Journal: Cell Death & Disease

Article Title: TDAG51 induces renal interstitial fibrosis through modulation of TGF-β receptor 1 in chronic kidney disease

doi: 10.1038/s41419-021-04197-3

Figure Lengend Snippet: A Rat renal fibroblasts were treated with TM and the IRE1 endonuclease inhibitor 4μ8c. TM-induced splicing of XBP1, which was prevented by 4μ8c treatment. B Renal fibroblasts were treated with TGF-β1 (Tβ) and ascorbic acid (AA2P; AA) to induce XBP1 splicing. Individually, TGF-β1 and AA2P increased XBP1 splicing, but the effect was increased with combined treatment. Co-treatment with 4μ8c was able to prevent TGF-β1-mediated XBP1 splicing. C Renal fibroblasts treated with TGF-β1 and AA2P show increased collagen deposition, which was inhibited with 4μ8c co-treatment. Bar = 100 μm. * P < 0.05 vs veh; *** P < 0.001 vs veh; #, P < 0.05 vs AA2P + TGF-β1. D Western blotting demonstrates that expression of TGF-β1 receptor (TGFβ-R1) is significantly reduced in kidneys from WT mice with CKD. Kidneys from TDKO mice with or without CKD do not express TGFβ-R1. * P < 0.05 vs sham; #, P < 0.05 vs WT. E Renal fibroblasts were treated with TGF-β1 and AA2P with or without SB431542 (SB), a TGFβ-R1 inhibitor. While TGF-β1 and AA2P increased mRNA levels of spliced XBP1, co-treatment with SB431542 inhibited XBP1 splicing. * P < 0.05 vs veh; #, P < 0.05 vs Tβ + AA. F HK-2 cells were treated with thapsigargin for varying time points (0, 18, 24, 48 h) and subsequently underwent western blotting. Thapsigargin induced TDAG51 expression at 18 and 24 h, but not 48 h. TGFβ-R1 was increased after 18 h of TG, but the expression was reduced at 24 and 48 h. * P < 0.05 vs 0 h.

Article Snippet: Antibodies to TDAG51 (sc-23866; Santa Cruz Biotechnology; Santa Cruz, CA, USA) and CHOP (sc-7351; Santa Cruz) were diluted 1:200, KDEL (SPA-827; Stressgen; Burlington, Canada) was diluted 1:1000 (used against the KDEL amino acid sequence of GRP78), TGFβ-R1 (MAB587; R&D systems; Oakville, Canada) was diluted 1:500, and β-actin (66009-1; ProteinTech; Rosemont, IL, USA) was diluted 1:5000.

Techniques: Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: TDAG51 induces renal interstitial fibrosis through modulation of TGF-β receptor 1 in chronic kidney disease

doi: 10.1038/s41419-021-04197-3

Figure Lengend Snippet: The induction of CKD, caused by reduced renal mass, increased angiotensin II and aldosterone, and increased salt consumption, triggers endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR). This amplifies TDAG51 expression in the kidney. The elevated levels of TDAG51 triggers a pro-apoptotic signaling pathway and a pro-fibrotic signaling pathway. In the pro-apoptotic pathway, TDAG51 induces expression of CHOP, which activates pro-apoptotic genes and, eventually, programmed cell death. In the pro-fibrotic pathway, TDAG51 increases expression of TGF-β receptor 1, which leads to the splicing of XBP1. Spliced XBP1 leads to the activation of pro-fibrotic genes, and the development of renal interstitial fibrosis.

Article Snippet: Antibodies to TDAG51 (sc-23866; Santa Cruz Biotechnology; Santa Cruz, CA, USA) and CHOP (sc-7351; Santa Cruz) were diluted 1:200, KDEL (SPA-827; Stressgen; Burlington, Canada) was diluted 1:1000 (used against the KDEL amino acid sequence of GRP78), TGFβ-R1 (MAB587; R&D systems; Oakville, Canada) was diluted 1:500, and β-actin (66009-1; ProteinTech; Rosemont, IL, USA) was diluted 1:5000.

