The auto-phosphorylation of murine receptor-interacting protein 3 (Rip3) on Thr 231 and Ser 232 in the necrosome is required to trigger necroptosis. However, how Rip3 phosphorylation is regulated is still largely unknown. Here we identified protein phosphatase 1B (Ppm1b) as a Rip3 phosphatase and found that Ppm1b restricts necroptosis in two settings: spontaneous necroptosis caused by Rip3 auto-phosphorylation in resting cells, and tumour necrosis factor-α (TNF)-induced necroptosis in cultured cells. We revealed that Ppm1b selectively suppresses necroptosis through the dephosphorylation of Rip3, which then prevents the recruitment of mixed lineage kinase domain-like protein (Mlkl) to the necrosome. We further showed that Ppm1b deficiency (Ppm1bd/d) in mice enhanced TNF-induced death in a Rip3-dependent manner, and the role of Ppm1b in inhibiting necroptosis was evidenced by elevated Rip3 phosphorylation and tissue damage in the caecum of TNF-treated Ppm1bd/d mice. These data indicate that Ppm1b negatively regulates necroptosis through dephosphorylating Rip3 in vitro and in vivo.

The epithelial-to-mesenchymal transition (EMT) is a process by which differentiated epithelial cells reprogram gene expression, lose their junctions and polarity, reorganize their cytoskeleton, increase cell motility and assume a mesenchymal morphology. Despite the critical functions of the microtubule (MT) in cytoskeletal organization, how it participates in EMT induction and maintenance remains poorly understood. Here we report that acetylated ␣-tubulin, which plays an important role in microtubule (MT) stabilization and cell morphology, can serve as a novel regulator and marker of EMT. A high level of acetylated ␣-tubulin was correlated with epithelial morphology and it profoundly decreased during TGF-␤-induced EMT. We found that TGF-␤ increased the activity of HDAC6, a major deacetylase of ␣-tubulin, without affecting its expression levels. Treatment with HDAC6 inhibitor tubacin or TGF-␤ type I receptor inhibitor SB431542 restored the level of acetylated ␣-tubulin and consequently blocked EMT. Our results demonstrate that acetylated ␣-tubulin can serve as a marker of EMT and that HDAC6 represents an important regulator during EMT process.

TGFβ signaling inhibits cell proliferation to block cancer initiation, yet it also enhances metastasis to promote malignancy during breast cancer development. The mechanisms underlying these differential effects are still unclear. Here, we report that HER2/EGFR signaling switches TGFβ function in breast cancer cells from antiproliferation to cancer promotion. Inhibition of HER2/EGFR activity attenuated TGFβ-induced epithelial-mesenchymal transition and migration but enhanced the antiproliferative activity of TGFβ. Activation of HER2/EGFR induced phosphorylation of Smad3 at Ser208 of the linker region through AKT, which promoted the nuclear accumulation of Smad3 and subsequent expression of the genes related to EMT and cell migration. In contrast, HER2/EGFR signaling had no effects on the nuclear localization of Smad2. Knockdown of Smad3, but not Smad2, blocked TGFβ-induced breast cancer cell migration. We observed a positive correlation between the nuclear localization of Smad3 and HER2 activation in advanced human breast cancers. Our results demonstrate a key role for HER2/EGFR in differential regulation of Smad3 activity to shift TGFβ function from antitumorigenic to protumorigenic during breast cancer development. TGFβ signaling can shift from inhibiting to promoting breast cancer development via HER2/EGFR AKT-mediated phosphorylation of Smad3 at S208, enhancing its nuclear accumulation and upregulation of EMT-related genes. http://cancerres.aacrjournals.org/content/canres/78/21/6073/F1.large.jpg .

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Smad7 is a negative feedback product of TGF-β superfamily signaling and fine tunes a plethora of pleiotropic responses induced by TGF-β ligands. However, its noncanonical functions independent of TGF-β signaling remain to be elucidated. Here, we show that Smad7 activates signal transducers and activators of transcription 3 (STAT3) signaling in maintaining mouse embryonic stem cell pluripotency in a manner independent of the TGF-β receptors, yet dependent on the leukemia inhibitory factor (LIF) coreceptor glycoprotein 130 (gp130). Smad7 directly binds to the intracellular domain of gp130 and disrupts the SHP2-gp130 or SOCS3-gp130 complex, thereby amplifying STAT3 activation. Consequently, Smad7 facilitates LIF-mediated self-renewal of mouse ESCs and is also critical for induced pluripotent stem cell reprogramming. This finding illustrates an uncovered role of the Smad7-STAT3 interplay in maintaining cell pluripotency and also implicates a mechanism involving Smad7 underlying cytokine-dependent regulation of cancer and inflammation.