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First published online 4 July 2007
doi: 10.1242/dev.003194

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The IL-4/IL-13/Stat6 signalling pathway promotes luminal mammary epithelial cell development

Walid T. Khaled¹, Eliot K. C. Read¹, Sandra E. Nicholson², Fiona O. Baxter¹, Amelia J. Brennan¹, Paul J. Came^1,*, Naomi Sprigg², Andrew N. J. McKenzie³ and Christine J. Watson^1,

¹ Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK.
² Division of Cancer and Haematology, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia.
³ Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, UK.

View larger version (62K): [in this window] [in a new window]   Fig. 1. Stat6 is upregulated and activated at the onset of pregnancy. (A) Protein was extracted from inguinal mammary glands from virgin mice; from mice at day 5, 10 and 15 of gestation; and from mice at day 0 (day of birth), 5 and 10 of lactation. Immunoblotting was performed for phosphorylated Stat6 (pStat6), Stat6, IL-4R, Gata3, c-Maf and -tubulin. pStat6 and Stat6 immunoblots were quantified using ImageJ software and plotted on a histogram (right). (B-I) Immunohistochemistry (IHC, right panels; left panels, merged with DAPI staining) was performed on mammary gland sections from wild-type mice for: pStat6 in virgin mice (B) and mice at day 5 of gestation (C); IL-4R in virgin mice (D) and mice at day 15 of gestation (E); Gata3 in mice at day 5 of gestation (F); and c-Maf in mice at day 15 of gestation (G). (H) pStat6 staining in Stat6-/- mice at day 5 of gestation. (I) Rabbit isotype control staining on wild-type mice at day 5 of gestation.

View larger version (87K): [in this window] [in a new window]   Fig. 2. Delay in lobuloalveolar development in Stat6-/- mice. (A-C) Whole mounts (left panels) and haematoxylin and Eosin (H&E)-stained sections (right panels) of mammary glands at day 5 (A), 10 (B) and 15 (C) of gestation from wild-type (WT) and Stat6-/- mice. Images shown are representative of at least three mice. (D) The number of alveoli was scored in H&E images from wild-type and Stat6-/- mice at 40x magnification and the area of the fat pad was measured using ImageJ software. The number of alveoli/mm2 was calculated and the percentage relative to wild type was plotted. Student's t-test was performed and *, ** and *** indicate statistical significance with P values of P<0.001, P=0.011 and P=0.052, respectively. (E) Proliferation rate was measured by scoring for Ki-67-positive cells from three different fields at 400x magnification. Error bars represent s.d. of three independent mice. Student's t-test was performed and *, ** and *** indicate statistical significance with P values of P<0.001, P<0.01 and P<0.003, respectively.

View larger version (41K): [in this window] [in a new window]   Fig. 3. Reduction in proliferation and in the levels of differentiation markers in Stat6-/- mice. (A) Immunoblots for Stat6, phosphorylated (p)Akt, Akt, pERK1/2, ERK1/2, ß-casein, pStat5, Stat5, Gata3, Pten, IL-4R and -tubulin were performed on wild-type (WT) and Stat6-/- tissue extracts at the indicated time points. (B) Immunoblots from A were quantified using ImageJ software and plotted on a histogram. (C) A microarray analysis of differentially regulated genes at day 5 of gestation comparing wild-type and Stat6-/- mammary glands. Genes listed displayed a statistically significant change across three biological replicates with a P value of <0.01.

