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ErbB4 signaling in the mammary gland is required for lobuloalveolar development and Stat5 activation during lactation.

Jones FE, Welte T, Fu XY, Stern DF - J. Cell Biol. (1999)

Bottom Line: However, whey acidic protein mRNA was reduced, and alpha-lactalbumin mRNA was undetectable.This phosphorylation required an intact Stat5 SH2 domain.In summary, our results demonstrate that ErbB4 signaling is necessary for mammary terminal differentiation and Stat5 activation at mid-lactation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, BML 342, Yale University School of Medicine, New Haven, Connecticut 06520-8023, USA.

ABSTRACT
Signaling by members of the epidermal growth factor receptor family plays an important role in breast development and breast cancer. Earlier work suggested that one of these receptors, ErbB4, is coupled to unique responses in this tissue. To determine the function of ErbB4 signaling in the normal mouse mammary gland, we inactivated ErbB4 signaling by expressing a COOH terminally deleted dominant-negative allele of ErbB4 (ErbB4DeltaIC) as a transgene in the mammary gland. Despite the expression of ErbB4DeltaIC from puberty through later stages of mammary development, an ErbB4DeltaIC-specific phenotype was not observed until mid-lactation. At 12-d postpartum, lobuloalveoli expressing ErbB4DeltaIC protein were condensed and lacked normal lumenal lactation products. In these lobuloalveoli, beta-casein mRNA, detected by in situ hybridization, was normal. However, whey acidic protein mRNA was reduced, and alpha-lactalbumin mRNA was undetectable. Stat5 expression was detected by immunohistochemistry in ErbB4DeltaIC-expressing tissue. However, Stat5 was not phosphorylated at Y694 and was, therefore, probably inactive. When expressed transiently in 293T cells, ErbB4 induced phosphorylation of Stat5. This phosphorylation required an intact Stat5 SH2 domain. In summary, our results demonstrate that ErbB4 signaling is necessary for mammary terminal differentiation and Stat5 activation at mid-lactation.

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In situ hybridization analysis of milk protein gene expression in ErbB4ΔIC-expressing mammary glands at 12-d postpartum. Sequential sections from a 12-d-postpartum mammary gland from an ErbB4ΔIC-expressing mouse were stained with hematoxylin/eosin (A) or stained by immunohistochemistry with anti-Flag antibody (B). Additional sequential sections were analyzed by in situ hybridization with β-casein sense (C) or antisense (D) riboprobes, WAP sense (E) or antisense (F) riboprobes, or α-lactalbumin sense (G) or antisense (H) riboprobes. Expanded secretory lobuloalveoli are indicated by arrows and condensed atypical lobuloalveoli are indicated by asterisks. Bar in G, 100 μm.
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Figure 3: In situ hybridization analysis of milk protein gene expression in ErbB4ΔIC-expressing mammary glands at 12-d postpartum. Sequential sections from a 12-d-postpartum mammary gland from an ErbB4ΔIC-expressing mouse were stained with hematoxylin/eosin (A) or stained by immunohistochemistry with anti-Flag antibody (B). Additional sequential sections were analyzed by in situ hybridization with β-casein sense (C) or antisense (D) riboprobes, WAP sense (E) or antisense (F) riboprobes, or α-lactalbumin sense (G) or antisense (H) riboprobes. Expanded secretory lobuloalveoli are indicated by arrows and condensed atypical lobuloalveoli are indicated by asterisks. Bar in G, 100 μm.

Mentions: ErbB4ΔIC expression at 12-d postpartum impaired lobuloalveolar development, resulting in condensed alveolar structures with pronounced lipid secretory activity. These structures resembled normal undifferentiated lobuloalveoli observed at late pregnancy and parturition. To determine if the ErbB4ΔIC-expressing lobuloalveoli were lactationally active, we performed in situ hybridization using antisense riboprobes specific for the milk genes β-casein, WAP, and α-lactalbumin. Serial paraffin sections containing both normal expanded lobuloalveolar structures and condensed lobuloalveoli were examined (Fig. 3 A, arrow and asterisks, respectively). ErbB4ΔIC expression within condensed lobuloalveoli was confirmed by anti-Flag immunohistochemistry (Fig. 3 B, asterisks). The sense probes for β-casein, WAP, and α-lactalbumin yielded similar levels of background hybridization in both expanded and condensed lobuloalveoli (Fig. 3C, Fig. E, and Fig. G, arrows and asterisks, respectively). With antisense probe, equivalent high levels of β-casein RNA expression was observed in both the normal and ErbB4ΔIC-expressing lobuloalveoli (Fig. 3 D, arrow and asterisks, respectively). However, the ErbB4ΔIC-expressing lobuloalveoli showed a moderate diminution in WAP expression (Fig. 3 F). Strikingly, α-lactalbumin expression was reduced to sense probe background levels in condensed areas, but not in normal areas of the same section (Fig. 3 H). The decrease in WAP and the absence of α-lactalbumin expression suggests that terminal differentiation in ErbB4ΔIC-expressing lobuloalveolar epithelium has been disrupted. Similar in situ hybridization analysis performed on mammary glands from female mice at 1-d postpartum yielded equivalent levels of expression of these genes in transgenic and nontransgenic sisters (data not shown).


