TSC-22 directly interacts with TβRI. (A) TGF-β enhances the TβRI-TSC-22 interaction at the endogenous protein level. HEK293 cells were treated with or without 100 pM TGF-β1 for 2 h and harvested for immunoprecipitation (IP) with control rabbit IgG or anti-TβRI antibody followed by anti-TSC-22 immunoblotting (IB). WCL, whole-cell lysate. (B) Direct interaction between TSC-22 and the intracellular domain (ICD) of TβRI. Purified recombinant GST-TSC-22 and TβRI-ICD (1 μg each) were subjected to GST pulldown assays. (C) TSC-22 associates with the type I receptors of the TGF-β family. After transfection with the indicated constructs (2 μg each) for 48 h, HEK293T cells were harvested for anti-hemagglutinin (anti-HA) immunoprecipitation (IP) followed by anti-Myc immunoblotting (IB). Protein expression was examined with whole-cell lysates (WCL) by immunoblotting. (D) TSC-22 associates with the type II receptors of the TGF-β family. (E) TGF-β and TβRI enhance the association between TSC-22 and TβRII. After transfection with indicated constructs for 48 h, HEK293T cells were treated with 100 pM TGF-β1 for 2 h before being harvested for coimmunoprecipitation. (F) TSC-22 forms a ternary complex with TβRI and TβRII. HEK293T cells transfected with indicated constructs were harvested for the first immunoprecipitation with anti-Flag antibody. The immunocomplex was then eluted by the use of Flag peptide and subjected to anti-HA immunoprecipitation followed by immunoblotting (right panel). Aliquots of whole-cell lysates and the first immunoprecipitants were analyzed by immunoblotting (left and middle panels). (G) TSC-22 substantially colocalizes with TβRI along the plasma membrane. HEK293T cells transfected with TSC-22-enhanced GFP (TSC-22-EGFP) and myc-TβRI were treated with 100 pM TGF-β1 for 2 h before being harvested for immunofluorescence analysis. TSC-22 localization was detected by direct EGFP immunofluorescence, and TβRI localization was determined indirectly using anti-myc antibody followed by a tetramethyl rhodamine isocyanate (TRITC)-conjugated second antibody and visualized under a fluorescence microscope. Colocalization was measured by IPP software; yellow coloring indicates the colocalization. (H) Schematic diagram of TSC-22 and its mutants. (I) The N terminus of TSC-22 mediates its interaction with ca-TβRI. The experiment was performed as described for panel A.

TSC-22 attenuates Smad7/Smurf-induced TβRI ubiquitination and degradation. (A) TSC-22 inhibits Smurf1/2-induced ubiquitination of TβRI. HEK293T cells transfected with the indicated constructs (2 μg each) were treated with 50 μM MG132 for 4 h before being harvested for precipitation with nickel beads followed by anti-HA immunoblotting. (B) TSC-22 attenuates Smad7/Smurf-induced turnover of TβRI. HEK293T cells were transfected with the indicated constructs, treated with 50 μg/ml cycloheximide (CHX) for the indicated time periods, and then harvested for anti-HA immunoblotting. The results were quantified by the use of Bandscan software. (C) TSC-22 knockdown enhances the association of Smad7 with TβRI. HEK293T cells were transfected with nonspecific or TSC-22 shRNA, treated with 100 pM TGF-β1 for 2 h, and harvested for coimmunoprecipitation. (D) TSC-22 impairs the association of Smad7 with TβRI. At 48 h posttransfection with the indicated constructs, HEK293T cells were harvested for coimmunoprecipitation assays. (E) Smad7 inhibits the association between TβRI and TSC-22. (F) TβRI decreases the binding of TSC-22 to Smad7. (G) TSC-22 associates with TβRI and Smad7 in different complexes. The experiment was performed as described for Fig. 2F. (H) TSC-22 impairs the association of Smurf2 (C716A) with TβRI in HEK293T cells. (F) Association of TSC-22 and Smad7 with TβRI. HaCaT cells treated with 100 pM TGF-β1 for the indicated time were harvested for coimmunoprecipitation assays.

TSC-22 is upregulated in ISO-induced myocardial fibrosis. (A) Masson staining analysis of the left ventricles of rat hearts. Blue staining indicates collagen fibers. (B) Upregulation of α-SMA and phospho-Smad2 in the left ventricles of ISO hearts as shown by immunohistochemistry. IHC, immunohistochemistry. (C) Upregulation of α-SMA, TSC-22, phospho-Smad2, and phospho-Smad3 in ISO hearts as shown by immunoblotting. (D) mRNA expression in the left ventricles of rat hearts. Total RNA isolated from the left ventricles (LV) was subjected to quantitative RT-PCR. Ctr, control; ColI, collagen I.

