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First published online 3 August 2006
doi: 10.1242/dev.02502

Development 133, 3485-3494 (2006)
Published by The Company of Biologists 2006
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IKKß/2 induces TWEAK and apoptosis in mammary epithelial cells

Fiona O. Baxter1, Paul J. Came1, Kathrine Abell1, Blandine Kedjouar1, Marion Huth2, Klaus Rajewsky3, Manolis Pasparakis2 and Christine J. Watson1,*

1 Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK.
2 EMBL Mouse Laboratory Programme, Via Ramarini 32, 00016 Monterotondo, Rome, Italy.
3 The CBR Institute for Biomedical Research, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA.


Figure 1
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  		Fig. 1. Analysis of IKK2 expression in mammary glands of floxed mice. (A) Southern blot analysis of Ikk2 locus. Lanes 1-4 controls: lane 1 wild-type DNA; lane 2 DNA from floxed/wild-type tissue (not recombined); lane 3 DNA from floxed/floxed tissue (not recombined); lane 4 DNA from floxed/deleted tissue. Lane 5 empty. Lanes 6-11: mammary gland DNA samples from Cre+/Ikk2fl/fl mice at 24 hours' involution, showing that varying levels of recombination have occurred (lanes 6-11, respectively: 37, 79, 43, 36, 31 and 37% recombination). (B) Protein levels of IKK2 were determined in mammary glands from Cre-, Cre+/Ikk2fl/wt or Cre+/Ikk2fl/fl mice by immunoblotting. At 24 hours' involution, reduced IKK2 levels were seen in Cre+/Ikk2fl/fl and Cre+/Ikk2fl/wt mice (loading control was {alpha}-tubulin). (C) EMSA of mammary gland tissue nuclear extracts for NF-{kappa}B and OCT1. NF-{kappa}B DNA-binding activity was determined by densitometry and normalised to OCT1. In the absence of IKK2 (Cre+/Ikk2fl/fl), NF-{kappa}B DNA-binding activity is reduced by approximately 50%. (D) DAB immunohistochemistry for NF-{kappa}B p50 and p65 subunits in mammary gland sections from Cre- or Cre+/Ikk2fl/fl mice at 24 hours' involution. Compared to Cre- glands, nuclear p50 staining was reduced in Cre+/Ikk2fl/fl glands (<). Nuclear p65 staining was weak in Cre- glands, and undetectable in glands from Cre+/Ikk2fl/fl mice. Percentage of positively staining nuclei is indicated in the lower right corner of each image. Scale bar: 100 µm.

 

Figure 2
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  		Fig. 2. Deletion of IKK2 in mammary gland results in reduced apoptosis and delayed involution. (A) Haematoxylin and eosin staining of mouse mammary gland sections from Cre-, Cre+/Ikk2fl/wt or Cre+/Ikk2fl/fl mice at 0dL, 24, 48, 72 or 144 hours' involution. Apoptosis is apparent in glands from Cre- control mice that subsequently underwent remodelling normally. Apoptotic cells are seen to accumulate in the open lumen of the lobuloalveolar structures (<). Involution and remodelling was delayed in the glands from Cre+/Ikk2fl/wt mice. This delay was more pronounced in Cre+/Ikk2fl/fl glands. By 6 days' involution very little difference is seen between mice of the different genotypes. (B) Cleavage of caspase 3 was determined by immunofluorescence in mammary gland sections from Cre-, Cre+/Ikk2fl/wt or Cre+/Ikk2fl/fl mice at 24, 48 or 72 hours' involution. (green: cleaved caspase-3-positive cells; red: DAPI-stained nuclei). TUNEL staining at 72 hours' involution showed a higher proportion of positive (green) cells in Cre- and heterozygous glands. (C) Cleaved caspase-3-positive cells were expressed as a percentage of total cell number and data presented in graphical format. Approximately 10,000 cells were counted. Cleaved caspase 3 levels peaked at 24 hours' involution in Cre- glands, was delayed by 48 hours in Cre+/Ikk2fl/wt mice and remained constant at all involution timepoints in Cre+/Ikk2fl/fl mice. Scale bars: 100 µm.

