Noncanonical STAT3 activation regulates excess TGF-β1 and collagen I expression in muscle of stricturing Crohn's disease.

Increased TGF-β1 and TGF-β1-dependent Collagen I production in intestinal mesenchymal cells result in fibrosis in patients with Montreal B2 fibrostenotic Crohn's disease. Numerous cytokines, including IL-6, are produced by activated mesenchymal cells themselves and activate STAT3. The aim of the current study was to determine the mechanisms by which STAT-3 activation might result in intestinal fibrosis. Cytokine levels were measured by ELISA. STAT3 and suppressor of cytokine signaling 3 protein levels were measured by immunoblot, STAT3-TGFB1 DNA-binding activity by chromatin immunoprecipitation, and TGFB1 transcriptional activity by luciferase reporter assay. TGF-β1 (TGFB1), Collagen1α1, and connective tissue growth factor (CTGF) gene expression was measured by quantitative RT-PCR. The role of STAT3 activation was determined using STAT3 inhibitor, Stattic, and by transfection of STAT3 mutants. Autocrine production of cytokines was increased in muscle cells of B2 phenotype patients from strictures and normal intestine in the same patient and compared with other Crohn's phenotypes, ulcerative colitis, and non-Crohn's patients. A unique pattern of STAT3 phosphorylation emerged: high STAT3(S727) and low STAT3(Y705) in strictures and the opposite in unaffected intestine. TGFB1 transcriptional activity was regulated by phospho-STAT3(S727) and was decreased by Stattic or dominant-negative STAT3(S727A). TGF-β1, COL1A1, and CTGF expression was inhibited by Stattic or dominant-negative STAT3(S727A). Treatment of normal muscle cells with IL-6 or expression of constitutively active STAT3(S727E) phenocopied muscle cells from strictured intestine. Neutralization of autocrine IL-6 reversed STAT3 phosphorylation and normalized expression of TGF-β1 in strictured intestinal muscle. The ability of Stattic to improve development of fibrosis was confirmed in mice with 2,4,6-trinitrobenzenesulfonic acid-induced colitis. We observed a unique phospho-STAT3(S727) response in patients with Montreal B2 Crohn's disease, particularly in response to IL-6 leading to increased TGF-β1, collagen, and CTGF production in ileal strictures.

Li C ，Iness A ，Yoon J ，Grider JR ，Murthy KS ，Kellum JM ，Kuemmerle JF ... -  《-》

Salirasib Inhibits the Expression of Genes Involved in Fibrosis in Fibroblasts of Systemic Sclerosis Patients.

Fibrosis is a principal sign of systemic sclerosis (SSc) which can affect several organs including the lung, heart, and dermis. Dermal fibroblasts of SSc patients are characterized by persistent and activated Ras and ERK1/2 signaling which stimulates extreme collagen and extracellular matrix synthesis. Salirasib is a Ras inhibitor that competitively prevents the adherence of GTP-bound Ras to the plasma membrane, that inhibits Ras signaling. This study intended to clarify whether salirasib can influence fibrotic mediators in SSc fibroblasts. Dermal fibroblasts from 10 SSc patients were treated with salirasib in the presence of TGF-β1, and mRNA levels of H-Ras and genes related to fibrosis, such as COL1A1, COL1A2, CTGF, TGF-β1, fibronectin, ACTA2, and MMP1 was measured by real-time PCR. The α-SMA protein expression was analyzed by immunofluorescence staining. In dermal fibroblasts of SSc patients, salirasib treatment, markedly downregulated the H-Ras gene expression. In addition, the protein expression of α-SMA and gene expression of ACTA2 were inhibited upon salirasib treatment. Salirasib also significantly reduced the expression of COL1A1, and COL1A2 genes and augmented the gene expression of MMP1. The mRNA levels of other genes related to fibrosis such as FN1, CTGF, and TGF-β1 were significantly decreased upon salirasib treatment. Considering salirasib significantly reduced the expression of genes related to the fibrosis process and α-SMA gene and protein expression, and given significant upregulation of MMP1 by salirasib, it can be considered as a new curative strategy for fibrotic diseases like SSc.

Sadeghi Shaker M ，Rokni M ，Kavosi H ，Enayati S ，Madreseh E ，Mahmoudi M ，Farhadi E ，Vodjgani M ... -  《Immunity Inflammation and Disease》

Angiotensin-converting Enzyme 2 Suppresses Pulmonary Fibrosis Associated with Wnt and TGF-β1 Signaling Pathways.

Tang Y ，Liu J ，Liu L 《-》

Biochanin A suppresses Klf6-mediated Smad3 transcription to attenuate renal fibrosis in UUO mice.

Renal fibrosis is a hallmark of chronic kidney disease (CKD). Smad3 serves as the principal transcription factor mediating the pro-fibrosis effects of TGF-β signaling in renal fibrosis. Biochanin A (BCA), a natural isoflavone, has been shown to attenuate renal fibrosis by inhibiting TGF-β signaling but the detailed mechanisms remain unresolved. This study aimed to elucidate the specific mechanisms by which BCA modulates TGF-β signaling. Renal fibrosis models were established both in vitro, using TGF-β1-stimulated mouse renal tubular TCMK1 cells, and in vivo, employing mice with unilateral ureter obstruction (UUO). RNA-seq was conducted to identify BCA-regulated genes. The AnimalTFDB4.0 database was utilized to predict transcription factors with potential binding to Smad3 promoter. The activities of TGF-β signaling and the cloned mouse Smad3 promoter were assessed using luciferase reporter assays. Plasmid transfection was performed using polyethylenimine in TCMK1 cells or ultrasound microbubbles in UUO kidneys. Gene expression was analyzed by RT-PCR, western blot, and immunohistochemistry assays. BCA significantly inhibited TGF-β signaling activity and suppressed TGF-β1-induced fibrotic gene expression in TCMK1 cells. RNA-seq and in silico analyses identified Smad3 as the key gene downregulated by BCA, while leaving Smad2 unaffected. This selective transcriptional suppression of Smad3 by BCA was validated by luciferase reporter assays using the cloned Smad3 promoter. Furthermore, transcription factor binding prediction identified that Klf6, a transcription factor downregulated by BCA, has binding potential to the Smad3 promoter and promotes Smad3 transcription. Klf6 expression was induced in TGF-β1-stimulated TCMK1 cells and UUO kidneys, but this induction was abolished upon BCA treatment. Importantly, Klf6 overexpression restored Smad3 expression and counteracted the anti-fibrosis effects of BCA in both TGF-β1-stimulated TCMK1 cells and UUO kidneys. TGF-β-responsive Klf6 transcriptionally transactivates Smad3 expression. BCA exerts anti-renal fibrosis effects by inhibiting the Klf6-Smad3 signaling axis, underscoring its therapeutic potential in the treatment of CKD.

Li YQ ，Yu XM ，Shang XM ，Lin JY ，Tan RZ ，Li JC ，Su HW ，Shen HP ，Wang HL ，Wang L ... -  《-》

Characterization of Epithelial-Mesenchymal and Neuroendocrine Differentiation States in Pancreatic and Small Cell Ovarian Tumor Cells and Their Modulation by TGF-β1 and BMP-7.

Ungefroren H ，von der Ohe J ，Braun R ，Gätje Y ，Lapshyna O ，Schrader J ，Lehnert H ，Marquardt JU ，Konukiewitz B ，Hass R ... -  《Cells》