Downregulation of Ski and SnoN co-repressors by anisomycin

Proteasome pathway regulates TGF-beta signaling; degradation of activated Smad2/3 and receptors turns TGF-beta signal off, while degradation of negative modulators such as Ski and SnoN maintains the signal. We have found that anisomycin is able to downregulate Ski and SnoN via proteasome as TGF-beta does, but through a mechanism independent of Smad activation. The mechanism used by anisomycin to downregulate Ski and SnoN is also independent of MAPK activation and protein synthesis inhibition. TGF-beta signal was the only pathway described causing Ski and SnoN degradation, thus this new effect of anisomycin on endogenous Ski and SnoN proteins suggests alternative processes to downregulate these negative modulators of TGF-beta signaling.     

SnoN co-repressor binds and represses smad7 gene promoter

SnoN and Ski oncoproteins are co-repressors for Smad proteins and repress TGF-beta-responsive gene expression. The smad7 gene is a TGF-beta target induced by Smad signaling, and its promoter contains the Smad-binding element (SBE) required for a positive regulation by the TGF-beta/Smad pathway. SnoN and Ski co-repressors also bind SBE but regulate negatively smad7 gene. Ski along with Smad4 binds and represses the smad7 promoter, whereas the repression mechanism by SnoN is not clear. Ski and SnoN overexpression inhibits smad7 reporter expression induced through TGF-beta signaling. Using chromatin immunoprecipitation assays, we found that SnoN binds smad7 promoter at the basal condition, whereas after a short TGF-beta treatment for 15-30 min SnoN is downregulated and no longer bound smad7 promoter. Interestingly, after a prolonged TGF-beta treatment SnoN is upregulated and returns to its position on the smad7 promoter, functioning probably as a negative feedback control. Thus, SnoN also seems to regulate negatively the TGF-beta-responsive smad7 gene by binding and repressing its promoter in a similar way to Ski.     

The anaphase-promoting complex mediates TGF-beta signaling by targeting SnoN for destruction.

Degradation of SnoN is thought to play an important role in the transactivation of TGF-beta responsive genes. We demonstrate that the anaphase-promoting complex (APC) is a ubiquitin ligase required for the destruction of SnoN and that the APC pathway is regulated by TGF-beta. The destruction box of SnoN is required for its degradation in response to TGF-beta signaling. Furthermore, the APC activator CDH1 and Smad3 synergistically regulate SnoN degradation. Under these circumstances, CDH1 forms a quaternary complex with SnoN, Smad3, and APC. These results suggest that APC(CDH1) and SnoN play central roles in regulating growth through the TGF-beta signaling system.     

Ski/Sno and TGF-beta signaling

Transforming growth factor-beta is a potent inhibitor of epithelial cell proliferation. Proteins involved in TGF-beta signaling are bona fide tumor suppressors and many tumor cells acquire the ability to escape TGF-beta growth inhibition through the loss of key signaling transducers in the pathway or through the activation of oncogenes. Recent studies indicate that there is a specific connection between the TGF-beta signaling pathway and the Ski/SnoN family of oncoproteins. We summarize evidence that Ski and SnoN directly associate with Smad proteins and block the ability of the Smads to activate expression of many if not all TGF-beta-responsive genes. This appears to cause abrogation of TGF-beta growth inhibition in epithelial cells.     

Proteomic analysis of the TGF-beta signaling pathway in pancreatic carcinoma cells using stable RNA interference to silence Smad4 expression

Smad4 is a tumor-suppressor gene that is lost or mutated in 50% of pancreatic carcinomas. Smad4 is also an intracellular transmitter of transforming growth factor-beta (TGF-beta) signals. Although its tumor-suppressor function is presumed to reside in its capacity to mediate TGF-beta-induced growth inhibition, there seems to be a Smad4-independent TGF-beta signaling pathway. Here, we succeeded in establishing Smad4 knockdown (S4KD) pancreatic cancer cell lines using stable RNA interference. Smad4 protein expression and TGF-beta-Smad4 signaling were impaired in S4KD cells, and we compared the proteomic changes with TGF-beta stimulation using two-dimensional gel electrophoresis (2-DE) and mass spectrometry. We identified five proteins that were up-regulated and seven proteins that were down-regulated; 10 of them were novel targets for TGF-beta. These proteins function in processes such as cytoskeletal regulation, cell cycle, and oxidative stress. Introducing siRNA-mediated gene silencing into proteomics revealed a novel TGF-beta signal pathway that did not involve Smad4.     

Decoding the quantitative nature of TGF-beta/Smad signaling

How transforming growth factor-beta (TGF-beta) signaling elicits diverse cell responses remains elusive, despite the major molecular components of the pathway being known. We contend that understanding TGF-beta biology requires mathematical models to decipher the quantitative nature of TGF-beta/Smad signaling and to account for its complexity. Here, we review mathematical models of TGF-beta superfamily signaling that predict how robustness is achieved in bone-morphogenetic-protein signaling in the Drosophila embryo, how changes in receptor-trafficking dynamics can be exploited by cancer cells and how the basic mechanisms of TGF-beta/Smad signaling conspire to promote Smad accumulation in the nucleus. These studies demonstrate the power of mathematical modeling for understanding TGF-beta biology.