Decreased IGF type 1 receptor signaling in mammary epithelium during pregnancy leads to reduced proliferation, alveolar differentiation, and expression of insulin receptor substrate (IRS)-1 and IRS-2 - PubMed (original) (raw)

Decreased IGF type 1 receptor signaling in mammary epithelium during pregnancy leads to reduced proliferation, alveolar differentiation, and expression of insulin receptor substrate (IRS)-1 and IRS-2

Zhaoyu Sun et al. Endocrinology. 2011 Aug.

Abstract

The IGFs and the IGF type 1 receptor (IGF-1R) are essential mediators of normal mammary gland development in mice. IGF-I and the IGF-1R have demonstrated functions in formation and proliferation of terminal end buds and in ductal outgrowth and branching during puberty. To study the functions of IGF-1R during pregnancy and lactation, we established transgenic mouse lines expressing a human dominant-negative kinase dead IGF-1R (dnhIGF-1R) under the control of the whey acidic protein promoter. We provide evidence that the IGF-1R pathway is necessary for normal epithelial proliferation and alveolar formation during pregnancy. Furthermore, we demonstrate that the whey acidic protein-dnhIGF-1R transgene causes a delay in alveolar differentiation including lipid droplet formation, lumen expansion, and β-casein protein expression. Analysis of IGF-1R signaling pathways showed a decrease in P-IGF-1R and P-Akt resulting from expression of the dnhIGF-1R. We further demonstrate that disruption of the IGF-1R decreases mammary epithelial cell expression of the signaling intermediates insulin receptor substrate (IRS)-1 and IRS-2. No alterations were observed in downstream signaling targets of prolactin and progesterone, suggesting that activation of the IGF-1R may directly regulate expression of IRS-1/2 during alveolar development and differentiation. These data show that IGF-1R signaling is necessary for normal alveolar proliferation and differentiation, in part, through induction of signaling intermediates that mediate alveolar development.

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Figures

Fig. 1.

Fig. 1.

WAP-dnhIGF-1R expression decreases mammary ductal branch outgrowth and alveolar density during pregnancy. A and B, Whole-mount stained glands at P10.5 showing no morphological difference between wild-type (WT) glands and WAP-dnhIGF-1R glands. C and D, Whole-mount stained glands from wild-type and WAP-dnhIGF-1R glands at P14.5. E–H, H&E-stained sections from wild-type and WAP-dnhIGF-1R glands at P14.5 (E and F) and P17.5 (G and H). I, Western blot analysis and quantification of CK8 expression in protein extracted from wild-type or WAP-dnhIGF-1R whole glands at P14.5, P17.5, and L5. Graph shows levels of CK8 adjusted to β-actin; n = 3 animals from each genotype; *, P < 0.03. Scale bars, 100 μm in A–D; 40 μm in E–I.

Fig. 2.

Fig. 2.

WAP-dnhIGF-1R expression decreases mammary epithelial cell proliferation during pregnancy. A, B, D, and E, Representative photomicrographs used for quantification of Ki67-positive cells in wild-type (WT) glands (A and D) and WAP-dnhIGF-1R glands (B and E) at P14.5 (A and B) and P17.5 (D and E). DAPI was used to detect nuclei (blue). C and F, Quantification of Ki67-positive cells at P14.5 (C) and P17.5 (F) expressed as a percentage of DAPI-positive epithelial cells. G and H, Immunofluorescent images showing reduced Ki67 staining along ducts at sties of alveolar bud outgrowths at P14.5. Scale bars, 50 μm (red autofluorescence of blood cells seen in panel G). **, P < 0.01; ***, P < 0.01.

Fig. 3.

Fig. 3.

WAP-dnhIGF-1R expression causes deficits in alveolar differentiation during pregnancy. A–D, Representative images of H&E-stained alveoli from wild-type (WT) (A and C) or dnhIGF-1R (B and D) glands at P14.5 (A and B) and at P17.5 (C and D). E and F, Images showing immunofluorescence for WGA (green) and DAPI (blue) in wild-type (E) or dnhIGF-1R (F) glands at P17.5. G and H, Quantification of alveolar lumen size at P14.5 and P17.5. Scale bar, 50 μm. *, P < 0.03.

Fig. 4.

Fig. 4.