 

Figure 3
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  		Fig. 3. Changes in gene and protein expression following loss of IKK2. (A) Western blot analysis for total and serine phosphorylated levels of AKT, FOXO3a and of STAT3 at 24 hours' involution in mammary glands from Cre-, Cre+/Ikk2fl/wt or Cre+/Ikk2fl/fl mice. Significantly increased levels of pAKT and pFOXO3a were seen in the absence of IKK2. Loading control was {alpha}-tubulin. (B) DAB immunohistochemistry for FOXO3a in mammary gland sections from Cre-, Cre+/Ikk2fl/wt or Cre+/Ikk2fl/fl mice at 24 hours' involution. Reduced nuclear FOXO3a staining was seen in Cre+/Ikk2fl/fl glands (<). Percentage of positively staining nuclei is indicated in the lower right corner of each image. (C) A representation of previously obtained microarray data showing expression of DR ligands throughout lactation and early involution (Clarkson et al., 2004Go). (D) qRT-PCR for the DR ligands: FASL, TNF, TRAIL and TWEAK and DR TNFR1. Significant reductions in TWEAK (** t<0.01), TNF (* t<0.05) and TNFR1 as determined by the paired t-test, were seen in glands from Cre+/Ikk2fl/fl mice compared with Cre- control mice. Levels are arbitrary units and have been normalised to cyclophilin A. (E) qRT-PCR for TWEAK of control and 15dPGJ2 treated undifferentiated KIM2 cells. Statistically significant decreases in TWEAK mRNA as determined by the paired t-test, were seen following 4 hours (**t<0.01) and 8 hours (***t<0.005) of 15dPGJ2 treatment. Levels are arbitrary units and have been normalised to cyclophilin A. Scale bar: 100 µm.

 

Figure 4
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  		Fig. 4. Expression profile of TWEAK in the mammary gland. (A) Expression of Fn14 in mammary epithelial cells. Fn14 was expressed in undifferentiated and 10-day differentiated KIM2 mammary epithelial cells, as determined by qRT-PCR analysis. (B) Immunoblotting for TWEAK demonstrated expression of full length (35 kDa) TWEAK in nuclear and cytoplasmic compartments in KIM2 cells, while soluble (18 kDa) TWEAK was exclusively cytoplasmic. Nuclear control was retinoblastoma protein (Rb). (C) DAB immunohistochemistry for TWEAK demonstrated nuclear staining at all mammary gland developmental time points examined. Cytoplasmic TWEAK staining was seen late in lactation (10 dl) and early in involution (12 and 24 hours' involution). (D) Confocal analysis of TWEAK localisation in KIM2 cells. Undifferentiated KIM2 cells were fixed and stained for TWEAK, using AlexaFlour 555. A single slice of the confocal analysis is shown, with nuclei visualised by DAPI staining. The bottom panel shows a merged image, with sectioning through a nuclear puncta. (E) Immunoblotting for TWEAK during mammary gland development showed that the soluble form of TWEAK was detected predominantly at 24-72 hours' involution. Scale bars: 100 µm in C; 20 µm in D.

 

Figure 5
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  		Fig. 5. DR ligands: promoter analysis and putative roles in apoptosis and involution. (A) Comparative promoter analysis for members of the TNF superfamily of DR ligands. Promoter analysis was performed as described in Materials and methods. The open boxes are 5'UTR regions of exon1 of the respective genes; the filled boxes are translated parts of the first exons. The dotted box in the Tweak promoter represents the putative 5'UTR from the potential transcription start site identified using the Eponine program. This figure shows promoter regions spanning 1 kb upstream of the predicted transcription start sites. Previously published NF-{kappa}B consensus-binding sequences are shown as filled circles: TNF (Drouet et al., 1991Go) and FASL (Matsui et al., 1998Go). The additional NF-{kappa}B-binding sites shown in white circles were identified using the Matinspector program. The FOXO3a consensus sequences used were RTAAAYA (Brunet et al., 1999Go) or CCAAACAA and TAAAACAA (Tran et al., 2002Go). (B) Proposed model of IKK2 induced apoptosis during early mammary gland involution.

 

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© The Company of Biologists Ltd 2006