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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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Immunohistochemical detection of ErbB4ΔIC protein in the mammary gland at 12-d postpartum. Paraffin-embedded section from a 12-d-postpartum nontransgenic sibling control stained with hematoxylin/eosin (A). Sequential sections (B–D) from a 12-d-postpartum mammary gland from an ErbB4ΔIC-expressing mouse were stained with hematoxylin/eosin (B), or stained by immunohistochemistry with rabbit IgG control antibody (C) or a rabbit anti-Flag antibody (D). Expanded secretory lobuloalveoli are indicated by arrows, and condensed atypical lobuloalveoli are indicated by asterisks. Bar in C, 100 μm.
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Figure 2: Immunohistochemical detection of ErbB4ΔIC protein in the mammary gland at 12-d postpartum. Paraffin-embedded section from a 12-d-postpartum nontransgenic sibling control stained with hematoxylin/eosin (A). Sequential sections (B–D) from a 12-d-postpartum mammary gland from an ErbB4ΔIC-expressing mouse were stained with hematoxylin/eosin (B), or stained by immunohistochemistry with rabbit IgG control antibody (C) or a rabbit anti-Flag antibody (D). Expanded secretory lobuloalveoli are indicated by arrows, and condensed atypical lobuloalveoli are indicated by asterisks. Bar in C, 100 μm.

Mentions: To determine the effects of ErbB4ΔIC expression on female mammary gland development, wholemounts and histological sections were examined from virgin mice at 3, 5, 6, 8, 10, and 19 wk of age; during early (12 d), mid- (16 d), and late (19 d) pregnancy; after parturition at days 3, 6, 9, 12, 15, or 18; and 2–4 d after weaning. At least three mice were analyzed at each time point. Despite the extensive time frame for transgene expression, and the fact that expression was highest shortly after parturition (Fig. 1), the only identifiable phenotypes were detected on day 12 postpartum. The fat pad of a nontransgenic mouse at 12-d postpartum is completely invested with engorged lobuloalveoli displacing stromal adipose cells. Secretory activity is demonstrated by lumens lined with protruding secretory epithelium (Fig. 2 A, arrow). Engorged active secretory lobuloalveoli were also observed in ErbB4ΔIC-expressing mice at 12-d postpartum (Fig. 2 B, arrow). In some transgenic mice (3 out of 5 examined), however, a subpopulation of lobuloalveoli failed to expand and contained an unusually high level of lumenal secretory lipids (Fig. 2 B, asterisk). Adipose cells were still abundant in this region of the mammary gland fat pad. The condensed lobuloalveoli resembled undifferentiated lobuloalveoli that are normally predominant during late pregnancy. We next used anti-Flag immunohistochemistry to determine if the condensed lobuloalveoli expressed the Flag-tagged ErbB4ΔIC transgene. Intense cytoplasmic immunostaining of epithelium within condensed lobuloalveoli was observed (Fig. 2 D, asterisks). Anti-Flag immunostaining was not observed in distended lobuloalveoli in the same tissue sections (Fig. 2 D, arrow). The lack of detectable transgene expression in this subpopulation of lobuloalveoli may be a result of variegated transgene expression. Variegated promoter expression within the mouse mammary gland has been reported for several mammary specific promoters, including the MMTV LTR promoter used in this study (Faerman et al. 1995; Deckard-Janatpour et al. 1997; Jones and Stern 1999).


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)

Immunohistochemical detection of ErbB4ΔIC protein in the mammary gland at 12-d postpartum. Paraffin-embedded section from a 12-d-postpartum nontransgenic sibling control stained with hematoxylin/eosin (A). Sequential sections (B–D) from a 12-d-postpartum mammary gland from an ErbB4ΔIC-expressing mouse were stained with hematoxylin/eosin (B), or stained by immunohistochemistry with rabbit IgG control antibody (C) or a rabbit anti-Flag antibody (D). Expanded secretory lobuloalveoli are indicated by arrows, and condensed atypical lobuloalveoli are indicated by asterisks. Bar in C, 100 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2164978&req=5

Figure 2: Immunohistochemical detection of ErbB4ΔIC protein in the mammary gland at 12-d postpartum. Paraffin-embedded section from a 12-d-postpartum nontransgenic sibling control stained with hematoxylin/eosin (A). Sequential sections (B–D) from a 12-d-postpartum mammary gland from an ErbB4ΔIC-expressing mouse were stained with hematoxylin/eosin (B), or stained by immunohistochemistry with rabbit IgG control antibody (C) or a rabbit anti-Flag antibody (D). Expanded secretory lobuloalveoli are indicated by arrows, and condensed atypical lobuloalveoli are indicated by asterisks. Bar in C, 100 μm.
Mentions: To determine the effects of ErbB4ΔIC expression on female mammary gland development, wholemounts and histological sections were examined from virgin mice at 3, 5, 6, 8, 10, and 19 wk of age; during early (12 d), mid- (16 d), and late (19 d) pregnancy; after parturition at days 3, 6, 9, 12, 15, or 18; and 2–4 d after weaning. At least three mice were analyzed at each time point. Despite the extensive time frame for transgene expression, and the fact that expression was highest shortly after parturition (Fig. 1), the only identifiable phenotypes were detected on day 12 postpartum. The fat pad of a nontransgenic mouse at 12-d postpartum is completely invested with engorged lobuloalveoli displacing stromal adipose cells. Secretory activity is demonstrated by lumens lined with protruding secretory epithelium (Fig. 2 A, arrow). Engorged active secretory lobuloalveoli were also observed in ErbB4ΔIC-expressing mice at 12-d postpartum (Fig. 2 B, arrow). In some transgenic mice (3 out of 5 examined), however, a subpopulation of lobuloalveoli failed to expand and contained an unusually high level of lumenal secretory lipids (Fig. 2 B, asterisk). Adipose cells were still abundant in this region of the mammary gland fat pad. The condensed lobuloalveoli resembled undifferentiated lobuloalveoli that are normally predominant during late pregnancy. We next used anti-Flag immunohistochemistry to determine if the condensed lobuloalveoli expressed the Flag-tagged ErbB4ΔIC transgene. Intense cytoplasmic immunostaining of epithelium within condensed lobuloalveoli was observed (Fig. 2 D, asterisks). Anti-Flag immunostaining was not observed in distended lobuloalveoli in the same tissue sections (Fig. 2 D, arrow). The lack of detectable transgene expression in this subpopulation of lobuloalveoli may be a result of variegated transgene expression. Variegated promoter expression within the mouse mammary gland has been reported for several mammary specific promoters, including the MMTV LTR promoter used in this study (Faerman et al. 1995; Deckard-Janatpour et al. 1997; Jones and Stern 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.

Show MeSH
Related in: MedlinePlus