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First published online December 15, 2003
doi: 10.1242/10.1242/jcs.00855

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A role for the cytoskeleton in prolactin-dependent mammary epithelial cell differentiation

¹ School of Biological Sciences, University of Manchester, Stopford Building, Oxford Road, Manchester, M13 9PT, UK
² CRC Structural Cell Biology Group, Paterson Institute for Cancer Research, Wilmslow Road, Manchester, M20 9BX, UK

View larger version (137K): [in a new window]   Fig. 1. Cytochalasin D disrupts microfilament networks in primary mammary epithelial cells. (A) First-passage mammary epithelial cells were plated on basement-membrane-coated coverslips in DMEM-F12 supplemented with insulin and hydrocortisone for 24 hours. Under these conditions, the cells form aggregates and become completely surrounded by basement membrane, developing into hollow structures resembling alveoli (Aggeler et al., 1991). Most epithelial cells interact directly with basement membrane (Streuli et al., 1991). The micrographs depict sections at the edges of hollow `alveoli', where more cells are available for inspection, but the lumens are not visible. Confocal micrographs of control cultures that were fixed and stained for the presence of actin using TRITC-conjugated phalloidin revealed a cortical actin network (arrows) that was not disrupted after 30 minutes or 6 hours of treatment with colchicine (col). By contrast, the cortical actin network was completely disrupted within 30 minutes of cytochalasin-D treatment (CD), and the staining became punctate (arrowheads). Scale bar, 10 µm. (B) Mammary cells were plated on collagen-I-coated coverslips and were either left untreated as controls or treated with 2 µM cytochalasin D (CD) for 30 minutes, then rinsed and harvested at 4 hours following cytochalasin-D removal (washout). Cells were stained for microfilaments using FITC-conjugated phalloidin. Similar experiments using cells plated on basement membrane indicate that the cortical actin organization can be restored after washing out cytochalasin D for several hours (data not shown).

View larger version (58K): [in a new window]   Fig. 2. Cytoskeletal inhibitors affect ß-casein expression in primary mammary epithelial cells First-passage mammary epithelial cells were plated on basement membrane in DMEM-F12 supplemented with insulin and hydrocortisone. Cytoskeletal inhibitors and prolactin were added for the indicated times and cells were extracted for protein and RNA analysis. (A-C) Cells were 35S-methionine labelled for 1 hour. Equal amounts of trichloroacetic-acid-precipitable counts were either directly analysed by gel electrophoresis to detect newly synthesized proteins (A) or immunoprecipitated with rabbit anti-mouse milk antiserum before SDS-PAGE, for ß-casein detection (B,C). Notice that the overall spectrums of newly synthesized proteins in (A) are not significantly affected by drug treatment. The asterisk in (A) corresponds to ß-casein, the levels of which are sufficient to be visualized within the total cell proteins 24 hours after inducing differentiation. Notice also that the exposure time for the gel in (C) is considerably longer than for that in (B). (D) RNA was extracted from cells and 5 µg total RNA was separated by agarose gel electrophoresis before northern blotting for ß-casein mRNA levels. The blot was reprobed with an 18S cDNA probe. L9 represents mammary tissue extract from day 9 of lactation, used as a control.

View larger version (26K): [in a new window]   Fig. 3. Blockade of Stat5 activation by cytochalasin D. Primary mammary epithelial cells were cultured on basement membrane and exposed to cytochalasin D for the indicated times. Prolactin was added to the cultures 15 minutes before preparing nuclear extracts for electrophoretic mobility shift assay (EMSA) (A,B) or detergent-soluble lysates for immunoprecipitation of prolactin signalling components (C). (A) Control cultures were either left untreated or were incubated with prolactin 15 minutes before harvesting. Extracts were subjected to EMSA with Stat5 oligodeoxynucleotides, in either the absence or the presence of 2 µg antibodies to Stat1, Stat3, Stat5 or control IgG. Notice that the mobility of the Stat5 band becomes almost completely supershifted in the presence of Stat5 antibodies. (B) Nuclear extracts of cells cultured with cytochalasin D or its carrier DMSO were assessed by EMSA for their ability to recognize Stat5 probe. Protein-DNA complexes were visualized by autoradiography (top) and quantified following storage phosphor image analysis (bottom). In the quantitative analysis, the levels of radioactivity in the samples of cytochalasin-treated cells are compared with those in extracts of cells treated with vehicle alone for the equivalent time and are plotted relative to the signal in control cultures treated with prolactin only. The data were obtained from three independent experiments. (C) Cell lysates were immunoprecipitated (IP) with antibodies to Stat5a or Stat5b. After separation by 6.25% SDS-PAGE, precipitated proteins were analysed by immunoblotting with antibodies for phosphotyrosine (4G10) or the appropriate precipitating antibody. The complete absence of Stat5 phosphorylation after 24 hours of cytochalasin-D treatment, in comparison to its residual DNA binding activity in (B), might reflect differences in the sensitivity of the assays.

View larger version (35K): [in a new window]   Fig. 4. Cytochalasin D does not compromise proximal events in prolactin signalling or tyrosine phosphorylation of FAK. Cells were cultured as in Fig. 3. (A) Lysates were immunoprecipitated with antibodies to Jak2 or PrlR and then blotted with antibodies for phosphotyrosine (4G10) or the appropriate precipitating antibody. (B) Lysates were immunoprecipitated with antibodies to FAK and then blotted with antibodies for phosphotyrosine (PY20) or the appropriate precipitating antibody. In control experiments, primary mammary epithelial cells were removed to the non-adhesive substratum polyHEMA for 1 hour. Under these conditions, FAK becomes dephosphorylated, in contrast to the cells on basement membrane, in which FAK phosphorylation remains even after cytochalasin-D treatment.

View larger version (29K): [in a new window]   Fig. 5. Inhibition of the prolactin signalling pathway by colchicine. Primary mammary epithelial cells were cultured on basement membrane and exposed to colchicine for the indicated times. Prolactin was added to the cultures 15 minutes before harvesting the cells as in Fig. 3. (A) Nuclear extracts were analysed by electrophoretic mobility shift assay for Stat5-DNA interactions. (B-E) Detergent lysates were immunoprecipitated for Stat5 (B,C), Jak2 (D) or PrlR (E) and immunoblotted with antibodies for phosphotyrosine (4G10) or the appropriate precipitating antibody.

View larger version (13K): [in a new window]   Fig. 6. Modelling a requirement for the cytoskeleton in prolactin signalling. (A) Our initial hypothesis suggested that the actin cytoskeleton is necessary for crosstalk between integrin-containing adhesion complexes and prolactin (P)-mediated signal transduction (magenta arrow), but the data in this paper suggest that this model is not correct. (B) Disruption of the microfilaments for up to 4 hours has no effect on prolactin signalling and activation of Stat5. This indicates that the signals downstream of PrlR are independent of the cortical actin network. Because Prl signalling is ECM dependent (Edwards et al., 1998), ligand-activated prolactin receptors might accumulate within multiprotein clusters of proteins localized to focal adhesions or, alternatively, that integrin-regulated adhesion complexes activate PrlR through long-range signals. (C) Disruption of the microfilaments for more than 4 hours leads to delayed restriction in Stat5 activation. Phosphorylation of PrlR, but not Stat5, can still be induced by ligand. Loss of microfilaments might lead to a delayed activation or synthesis of phosphatases or other inhibitors of cytokine signalling (green inhibitory arrow).

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