TSC-22 promotes TGF-β signaling. (A to C) TSC-22 increases TGF-β-induced expression of reporter genes in HaCaT cells. HaCaT cells transfected with various reporters (CAGA-luciferase [CAGA-Luc] [200 ng] [A], ARE-luciferase [300 ng]/FAST2 [150 ng] [B], and 3TP-luciferase [200 ng] [C]) together with Renilla luciferase (20 ng) and TSC-22 (100 or 200 ng) were treated with 100 pM TGF-β1 for 20 h and harvested for luciferase activity measurements. (D) TSC-22 increases TGF-β-induced expression of ARE-luciferase reporter in HEK293 cells. (E) TSC-22 enhances expression of TGF-β target genes in HaCaT cells. After infection with TSC-22 lentivirus, HaCaT cells were treated with TGF-β1 for 20 h and then harvested for quantitative RT-PCR. (F) TSC-22 enhances TGF-β-induced phospho-Smad2/3 levels. After infection with TSC-22 lentivirus, HaCaT cells were treated with TGF-β1 for the indicated times. Phospho-Smad2/3 and protein expression levels were analyzed by immunoblotting. (G) TSC-22 knockdown reduces TGF-β-induced expression of reporters. HEK293 cells were transfected with various reporters as described for panels A and B and with nonspecific (NS) shRNA or TSC-22-specific shRNA (50 ng). After treatment with 100 pM TGF-β1 for 20 h, the cells were harvested for luciferase activity measurements. (H) TSC-22 knockdown reduces TGF-β-mediated phospho-Smad2 level in HEK293 cells. (I) TSC-22 increases BMP4-induced expression of BRE-luciferase in HEK293 cells. HEK293 cells transfected with plasmids encoding BRE-luciferase (200 ng), Renilla luciferase (20 ng), and TSC-22 (100 ng or 200 ng) were treated with 10 ng of BMP4/ml for 20 h and harvested for luciferase determinations. (J) TSC-22 enhances reporter expression induced by TβRI but not by Smads. L17 cells transfected with the reporter and wt-TβRI, ca-TβRI (50 ng), Smad3/4 (25 ng each), or TSC-22 (100 or 200 ng) were treated with 100 pM TGF-β1 for 20 h before being harvested for luciferase determinations. In all of the reporter assay experiments, empty vectors were used to equalize the total amounts of plasmids in each sample. Each experiment was performed in triplicate, and the data are presented as means ± SD after normalization to Renilla activity.

TSC-22 interferes with the inhibitory effect of Smad7/Smurfs on TGF-β signaling. (A and B) TGF-β/BMP enhances the interaction of TSC-22 with Smad6/7. HEK293T cells transfected with the indicated constructs (2 μg each) were treated with 100 pM TGF-β1 or 400 pM BMP4 for 2 h and harvested for coimmunoprecipitation. (C) TGF-β induces endogenous TSC-22-Smad7 interaction. HEK293 cells were treated with or without 100 pM TGF-β1 for 2 h before being harvested for coimmunoprecipitation. (D) TSC-22 attenuates the inhibitory effect of Smad7/Smurfs on TGF-β-induced expression of CAGA-luciferase. HEK293 cells were transfected with plasmids encoding CAGA-luciferase (200 ng), Renilla luciferase (20 ng), Smad7 (50 ng), Smurf1 (20 ng), Smurf2 (20 ng), and TSC-22 (100 or 200 ng) and then treated with 100 pM TGF-β1 for 20 h before being harvested for luciferase assays. (E) Smad7 is required by TSC-22 to promote TGF-β signaling. HEK293 cells were transfected with reporters, shRNA (50 ng), and TSC-22 (100 or 200 ng) and treated with or without 100 pM TGF-β1 for 20 h and harvested for luciferase assays. NS, nonspecific. (F) The function of TSC-22 in regulating TGF-β signaling depends on Smad7. Reporter assays were carried out with Smad7^+/+ or Smad7^−/− MEFs. (G and H) HEK293T cells were transfected with the indicated constructs for 48 h before being harvested for coimmunoprecipitation assays. (I) Reporter assays were performed as described for panel D.

TSC-22 promotes TGF-β-induced cardiac myofibroblast differentiation. (A) TGF-β upregulates the expression of TSC-22, PAI-1, and α-SMA in primary cardiac fibroblasts from neonatal rats. After treatment with TGF-β1 (100 or 300 pM) for 20 h, the cells were harvested for quantitative RT-PCR. (B) TGF-β enhances the protein levels of TSC-22, PAI-1, and α-SMA in primary rat cardiac fibroblasts. Cells were treated with 100 pM TGF-β1 for 20 h and harvested for immunoblotting. (C) Overexpression of TSC-22 promotes TGF-β-induced cardiac myofibroblast differentiation. Primary rat cardiac fibroblasts were infected with TSC-22 lentivirus. After 24 h, the cells were treated with 100 pM TGF-β1 for 20 h and harvested for quantitative RT-PCR. (D) Knockdown of TSC-22 inhibits TGF-β-induced cardiac myofibroblast differentiation. Primary rat cardiac fibroblasts were transfected with nonspecific or TSC-22 shRNA. At 24 h posttransfection, the cells were treated with TGF-β1 and harvested for quantitative RT-PCR. ColI, collagen I.