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Differential regulation of Smad3 and of the type II transforming growth factor-? receptor in mitosis: implications for signaling.

ABSTRACT: The response to transforming growth factor-? (TGF-?) depends on cellular context. This context is changed in mitosis through selective inhibition of vesicle trafficking, reduction in cell volume and the activation of mitotic kinases. We hypothesized that these alterations in cell context may induce a differential regulation of Smads and TGF-? receptors. We tested this hypothesis in mesenchymal-like ovarian cancer cells, arrested (or not) in mitosis with 2-methoxyestradiol (2ME2). In mitosis, without TGF-? stimulation, Smad3 was phosphorylated at the C-terminus and linker regions and localized to the mitotic spindle. Phosphorylated Smad3 interacted with the negative regulators of Smad signaling, Smurf2 and Ski, and failed to induce a transcriptional response. Moreover, in cells arrested in mitosis, Smad3 levels were progressively reduced. These phosphorylations and reduction in the levels of Smad3 depended on ERK activation and Mps1 kinase activity, and were abrogated by increasing the volume of cells arrested in mitosis with hypotonic medium. Furthermore, an Mps1-dependent phosphorylation of GFP-Smad3 was also observed upon its over-expression in interphase cells, suggesting a mechanism of negative regulation which counters increases in Smad3 concentration. Arrest in mitosis also induced a block in the clathrin-mediated endocytosis of the type II TGF-? receptor (T?RII). Moreover, following the stimulation of mitotic cells with TGF-?, the proteasome-mediated attenuation of TGF-? receptor activity, the degradation and clearance of T?RII from the plasma membrane, and the clearance of the TGF-? ligand from the medium were compromised, and the C-terminus phosphorylation of Smad3 was prolonged. We propose that the reduction in Smad3 levels, its linker phosphorylation, and its association with negative regulators (observed in mitosis prior to ligand stimulation) represent a signal attenuating mechanism. This mechanism is balanced by the retention of active TGF-? receptors at the plasma membrane. Together, both mechanisms allow for a regulated cellular response to TGF-? stimuli in mitosis.

SUBMITTER: Hirschhorn T

PROVIDER: S-EPMC3425481 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Differential regulation of Smad3 and of the type II transforming growth factor-β receptor in mitosis: implications for signaling.

Hirschhorn Tal T Barizilay Lior L Smorodinsky Nechama I NI Ehrlich Marcelo M

PloS one 20120822 8

The response to transforming growth factor-β (TGF-β) depends on cellular context. This context is changed in mitosis through selective inhibition of vesicle trafficking, reduction in cell volume and the activation of mitotic kinases. We hypothesized that these alterations in cell context may induce a differential regulation of Smads and TGF-β receptors. We tested this hypothesis in mesenchymal-like ovarian cancer cells, arrested (or not) in mitosis with 2-methoxyestradiol (2ME2). In mitosis, wit ...[more]

PMID: 22927969

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Project description:BACKGROUND & AIMS:Chronic pancreatitis (CP) is characterized by pancreatic inflammation and fibrosis, associated with increased pancreatic expression of transforming growth factor beta (TGFB). It is not clear how these might contribute to pain. We investigated whether TGFB signaling via SMAD induces sensitization of pancreatic sensory neurons to increase nociception. METHODS:CP was induced in Sprague-Dawley rats by infusion of trinitrobenzene sulfonic acid; some rats were given intrathecal infusions of TGFB1. CP was induced in control mice by administration of cerulein; we also studied ?1glo/Ptf1acre-ER mice, which on induction overexpress TGFB1 in pancreatic acinar cells, and TGFBr1f/f-CGRPcreER mice, which have inducible disruption of TGFBr1 in calcitonin gene-related peptide-positive neurons. Dominant negative forms of human TGFBR2 and SMAD3 were overexpressed from viral vectors in rat pancreas. Some rats were given the SMAD3 inhibitors SIS3 or halofuginone. After induction of CP, mice were analyzed for pain in behavior tests or electrophysiologic studies of sensory neurons. Pancreatic nociceptor excitability was examined by patch-clamp techniques and nociception was measured by Von Frey Filament tests for referred somatic hyperalgesia and behavioral responses to pancreatic electrical stimulation. Pancreata were collected from mice and rats and analyzed histologically and by enzyme-linked immunosorbent assay and immunohistochemistry. RESULTS:Overexpression of TGFB in pancreatic acinar cells of mice and infusion of TGFB1 into rats resulted in sensory neuron hyperexcitability, SMAD3 activation, and increased nociception. This was accompanied by a reduction in the transient A-type current in pancreas-specific sensory neurons in rats, a characteristic of nociceptive sensitization in animal models of CP. Conversely, pancreata from TGFBr1f/f-CGRPcreER mice, rats with pancreatic expression of dominant negative forms of human TGFBR2 or SMAD3, and rats given small molecule inhibitors of SMAD3 had attenuated neuronal sensitization and pain behavior following induction of CP. In contrast to findings from peripheral administration of TGFB1, intrathecal infusion of TGFB1 reduced hyperalgesia in rats with CP. CONCLUSIONS:In pancreata of mice and rats, TGFB promotes peripheral nociceptive sensitization via a direct effect on primary sensory neurons mediated by intra-neuronal SMAD3. This is distinct from the central nervous system, where TGFB reduces nociception. These results provide an explanation for the link between fibrosis and pain in patients with CP. This signaling pathway might be targeted therapeutically to reduce pain in patients with CP.

Dataset Information

Differential regulation of Smad3 and of the type II transforming growth factor-? receptor in mitosis: implications for signaling.

Publications

Differential regulation of Smad3 and of the type II transforming growth factor-β receptor in mitosis: implications for signaling.

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