Techniques: Activation Assay, Expressing

Journal: Cancer medicine

Article Title: LY6K depletion modulates TGF-β and EGF signaling.

doi: 10.1002/cam4.5940

Figure Lengend Snippet: FIGURE 3 Lymphocyte antigen 6 complex locus K (LY6K) binds TGF-β receptor-I (TβRI) but not EGF receptor (EGFR) and regulates transforming growth factor-β (TGF-β) and epidermal growth factor (EGF) signaling pathways. (A) Time course of changes in Smad signaling in LY6K-depleted cells. The intensities of p-Smad2 and 3 band were quantified with ImageJ software. (B) Cells transfected with 20 ng/L of siLY6K for 48 h were incubated with 5 ng/mL of TGF-β and 10 ng/mL of EGF for 1 and 24 h. The intensities of p-EGFR, p-AKT, p-ERK, p-Smad2, and LY6K band were quantified with ImageJ software. (C) HEK293T cells were transfected with Myc-LY6K or/and mCherry- TβRI and LY6K immunoprecipitated with anti-LY6K antibody; levels of LY6K and TβRI were examined. (D, E) HeLa and SiHa cells were incubated with 5 ng/mL of TGF-β (D) and 10 ng/mL of EGF (E) for 1 h, and LY6K immunoprecipitated with anti-LY6K antibody. LE, long exposure; SE, short exposure.

Article Snippet: Human TGFβR1 cloned in mCherry- N1 vector (#54969) was purchased from Addgene. pCMV6- LY6K was a gift from Prof. Jong Hoon Park, Sookmyung Women's University.

Techniques: Protein-Protein interactions, Software, Transfection, Incubation, Immunoprecipitation

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Mechanistic interrogation of mutation-independent disease modulators of RDEB identifies the small leucine-rich proteoglycan PRELP as a TGF-β antagonist and inhibitor of fibrosis.

doi: 10.1016/j.matbio.2022.06.007

Figure Lengend Snippet: Fig. 6. PRELP counteraction of the pro-fibrotic and activated phenotype in RDEBFs by TGF-b1 signaling inhibition. (A) Interaction of PRELP with TGF-b1, TGF-b receptor 1 (TGFBRI), and TGF-b receptor 2 (TGFBRII) was analyzed by a solid-phase binding assay (n = 3 technical replicates). Endorepellin/Perlecan was used as a positive control. Results are shown according to the absorbance values (left panel) and the percentage of maximum possible binding (right panel). The molar concentration required for 50% maximal binding of the soluble partner to the immobilized protein is representa- tive of the binding affinity (Ka). (B) Western blot analysis of p-Smad2/3 and TNC induction shows a decreased TGF-b1 response in S-RDEBFs overexpressing PRELP (lv.PRELP), as compared to fibroblasts transduced with a control lentivi- ral vector (lv.GFP). A representative WB (upper panel) and densitometric quantification (lower panel) of independent

Article Snippet: PRELPbound partners were detected using antibodies against TGF-b1 (sc-146, Santa Cruz Biotechnology), TGFBRI (AF3025, R&D Systems), TGFBRII (AF-241, R&D Systems), and Endorepellin/Perlecan (AF2364, R&D Systems).

Techniques: Inhibition, Binding Assay, Positive Control, Concentration Assay, Western Blot, Transduction, Control, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation

