FKBP38 Alleviates Osteoarthritis Progression by Inhibiting Chondrocyte Senescence.

Phosphatidic acid (PA) is a critical mediator of mitogenic activation of mammalian target of rapamycin complex 1 (mTORC1) signaling, a master regulator of mammalian cell growth and proliferation. The mechanism by which PA activates mTORC1 signaling has remained unknown. Here, we report that PA selectively stimulates mTORC1 but not mTORC2 kinase activity in cells and in vitro. Furthermore, we show that PA competes with the mTORC1 inhibitor, FK506 binding protein 38 (FKBP38), for mTOR binding at a site encompassing the rapamycin-FKBP12 binding domain. This leads to PA antagonizing FKBP38 inhibition of mTORC1 kinase activity in vitro and rescuing mTORC1 signaling from FKBP38 in cells. Phospholipase D 1, a PA-generating enzyme that is an established upstream regulator of mTORC1, is found to negatively affect mTOR-FKBP38 interaction, confirming the role of endogenous PA in this regulation. Interestingly, removal of FKBP38 alone is insufficient to activate mTORC1 kinase and signaling, which require PA even when the FKBP38 level is drastically reduced by RNAi. In conclusion, we propose a dual mechanism for PA activation of mTORC1: PA displaces FKBP38 from mTOR and allosterically stimulates the catalytic activity of mTORC1.

ObjectiveOsteoarthritis (OA) is characterized by the chronic and progressive deterioration of articular cartilage. Chondrocyte senescence could lead to a shift in the balance between extracellular matrix (ECM) component synthesis and degradation. Small noncoding RNAs (sncRNAs), including microRNAs (miRNAs), P-element-induced wimpy testis-(PIWI-) interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs), and repeat-associated siRNAs (rasiRNAs), are a class of important epigenetic molecules. We aimed to gain insights into the changes and roles of sncRNA in chondrocyte senescence.DesignHealthy mouse postnatal chondrocytes were isolated, and a replicative aging model was constructed. We used small RNA sequencing (small RNA-seq) to generate extensive small RNA data. We identified differentially expressed sncRNAs and performed tissue-specific analysis using real-time quantitative polymerase chain reaction (qRT-PCR). β-galactosidase staining was used to detect chondrocyte senescence. The results showed that the expression profiles of sncRNA in passage 5 chondrocytes were significantly different from those in passage 0 chondrocytes. The expression of sncRNA was tissue specific. We found that 40 miRNAs were upregulated and 70 miRNAs were downregulated during chondrocyte senescence, and that miR-132-5p expression inhibition prevented chondrocyte senescence. We found that 8 piRNAs were upregulated and 17 piRNAs were downregulated during chondrocyte senescence, and that piRNA piR_025576 overexpression delayed chondrocyte senescence. We found that 24 snoRNAs were upregulated and 28 snoRNAs were downregulated during chondrocyte senescence, and that snoRNA ENSMUSG00000087935 overexpression delayed chondrocyte senescence. We found that 5 snRNAs were upregulated and 6 snRNAs were downregulated during chondrocyte senescence, and that snRNA ENSMUSG00000064682 overexpression delayed chondrocyte senescence. We found that 1 rasiRNA was upregulated and 4 rasiRNAs were downregulated during chondrocyte senescence.ConclusionsThese findings might provide novel insights into OA pathogenesis and contribute to the development of candidates for targeted therapeutics in OA.

Hypothalamic mTORC1 Signaling Controls Sympathetic Nerve Activity and Arterial Pressure and Mediates Leptin Effects

The mammalian target of rapamycin (mTOR) Ser/Thr kinase signals in at least two multiprotein complexes distinguished by their different partners and sensitivities to rapamycin. Acute rapamycin inhibits signaling by mTOR complex 1 (mTORC1) but not mTOR complex 2 (mTORC2), which both promote cell growth, proliferation, and survival. Although mTORC2 regulation remains poorly defined, diverse cellular mitogens activate mTORC1 signaling in a manner that requires sufficient levels of amino acids and cellular energy. Before the identification of distinct mTOR complexes, mTOR was reported to autophosphorylate on Ser-2481 in vivo in a rapamycin- and amino acid-insensitive manner. These results suggested that modulation of mTOR intrinsic catalytic activity does not universally underlie mTOR regulation. Here we re-examine the regulation of mTOR Ser-2481 autophosphorylation (Ser(P)-2481) in vivo by studying mTORC-specific Ser(P)-2481 in mTORC1 and mTORC2, with a primary focus on mTORC1. In contrast to previous work, we find that acute rapamycin and amino acid withdrawal markedly attenuate mTORC1-associated mTOR Ser(P)-2481 in cycling cells. Although insulin stimulates both mTORC1- and mTORC2-associated mTOR Ser(P)-2481 in a phosphatidylinositol 3-kinase-dependent manner, rapamycin acutely inhibits insulin-stimulated mTOR Ser(P)-2481 in mTORC1 but not mTORC2. By interrogating diverse mTORC1 regulatory input, we find that without exception mTORC1-activating signals promote, whereas mTORC1-inhibitory signals decrease mTORC1-associated mTOR Ser(P)-2481. These data suggest that mTORC1- and likely mTORC2-associated mTOR Ser-2481 autophosphorylation directly monitors intrinsic mTORC-specific catalytic activity and reveal that rapamycin inhibits mTORC1 signaling in vivo by reducing mTORC1 catalytic activity.

