Smad Signaling Antagonizes STAT5-mediated Gene Transcription and Mammary Epithelial Cell Differentiation

Abstract

Both the transforming growth factor-␤ (TGF␤)/Smad and the prolactin/JAK/STAT pathway are critical to the proper development, maintenance, and function of the mammary epithelial tissue. Interestingly, opposing physiological effects between these two signaling pathways are prominent in the regulation of mammary gland development. However, the exact nature of the biological network existing between the Smad and STAT signal transduction pathways has remained elusive. We identified a novel regulatory cross-talk mechanism by which TGF␤-induced Smad signaling acts to antagonize prolactin-mediated JAK/ STAT signaling and expression of target genes. Furthermore, we found activin, another member of the TGF␤ family, to also efficiently block STAT5 signaling and ␤-casein expression in mammary epithelial cells. Our results indicate that ligand-induced activation of Smad2, -3, and -4 by activin and TGF␤ leads to a direct inhibition of STAT5 transactivation and STAT5-mediated transcription of the downstream target genes, ␤-casein and cyclin D1, thereby blocking vital processes for mammary gland growth and differentiation. Finally, we unveiled the mechanism by which these two signaling cascades antagonize their effects, and we found that activated Smads inhibit STAT5 association with its co-activator CREB-binding protein, thus blocking STAT5 transactivation of its target genes and leading to inhibition of mammary gland differentiation and lactation.Mammary gland growth and differentiation are complex processes regulated by steroids, polypeptide hormones, and growth factors. Among them, prolactin and TGF␤ 5 family members play a major role in the regulation of mammary gland development. Prolactin is required for lobuloalveolar formation and functional differentiation of mammary epithelial cells.TGF␤ has an opposite effect, inducing apoptosis during mammary gland involution and inhibiting expression of the milk proteins (1, 2). TGF␤ is expressed and plays critical roles in every phase of post-natal mammary gland development (3). TGF␤ has been shown to inhibit alveolar formation and synthesis of milk proteins and to induce apoptosis during involution of the mammary gland (4 -6). Together, these data suggest that TGF␤ would antagonize prolactin (PRL)-induced signals in mammary cells (7,8). The effect of activin, another member of the TGF␤ family, on the development of the mammary gland stems from the activin ␤b subunit knock-out mouse model. Deletion of the activin ␤b subunit, through ablation of three of the dimeric ␤ molecules (activin B, activin AB, and inhibin B), results in mice with the phenotype of incomplete mammary development and an absence of lactation, suggesting that activin/inhibin may play an important role in this process (9). In summary, although TGF␤ and activin clearly play important roles in mammary gland development, their mechanism of action in mammary epithelial functional differentiation has yet to be fully elucidated.Prolactin signal transduction is induced by formation of a homodimeric complex of two molec...

Tif1γ is essential for the terminal differentiation of mammary alveolar epithelial cells and for lactation through SMAD4 inhibition

“…The effect of Tif1g silencing on STAT5 phosphorylation depends on SMAD4 and TGF pathways TGFβ had previously been shown to inhibit PRL/STAT5 signalling via mechanisms that involve SMAD proteins (Cocolakis et al, 2008;Wu et al, 2008) and Tif1 is known to inhibit the SMAD4-dependent TGFβ pathway (Dupont et al, 2009;Morsut et al, 2010). Accordingly, we observed that co-treatment with TGFβ1 counteracted STAT5 phosphorylation induced by PRL (Fig.…”

“…It has previously been shown that TGFβ signalling -via SMAD3/4 -could block the association of STAT5 with its co-activator CBP (CREB-binding protein), leading to inhibition of the transactivation of STAT5 target genes (Cocolakis et al, 2008). We note that the authors of this study did not report a modification of PRL-induced STAT5 phosphorylation by TGFβ.…”

“…-Casein mRNA expression was strongly induced in control cells treated with PRL (si-ctrl, DIP lane). This induction was strongly counteracted by TGFβ1 (si-ctrl, DIPT lane), confirming the antagonistic actions of PRL and TGFβ on target genes encoding milk proteins (Mieth et al, 1990;Cocolakis et al, 2008). We next transiently silenced Tif1g expression in HC11 cells by siRNAmediated knockdown.…”

BMP-6 in Renal Cell Carcinoma Promotes Tumor Proliferation through IL-10–Dependent M2 Polarization of Tumor-Associated Macrophages

“…However, one STAT-binding motif was identified. Previously, it has been reported that STAT5 interacts with Smad2 of the TGF-b signaling pathway (31). Of the known subtypes of STATs, STAT3 has been reported regulate IL-10 expression (32).…”

An Rb family–independent E2F3 transcription factor variant impairs STAT5 signaling and mammary gland remodeling during pregnancy in mice

“…Interestingly, knockout of Stat5a alone caused lactation defects (25), and inactivation of both Stat5a and Stat5b caused complete absence of lobuloalveolar development, similar to the effect of inactivating PRLR (26). STAT5 activation can also be modulated by several negative regulators, including CAV1, SOCS3, and TGFβ (27)(28)(29). CAV1 is the main structural component of Caveolae, which are 50-100 nm invagination of the plasma membrane involved in signal transduction, transportation and endocytosis.…”