ErbB4 signaling in the mammary gland is required for lobuloalveolar development and Stat5 activation during lactation.

Jones FE, Welte T, Fu XY, Stern DF - J. Cell Biol. (1999)

In situ hybridization analysis of milk protein gene expression in ErbB4ΔIC-expressing mammary glands at 12-d postpartum. Sequential sections from a 12-d-postpartum mammary gland from an ErbB4ΔIC-expressing mouse were stained with hematoxylin/eosin (A) or stained by immunohistochemistry with anti-Flag antibody (B). Additional sequential sections were analyzed by in situ hybridization with β-casein sense (C) or antisense (D) riboprobes, WAP sense (E) or antisense (F) riboprobes, or α-lactalbumin sense (G) or antisense (H) riboprobes. Expanded secretory lobuloalveoli are indicated by arrows and condensed atypical lobuloalveoli are indicated by asterisks. Bar in G, 100 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2164978&req=5

Figure 3: In situ hybridization analysis of milk protein gene expression in ErbB4ΔIC-expressing mammary glands at 12-d postpartum. Sequential sections from a 12-d-postpartum mammary gland from an ErbB4ΔIC-expressing mouse were stained with hematoxylin/eosin (A) or stained by immunohistochemistry with anti-Flag antibody (B). Additional sequential sections were analyzed by in situ hybridization with β-casein sense (C) or antisense (D) riboprobes, WAP sense (E) or antisense (F) riboprobes, or α-lactalbumin sense (G) or antisense (H) riboprobes. Expanded secretory lobuloalveoli are indicated by arrows and condensed atypical lobuloalveoli are indicated by asterisks. Bar in G, 100 μm.
Mentions: ErbB4ΔIC expression at 12-d postpartum impaired lobuloalveolar development, resulting in condensed alveolar structures with pronounced lipid secretory activity. These structures resembled normal undifferentiated lobuloalveoli observed at late pregnancy and parturition. To determine if the ErbB4ΔIC-expressing lobuloalveoli were lactationally active, we performed in situ hybridization using antisense riboprobes specific for the milk genes β-casein, WAP, and α-lactalbumin. Serial paraffin sections containing both normal expanded lobuloalveolar structures and condensed lobuloalveoli were examined (Fig. 3 A, arrow and asterisks, respectively). ErbB4ΔIC expression within condensed lobuloalveoli was confirmed by anti-Flag immunohistochemistry (Fig. 3 B, asterisks). The sense probes for β-casein, WAP, and α-lactalbumin yielded similar levels of background hybridization in both expanded and condensed lobuloalveoli (Fig. 3C, Fig. E, and Fig. G, arrows and asterisks, respectively). With antisense probe, equivalent high levels of β-casein RNA expression was observed in both the normal and ErbB4ΔIC-expressing lobuloalveoli (Fig. 3 D, arrow and asterisks, respectively). However, the ErbB4ΔIC-expressing lobuloalveoli showed a moderate diminution in WAP expression (Fig. 3 F). Strikingly, α-lactalbumin expression was reduced to sense probe background levels in condensed areas, but not in normal areas of the same section (Fig. 3 H). The decrease in WAP and the absence of α-lactalbumin expression suggests that terminal differentiation in ErbB4ΔIC-expressing lobuloalveolar epithelium has been disrupted. Similar in situ hybridization analysis performed on mammary glands from female mice at 1-d postpartum yielded equivalent levels of expression of these genes in transgenic and nontransgenic sisters (data not shown).

Bottom Line: However, whey acidic protein mRNA was reduced, and alpha-lactalbumin mRNA was undetectable.This phosphorylation required an intact Stat5 SH2 domain.In summary, our results demonstrate that ErbB4 signaling is necessary for mammary terminal differentiation and Stat5 activation at mid-lactation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, BML 342, Yale University School of Medicine, New Haven, Connecticut 06520-8023, USA.

ABSTRACT
Signaling by members of the epidermal growth factor receptor family plays an important role in breast development and breast cancer. Earlier work suggested that one of these receptors, ErbB4, is coupled to unique responses in this tissue. To determine the function of ErbB4 signaling in the normal mouse mammary gland, we inactivated ErbB4 signaling by expressing a COOH terminally deleted dominant-negative allele of ErbB4 (ErbB4DeltaIC) as a transgene in the mammary gland. Despite the expression of ErbB4DeltaIC from puberty through later stages of mammary development, an ErbB4DeltaIC-specific phenotype was not observed until mid-lactation. At 12-d postpartum, lobuloalveoli expressing ErbB4DeltaIC protein were condensed and lacked normal lumenal lactation products. In these lobuloalveoli, beta-casein mRNA, detected by in situ hybridization, was normal. However, whey acidic protein mRNA was reduced, and alpha-lactalbumin mRNA was undetectable. Stat5 expression was detected by immunohistochemistry in ErbB4DeltaIC-expressing tissue. However, Stat5 was not phosphorylated at Y694 and was, therefore, probably inactive. When expressed transiently in 293T cells, ErbB4 induced phosphorylation of Stat5. This phosphorylation required an intact Stat5 SH2 domain. In summary, our results demonstrate that ErbB4 signaling is necessary for mammary terminal differentiation and Stat5 activation at mid-lactation.

Show MeSH
Related in: MedlinePlus