ABSTRACT
Germline loss-of-function variation in TNFAIP3, encoding A20, has been implicated in a wide variety of autoinflammatory and autoimmune conditions, with acquired somatic missense mutations linked to cancer progression. Furthermore, human sequence data reveals that the A20 locus contains ~ 400 non-synonymous coding variants, which are largely uncharacterised. The growing number of A20 coding variants with unknown function, but potential clinical impact, poses a challenge to traditional mouse-based approaches. Here we report the development of a novel functional genomics approach that utilizes a new A20-deficient zebrafish (Danio rerio) model to investigate the impact of TNFAIP3 genetic variants in vivo. A20-deficient zebrafish are hyper-responsive to microbial immune activation and exhibit spontaneous early lethality. Ectopic addition of human A20 rescued A20-null zebrafish from lethality, while missense mutations at two conserved A20 residues, S381A and C243Y, reversed this protective effect. Ser381 represents a phosphorylation site important for enhancing A20 activity that is abrogated by its mutation to alanine, or by a causal C243Y mutation that triggers human autoimmune disease. These data reveal an evolutionarily conserved role for TNFAIP3 in limiting inflammation in the vertebrate linage and show how this function is controlled by phosphorylation. They also demonstrate how a zebrafish functional genomics pipeline can be utilized to investigate the in vivo significance of medically relevant human TNFAIP3 gene variants.
Subject(s)
Tumor Necrosis Factor alpha-Induced Protein 3/genetics , Tumor Necrosis Factor alpha-Induced Protein 3/physiology , Zebrafish Proteins/genetics , Zebrafish Proteins/physiology , Zebrafish/genetics , Amino Acid Substitution , Animals , Animals, Genetically Modified , Autoimmune Diseases/etiology , Autoimmune Diseases/genetics , Conserved Sequence , Evolution, Molecular , Genetic Variation , Humans , Inflammation/etiology , Inflammation/genetics , Macrophages/immunology , Macrophages/metabolism , Models, Animal , Models, Genetic , Mutation, Missense , NF-kappa B/metabolism , Phosphorylation , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Tumor Necrosis Factor alpha-Induced Protein 3/deficiency , Zebrafish/physiology , Zebrafish Proteins/deficiencyABSTRACT
Secreted factors are a key component of stem cell niche and their dysregulation compromises stem cell function. Legumain is a secreted cysteine protease involved in diverse biological processes. Here, we demonstrate that legumain regulates lineage commitment of human bone marrow stromal cells and that its expression level and cellular localization are altered in postmenopausal osteoporotic patients. As shown by genetic and pharmacological manipulation, legumain inhibited osteoblast (OB) differentiation and in vivo bone formation through degradation of the bone matrix protein fibronectin. In addition, genetic ablation or pharmacological inhibition of legumain activity led to precocious OB differentiation and increased vertebral mineralization in zebrafish. Finally, we show that localized increased expression of legumain in bone marrow adipocytes was inversely correlated with adjacent trabecular bone mass in a cohort of patients with postmenopausal osteoporosis. Our data suggest that altered proteolytic activity of legumain in the bone microenvironment contributes to decreased bone mass in postmenopausal osteoporosis.
Subject(s)
Cell Differentiation , Cysteine Endopeptidases/metabolism , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Osteoporosis, Postmenopausal/etiology , Osteoporosis, Postmenopausal/metabolism , Adipocytes/cytology , Adipocytes/metabolism , Animals , Calcification, Physiologic/genetics , Cell Line , Cells, Cultured , Cellular Microenvironment , Cysteine Endopeptidases/blood , Cysteine Endopeptidases/genetics , Disease Models, Animal , Enzyme Activation , Female , Gene Knockdown Techniques , Humans , Mice , Osteoblasts/cytology , Osteoblasts/metabolism , Osteogenesis/genetics , Osteoporosis, Postmenopausal/pathologyABSTRACT
The bone marrow microenvironment plays a decisive role in multiple myeloma progression and drug resistance. Chemokines are soluble mediators of cell migration, proliferation and survival and essentially modulate tumor progression and drug resistance. Here we investigated bone marrow-derived chemokines of naive and therapy-refractory myeloma patients and discovered that high levels of the chemokine CCL27, known so far for its role in skin inflammatory processes, correlated with worse overall survival of the patients. In addition, chemokine levels were significantly higher in samples from patients who became refractory to bortezomib at first line treatment compared to resistance at later treatment lines.In vitro as well as in an in vivo model we could show that CCL27 triggers bortezomib-resistance of myeloma cells. This effect was strictly dependent on the expression of the respective receptor, CCR10, on stroma cells and involved the modulation of IL-10 expression, activation of myeloma survival pathways, and modulation of proteasomal activity. Drug resistance could be totally reversed by blocking CCR10 by siRNA as well as blocking IL-10 and its receptor.From our data we suggest that blocking the CCR10/CCL27/IL-10 myeloma-stroma crosstalk is a novel therapeutic target that could be especially relevant in early refractory myeloma patients.