred in the absence of IRF-1 the cross sectional area of alveoli in the gland was measured (Lund et al., 1996), Figure 2A shows the distribution of the alveolar area. At 24 h a range of alveolar areas were recorded, a small number of alveoli were collapsed but the variation in size also reflects the position in space of the alveoli when the section was cut. There was no significant difference between control and knockout mice at this point. By 48 h the percentage of alveoli in the IRF-1 null glands with an area of less than 2 μm2×103 had significantly increased from 28% (±13%) at 24 h to 81% (±5%, n=3 mean±s.e.m., Mann–Whitney U test P<0.05) whereas alveoli in the control glands had a similar size distribution to that observed at 24 h. As expected, IRF-1 null glands also had significantly fewer alveoli larger than 2 μm2×103 compared with control glands at 48 h. By 72 h, alveoli in the control glands had also started to collapse with a marked increase seen in the percentage of alveoli with an area of less than 2 μm2×103, by this stage there was no significant difference between control and null animals.
During the later stages of involution there is a reappearance of fat tissue (see Figure 1G,H), the area of the gland occupied by adipocytes was measured as previously described (Chapman et al., 1999) and is shown in Figure 2B. Less than 5% of the area of the gland was occupied by adipocytes after 24 h of involution, a small increase was observed at 48 h and 72 h and by 96 h more than 50% of the gland area was filled with adipocytes. No significant difference was seen between control and IRF-1 null animals at any of the time points measured (Mann–Whitney U test, P>0.05 n=3). This supports our observations that early stages of involution are accelerated in the absence of IRF-1 but later stages occur with the same kinetics as in control mice.
During the early stages of involution, prior to generalized degradation of the gland, the secretory epithelial cells undergo apoptosis and are shed from the epithelial wall (Walker et al., 1989; Strange et al., 1992). IRF-1 has been shown to be required for apoptosis in some systems (Tamura et al., 1995; Horiuchi et al., 1997; Kirchhoff and Hauser, 1999) but the accelerated involution seen in the absence of this factor could be due to premature engagement of the apoptotic programme. Apoptosis was scored by both morphological assessment of condensed chromatin (Figure 3A) and TUNEL analysis of DNA strand breaks (Figure 3B). At 24 h of involution there were very few apoptotic cells in the control and IRF-1 null glands scored by either method. By 48 h a small increase in the number of cells undergoing apoptosis was seen in control glands (morphology 1.6±0.2%, TUNEL 1.9±0.5%). However, a significantly greater number of cells were apoptotic in the absence of IRF-1 scored by both morphology (3.9±0.5%) and TUNEL (6.5±0.8%, mean±s.e.m., n=3, P<0.05 Mann–Whitney U test). After 72 h the number of apoptotic cells increased in the control glands to similar levels found in the IRF-1 null glands. Thus the accelerated involution seen in the absence of IRF-1 at 48 h is characterized by premature apoptosis of the epithelial cells resulting in the breakdown of the lobular-alveolar structure.
In order to investigate the mechanism of this accelerated involution we analysed a series of apoptosis-regulatory proteins which are known to be altered during involution and which therefore might have been predicted to mediate the IRF-1 dependent survival (see Philp et al., 1996; Liu et al., 1996; Strange et al., 1992; Lund et al., 1996; Heermeier et al., 1996; Jerry et al., 1998; Tonner et al., 1997). Figure 4 shows Western blot analysis. No differences were seen between control and IRF-1 null glands in the regulation of Stats1 and 3, SGP-2, Bcl-xL, Bax (not shown), p53, p21 or c-myc. Levels of IGFBP-5, which is proposed to induce apoptosis by sequestering IGF-1, started to increase in the IRF-1 null glands at 24 h. However, the variation between mice (a common feature of IGFBP-5) meant that the difference between wild type (11 385, 20 372 and 27 556 arbitrary units) and IRF-1 null glands (19 791, 41 495 and 140 226 arbitrary units) was not significant. By 48 h higher levels of IGFBP-5 were actually found in the wild type glands than the IRF-1 null glands – this result probably reflects the earlier engagement of the apoptotic pathway and collapse of the glands seen in the absence of IRF-1. Transcriptional inhibition of IGFBP-5 may be the mechanism by which IRF-1 suppresses apoptosis – delayed involution in the absence of Stat3 was accompanied by significantly lower levels of IGFBP-5 (Chapman et al., 1999) – however more experiments will need to be performed to clarify whether there are early significant changes in IGFBP-5.
In the absence of any significant changes in involution-associated proteins IRF-1 may be acting by a novel mechanism – this hypothesis is more consistent with the lack of any detectable IRF-1 DNA binding activity during involution. Mice transgenic for Bcl-2 displayed delayed apoptosis during involution but here also none of the associated molecular events were altered suggesting that if these signals are important for driving apoptosis then Bcl-2 must act downstream of them (Jager et al., 1997). Further work will focus on the possible mechanism of action of IRF-1 in the suppression of apoptosis.
In summary we have used an IRF-1 knockout mouse to examine the role of IRF-1 in the mammary gland. The absence of IRF-1 resulted in accelerated apoptosis of epithelial cells leading to premature involution following weaning. IRF-1 was found to be expressed in KIM-2 mammary epithelial cells in culture but it is possible that in the mammary gland IRF-1 may not be having a direct effect in epithelial cells. Stromal cells contribute to involution through the production of proteases which degrade the extracellular matrix resulting in collapse of the lobuloalveolar structure (Lund et al., 1996). However, epithelial apoptosis occurs prior to and independently of the production of these proteases suggesting that IRF-1 is not exerting its effect in these cells. It is also possible that IRF-1 is mediating its effect through the lymphocytes and macrophages that are found in the gland (Walker et al., 1989). However, this possibility again seems unlikely as the infiltration of inflammatory cells seen during involution is delayed, occurring in conjunction with the breakdown of the lobuloalveolar structure suggesting that these cells do not contribute significantly to the initial phase of involution.
The timing of involution is dependent on the balance between apoptosis-inducing and apoptosis-suppressing signals (Li et al., 1997). IRF-1 is therefore an important component of the survival signal provided to mammary epithelial cells at the start of involution which allows the process to be reversed during the first 24–48 h. This is a novel function for this tumour suppressor gene and is in stark contrast to its previously reported role in initiating apoptosis either directly or following DNA damage or serum withdrawal (Nguyen et al., 1997; Tamura et al., 1995; Tanaka et al., 1994; Horiuchi et al., 1997).
This study identifies a new anti-apoptotic role for IRF-1. We therefore show that in different circumstances IRF-1 can act in apparently contradictory ways (pro and anti apoptotic) and the challenge now will be to understand the changes in context which lead to such dramatically different endpoints.
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This work was supported by an AICR grant. AR Clarke is a Royal Society University Research Fellow and CJ Watson is funded by the Cancer Research Campaign.
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Chapman, R., Duff, E., Lourenco, P. et al. A novel role for IRF-1 as a suppressor of apoptosis. Oncogene 19, 6386–6391 (2000). https://doi.org/10.1038/sj.onc.1204016
- mammary gland
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