Noncanonical WNT5A controls the activation of latent TGF-ß to drive fibroblast activation and tissue fibrosis.

Transforming growth factor ß (TGF-ß) signaling is a core pathway of fibrosis, but the molecular regulation of the activation of latent TGF-ß remains incompletely understood. Here, we demonstrate a crucial role of WNT5A/JNK/ROCK signaling that rapidly coordinates the activation of latent TGF-ß in fibrotic diseases. WNT5A was identified as a predominant noncanonical WNT ligand in fibrotic diseases such as systemic sclerosis, sclerodermatous chronic graft-versus-host disease, and idiopathic pulmonary fibrosis, stimulating fibroblast-to-myofibroblast transition and tissue fibrosis by activation of latent TGF-ß. The activation of latent TGF-ß requires rapid JNK- and ROCK-dependent cytoskeletal rearrangements and integrin αV (ITGAV). Conditional ablation of WNT5A or its downstream targets prevented activation of latent TGF-ß, rebalanced TGF-ß signaling, and ameliorated experimental fibrosis. We thus uncovered what we believe to be a novel mechanism for the aberrant activation of latent TGF-ß in fibrotic diseases and provided evidence for targeting WNT5A/JNK/ROCK signaling in fibrotic diseases as a new therapeutic approach.

Attenuation of fibroblast activation and fibrosis by adropin in systemic sclerosis.

Fibrotic diseases impose a major socioeconomic challenge on modern societies and have limited treatment options. Adropin, a peptide hormone encoded by the energy homeostasis-associated (ENHO) gene, is implicated in metabolism and vascular homeostasis, but its role in the pathogenesis of fibrosis remains enigmatic. Here, we used machine learning approaches in combination with functional in vitro and in vivo experiments to characterize adropin as a potential regulator involved in fibroblast activation and tissue fibrosis in systemic sclerosis (SSc). We demonstrated consistent down-regulation of adropin/ENHO in skin across multiple cohorts of patients with SSc. The prototypical profibrotic cytokine TGFß reduced adropin/ENHO expression in a JNK-dependent manner. Restoration of adropin signaling by therapeutic application of bioactive adropin34-76 peptides in turn inhibited TGFß-induced fibroblast activation and fibrotic tissue remodeling in primary human dermal fibroblasts, three-dimensional full-thickness skin equivalents, mouse models of bleomycin-induced pulmonary fibrosis and sclerodermatous chronic graft-versus-host-disease (sclGvHD), and precision-cut human skin slices. Knockdown of GPR19, an adropin receptor, abrogated the antifibrotic effects of adropin in fibroblasts. RNA-seq demonstrated that the antifibrotic effects of adropin34-76 were functionally linked to deactivation of GLI1-dependent profibrotic transcriptional networks, which was experimentally confirmed in vitro, in vivo, and ex vivo using cultured human dermal fibroblasts, a sclGvHD mouse model, and precision-cut human skin slices. ChIP-seq confirmed adropin34-76-induced changes in TGFß/GLI1 signaling. Our study characterizes the TGFß-induced down-regulation of adropin/ENHO expression as a potential pathomechanism of SSc as a prototypical systemic fibrotic disease that unleashes uncontrolled activation of profibrotic GLI1 signaling.

X-linked inhibitor of apoptosis protein (XIAP) inhibition in systemic sclerosis (SSc).

