Latent transforming growth factor-beta activation in mammary gland: regulation by ovarian hormones affects ductal and alveolar proliferation - PubMed (original) (raw)
Latent transforming growth factor-beta activation in mammary gland: regulation by ovarian hormones affects ductal and alveolar proliferation
Kenneth B Ewan et al. Am J Pathol. 2002 Jun.
Abstract
Transforming growth factor-beta1 (TGF-beta 1) is a pluripotent cytokine that can inhibit epithelial proliferation and induce apoptosis, but is also widely implicated in breast cancer progression. Understanding its biological action in mammary development is critical for understanding its role in cancer. TGF-beta 1 is produced as a latent complex that requires extracellular activation before receptor binding. To better understand the spatial and temporal regulation of its action during mammary gland development, we examined the pattern of activation in situ using antibodies selected to distinguish between latent and active TGF-beta. Activation was highly restricted. TGF-beta 1 activation was localized primarily to the epithelium, and within the epithelium it was restricted to luminal epithelial cells but absent from either cap or myoepithelial cells. Within the luminal epithelium, we noted a further restriction. During periods of proliferation (ie, puberty, estrus and pregnancy), which are stimulated by ovarian hormones, TGF-beta 1 activation decreased in some cells, consistent with preparation for proliferation. Paradoxically, other cells simultaneously increase TGF-beta 1 immunoreactivity, which suggests that TGF-beta 1 differentially restrains epithelial subpopulations from responding to hormonal signals to proliferate. These data suggest that endogenous TGF-beta 1 activation and thus activity are regulated by ovarian hormones. To determine the specific consequences of TGF-beta 1 activity, we manipulated TGF-beta 1 levels in vivo using Tgfbeta 1 knockout mice and undertook tissue recombination experiments with heterozygous tissue. In Tgfbeta 1 heterozygous mice, which have <10% wild-type levels of TGF-beta1, ductal development during puberty and alveolar development during pregnancy were accelerated, consistent with its role as a growth inhibitor. The proliferative index of Tgfbeta 1+/- epithelium was increased approximately twofold in quiescent tissue and fourfold in proliferating tissue but both ducts and alveoli were grossly and histologically normal. To test whether epithelial TGF-beta1 was critical to the proliferative phenotype, Tgfbeta 1+/+ and +/- epithelium were transplanted into +/+ mammary stroma. The outgrowth of Tgfbeta 1+/- epithelium was accelerated in wild-type hosts, indicating that the phenotype was intrinsic to the epithelium. Moreover, proliferation was 15-fold greater in Tgfbeta 1+/- than wild-type mice after ovariectomy and treatment with estrogen and progesterone, suggesting that TGF-beta 1 acts in an autocrine or juxtacrine manner to regulate epithelial proliferation. Together these data indicate that ovarian hormones regulate TGF-beta 1 activation, which in turn restricts proliferative response to hormone signaling.
Figures
Figure 1.
Mammary epithelial LAP and TGF-β1 immunostaining are regulated during ductal morphogenesis. A: Nuclear DAPI staining (top) and dual immunolocalization of active TGF-β1 (middle) and LAP (bottom) of endbuds (left) and distal ducts (right). During ductal morphogenesis, most body cells in endbuds showed LAP and strong TGF-β1 immunostaining, but some lacked active TGF-β1 (arrowhead). Cap cells (arrows) at the interface between the endbud and adipose stroma were negative for TGF-β1 even though they stained with antibodies to LAP. Ductal epithelium proximal to the region of endbuds exhibited less TGF-β1 immunostaining. Tissue was from FVB mice. Scale bar, 20 μm. B: The mean (±SD) intensity of TGF-β1 and LAP were quantified in the peri-epithelial stroma of the ducts (Sd) and endbuds (Se) and for epithelial cells from the cap cell layer (C), endbud body cell (B), and distal ducts (D). Endbud body cells had significantly more TGF-β1 immunoreactivity than any other population. C: To evaluate the frequency of TGF-β1-positive cells in different epithelial populations, cells with immunoreactivity greater than the mean +2 SD of the stromal cell TGF-β1 intensity were defined as TGF-β1-positive. The majority of endbud body and duct epithelial cells were TGF-β1-positive, whereas cap cells were rarely positive.
Figure 2.
