Project description:Study questionDo all 10 human pregnancy-specific beta 1-glycoproteins (PSGs) and murine PSG23 activate latent transforming growth factor-β1 (TGF-β1)?Summary answerAll human PSGs and murine PSG23 activated latent TGF-β1.What is known alreadyTwo of the 10 members of the PSG1 family, PSG1 and PSG9, were previously shown to activate the soluble small latent complex of TGF-β1, a cytokine with potent immune suppressive functions.Study design, size, durationRecombinant PSGs were generated and tested for their ability to activate the small latent complex of TGF-β1 in a cell-free ELISA-based assay and in a bioassay. In addition, we tested the ability of PSG1 and PSG4 to activate latent TGF-β bound to the extracellular matrix (ECM) or on the membranes of the Jurkat human T-cell line.Participants/materials, setting, methodsRecombinant PSGs were generated by transient transfection and purified with a His-Trap column followed by gel filtration chromatography. The purified PSGs were compared to vehicle (PBS) used as control for their ability to activate the small latent complex of TGF-β1. The concentration of active TGF-β was measured in an ELISA using the TGF-β receptor II as capture and a bioassay using transformed mink epithelial cells that express luciferase in response to active TGF-β. The specificity of the signal was confirmed using a TGF-β receptor inhibitor. We also measured the binding kinetics of some human PSGs for the latent-associated peptide (LAP) of TGF-β using surface plasmon resonance and determined whether PSG1 and PSG4 could activate the large latent complex of TGF-β1 bound to the ECM and latent TGF-β1 bound to the cell membrane. All experiments were performed in triplicate wells and repeated three times.Main results and the role of chanceAll human PSGs activated the small latent complex of TGF-β1 (P < 0.05 vs. control) and showed similar affinities (KD) for LAP. Despite the lack of sequence conservation with its human counterparts, the ability to activate latent TGF-β1 was shared by a member of the murine PSG family. We found that PSG1 and PSG4 activated the latent TGF-β stored in the ECM (P < 0.01) but did not activate latent TGF-β1 bound to glycoprotein A repetitions predominant (GARP) on the surface of Jurkat T cells.Limitations, reasons for cautionThe affinity of the interaction of LAP and PSGs was calculated using recombinant proteins, which may differ from the native proteins in their post-translational modifications. We also utilized a truncated form of murine PSG23 rather than the full-length protein. For the studies testing the ability of PSGs to activate membrane-bound TGF-β1, we utilized the T-cell line Jurkat and Jurkat cells expressing GARP rather than primary T regulatory cells. All the studies were performed in vitro.Wider implications of the findingsHere, we show that all human PSGs activate TGF-β1 and that this function is conserved in at least one member of the rodent PSG family. In vivo PSGs could potentially increase the availability of active TGF-β1 from the soluble and matrix-bound latent forms of the cytokine contributing to the establishment of a tolerogenic environment during pregnancy.Large-scale dataNone.Study funding/competing interest(s)The research was supported by a grant from the Collaborative Health Initiative Research Program (CHIRP). No conflicts of interests are declared by the authors.

Project description:Background: Increased Galectin 3-binding protein (Lgals3bp) serum levels have been used to assess hepatic fibrosis stages and the severity of hepatocellular carcinoma (HCC). Considering the crucial role of transforming growth factor-β1 (TGF-β1) in the emergence of these diseases, the present study tested the hypothesis that Lgals3bp regulates the TGF-β1 signaling pathway. Methods: The expression levels of Lgals3bp and TGF-β1 were analyzed in patients with non-alcoholic steatohepatitis (NASH) and HCC. Multiple omics techniques, such as RNA-sequencing, transposase-accessible chromatin-sequencing assay, and liquid chromatography-tandem mass spectrometry proteomics, were used to identify the regulatory mechanisms for the Lgals3bp-TGF-β1 axis. Moreover, the effects of altered TGF-β1 signaling in chronic inflammatory conditions were investigated in conditional Lgals3bp-knockin and Lgals3bp-knockout mice. Results: In patients with NASH and HCC, the levels of Lgals3bp and TGF-β1 exhibited positive correlations. Stimulation of Lgals3bp by the inflammatory cytokine interferon α in HCC cells or ectopic overexpression of Lgals3bp in hepatocytes promoted the expression levels of TGFB1. Aggravated fibrosis was observed in the livers of hepatocyte-specific Lgals3bp knock-in mice, with increased TGF-β1 levels. Lgals3bp directly bound to and assembled integrin αV, an integral mediator required for releasing active TGF-β1 from extracellular latent complex with the rearranged F-actin cytoskeleton. The released TGF-β1 activated JunB transcription factor, which in turn promoted the TGF-β1 positive feedback loop. Lgals3bp deletion in the hepatocytes downregulated TGF-β1 signaling and CCl4 induced fibrosis. Finally, Lgals3bp depletion hindered hepatocarcinogenesis by limiting the availability of fibrogenic TGF-β1. Conclusion: Lgals3bp plays a crucial role in hepatic fibrosis and carcinogenesis by controlling the TGF-β1 signaling pathway, making it a promising therapeutic target in TGF-β1-related diseases.

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Profiling of concanavalin A-binding glycoproteins in human hepatic stellate cells activated with transforming growth factor-β1.

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