Accelerated apoptosis in the Timp-3–deficient mammary gland
Jimmie E. Fata, Kevin J. Leco, Evelyn B. Voura, Hoi-Ying E. Yu, Paul Waterhouse, Gillian Murphy, Roger A. Moorehead, Rama Khokha
Jimmie E. Fata, Kevin J. Leco, Evelyn B. Voura, Hoi-Ying E. Yu, Paul Waterhouse, Gillian Murphy, Roger A. Moorehead, Rama Khokha
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Article

Accelerated apoptosis in the Timp-3–deficient mammary gland

Abstract

The proapoptotic proteinase inhibitor TIMP-3 is the only molecule of this family thought to influence cell death. We examined epithelial apoptosis in TIMP-3–deficient mice during mammary gland involution. Lactation was not affected by the absence of TIMP-3, but glandular function, as measured by gland-to-body weight ratio and production of β-casein, was suppressed earlier during post-lactational involution than in controls. Histological examination revealed accelerated lumen collapse, alveolar-epithelial loss, and adipose reconstitution in Timp-3–/– females. Epithelial apoptosis peaked on the first day of involution in Timp-3–null glands but at day 3 in wild-type littermates. Unscheduled activation of gelatinase-A was evident by zymography and correlated with earlier fragmentation of fibronectin in Timp-3–/– mammary. To obtain independent evidence of the proapoptotic effects of TIMP-3 deficiency, we introduced recombinant TIMP-3–releasing pellets into regressing Timp-3–/– mammary tissue and showed that this treatment rescued lumen collapse and epithelial apoptosis. Ex vivo, involuting Timp-3–/– mammary tissue demonstrated accelerated epithelial apoptosis that could be reduced by metalloproteinase inhibition. The physiological relevance of TIMP-3 became apparent as Timp-3–/– dams failed to reestablish lactation after brief cessation of suckling. Thus, TIMP-3 is a critical epithelial survival factor during mammary gland involution.

Authors

Jimmie E. Fata, Kevin J. Leco, Evelyn B. Voura, Hoi-Ying E. Yu, Paul Waterhouse, Gillian Murphy, Roger A. Moorehead, Rama Khokha

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TIMP-3 regulates kinetics of mammary gland involution. (a–p) H&E–sta...
TIMP-3 regulates kinetics of mammary gland involution. (a–p) H&E–stained involuting mammary glands from wild-type (a–d and i–l) and Timp-3–null (e–h and m–p) female mice. At 1di, more cell shedding into the lumen was evident in Timp-3–deficient mammary glands (e and m) compared with controls (a and i). Lumen regression and adipose reconstitution occurred at 3di in mammary glands from wild-type mice (c and k). In contrast, lumen regression and adipose reconstitution occurred as early as 2di in mammary glands from Timp-3–null mice (f and n). Epithelial chords (epith. chord) were evident 1 day earlier (3di) in Timp-3–deficient mammary glands compared with wild-type mammary glands (compare g and o with d and l). (q) Histomorphometric analysis revealed lumen regression (solid line) was rapid between 2di and 3di in Timp-3–null mammary glands, whereas regression of lumens occurred between 3di and 4di in wild-type mammary glands. Adipose reconstitution (dotted line) was approximately 70% of the tissue by 4di in Timp-3–null mammary glands. This value was not reached until 7di in wild-type mammary glands. (r–u) At 10 days of lactation (10L) electron microscopy of secretory epithelial cells revealed that apical secretions into the lumen occurred in both wild-type and Timp-3–null mammary glands. However, at 1di these secretions (red arrows) were evident on the basal side of Timp-3–null epithelial cells within the interstitial matrix, whereas they were still only found apically in wild-type tissue. Scale bars in a–h, 100 μm, and in i–p, 50 μm; ×9,000 in r–u.