Abstract
Recent data investigating the role of the Smad anchor for receptor activation (SARA) in TGF-beta signaling have suggested that it has a crucial function in both aiding the recruitment of Smad to the TGF-beta receptor, and ensuring appropriate subcellular localization of the activated receptor-bound complex. The FYVE domain in SARA directs its localization to early endosomal compartments where it can interact with both the TGF-beta receptors and Smads. However, the necessity of endocytosis in the TGF-beta response remains controversial. We sought to examine the role of internalization in TGF-beta/Smad signaling in human kidney mesangial cells. Using co-immunoprecipitation studies, we show that endogenous Smad2 interacts with SARA after TGF-beta1 stimulation. Inhibition of clathrin-mediated internalization only slightly affects TGF-beta1-stimulated association between SARA and Smad2, Smad2 phosphorylation, or Smad2 interaction with Smad4. However, endocytosis inhibition decreases TGF-beta1-induced Smad2 nuclear translocation and thus abrogates Smad2-dependent transcriptional responses. The TGF-beta1-stimulated association between SARA and Smad2 peaks at 30 min followed by separation of the complex components. However, under conditions of inhibited endocytosis, Smad2 remains bound to SARA for at least 6 h without a significant decline in associated levels. This lack of complex dissociation correlates with a lack of Smad2 nuclear accumulation and reduction of Smad2-dependent ARE-Luc reporter activity. Our data therefore suggest that endocytosis plays a critical role in TGF-beta signaling in mesangial cells, and that internalization enhances the dissociation of Smad2 from the TGF-beta receptor-SARA complex, allowing Smad2 to accumulate in the nucleus and modulate target gene transcription.
Highlights
Transforming growth factor (TGF)1- is a ubiquitously expressed cytokine that has varied roles, affecting cellular processes including proliferation, differentiation, apoptosis, fibro
Recent data investigating the role of the Smad anchor for receptor activation (SARA) in TGF- signaling have suggested that it has a crucial function in both aiding the recruitment of Smad to the TGF- receptor, and ensuring appropriate subcellular localization of the activated receptor-bound complex
Our data suggest that endocytosis plays a critical role in TGF- signaling in mesangial cells, and that internalization enhances the dissociation of Smad2 from the TGF- receptor-SARA complex, allowing Smad2 to accumulate in the nucleus and modulate target gene transcription
Summary
Transforming growth factor (TGF)1- is a ubiquitously expressed cytokine that has varied roles, affecting cellular processes including proliferation, differentiation, apoptosis, fibro-. Our data suggest that endocytosis plays a critical role in TGF- signaling in mesangial cells, and that internalization enhances the dissociation of Smad2 from the TGF- receptor-SARA complex, allowing Smad2 to accumulate in the nucleus and modulate target gene transcription. Some reports indicate that TGF--mediated, Smad2-dependent transcription is reduced by inhibition of endocytosis (9 –12), others find no effect of either inhibiting vesicle formation or interrupting SARA association on Smad2 [13] or Smad3 [14] responses at either the phosphorylation or transcriptional level.
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