Mammary Epithelial-specific Deletion of the Focal Adhesion Kinase Gene Leads to Severe Lobulo-Alveolar Hypoplasia and Secretory Immaturity of the Murine Mammary Gland

Tamas Nagy,Huijun Wei,Tang-Long Shen,Xu Peng,Chun-Chi Liang,Boyi Gan,Jun-Lin Guan

doi:10.1074/jbc.m705403200

Tamas Nagy, Huijun Wei + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m705403200

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Abstract

Integrin-mediated cell adhesion and signaling is required for mammary gland development and functions. As a major mediator of integrin signaling, focal adhesion kinase (FAK) has been implicated to play a role in the survival, proliferation, and differentiation of mammary epithelial cells in previously studies in vitro. To assess the role of FAK in vivo, we created mice in which FAK is selectively deleted in mammary epithelial cells. The mammary gland FAK conditional knock-out (MFCKO) mice are viable, fertile, and macroscopically indistinguishable from the control littermates. In virgin MFCKO mice, mammary ductal elongation is retarded at 5 weeks of age but reaches the full extent by 8 weeks of age compared with the control mice. However, the MFCKO females are unable to nurse their pups due to severe lobulo-alveolar hypoplasia and secretory immaturity during pregnancy and lactation. Analysis of the mammary epithelial cells in MFCKO mice showed reduced Erk phosphorylation, expression of cyclin D1, and a corresponding decrease in proliferative capability compared with the littermate controls. In addition, phosphorylation of STAT5 and expression of whey acidic protein are significantly reduced in the mammary glands of MFCKO mice, suggesting defective secretory maturation in these mice. Therefore, the combination of the severe lobulo-alveolar hypoplasia and defective secretory differentiation is responsible for the inability of the MFCKO females to nurse their pups. Together, these results provide strong support for a role of FAK in the mammary gland development and function in vivo.

Highlights

Ductal elongation commences [1]
We found that specific inactivation of Focal adhesion kinase (FAK) in mammary epithelial cells led to severe lobulo-alveolar hypoplasia and inhibition of secretory maturation during pregnancy
The individual alveoli in the mammary gland FAK conditional knock-out (MFCKO) mammary glands were smaller, lined by cuboidal epithelial cells, and had very little secretum within (Fig. 5D). Together these results suggested that deletion of FAK in the mammary epithelial cells significantly impaired mammary gland morphogenesis and function as a result of severe lobulo-alveolar hypoplasia during pregnancy and lactation

Summary

EXPERIMENTAL PROCEDURES

Antibodies, and Other Materials—Carmine and aluminum potassium sulfate were purchased from Sigma-Aldrich. Tissues were washed in PBS for 20 min for 3 times at room temperature, transferred into 65% ethanol (30 min), and transferred to 70% ethanol for storage. Unstained tissue sections were first deparaffinized in 2 washes of xylene (5 min each) and were rehydrated in graded ethanol solutions (100, 95, and 70%). Sections were incubated with horseradish peroxidase-streptavidin (ABC Elite kit, Vector Laboratories) for 15 min at room temperature in a humid chamber and washed with PBS as before. Euthanasia, necropsy, and tissue processing for histology have been described in detail in previous sections of this report. Unstained tissue sections were depraffinized, rehydrated as previously described, and stained using a BrdUrd staining kit (Zymed Laboratories Inc.௡). BrdUrd injection, euthanasia, necropsy, and tissue processing have been described previously in this report. An Enhanced Chemiluminescent (ECL) kit was used to visualize the signal (Pierce)

RESULTS

DISCUSSION

Previous studies have established