The consequences of endogenous TGF-β activity in the mammary gland have been evaluated by using exogenous delivery of TGF-β or neutralizing antibodies. The pivotal observations by Daniel and coworkers [4
], who administered exogenous TGF-β via diffusion from miniature inorganic pellets, showed that end-buds undergo reversible regression during puberty, whereas alveolar buds in pregnancy, which are also actively proliferating, do not. During puberty, end-bud DNA synthesis was profoundly inhibited within 12 h of exposure to TGF-β-containing implants, and by 48 h this resulted in an 80-90% decrease in end-bud number [4
]. In contrast, DNA synthesis in distal ductal epithelium and fibroblasts was unaffected, indicating an underlying difference in responses and/or access to these cells. End-bud regression was preceded by the deposition of a thick, collagen- and glycosoaminoglycan-rich ECM, which was postulated to force premature differentiation [49
]. This selective regression of end-buds was reversible if the implant was removed, even after long-term exposures [5
], indicating that the gland did not become refractory or lose its multipotent stem-cell population. In contrast, exogenous TGF-β did not inhibit lobuloalveolar growth, morphogenesis or differentiation. This stage specificity belies a simple role for TGF-β as a DNA synthesis inhibitor and points to the possible differences in how these two actively proliferating structures are regulated. It may also indicate that endogenous TGF-β activity is differentially regulated during these distinct phases of concomitant mammary gland growth and differentiation.
Transgenic manipulation of TGF-β in mammary gland underscores the importance of defining when and where the protein is active. As mentioned above, overexpression of LTGF-β does not produce a mammary phenotype. TGF-β can be produced in a constitutively active form by expression of a construct in which the cysteines at positions 223 and 225 are mutated to serines, however, preventing dimerization of LAP [50
]. Two transgenic models have been created with dramatically different phenotypes, depending on the promoter that is used to drive expression of the transgene. Murine mammary tumor virus (MMTV)-long terminal repeat promoter is used to direct transgene expression to the mammary epithelium. When TGF-β223-225
is expressed on an MMTV promoter, the gland is transiently hypoplastic during ductal morphogenesis, but recovers and is able to lactate and support offspring without apparent difficulty [11
]. It has been suggested by Smith [51
] that additional expression of the MMTV-driven transgene in the salivary gland may influence this phenotype. If the mutated constitutively active TGF-β is driven by the whey acidic protein (WAP) promoter, however, a milk protein that is highly expressed during pregnancy and lactation, ductal morphogenesis is unaffected, but alveolar development is greatly compromised [52
Subsequent experiments demonstrated that proliferation was uninhibited but that apoptosis was stimulated during both estrous and pregnancy [53
]. Furthermore, transplant experiments indicated that the action of TGF-β was intrinsic to the epithelial cells and acted in an autocrine manner. For example, wild-type epithelium was unaffected and differentiated normally [53
]. Taking full advantage of the mammary model, Kordon et al
] used serial transplantation of WAP-TGF-β223-225
epithelium to show that the mammary stem cells were significantly compromised by the transgene expression. Because endogenous WAP protein or reporter expression is restricted to specific luminal epithelial cells and is regulated by the estrus cycle, those investigators postulated that WAP-TGF-β223-225
expression targeted the immediate daughters of mammary epithelial cells, causing inappropriate apoptosis and subsequent depletion of this compartment. The severity of the MMTV-TGF-β223-225
phenotype may also be influenced by the timing or cell specificity of expression relative to that of endogenous LTGF-β activation. Our preliminary data using antibodies that discriminate between active and latent TGF-β supports this concept in that TGF-β activation is suppressed by mid to late pregnancy. Thus, WAP-β223-225
would be driving inappropriate TGF-β activity at a point when it is normally inhibited.
The question of how mammary glands of TGF-β-null mice develop has not yet been addressed because the null mice die around the time of mammary gland development [54
]. These mice have provided evidence of a previously unrecognized aspect of TGF-β biology, however. Letterio et al
] demonstrated that homozygous TGF-β-null off-spring are protected from the gross inflammation that leads to their early death by maternal transfer of TGF-β via the placenta and milk. Another surprising aspect of the TGF-β1
-null mouse is that the haploid genotype leads to greatly reduced TGF-β production, which is reflected by serum levels that are 11% of the levels in wild-type animals, indicating that TGF-β regulates its own production and/or stability. This chronic depletion of the latent complex pools presumably restricts TGF-β activity, and results in a subtly altered proliferative phenotype in liver and lung. A further consequence is increased tumor development, indicating that TGF-β is sufficiently depleted to impede its action as a tumor suppressor [12
We recently evaluated the mammary gland phenotype of TGF-β-null heterozygote mutant mice (Barcellos-Hoff MH, et al
, unpublished data). Mammary ductal outgrowth during puberty was accelerated, and mammary epithelial proliferation was increased in young TGF-β1
haploid genotype animals. This effect appeared to be stage-specific in that mammary gland whole mounts from adult nulliparous mice were grossly normal in morphology and functionally intact. Despite the apparently normal morphology, we found that the frequency of proliferating cells of the mammary epithelium is significantly elevated in mammary gland, as occurs in other tissues like liver from TGF-β-null mutant heterozygotes [12
It is unknown at this time whether systemic, stromal, epithelial or all sources of TGF-β are critical in defining the rate of growth in mammary gland. Gorska et al
] addressed this question by overexpressing a dominant-negative kinase-deficient TGF-β type II receptor driven by MMTV-long terminal repeat, resulting in primarily epithelial expression, or metallothionein-derived promoter, which is expressed in the stroma. The dominant-negative receptor impedes signaling by all TGF-β isoforms, but circumvents the issues raised by constitutively active TGF-β transgenes. Blunting the response to TGF-β in the mammary epitheliuem resulted in alveolar hyperplasia and premature functional differentiation. In contrast, inhibition of the stromal response to TGF-β by this means resulted in increased ductal branching [57
]. Thus, both stromal and epithelial responses to TGF-β activity play a role in defining the maturation of mammary tissue.
The question of when and where LTGF-β is activated during normal development and homeostasis remains to be addressed, however. We recently developed dual immunofluorescence of LAP antibodies and activation-specific chNTGF-β antibodies [27
] and have begun to study the pattern of localization during mouse mammary gland development and differentiation (Fig. ). Although LAP immunoreactivity (green) is relatively uniform, a heterogeneous pattern of TGF-β staining (yellow-orange) is observed in the epithelium of adult mammary glands (Barcellos-Hoff MH et al
, unpublished data). We have examined mammary glands from a variety of developmental states to determine the physiologic correlates of this pattern. We found that mammary epithelial TGF-β immunoreactivity was heterogeneous and most intense during periods of proliferation and morphogenesis, suggesting the presence of a distinct subpopulation. Further characterization of these cells and determination of their ultimate fate in terms of proliferation and differentiation may be informative regarding the cellular mechanisms by which pattern and function are established during mammary gland development.
Figure 3 Dual immunolocalization of latency-associated peptide (LAP) and transforming growth factor (TGF)-β in murine mammary gland. Antibodies to LAP (green) to localize latent TGF (LTGF)-β, antigen-purified TGF-β antibodies that specifically (more ...)