ABSTRACT
Osteoarthritis is the most prevalent and crippling joint disease, and lacks curative treatment, as the underlying molecular basis is unclear. Here, we show that DOT1L, an enzyme involved in histone methylation, is a master protector of cartilage health. Loss of DOT1L disrupts the molecular signature of healthy chondrocytes in vitro and causes osteoarthritis in mice. Mechanistically, the protective function of DOT1L is attributable to inhibition of Wnt signalling, a pathway that when hyper-activated can lead to joint disease. Unexpectedly, DOT1L suppresses Wnt signalling by inhibiting the activity of sirtuin-1 (SIRT1), an important regulator of gene transcription. Inhibition of SIRT1 protects against osteoarthritis triggered by loss of DOT1L activity. Modulating the DOT1L network might therefore be a therapeutic approach to protect the cartilage against osteoarthritis.
Subject(s)
Cartilage/metabolism , Methyltransferases/metabolism , Osteoarthritis/pathology , Animals , Benzimidazoles/pharmacology , Benzimidazoles/toxicity , Cartilage/pathology , Cells, Cultured , Chondrocytes/drug effects , Chondrocytes/metabolism , Female , Gene Expression Regulation , Histone-Lysine N-Methyltransferase , Homeostasis , Male , Methylation , Methyltransferases/genetics , Mice, Inbred C57BL , Mice, Knockout , Osteoarthritis/chemically induced , Osteoarthritis/metabolism , Sirtuin 1/genetics , Sirtuin 1/metabolism , Wnt Signaling PathwayABSTRACT
The pleiotropic drug response (PDR) or multidrug resistance (MDR) are cellular defence mechanisms present in all species to deal with potential toxicity from environmental small molecule toxins or bioactives. The rapid induction of MDR by xenobiotics in mammalian cells and PDR in budding yeast (S. cerevisiae) has been well studied but how pathway specificity is achieved across different structural classes of xenobiotics is not well understood. As a novel approach to this problem we investigated the genome-wide network of genes modulating the yeast PDR. Fluorescently-tagged ABC pumps Pdr5p-GFP and Yor1p-GFP were used as real-time reporters for the Pdr1p/Pdr3p controlled response. Using the yeast non-essential gene deletion set fifty-four gene deletions that suppressed up-regulation of reporter fluorescence to the cell surface in the presence of atorvastatin were identified by high content confocal automated microscopy. Secondary validation using spot dilution assays to known PDR substrates and Western blot assays of Pdr5p expression confirmed 26 genes able to modulate the PDR phenotype. By analysis of network connectivity, an additional 10 genes that fell below the primary screen cut-off were predicted to be involved in PDR and confirmed as above. The PDR modulating genes taken together were enriched in signalling (Rho-GTPase, MAPK), Mediator complexes, and chromatin modification (subunits of ADA and SAGA complexes). Many of the gene deletions cause extra sensitivity in Δpdr1Δpdr3 strains strongly suggesting that there are alternative pathways to upregulate PDR, independently of Pdr1p/Pdr3p. We present here the first high-content microscopy screening for PDR modulators, and identify genes that are previously unsuspected regulators of PDR apparently contributing via network interactions.
Subject(s)
Drug Resistance, Multiple/genetics , Gene Regulatory Networks , Saccharomyces cerevisiae Proteins/biosynthesis , Signal Transduction/genetics , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Atorvastatin , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Deletion , Gene Expression Regulation, Fungal/drug effects , Heptanoic Acids/pharmacology , Pyrroles/pharmacology , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic/geneticsABSTRACT
Neothyonidioside is a triterpene glycoside (TG) isolated from the sea cucumber, Australostichopus mollis, that is potently cytotoxic to S. cerevisiae, but does not permeabilize cellular membranes. We mutagenized S. cerevisiae and isolated a neothionidioside-resistant (neo(R)) strain. Using synthetic genetic array mapping and sequencing, we identified NCP1 as the resistance locus. Quantitative HPLC revealed that neo(R)/ncp1 mutants have reduced ergosterol content. Ergosterol added to growth media reversed toxicity, demonstrating that neothionidioside binds directly to ergosterol, similar to the polyene natamycin. Ergosterol synthesis inhibitors ketoconazole and atorvastatin conferred resistance to neothionidioside in a dose-dependent manner showing that a threshold ergosterol concentration is required for toxicity. A genome-wide screen of deletion mutants against neothionidioside revealed hypersensitivity of many of the component genes in the ESCRT complexes relating to multivesicular body formation. Confocal microscopy of cells stained with a vital dye showed blockage at this step. Thus, we propose neothionidioside may affect membrane curvature and fusion capability in the endosome-vacuole pathway.