OBJECTIVE: X-linked inhibitor of apoptosis protein (XIAP) is a multifunctional protein with important functions in apoptosis, cellular differentiation and cytoskeletal organisation and is emerging as potential target for the treatment of various cancers. The aim of the current study was to investigate the role of XIAP in the pathogenesis of systemic sclerosis (SSc). METHODS: The expression of XIAP in human skin samples of patients with SSc and chronic graft versus host disease (cGvHD) and healthy individuals was analysed by quantitative PCR, immunofluorescence (IF) and western blot. XIAP was inactivated by siRNA-mediated knockdown and pharmacological inhibition. The effects of XIAP inactivation were analysed in cultured fibroblasts and in the fibrosis models bleomycin-induced and topoisomerase-I-(topoI)-induced fibrosis and in Wnt10b-transgenic mice. RESULTS: The expression of XIAP, but not of other inhibitor of apoptosis protein family members, was increased in fibroblasts in SSc and sclerodermatous cGvHD. Transforming growth factor beta (TGF-ß) induced the expression of XIAP in a SMAD3-dependent manner. Inactivation of XIAP reduced WNT-induced fibroblast activation and collagen release. Inhibition of XIAP also ameliorated fibrosis induced by bleomycin, topoI and overexpression of Wnt10b in well-tolerated doses. The profibrotic effects of XIAP were mediated via WNT/ß-catenin signalling. Inactivation of XIAP reduces binding of ß-catenin to TCF to in a TLE-dependent manner to block WNT/ß-catenin-dependent transcription. CONCLUSIONS: Our data characterise XIAP as a novel link between two core pathways of fibrosis. XIAP is overexpressed in SSc and cGvHD in a TGF-ß/SMAD3-dependent manner and in turn amplifies the profibrotic effects of WNT/ß-catenin signalling on fibroblasts via transducin-like enhancer of split 3. Targeted inactivation of XIAP inhibits the aberrant activation of fibroblasts in murine models of SSc.

Targeting of canonical WNT signaling ameliorates experimental sclerodermatous chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD) is a major life-threatening complication of allogeneic hematopoietic stem cell transplantation. The molecular mechanisms underlying cGVHD remain poorly understood, and targeted therapies for clinical use are not well established. Here, we examined the role of the canonical WNT pathway in sclerodermatous cGVHD (sclGVHD). WNT signaling was activated in human sclGVHD with increased nuclear accumulation of the transcription factor ß-catenin and a WNT-biased gene expression signature in lesional skin. Treatment with the highly selective tankryase inhibitor G007-LK, the CK1α agonist pyrvinium, or the LRP6 inhibitor salinomycin abrogated the activation of WNT signaling and protected against experimental cGVHD, without a significant impact on graft-versus-leukemia effect (GVL). Treatment with G007-LK, pyrvinium, or salinomycin almost completely prevented the development of clinical and histological features in the B10.D2 (H-2d) → BALB/c (H-2d) and LP/J (H-2b) → C57BL/6 (H-2b) models of sclGVHD. Inhibition of canonical WNT signaling reduced the release of extracellular matrix from fibroblasts and reduced leukocyte influx, suggesting that WNT signaling stimulates fibrotic tissue remodeling by direct effects on fibroblasts and by indirect inflammation-dependent effects in sclGVHD. Our findings may have direct translational potential, because pyrvinium is in clinical use, and tankyrase inhibitors are in clinical trials for other indications.

Pichia pastoris versus Saccharomyces cerevisiae: a case study on the recombinant production of human granulocyte-macrophage colony-stimulating factor.

BACKGROUND: Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) is a glycoprotein that has been approved by the FDA for the treatment of neutropenia and leukemia in combination with chemotherapies. Recombinant hGM-CSF is produced industrially using the baker's yeast, Saccharomyces cerevisiae, by large-scale fermentation. The methylotrophic yeast, Pichia pastoris, has emerged as an alternative host cell system due to its shorter and less immunogenic glycosylation pattern together with higher cell density growth and higher secreted protein yield than S. cerevisiae. In this study, we compared the pipeline from gene to recombinant protein in these two yeasts. RESULTS: Codon optimization in silico for both yeast species showed no difference in frequent codon usage. However, rhGM-CSF expressed from S. cerevisiae BY4742 showed a significant discrepancy in molecular weight from those of P. pastoris X33. Analysis showed purified rhGM-CSF species with molecular weights ranging from 30 to more than 60 kDa. Fed-batch fermentation over 72 h showed that rhGM-CSF was more highly secreted from P. pastoris than S. cerevisiae (285 and 64 mg total secreted protein/L, respectively). Ion exchange chromatography gave higher purity and recovery than hydrophobic interaction chromatography. Purified rhGM-CSF from P. pastoris was 327 times more potent than rhGM-CSF from S. cerevisiae in terms of proliferative stimulating capacity on the hGM-CSF-dependent cell line, TF-1. CONCLUSION: Our data support a view that the methylotrophic yeast P. pastoris is an effective recombinant host for heterologous rhGM-CSF production.