doi: 10.1074/jbc.M117.805747

Figure Lengend Snippet: Identification of FZD4, FZD6, and TGFBR1 as miR-101 targets. a, 3′-UTR reporter assay: FZD4-UTR-S1 (position 1–1,432) containing miR-101-binding site 1 (position 537–543), FZD4-UTR-S2 (position 4,178–5,463) containing miR-101-binding site 2 (position 1,240–1,246), full length of FZD6-UTR, TGFBR1-UTR-S1 (position 66–1,524) containing miR-101-binding site 1 (position 460–466), or TGFBR1-UTR-S2 (position 3,210–4,749) containing miR-101-binding site 2 (position 3,993–3,999) were co-transfected into HEK 293T cells with miR-101-1. The relative luciferase activities were measured using the Dual-Luciferase® reporter assay system. n = 3. b and c, effect of overexpressing or knockdown miR-101 on the protein expression of FZD4, FZD6, and TGFBR1 in fibroblasts. LL29 cells were treated with a lentivirus expressing miR-101-1 or the virus control (VC) at an m.o.i. of 50 for 48 h (b) or CCD-8Lu fibroblasts were treated with a lentivirus expressing anti-miR-101 or the virus control (Anti-CON) at an m.o.i. of 50 for 72 h. c, Western blotting was performed to determine the protein expression of FZD4, FZD6, and TGFBR1. The protein samples used here were the same as mentioned in Fig. 7c. The GAPDH immunoblot shown in Fig. 7c was used again here. d, overexpression of miR-101 inhibits FZD4, FZD6, and TGFBR1 mRNA expression in fibroblasts. LL29 cells were treated with lentiviral miR-101 or the virus control at an m.o.i. of 50 for 48 h. The mRNA expression of FZD4, FZD6, and TGFBR1 was determined by real-time PCR and normalized to GAPDH. The results are presented as the mean ± S.E. n = 3. e, real-time PCR showing the increased mRNA levels of FZD4, FZD6, and TGFBRR1 by anti-miR-101. CCD-8Lu fibroblasts were treated with lentiviral anti-miR-101 or the virus control at an m.o.i. of 50 for 72 h. The expression levels were relative to GAPDH from two cell preparations, each performed in duplicate. f–h, mRNA expression of FZD4, FZD6, and TGFBR1 in IPF patient lungs. The mRNA levels of FZD4, FZD6, and TGFBR1 were determined by real-time PCR and normalized to β-actin. n = 8–10. The results are presented as the mean ± S.E. Statistical analyses were performed by using Student's t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: FZD4, FZD6, and TGFBR1 expression vectors The open reading frame of FZD4, FZD6, and TGFBR1 was amplified by PCR using specific primers ( ) from mouse cDNA clones purchased from OriGene (FZD4, catalogue no. MC203860; FZD6, catalogue no. MC204555; TGFBR1, catalogue no. MR20891).

Techniques: Reporter Assay, Binding Assay, Transfection, Luciferase, Knockdown, Expressing, Virus, Control, Western Blot, Over Expression, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation

doi: 10.1074/jbc.M117.805747

Figure Lengend Snippet: Overexpression of FZD4, FZD6, or TGFBR1 rescues miR-101-mediated inhibition of fibroblast proliferation and activation. a–d, knockdown of TGFBR1 repressed the TGF-β1-induced mRNA expression of α-SMA, COL1A1, COL3A1, and COL4A1 in fibroblasts. LL29 cells were treated with lentivirus shRNA-TGFBR1 or the virus control at an m.o.i. of 50 for 48 h. Then, the cells were stimulated with 5 ng/ml TGF-β1 for 48 h. The α-SMA, COL1A1, COL3A1, and COL4A1 mRNA expression levels were determined by real-time PCR. The expression levels were relative to GAPDH from two cell preparations, each performed in duplicate. The results are presented as the mean ± S.D. **, p < 0.01. e and f, overexpression of FZD4 or FZD6 rescues the miR-101-mediated inhibition of fibroblast proliferation. LL29 cells were split into 96-well plates at 3,000 cells per well. After a 24-h culture, cells were infected with lentiviruses at a total of m.o.i. = 50 (miR-Con miR-101, GFP, FZD4, and FZD6 at m.o.i. = 25 each, virus control (VC) = miR-Con + GFP) for 48 h. Cells were starved for another 24 h and stimulated with WNT5a (1 μg/ml) for 12 h. FZD4 and FZD6 expression was determined by Western blotting by using anti-GFP antibody. Cell proliferation was determined by BrdU assay. Data were presented as mean ± S.E. Statistical analysis was performed by ANOVA and followed by Tukey's HSD test. n = 4. **, p < 0.01. g–j, overexpression of TGFBR1 rescues miR-101-mediated inhibition of α-SMA, COL1A1, and COL3A1 mRNA expression. LL29 cells were infected with lentiviruses at a total of m.o.i. = 50 (miR-Con, miR-101, GFP, and TGFBR1 at m.o.i. = 25 each, virus control = miR-Con + GFP) for 48 h. Cells were then stimulated with TGFβ1 (5 ng/ml) for 48 h. Cells were collected for real-time PCR. The results are presented as the mean ± S.E., n = 3. ANOVA followed by Tukey's HSD test was performed. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: FZD4, FZD6, and TGFBR1 expression vectors The open reading frame of FZD4, FZD6, and TGFBR1 was amplified by PCR using specific primers ( ) from mouse cDNA clones purchased from OriGene (FZD4, catalogue no. MC203860; FZD6, catalogue no. MC204555; TGFBR1, catalogue no. MR20891).