LAP and TGF-β1 immunostaining is highly heterogeneous during the estrus cycle. A–C: Mammary epithelial immunoreactivity of nulliparous FVB animals as a function of the estrus cycle; DAPI-stained nuclei (A), LAP (B) and active TGF-β1 (TGF-β) (C) immunoreactivity. In diestrus, most epithelial cells stained with both antibodies. During proestrus, a transition occurs in which epithelial ducts show heterogeneous TGF-β1 staining. During estrus, the heterogeneity of TGF-β1 immunoreactivity increased, such that the epithelium contains TGF-β1-negative cells adjacent to positive cells. LAP is more homogeneous. Occasional variation was observed in that a few epithelial ducts from estrus exhibited low homogeneous staining similar to that seen in diestrus (not shown). D: Quantitative image analysis of the intensity of LAP and TGF-β1 immunoreactivity per cell. In diestrus, the relative intensity of LAP per cell was linearly correlated with TGF-β1 in most cells. In proestrus, a population of cells with very low TGF-β1 is evident. At estrus, an additional population appears consisting of cells exhibiting high TGF-β1 and low LAP immunoreactivity. These cells correspond to those with intense TGF-β1 shown above and in E. E: False-color digital micrographs of the dual immunolocalization of antigen-purified TGF-β1 antibodies (red) and LAP antibodies (green) visualized simultaneously with DAPI-stained nuclei (blue). Mammary gland tissue was obtained from animals sacrificed at estrus. LAP immunoreactivity (green) was relatively uniform in the peri-epithelial stroma and adipose stroma, whereas immunoreactive TGF-β1 was not evident in the stroma. Concordant TGF-β1 and LAP staining appeared yellow-orange. Note that all cells stain with LAP. The discordance of LAP and TGF-β1 immunoreactivity suggests that this TGF-β1 epitope is masked in the majority of stromal cells and in some epithelial cells. This highly localized TGF-β1 is indicative of restricted activation. Scale bar, 10 μm.
Figure 3.
Transition of adult mammary gland from estrus cycle to lobular-alveolar differentiation during pregnancy is accompanied by progressive loss of both active and latent TGF-β. A: During diestrus, chNTGF-β1 immunoreactivity is relatively homogeneous in nulliparous mammary gland. B: In contrast the epithelium was distinctly heterogeneous during estrus. C: Mammary tissue from early (6 day) pregnant mice exhibited less intense LAP immunoreactivity and less chNTGF-β, suggesting that gradual loss of both TGF-β1 production and activation is reduced. D: Epithelium undergoing functional differentiation during late (18 day) pregnancy exhibited barely detectable TGF-β1 and LAP immunostaining. The tissue sections were stained and images acquired together. False-color digital micrographs of the dual immunolocalization of antigen-purified TGF-β1 antibodies (red) and LAP antibodies (green) visualized simultaneously with DAPI-stained nuclei (blue). The images are scaled identically to allow comparison within the figure of the pattern of TGF-β1 and LAP immunoreactivity. Scale bar, 20 μm.
Figure 4.
LAP and TGF-β1 immunoreactivity are both decreased in Tgfβ1+/− mice compared to +/+ mice. Comparison of relative LAP (A and C) and TGF-β1 (B and D) immunoreactivity intensity in the epithelium of Tgfβ1+/+ (shaded bars) and +/− (open bars) littermates. A and B: The staining intensity of LAP and TGF-β1 in the distal ducts at puberty was significantly decreased (P < 0.01, Kolmogorov-Smirnov test) in Tgfβ1 heterozygotes. C and D: Similarly, both LAP and TGF-β1 were decreased in Tgfβ1+/− mice in the mammary epithelium of adult mice in estrus.
Figure 5.
Accelerated mammary ductal growth in Tgfβ1+/− mice. Mammary gland whole mounts and histology from Tgfβ1+/+ (A, C, and E) and +/− (B, D, and F) littermates. A and B: Low-magnification views of mammary gland whole mounts from 6-week-old mice. In wild-type mice, epithelial outgrowth has reached the lymph node (LN), whereas in the heterozygotes the fat pad is two-thirds full. The nipple is to the left in both cases. C and D: The endbud epithelium of both genotypes is similar and well organized (H&E stain). Asterisks indicate pyknotic nuclei. E and F: High-magnification photomicrographs of mammary gland whole mounts show similar branching patterns in wild-type and heterozygote mice.
Figure 6.
Schematic of the pattern of TGF-β1 immunoreactivity and proliferation in mammary epithelium relative to ovarian hormones during the estrus cycle. A: Characteristic patterns of active TGF-β in mammary epithelium are represented as a shaded bar representing the relative intensity of epithelial TGF-β1 as a function of mammary gland development. The pattern is heterogeneous during periods of proliferation and relatively homogeneous during quiescent stages. B: During the estrus cycle, highly heterogeneous TGF-β1 immunoreactivity at estrus correlates with the phenotype of increased proliferation and decreased apoptosis (not shown) in Tfgβ1+/− mice. The relative levels of estrogen (dotted line) and progesterone (solid line) serum concentrations are graphed and the proliferative indices of Tfgβ1+/+ and +/− mammary epithelium are summarized in the bar graph below. The functional link between the role of TGF-β as a growth inhibitor and ovarian hormones regulating proliferation in the mammary gland is supported by the dramatic proliferative response of ovarectomized Tfgβ1+/− mice to estrogen and progesterone.
References
- Moses HL, Coffey RL, Jr, Leof EB, Lyons RM, Keski-Oja J: Transforming growth factor β regulation of cell proliferation J Cell Physiol 1987, 5:S1-S7 - PubMed
- Roberts AB: Molecular and cell biology of TGF-beta. Miner Electrolyte Metab 1998, 24:111-119 - PubMed
- Ewen ME, Sluss HK, Whitehouse LL, Livingston DM: TGFβ inhibition of Cdk4 synthesis is linked to cell cycle arrest. Cell 1993, 74:1009-1020 - PubMed
- Fynan TM, Reiss M: Resistance to inhibition of cell growth by transforming growth factor-β and its role in oncogenesis. Crit Rev Oncog 1993, 4:493-540 - PubMed
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