Localization Of TGF Beta (TGF-b) Receptors Within The Nuclei Of Human Airway Smooth Muscle Cells

Abstract

4Activation of the TGF-b signaling pathway is thought to play a key role in the airway remodeling that is characteristic of asthma. Binding of TGF-b to the cell membrane type I (TbRI) and type II (TbRII) TGF-b receptors induces the phosphorylation and activation of the signaling proteins Smad2 and Smad3. Phosphorylated Smad2/3 form a complex with Smad4, and they translocate into the nucleus wherein, in association with other co-factors, they bind the regulatory region of TGF-b responsive genes. Using confocal immunofluorescence microscopy of cultured HASM cells we observed that TbRI and TbRII but not caveolin-1 are present within the nuclear compartment. Based on this finding we hypothesized that the localization of TbRs within the nucleus is mediated through their association with Smad2/3. We speculated that Smad2/3 could be responsible, at least in part, for this localization because they associate with the TbRs outside the nuclei, and because of the ability of Smad2/3 to move into the nucleus upon activation. To test this hypothesis, we prepared nuclear (NE) and cytosolic (CE) extracts of HASM cells treated with and without TGF-b (2.5 ng/ml, 24 hrs), and examined the localization of TbRI, TbRII and Smad1/2/3 by western analysis. As expected, TbRI and TbRII are abundantly present in the CE. However, these receptors were also localized in the NE. Interestingly, Smad2/3 was observed in the CE and in the NE of untreated cells, which is consistent with a basal stimulation of the TGF-b signaling pathway in ASM cells, likely due to an autocrine mechanism. To confirm these findings, we performed immunoprecipitation with NE and CE of HEK293 transfected cells that express recombinant TbRI, TbRII and Smad3. We demonstrate that TbRI and TbRII were able to associate with Smad2/3 within the nuclei of non-ASM cells. We conclude that type I and type II TGF-b receptors are present within the cell nucleus in association with Smad2/3. Although a common mechanism in signal transduction involves the mobilization of receptors from the cell membrane into the cell after ligand binding, there are a very few examples of cell membrane receptors that are found also within the nuclei. At present, the potential role of the nuclear localization of these receptors as well as of the TbRI and TbRII reported here remains unknown. We speculate that mobilization of TbRs into the nuclei may represent a novel modulatory mechanism of TGF-b activities that are dysregulated in several human diseases, including asthma. This abstract is funded by: K01HL092588, CTSAUL1RR024999, RHAMC, R34AI080962 Am J Respir Crit Care Med