WAP-dnhIGF-1R expression alters myoepithelial cell morphology and CK14 expression but does not effect E-cadherin and ZO-1 expression in alveoli. A and B, Immunofluorescence for CK14 in wild-type (WT) (A) and dnhIGF-1R (B) glands at P17.5. C, Western blot analysis of CK14 expression in isolated MECs from wild-type and dnhIGF-1R glands at P17.5 after adjusting to levels of β-actin. D–G, Immunofluorescence for E-cadherin (E-cad; red, D and E) or ZO-1 (green; F and G) on sections from wild-type (D and F) or dnhIGF-1R (E and G) glands at P17.5. Sections were stained with DAPI to detect nuclei (blue). Scale bars, 50 μm. *, P < 0.05.

Fig. 5.

Fig. 5.

WAP-dnhIGF-1R expression delays alveolar differentiation during pregnancy but does not effect pup weight gain during lactation. A–D, Sections from wild-type (WT) (A and C) and dnhIGF-1R (B and D) glands at L2 were analyzed for lipid droplet formation by ADPH immunofluorescence (red, A and B) or by H&E-staining (C and D). E and F, ADPH immunofluorescence (red) in sections from wild-type (E) and dnhIGF-1R (F) glands at L5. G, Analysis of average pup weight gain from PPD 2–PPD 19 litters from wild-type (n = 5) and dnhIGF-1R (n = 14) dams. H, Relative Q-PCR analysis showing human IGF-1R transgene expression in purified MECs during pregnancy at P10.5, P12.5, P14.5, P17.5, and lactation at L5 after normalization to β-actin. n = 2 for P10.5 and n = 3 for all other time points. Scale bars in A–F, 50 μm. DAPI (blue) was used to detect nuclei in A and B and E and F.

Fig. 6.

Fig. 6.

WAP-dnhIGF-1R expression reduces P-IGF-1R and P-Akt in late pregnant MECs. A–D, Immunoprecipitation using anti-P-tyrosine (100) and Western blot analyses to detect IGF-1Rβ (A and B) or IRβ (C and D) after acute (15 min) IGF-I or insulin stimulation of purified MECs from midpregnant (P12.5–P14.5; A and C) and late pregnant (P15.5–P18.5; B and D) glands. Graphs show quantification after normalization to IgG bands. E and F, Western blot analysis and quantification of P-Akt/total Akt expression in MEC samples shown in panels A–D. Control samples were treated with saline in the absence of ligand. n = 3 for each genotype. For midpregnant samples, each lane represents MECs isolated from one to two animals; for late-pregnant samples, each lane represents MECs isolated from individual animals. Panels B and F, *, P < 0.03; panel C, *, P < 0.02. WT, Wild type.

Fig. 7.

Fig. 7.

WAP-dnhIGF-1R expression decreases IRS-1 and IRS-2 protein and mRNA expression at P14.5. A, Western blot analysis and quantification of total IRS-1 and P-IRS-1(tyr612)/total IRS-1 in IGF-I stimulated MECs from P12.5–P14.5. B and C, Western blot analysis and quantification of protein levels (B) and Q-PCR analysis of mRNA expression (C) of IRS-1 in isolated MECs at P14.5 after normalization to β-actin. D and E, Western blot analysis and quantification of protein levels (D) and Q-PCR analysis of mRNA expression (E) of IRS-2 in isolated MECs at P14.5 after normalization to β-actin. n = 3 for each genotype; **, P < 0.008 (A); *, P < 0.04 (B); *, P < 0.03 (C); *, P < 0.02 (D); *, P < 0.05 (E). WT, Wild type.

Fig. 8.

Fig. 8.

WAP-dnhIGF-1R expression decreases β-casein protein expression and STAT5A mRNA expression at P14.5. A and B, Immunofluorescence staining for β-casein on sections from wild-type (WT) (A) and dnhIGF-1R (B) glands at P14.5. C and D, Western blot analysis and quantification of β-casein protein in whole glands at P14.5 after normalization to β-actin expression (C) and at P17.5 after normalization to CK8 to adjust for reduced number of epithelial cells at this time (D). E and F, Q-PCR analysis of β-casein (F) and STAT5A (G) mRNA expression in isolated MECs at P14.5 after normalization to β-actin. G, Western blot analysis and quantification of STAT5A protein in isolated MECs at P14.5 after normalization to β-actin expression. H, Q-PCR analysis of RANKL mRNA expression in isolated MECs at P14.5 after normalization to β-actin. n = 3 for each genotype; *, P < 0.05 (C and F); *, P < 0.02 (H).

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