Control of cartilage homeostasis and osteoarthritis progression by mTORC1/4E-BP/eIF4E axis

Abstract

As world population growing older, burden of age-related conditions soars. One of them is osteoarthritis (OA), a debilitating joint disease with no effective treatment. Articular cartilage degeneration is a central event in OA, and changes in expression of many genes in OA cartilage are well-documented. Still, the specific mechanisms are rarely known. We showed that in OA cartilage the increased abundance of many proteins, including extracellular matrix protein Fibronectin (Fn1) and an orphan nuclear receptor Nr4a1 is translationally controlled and requires inactivation of 4EBP, an inhibitor of cap-dependent translation. Importantly, intra-articular injection of the translation inhibitor 4E1RCat reduces Fn1 and Nr4a upregulation in a rodent OA model and delays cartilage degeneration. Our results support the hypothesis that maintaining proper translational control is an important homeostatic mechanism, the loss of which contributes to OA development.Recent publications from two independent groups have highlighted the importance of IL-1 signaling in various small and larger animal models of posttraumatic OA (22)(23)(24)(25). For example, IL-1 receptor antagonist therapy significantly improved cartilage, synovial membrane parameters, and disease-modifying effects in the equine OA model (26). The role of translation control in the inflammatory response was examined in macrophages (27), but never in chondrocytes. We therefore used polysome profiling to assess the translational landscape of IL-1β treated primary articular chondrocytes. In polysomal profiling, actively translated mRNAs bound by several ribosomes (polysomes) are separated from "free" mRNA and the 80S monosomes by sucrose gradient centrifugation. This allows determining the translational status of a specific mRNA. Potential clinical relevance of several identified translationally controlled targets, such as Fn and an orphan receptor Nr4a1, was tested in diseased cartilage and in a rodent model of post-traumatic OA. To demonstrate the importance of translational control for cartilage homeostasis, we injected intra-articularly an inhibitor of cap-dependent translation. This treatment significantly delays OA progression. Our findings therefore reveal that translational control, acting at Fn and Nr4a1 among other targets, is essential for maintaining cartilage homeostasis.Moreover, targeting translational apparatus represents a new strategy to treat/prevent OA, particularly as it opens up the possibility to target simultaneously a pool of functionally distinct proteins. MATERIALS AND METHODSIsolation and characterization of translationally active pool of mRNAs by polysome fraction analysis, library preparation, and sequencing. RAC was isolated and treated at the first passage as described previously (18). For 10 final minutes, cells were incubated with Cyclohexamide (100ug/ml) at 37 o C.Cells were collected, washed twice with ice-cold PBS supplemented with Cyclohexamide (100ug/ml) and lysed on ice for 10min in buffer "A" (SI). 2OD units of extract (3 independ...