Expression Levels Of Smad7 Research Articles

Effects of Arkadia on airway remodeling through enhancing TGF-β signaling in allergic rats

Upregulation of transforming growth factor-β (TGF-β) signaling is interrelated with the development of airway remodeling. In this study, we examined the role of two E3 ubiquitin ligases, Arkadia and Smurf2, which are critically required for TGF-β signaling in airway remodeling. Rats were immunized with ovalbumin (OVA) and then challenged with an OVA aerosol. In in vitro experiments, normal human bronchial epithelial cells were stimulated with TGF-β1 with or without the preincubation of Arkadia/Smurf2 small interfering RNA (siRNA) or lactacystin (an inhibitor of proteasomal degradation). In the lungs of OVA-treated rats, a large number of inflammatory cells were present near the airways. An increased subepithelial collagen deposition was associated with high expression levels of Smad7, SnoN and Ski mRNAs, Arkadia, Smurf2, and TGF-β type I receptor (TβRI), but low expression levels of Smad7, SnoN and Ski proteins. Smad7, SnoN and Ski interacted with both Arkadia and Smurf2 while TβRI only interacted with Smurf2 but not with Arkadia. In in vitro experiments, the inhibitory effect of TGF-β1 on the expression of Smad7, SnoN and Ski was reversed by Arkadia siRNA and lactacystin, whereas the stimulatory effect of TGF-β1 on the expression of TβRI protein and Smad7/SnoN/Ski mRNAs was not affected. In contrast, Smurf2 siRNA did not influence the effects of TGF-β1 on the expression of the above proteins. Our results suggest that Arkadia may contribute to the pathogenesis of airway remodeling through enhancing TGF-β signaling by inducing the reduction of Smad7, SnoN and Ski proteins in OVA-sensitized and -challenged rats.

Expression of tenascin-X in alcoholic cardiomyopathy and relation thereof to myocardial fibrosis: experiment with rats

To explore the protein expression of tenascin-X (TN-X) and the relation between TN-X and cardiac remodeling and myocardial fibrosis in alcoholic cardiomyopathy (ACM). 60 Wistar rats were randomly divided into 2 equal groups: alcohol-fed group fed with alcohol to establish ACM models and control group fed with common food. 6 months later the rats were killed and the cardiac structure and function, and myocardial collagen content were measured. Western blotting and immunohistochemistry were used to detect the protein expression of TN-X, and smad-3 and smad-7, the signal transduction proteins. The levels of left ventricle ejection fraction (LVEF) and fraction shortening (FS) of the alcohol-fed group were both significantly lower, and the levels of left ventricle end-diastolic diameter (LVEDd) and collagen volume fraction (CVF) were both higher than those of the control group (both P < 0.05). The protein expression levels of TN-X and smad-3 of the alcohol-fed group were (88% +/- 6%, 82% +/- 8%), both significantly higher than those of the control group (33% +/- 11%, 29% +/- 8%) (both P < 0.01), and the protein expression level of smad-7 of the alcohol-fed group were (37% +/- 9%), both significantly lower than those of the control group (79% +/- 10%, P < 0.01). The protein expression of TN-X was significantly negatively correlated with LVEF, FS, and smad-7, and positively correlated with LVEDd, CVF, and smad-3 (all P < 0.01). Protein expression of TN-X is upregulated in alcoholic cardiomyopathy which may facilitate the myocardial fibrosis in ACM.

The differential regulation of Smad7 in kidney tubule cells by connective tissue growth factor and transforming growth factor‐beta1

