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doi: 10.1242/10.1242/dev.00578

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A dual, non-redundant, role for LIF as a regulator of development and STAT3-mediated cell death in mammary gland

¹ Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK
² Clinical Research Department, Christie Hospital NHS Trust, Manchester M20 4BX, UK

View larger version (18K): [in a new window]   Fig. 1. LIF activates STAT3 and induces apoptosis in vitro and is differentially regulated in vivo. (A) LIF activates STAT3 in KIM-2 cells. Western blot analysis of KIM-2 cells treated with increasing concentrations of LIF (1-100 ng/ml) for 30 minutes. Two western blots show pSTAT3 and total STAT3. (B) LIF induces apoptosis in KIM-2 cells. Flow cytometry analysis of annexin V stained KIM-2 cells treated with 100 ng/ml LIF in growth medium (MM + LIF) for 24 hours and MM alone. The graph shown represents fold induction of annexin V-positive cells calculated relative to the negative control, mean±s.e.m, n=3. (C) LIF does not activate STAT5 in mammary epithelial cells. Western blot analysis of KIM-2 cells untreated or treated with 100 ng/ml LIF for 24 hours in MM. The blot was probed with antibodies against pSTAT3, total STAT3 and pSTAT5. KIM-2 cells treated with prolactin for 30 minutes were used as positive control for pSTAT5. (D) Distinct expression profiles of LIF, LIF receptor and gp130 during mammary gland developmental cycle. RNA was extracted from mammary glands harvested at different time points during mammary gland development. RT-PCR analysis was performed using the primers indicated and Cyclophilin (Cyclo.) was used as an internal control. Each gel is representative of three independent mammary gland time courses. wc, water control; d Preg, day pregnancy; d Lac, day lactation; hrs Inv, hours involution. (E) Taqman quantitative PCR data for LIF, performed on a fourth independent set of mammary gland samples, normalised to 18s RNA and displayed as the mean of three PCR reactions.

View larger version (67K): [in a new window]   Fig. 2. Morphological and molecular changes associated with LIF deficiency in involuting mammary glands. (A) Delayed involution in Lif-;/-; mammary glands compared with controls. Haematoxylin and Eosin staining of mammary gland sections from Lif-;/-; and Lif+/-; mice at day 2 involution. (B) Absence of pSTAT3 in involuting glands from LIF-deficient animals. Immunohistochemistry on sections of mammary glands from animals at 2 day involution using antibodies against pSTAT3. (C) LIF deficiency at 2 day involution is associated with changes in pSTAT3 and cleaved caspase 3 levels. Levels of pSTAT3, STAT3, cleaved caspase3 and the epithelial marker protein cytokeratin 18 (Ker18) were assessed by western blot analysis. Graphs indicate densitometry analysis for three independent mice, mean±s.e.m.; Lif-;/-;, black bars; Lif+/-;, white bars. (D) Regulation of C/EBP transcription in glands from LIF-deficient mice at day 2 of involution. RT-PCR analysis of C/EBP expression in glands from wild-type mice (lane 1-3), Stat3-;/-; mice (lane 4), Lif+/-; control mice (lane 5) and Lif-;/-; mice (lane 6). The glands were harvested at 5 day lactation (lane 1), 24 hours involution (lane 2) and 48 hours involution (lane 4-6). Cyclophilin (Cyclo.) was used as an internal control. Graph indicates densitometry analysis normalised to cyclophilin, mean±s.e.m., n=3.

View larger version (35K): [in a new window]   Fig. 3. Expression of apoptosis regulators in Lif-;/-; and wild-type mammary gland. (A) Regulation of STAT proteins, milk protein and apoptosis related protein expression in involuting glands in absence of LIF. The levels of pSTAT5, total STAT5, pSTAT1, STAT1, ß-casein, Bcl-x, Bax, p53, p21 and ERK1/2 in mammary glands from 2 day involuting mice were determined by western blot analysis. Extract from interferon gamma-treated KIM-2 cells was used as positive control for pSTAT1 and STAT1. Graphs indicate densitometry analysis for three independent mice, mean±s.e.m.. Lif-;/-;, black bars; Lif+/-;, white bars. (B) Expression and activation profiles of STAT3 and ERK1/2 during mammary gland development. Western blot analysis of wild-type mammary glands harvested at indicated time points The blot was probed with antibodies specific for pSTAT3, total STAT3, pERK1/2 and total ERK1/2. d Preg, day pregnancy; d Lac, day lactation; h Involution, hours involution.

View larger version (52K): [in a new window]   Fig. 4. Effect of LIF deficiency in early mammary gland development. (A) Whole-mount staining of mammary glands from pregnancy day 8 Lif-;/-; (1,3,5) and Lif+/-; (2,4,6) mice at three magnifications. (B) ERK, STAT5 and STAT3 activation in early development. Glands from pregnancy day 8 Lif-;/-; and Lif+/-; control mice were subjected to western blot analysis using specific antibodies against pSTAT3, total STAT3, pSTAT5, total STAT5, pERK1/2 and the epithelial marker protein Cytokeratin 18 (Ker18). Graphs indicates densitometry analysis for three independent mice, mean±s.e.m. Lif-;/-;, black bars; Lif+/-;, white bars. (C) Whole-mount staining of mammary glands from Lif knockout (1,3) and control Lif heterozygous (2,4) virgin animals. Whole-mount staining of the glands from 4-week-old virgin mice (1,2), and 7-week-old virgin mice (3,4).

View larger version (77K): [in a new window]   Fig. 5. ERK1/2 is activated by LIF and protects against LIF/STAT3-induced apoptosis. (A) Western blot analysis of differentiated KIM-2 cells treated with 100 ng/ml LIF for the indicated times (30 minutes, 1 hour, 2 hours and 4 hours) in differentiation medium (DM). Immunoblotting was performed using specific antibodies against pSTAT3, total STAT3, pERK1/2 and total ERK1/2. The western blot is representative of three independent experiments. (B) Increased levels of activated ERK1/2 in mammary glands from involuting STAT3-deficient mice. Levels of phospho-ERK1/2 and total ERK1/2 in 2 day involuting glands from Stat3-;/-; and Stat3+/-; control mice was assessed by western blot analysis. Graphs indicate densitometry analysis for three independent Stat3-;/-; mice, mean±s.e.m. (black bars) and two independent Stat3+/-; mice (white bars). (C) Phase-contrast (left) and annexin V-FITC in situ fluorescence microscopy (right) of KIM-2 cells treated with 100 ng/ml LIF in the presence or absence of 10 µM U0126 for 24 hours (bottom row) and with U0126 alone (top row). MM, time-matched control in growth medium. The results are representative of three independent experiments.