Signal transducer and activator of transcription (Stat) 5 controls the proliferation and differentiation of mammary alveolar epithelium

Abstract

Functional development of mammary epithelium during pregnancy depends on prolactin signaling. However, the underlying molecular and cellular events are not fully understood. We examined the specific contributions of the prolactin receptor (PrlR) and the signal transducers and activators of transcription 5a and 5b (referred to as Stat5) in the formation and differentiation of mammary alveolar epithelium. PrlR- and Stat5-null mammary epithelia were transplanted into wild-type hosts, and pregnancy-mediated development was investigated at a histological and molecular level. Stat5-null mammary epithelium developed ducts but failed to form alveoli, and no milk protein gene expression was observed. In contrast, PrlR-null epithelium formed alveoli-like structures with small open lumina. Electron microscopy revealed undifferentiated features of organelles and a perturbation of cell-cell contacts in PrlR- and Stat5-null epithelia. Expression of NKCC1, an Na-K-Cl cotransporter characteristic for ductal epithelia, and ZO-1, a protein associated with tight junction, were maintained in the alveoli-like structures of PrlR- and Stat5-null epithelia. In contrast, the Na-Pi cotransporter Npt2b, and the gap junction component connexin 32, usually expressed in secretory epithelia, were undetectable in PrlR- and Stat5-null mice. These data demonstrate that signaling via the PrlR and Stat5 is critical for the proliferation and differentiation of mammary alveoli during pregnancy.

Figure 1.

Pregnancy-mediated mammary gland development depends on the PrlR and Stat5. (A) Whole mount analyses of wild-type, PrlR-, and Stat5-null mammary epithelia at parturition. The lower panel represents a higher magnification. Wild-type epithelium filled the fat pad and developed lobuloalveolar structures (*). PrlR- and Stat5-null mammary epithelia were severely underdeveloped. PrlR-null epithelium exhibited wide ducts (black arrow), short branches, and few alveoli-like structures. Stat5-null epithelium displayed normal branches with small decorations (black arrowhead). (B) Histological sections of the whole mounts shown in A. The lower panel represents a 6× higher magnification. Wild-type epithelium was fully expanded, filled the fat pad and alveolar lumina contained milk (*). However, all null epithelia were sparse, and the alveoli-like structures (black arrowhead) did not contain milk. The epithelial cells are cuboidal in shape (white arrow). Note the presence of open lumina in the alveoli-like structures in PrlR-, but not Stat5-null epithelium. (C) Whole mount analyses of wild-type and PrlR- and Stat5-null virgin mammary epithelia 8 wk after transplantation. All transplanted epithelia completely filled the fat pad.

Figure 2.

Progesterone- and estrogen-induced proliferation is more severely impaired in PrlR-null than in Stat5a/b–null mammary epithelium. 9 wk after transplantation, mice were given an acute, 2-d E+P treatment. Mammary glands were removed and proliferation was evaluated by BrdU immunostaining. (A) Green staining represents BrdU-positive cells and DAPI-stained nuclei are blue. Merging the images shows the proliferation occurring mainly in the ductal epithelium. The number of BrdU-positive cells was clearly decreased in both PrlR- and Stat5-null transplants, as compared with the control #3 gland. (B) Quantitation of BrdU-positive ductal cells (mean percentage ± SEM) is shown in the bar graph. The P values comparing PrlR- or Stat5-null to control (*) and Stat5-null to PrlR-null (**) were <0.001 as determined by Mann-Whitney paired t test.

Figure 3.

Milk protein expression is impaired in Stat5- and PrlR-null mammary epithelia. Northern blot analysis of milk protein mRNAs (β-casein, WAP, WDNM1) and keratin 18 (K18) mRNA in mammary tissue of wild-type mice (WT) and Stat5a-, 5a/b-, and PrlR-null epithelial transplants. V, virgin (5 wk); P13, pregnancy day 13; Part, after parturition.

Figure 4.

