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doi: 10.1242/10.1242/jcs.00523

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Collagen-IV and laminin-1 regulate estrogen receptor expression and function in mouse mammary epithelial cells

¹ Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
² Department of Anatomy, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada

View larger version (69K): [in a new window]   Fig. 1. Reconstituted basement membrane (rBM) allows partial maintenance of ER protein levels in primary mammary epithelial cells, but has no effect in primary fibroblasts. (A) Morphology of (a) freshly isolated mammary organoids derived from mouse mammary glands (time 0); (b) primary epithelial cells grown for 2 days on plastic; or (c) on top rBM. Bar, 100 µm. (B) Representative western blots showing ER and ß-casein protein expression in cell extracts derived from primary mammary epithelial cells grown in the presence of lactogenic hormones for 10 days on plastic (a) or on top of rBM (b). Total E-cadherin levels do not change in these experiments and were used for loading control. Quantification was by densitometry of ER protein levels from mammary epithelial cells grown on plastic (solid line) or on top of rBM (dotted line) in the presence (c) or absence (d) of lactogenic hormones. ER was expressed in relative units (day of isolation of the organoids, time 0, corresponds to 1). Results are mean and s.e.m. of five different primary culture preparations using a pool of bilateral 4th inguinal mammary glands from ten nulliparous Balb/c mice in each preparation. *P<0.05 vs. plastic. (C) Morphology of mammary fibroblasts grown for 6 days on plastic (a) or on top of rBM (b). Bar, 100 µm. (D) ER protein expression in epithelial cells (ep) and fibroblasts just after isolation from the gland (t0), and in fibroblasts grown for 6 days on plastic (pl) or on top of rBM (rBM).

View larger version (58K): [in a new window]   Fig. 2. rBM increases ER protein levels in a clonal mammary epithelial cell line. (A) Morphological changes in Scp2 cells. Cells grown on plastic formed a flattened monolayer (a), whereas cells grown on top of rBM aggregated and formed acinus-like structures (b). Bar, 100 µm. They progressively become rounded when cultured in the presence of increasing concentrations of rBM (c-e). Bar, 100 µm. (B) In Scp2 cells grown as indicated in A for 3 days, ER protein levels were determined by western blot (a, representative gel) quantified by densitometry and expressed relative to the values measured on plastic (a, bar graph). In Scp2 cells grown for the indicated hours after the addition of serum-free medium containing lactogenic hormones in the presence (grey bars) or absence (black bars) of 2% rBM (b), ER protein levels were determined by western blot and expressed relative to the values measured on plastic 2 hours after the addition of the new medium. n=5; *P<0.05. (C) Immunofluorescence of ER in Scp2 cells grown for 2 days on plastic (a,b) or in the presence of 2% rBM (c,d). Note that not all the cells (DAPI staining of DNA; blue) stained for ER (green), and that the staining for ER was predominantly nuclear. Bar, 20 µm. The table (e) shows mean ± s.e.m. corresponding to percentages of ER-positive cells and to ER expression levels quantified in single cells grown on plastic or in the presence of rBM. Intensity of fluorescence was expressed as arbitrary units (A.U.). At least 100 cells were analyzed for each culture condition. The proportion of ER-positive cells was higher in the presence of rBM.

View larger version (50K): [in a new window]   Fig. 3. rBM increases ER-mediated response independently of estradiol. (A) Morphological changes in Scp2-ERE-TK-CAT cells grown on plastic (a) or on top rBM (b). Bar, 25 µm. (B) ER-mediated transcriptional activity (measured using CAT as reporter gene) was quantified in cells grown for 3 days on plastic or on top rBM, in the presence or absence of 17ß-estradiol 10-8 M, or the antagonist ICI 182,780 10-7 M for the last 48 hours in culture. The reporter activity was expressed relative to values measured in cells grown on plastic in the presence of the vehicle ethanol. n=4; *P<0.01 versus ethanol for each substratum.

View larger version (23K): [in a new window]   Fig. 4. rBM increases ER mRNA levels in mammary epithelial cells. ER mRNA was analyzed by quantitative RT-PCR 2 and 6 days after isolating primary cells from the mammary gland (A), or in Scp2 cells cultured for 3 days (B) on plastic or in the presence of rBM (dripped or on top). Values were normalized with respect to GAPDH mRNA. Three independent experiments comprising different primary culture preparations or Scp2 cultures were used to create the graph. *P<0.05 vs. plastic for each condition.

View larger version (23K): [in a new window]   Fig. 5. The regulatory effect of rBM on ER expression is not due to changes in growth rate. (A) Proliferation of Scp2 cells grown for 3 days on plastic or in the presence of rBM after adding different concentrations of insulin (in the absence of serum or other growth factor). Proliferation was determined as the percentage of BrdU-labeled cells by immunofluorescence. (B) ER protein levels in Scp2 cells grown in the presence of different insulin concentrations as indicated in A were evaluated by western blot (a, representative gel) and quantified by densitometry (b). ER expression did not correlate with the proliferation status of the cells, regardless of the substrata.n=4; *P<0.05 versus no insulin.

View larger version (56K): [in a new window]   Fig. 6. The regulatory effect of rBM on ER levels is independent of cell shape changes. (A) Primary cells (a) and Scp2 cells (b) form typical flattened monolayers on plastic. Morphological change (cell rounding and aggregation) can be induced by the non-adherent substratum polyHEMA in primary cells (c) and in Scp2 cells (d). The addition of rBM on polyHEMA did not affect this cell rounding (e,f). Bar, 100 µm. (B) The densitometric analysis of ER protein levels determined by western blot in primary mammary epithelial cells at days 2 or 6 after isolation (a) and in Scp2 cells at day 3 in culture (b) shows that addition of rBM to pre-rounded cells was necessary to upregulate ER over basal levels detected on plastic. n=3; *P<0.05.

View larger version (50K): [in a new window]   Fig. 7. The regulatory effect of rBM on ER levels is reproduced by purified collagen-IV and laminin-1. (A) Isolated mouse mammary epithelial organoids (a) were grown in the presence of different matrix components dripped into the medium: collagen-I (b), collagen-IV (c), laminin-1 (c) or a mixture of laminin-1 and collagen-IV (e). In all cases the organoids were attached and spread on the plastic after 2 days in culture. Bar, 100 µm. ER protein levels were measured in these different culture conditions by western blot at days 2 and 6 after culture, and expressed as relative units to levels at time 0 (f). Results are the average of four different primary culture preparations. *P<0.05 vs. plastic. (B) Scp2 cells formed monolayers after dripping collagen-I (a) or collagen-IV (b), and adopted a rounded morphology when laminin-1 was dripped into the medium (c). Bar, 100 µm. ER protein levels were determined by western blot at day 3 in culture, quantified by densitometry and were expressed relative to the values measured on plastic (d). n=5; *P<0.05.

View larger version (25K): [in a new window]   Fig. 8. The regulatory effect of collagen-IV and laminin-1 on ER levels is mediated by 2, 6 and ß1 integrin subunits. ER protein levels were determined by western blot in primary mammary epithelial cells (A) and in Scp2 cells (B) after 3 days adding mouse IgG (control, c), or 10 µg/ml of 1, 2 or 6 or 5 µg/ml of ß1 integrin blocking antibodies to cells grown on plastic or in the presence of collagen-IV, laminin-1 or rBM. n=3; *P<0.05 versus control (IgG addition).

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