Smad7 is an inhibitory Smad that regulates transforming growth factor-beta (TGF-beta) signaling. Connective tissue growth factor (CTGF) is recognized as a potent downstream mediator of the fibrogenic effects of TGF-beta1. SMAD binding sites have been identified in both TGF-beta and CTGF promoters. The effect of CTGF on Smad7 expression and its role in the regulation of Smad7 induced by TGF-beta1 in renal tubular cells is unknown. Human model of proximal tubular cells (HK-2 cells) was used and confirmed using a diabetic rat model. RT-PCR was performed to measure Smad7, TGF-beta1 and Smad2 and ELISA was performed to measure active TGF-beta1. CTGF or TGF-beta1 was silenced in HK-2 cells using siRNA methodology. TGF-beta1 induced Smad7 in a time-dependent manner, peaking at 30 min (P<0.0005) but sustained up to 24 hrs (p<0.005). Conversely, CTGF reduced Smad7, which was maximal at 24 hrs (p<0.05). This was supported by our in vivo data demonstrating that CTGF protein significantly increased while Smad7 mRNA level was reduced in a diabetic rat model. The basal expression level of Smad7 decreased in TGF-beta1 silenced cells compared to cells transfected with non-specific siRNA (p<0.0005). The basal expression level of Smad7 increased in CTGF silenced cells (p<0.05), which was increased by TGF-beta1 (p<0.005). Both mRNA and protein levels of TGF-beta1 decreased in CTGF silenced cells (p<0.05 and p<0.005 respectively) accompanied by reduction in Smad2 mRNA level in CTGF silenced cells. Smad7 is induced rapidly by TGF-beta1 limiting the response to TGF-beta1. CTGF likely plays a key role in promoting TGF-beta1 activity by decreasing the availability of Smad7 and increasing Smad2.

The novel E3 ubiquitin ligase Tiul1 associates with TGIF to target Smad2 for degradation.

Ubiquitin-dependent degradation plays an important role in the negative regulation of TGF-beta signaling. Here, we identify Tiul1 (for TGIF interacting ubiquitin ligase 1), a novel E3 ubiquitin ligase that inhibits TGF-beta signaling by targeting both the activated receptor and Smad2 for degradation. Tiul1 associates constitutively with Smad7 and induces degradation of the activated type I receptor without affecting the expression levels of Smad7. Tiul1 can also interact with Smad2 and the nuclear corepressor TGIF upon activation of TGF-beta signaling. Like Smad7, the steady-state levels of TGIF are not affected by Tiul1, but the interaction of Tiul1 with TGIF allows this ubiquitin ligase to target Smad2 for degradation. Consistent with this, overexpression of Tiul1 suppressed TGF-beta-induced growth arrest and transcriptional responses. In addition, silencing of Tiul1 or TGIF genes by siRNA resulted in suppression of the TGF-beta-dependent degradation of Smad2 and an enhancement of TGF-beta-mediated gene expression. These results reveal a new role for TGIF as a component of a ubiquitin ligase complex that mediates the degradation of Smad2 in response to TGF-beta signaling.

Expression of Smad7 in bronchial epithelial cells is inversely correlated to basement membrane thickness and airway hyperresponsiveness in patients with asthma

Smad7 is an intracellular antagonist of transforming growth factor beta (TGF-beta) signaling, which could determine the intensity or duration of the TGF-beta signal. Because TGF-beta has been implicated in the development of airway remodeling in asthma on the basis of its strong capacity to induce extracellular matrix production, it is possible that Smad7 also plays some roles in the regulation of the process. We sought to determine the relationships between Smad7 expression in bronchial biopsy samples from asthmatic subjects and clinicopathologic features. Bronchial biopsy specimens were obtained from 40 asthmatic subjects and 6 healthy control subjects. Expression levels of Smad7 on a histologic section were estimated by immunohistochemical staining. In addition, the roles of Smad7 in TGF-beta-mediated transcriptional responses were studied by in vitro studies. Smad7 immunoreactivity was detected mainly in bronchial epithelial cells in control and asthmatic subjects. Interestingly, asthmatic subjects exhibited less Smad7 immunoreactivity in bronchial epithelial cells than normal subjects. Expression levels of Smad7 in bronchial epithelial cells were inversely correlated with basement membrane thickness and airway hyperresponsiveness in asthmatic subjects. In addition, abrogation of endogenous Smad7 expression through use of an antisense oligonucleotide enhanced transcriptional responses to TGF-beta, whereas overexpression of Smad7 inhibited TGF-beta-induced plasminogen activator inhibitor 1production in a human bronchial epithelial cell line, BEAS2B cells. These findings suggest that Smad7 is a key molecule that defines the susceptibility of bronchial epithelial cells to TGF-beta action and that regulation of Smad7 expression in bronchial epithelial cells might be related to the development of airway remodeling and airway hyperresponsiveness in asthma.

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