Disorganized structures of Stat5- and PrlR-null epithelia. Stat5- and PrlR-null mammary epithelia at parturition were analyzed by electron microscopy. (A and D) Control mammary epithelium at lactation day 1 was fully differentiated and contained secreted milk proteins and lipid droplets. Golgi apparatus (white long arrow) and RER (white long arrow) were detected. Lu, lumen; L, lipid droplet; N, nucleus; black short arrow, β-casein micelles; black circle, tight junction. (B and E) Alveoli-like structures of PrlR-null epithelium were more organized than Stat5-null epithelia. Lumina were detected in alveolar-like structure (white circle). The centrosome was located close to the apical membrane (black arrow). Tight junctions were maintained (black circle). (C and F) Alveoli-like structures in Stat5-null epithelia were disorganized and cell–cell contacts were aberrant (black arrow). Frequently, two or more pseudo-lumina were detected in one alveolar-like structure (black circles). The cells near the basement membrane contained lipid droplet-like structures (white arrow). Active Golgi apparatus and a RER in Stat5- and PrlR-null epithelia were not apparent. Bars: (A–C) 2.4 μm; (D–F) 1.6 μm.

Figure 5.

Maintenance of ZO-1 expression but loss of Cx 32 expression. Immunohistochemical staining of ZO-1 (green) and E-cadherin (red) at parturition (A–C). Tight junctions (green dots) were present in PrlR- (A) and Stat5-null (B) the same as in wild-type epithelium (C). Arrows point to alveoli-like structures of PrlR- and Stat5-null epithelia. (D) RT-PCR analysis of Cx 32 mRNA. Total RNA from wild-type, Stat5-, and PrlR-null transplanted epithelia at parturition was reverse transcribed, and the cDNA was subjected to PCR. Cx 32 cDNA was detected in wild-type but not in PrlR- or Stat5-null samples. GAPDH levels were similar in all samples. M, marker.

Figure 7.

Npt2b expression was not detected in PrlR- and Stat5-null alveoli-like structures at parturition. Immunohistochemical staining of Npt2b (red) and E-cadherin (green) in mammary epithelia of virgin mice (A, C, and E) and after parturition (B, D, and F). Npt2b was detected in apical membranes of secreting epithelia from wild-type tissue (B, white arrow). PrlR- and Stat5-null epithelia at parturition did not contain Npt2b (D and F). E-cadherin was detected in the sub-apical/basolateral membrane of all samples (A–F). (G) Wild-type epithelia at pregnancy day 12 did not express Npt2b. Du, duct.

Figure 6.

NKCC1 and smooth muscle actin are expressed in PrlR- and Stat5-null alveoli-like structures at parturition, but not in wild-type mice. Immunohistochemical staining of NKCC1 (red) and smooth muscle actin (green) in mammary epithelia of virgin mice (A, C, and E) and after parturition (B, D, and F). NKCC1 levels were high in ductal epithelium of wild-type virgin mice (A) and sharply reduced in alveoli by pregnancy day 12 (G, circle) and at parturition (B). PrlR- and Stat5-null epithelia maintained high levels of NKCC1 at parturition (D and F, compare white arrows with white arrow in G).

Figure 8.

Epidermal growth factor and GH can activate Stat5a in PrlR-null epithelium. Immunohistochemical staining of Stat5a (green) and E-cadherin (red) in mammary epithelium at parturition (A) and in virgin tissue after hormone injection (B). Arrows show Stat5a nuclear staining. (A) In PrlR-null epithelium, some Stat5a nuclear staining was detected. In contrast, almost all cells in wild-type epithelia had Stat5a nuclear translocation. (B) Virgin mice carrying PrlR- and Stat5-null epithelia were injected with EGF or GH. Extensive nuclear localization of Stat5a was observed in PrlR-null epithelium but not in Stat5-null epithelium.

Figure 9.

PrlR and Stat5 control the cell fate of mammary alveolar epithelium. Wild-type mammary epithelium differentiates into functional alveoli during pregnancy. However, PrlR- and Stat5-null epithelia do not undergo alveolar development. The null epithelia maintain ductal features and cell proliferation at the branching points results in the development of “ductoli” (ductal feature but alveoli-like structure). In contrast to Stat5-null ductoli, PrlR-null ductoli contain open lumina. In the absence of Stat5 ductolar cells have impaired cell–cell contacts. The ductoli do not differentiate into functional alveoli. Blue line, ductal development; red line, alveolar development. The myoepithelial cells surrounding the alveoli are flat. The outer cell layer of ductoli is positive for smooth muscle actin and can thus be considered to be of myoepithelial nature.