No 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

IL-10 is an anti-inflammatory cytokine expressed mainly in blood cells. Both elevated and decreased levels of IL-10 were reported to be present in the bronchoalveolar lavage fluid (BALF) of asthmatic patients. Compared to wild type animals, IL-10 knockout mice exhibit attenuated lung inflammation after allergen sensitization and challenge. We showed that transgene overexpression of TGF-b from the airway epithelium of mice resulted in lung remodeling and airflow obstruction, and correlated with decreased IL-10 levels in BALF. Administration of lovastatin augmented IL-10 amounts in BALF and prevented development of airway remodeling in these transgenic mice. Thus, we hypothesize that TGF-b exposure inhibits IL-10 expression in human airway epithelial cells (HAEC), and lovastatin co-treatment prevents the inhibition of IL-10 promoter function induced by TGF-b. To test this hypothesis, we transfected HAEC with reporter plasmids in which luciferase is driven by either 1538, 938, 688, 336, 158 or 78 bp of the mouse IL-10 5' regulatory region. Transfected cells were treated with none or TGF-b (2.5ng/mL) +/-lovastatin (10 uM) in 1% FBS-containing medium for 24 hrs. We found robust promoter function in HAEC from constructs harboring sequences upstream of -336 bp. Stimulation with TGF-b significantly reduced promoter activity more than half. DNA sequences between -158 and -78 bp were responsive to TGF-b administration. The inhibitory effect of TGF-b was partially blocked by lovastatin; bp -938 to -688 were required for the lovastatin effect. Analysis of a Smad-dependent promoter function showed that the enhancing activity of lovastatin is independent of Smad protein. Modulation of IL-10 promoter was specific because neither the viral MSV promoter nor the promoter-less vector elicit the same response to TGF-b or lovastatin in HAEC. To our knowledge, this the first report that the IL-10 promoter is active in resident cells of the lung; that IL-10 promoter function is inhibited by TGF-b stimulation, and that lovastatin antagonizes the suppressing effect of TGF-b. We conclude that augmentation of IL-10 expression is a plausible mechanism by which lovastatin decreases airway remodeling, and we propose that IL10 activation may be beneficial for treating asthma. This abstract is funded by: K01HL092588, NIH-CTSA UL1RR024999, RHAMC, R34AI080962 Am J Respir Crit Care Med