View larger version (67K): [in this window] [in a new window]   Fig. 4. IL-4/IL-13 expression is reduced in Stat6-/- mammary glands and these cytokines are required for mammary gland development. (A) Quantitative real-time-PCR was performed using specific primers for Il4, Il13, Il5, Il12a and Gata3 on wild-type (WT) and Stat6-/- glands at day 5 and 15 of gestation. Error bar represents s.d. of three samples and is representative of at least two experiments. (B-D) Whole mounts (left panels) and H&E-stained sections (right panels) of inguinal mammary glands from wild-type (strain matched) and IL-4-/-/IL-13-/- mice at day 5 (B), 10 (C) and 15 (D) of gestation. (E) The number of alveoli was scored in H&E images from wild-type and IL-4-/-/IL-13-/- mice at 40x magnification and the area of the fat pad was measured using ImageJ software. The number of alveoli/mm2 was calculated and the percentage relative to wild-type was plotted. (F) Immunoblots for phosphorylated (p)Stat6 and Stat6 were performed on wild-type and IL-4-/-/13-/- mice at day 5 of gestation. The density of the bands was quantified using ImageJ software and the ratio of pStat6 to Stat6 plotted on a histogram.

View larger version (84K): [in this window] [in a new window]   Fig. 5. Socs5-/- mice exhibit accelerated mammary gland development. (A) Whole mounts and H&E-stained sections of inguinal mammary glands from wild-type (WT) and Socs5-/- mice at day 10 and 15 of gestation. (B) The number of alveoli was scored in H&E images from wild-type and Socs5-/- mice at 40x magnification and the area of the fat pad was measured using ImageJ software. The number of alveoli/mm2 was calculated and the percentage relative to wild-type was plotted. (C) Whole mounts and H&E staining of inguinal mammary glands from NOD and NOD-SCID mice at day 5 of gestation. (D) The number of alveoli/mm2 from wild-type, NOD, NOD-SCID, Stat6-/- and IL-4-/-/IL-13-/- mice at day 5 of gestation were plotted on a histogram for comparison.

View larger version (67K): [in this window] [in a new window]   Fig. 6. Stat6 signalling is intact in KIM-2 cells. KIM-2 cells respond to IL-4 (A) and IL-13 (B) in a dose-dependent manner, confirmed by immunoblotting using phosphorylated (p)Stat6 and Stat6 antibodies. (C) KIM-2 medium was changed and then cells were harvested at various time points after treatment with or without 40 ng/ml of recombinant murine IL-13 (rmIL-13). Quantitative real-time-PCR was performed for Il4r1 and Gata3; * denotes Student's t-test P value of <0.02. (D) Immunoblots for the same time course were performed for pStat6, Stat6 and -tubulin. (E-G) KIM-2 cells were treated with brefeldin A (10 µg/ml) for 4 hours, fixed and stained with anti-IL-4 (E) or anti-IL-13 (F) antibodies or no antibody (G).

View larger version (30K): [in this window] [in a new window]   Fig. 7. Th-2 cytokines are upregulated and secreted by differentiating KIM-2 cells. (A) Quantitative real-time-PCR was performed using specific primers for the Th1 cytokines Il12a, Tnfa and Ifng (right) and the Th2 cytokines Il4, Il13 and Il5 (left) on KIM-2 cells across a differentiation time course (DD, days of differentiation). Error bar represents s.d. of three samples and is representative of at least two experiments. (B) Cytokine protein array demonstrates Th2 cytokine production by differentiated KIM-2 cells at day 8 (right) compared to day 0 (left). Th2 cytokines are indicated in red. Green arrows indicate elevated levels of IL-4 secretion at day 8 of differentiation. Pos, positive control; Neg, negative control; GM-CSF, granulocyte-macrophage colony-stimulating factor; GCSF, granulocyte-colony stimulating factor.

View larger version (36K): [in this window] [in a new window]   Fig. 8. Model explaining the effect of Stat6/IL-4/IL-13 deficiency on mammary gland development. (A) Th2 cytokine production is orchestrated by Stat6-mediated Gata3 gene transcription. Gata3 induces a chromatin conformational change at the IL-4/IL-13/IL-5 locus, facilitating the binding of Stat6, Stat5 and c-Maf, which produces cytokines that trigger the differentiation process in undifferentiated cells. Gata3 is also activated by other pathways, such as Notch. (B) In the absence of Stat6, IL-4/IL-13/IL-5 production is reduced even in the presence of Gata3. We propose that this affects paracrine signalling between neighbouring cells and thus delays development.

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