Techniques: Over Expression, Inhibition, Activation Assay, Knockdown, Expressing, shRNA, Virus, Control, Real-time Polymerase Chain Reaction, Infection, Western Blot, BrdU Staining

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation

doi: 10.1074/jbc.M117.805747

Figure Lengend Snippet: Adenovirus-mediated miR-101 gene transfer attenuates bleomycin-induced pulmonary fibrosis. On day 1, an adenovirus expressing miR-101-1 or the control adenovirus (VC) (5 × 109 pfu per mouse) was delivered into the lungs of 6–8-week-old C57BL/6 mice by nasal instillation. On day 2, bleomycin (Bleo) (0.06 units per mouse) or the same volume of saline (Sal) was intranasally instilled into the lungs. On day 15, the mice were subjected to the analysis of respiratory mechanics by Flexivent and then sacrificed. The left lungs were collected for RNA and protein analysis, and the right lungs were fixed for histological analysis. a, real-time PCR analysis showing adenovirus-mediated overexpression of miR-101 in the mouse lung. The expression levels were relative to U6. b, H&E staining showing the fibrotic changes in the mouse lung induced by bleomycin. miR-101 attenuated the fibrotic changes in bleomycin-treated mouse lungs. Scale bar, 100 μm. c, Ashcroft score grade of pulmonary fibrosis. d, lung collagen content as determined using the QuickZyme hydroxyproline assay kit. e and f, real-time PCR analysis showing the increased COL1A1 and COL3A1 mRNA levels in bleomycin-treated mouse lungs and miR-101 suppression of the increase of COL1A1 and COL3A1 mRNA levels. The expression levels were relative to GAPDH. g and h, analysis of lung mechanics by Flexivent analysis. Elastance (Ers) was measured in a single-compartment model. h was measured in the Constant-Phase model. i and j, NFATc2, FZD4, FZD6, and TGFBR1 mRNA expression was increased in bleomycin-treated mouse lungs, and the increase was suppressed by miR-101 treatment as determined by real-time PCR analysis. The expression level was relative to GAPDH. n = 5 for saline + VC and saline + miR-101; n = 7–10 for bleomycin + VC and bleomycin + miR-101. The results are presented as the mean ± S.E. Statistical analyses were performed by using Student's t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: FZD4, FZD6, and TGFBR1 expression vectors The open reading frame of FZD4, FZD6, and TGFBR1 was amplified by PCR using specific primers ( ) from mouse cDNA clones purchased from OriGene (FZD4, catalogue no. MC203860; FZD6, catalogue no. MC204555; TGFBR1, catalogue no. MR20891).

Techniques: Expressing, Control, Saline, Real-time Polymerase Chain Reaction, Over Expression, Staining, Hydroxyproline Assay

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation

doi: 10.1074/jbc.M117.805747

Figure Lengend Snippet: Primers for the construction of plasmids FW is forward, and RE is reverse.

Article Snippet: FZD4, FZD6, and TGFBR1 expression vectors The open reading frame of FZD4, FZD6, and TGFBR1 was amplified by PCR using specific primers ( ) from mouse cDNA clones purchased from OriGene (FZD4, catalogue no. MC203860; FZD6, catalogue no. MC204555; TGFBR1, catalogue no. MR20891).

Techniques: Over Expression