Osteoarthritis (OA) results in pathologic changes in the joint tissue. The mechanisms driving disease progression remain largely unclear, and thus disease-modifying treatments are lacking. Pannexin 3 (Panx3) was identified as a potential mediator of cartilage degeneration in OA, and our previous study in mice indicated that deletion of the Panx3 gene delayed surgically induced cartilage degeneration. This study was undertaken to examine the role of Panx3 in other OA subtypes, particularly primary OA during aging, in a mouse model of aging-induced OA. Wild-type (WT) and Panx3-/- C57BL/6J (Black-6) mice, ages 18-24 months, were analyzed by micro-computed tomography to investigate bone mineral density and body composition. Joints were harvested from the mice, and histopathologic analysis of the joint tissue for OA development was conducted with a specific focus on changes in articular cartilage, subchondral bone, and synovial tissue. Global loss of Panx3 in aging mice was not associated with increased mortality or changes in body composition. Mice lacking Panx3 had shorter appendicular skeletons than WT mice, but overall the body compositions appeared quite similar. Panx3 deletion dramatically accelerated cartilage degeneration and subchondral bone thickening with aging in both 18-month-old and 24-month-old mice, while promoting synovitis in 18-month-old mice. These observations in a mouse model of OA suggest that Panx3 has a protective role against the development of primary aging-associated OA. It appears that Panx3 has opposing context-specific roles in joint health following traumatic injury versus that associated with aging. These data strongly suggest that there are differences in the molecular pathways driving different subtypes of OA, and therefore a detailed understanding of these pathways could directly improve strategies for OA diagnosis, therapy, and research.

Pharmacological and Genetic Evaluation of Proposed Roles of Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase (MEK), Extracellular Signal-regulated Kinase (ERK), and p90RSK in the Control of mTORC1 Protein Signaling by Phorbol Esters

The Ras-like small GTPase Rheb is an upstream activator of the mammalian target of rapamycin (mTOR). It has recently been shown that Rheb activates mTOR by binding to its endogenous inhibitor FKBP38 and preventing it from association with mTOR. The interaction of Rheb with FKBP38 is controlled by its guanine nucleotide binding states, which are responsive to growth factor and amino acid conditions. In this study, we show that Rheb interacts with FKBP38 through a section within its switch I region that is equivalent to the effector domain of other Ras-like small GTPases. We find that the ability for Rheb to interact with FKBP38 correlates with its activity for mTOR activation. Our findings suggest that FKBP38 is a bona fide effector of Rheb and that the ability to interact with FKBP38 is important for Rheb as an activator of mTOR.

Efficient Intrathymic Gene Transfer Following In Situ Administration of a rAAV Serotype 8 Vector in Mice and Nonhuman Primates

Objective Osteoarthritis (OA) is a progressive disease characterized by degeneration of cartilage and echinacoside (Ech) has anti-inflammatory and antioxidant effects in various human diseases. This study aimed to reveal the effect and potential mechanism of Ech on OA. Materials and methods The in vitro OA model was established by rat chondrocytes treated with IL-1β, and the in vivo OA model was established by anterior cruciate ligament transaction. The effect of Ech on the viability, inflammatory response, extracellular matrix (ECM) degradation, and oxidative stress of IL-1β-treated rat chondrocytes were evaluated by Cell Counting Kit-8 assay, enzyme-linked immunosorbent assay, quantitative real-time PCR, Western blot, and immunofluorescence assay. Meanwhile, the mechanism of Ech was assessed using Western blot, Cell Counting Kit-8 assay, enzyme-linked immunosorbent assay, and immunofluorescence analysis. Moreover, the function of Ech in vivo was analyzed in rat models of OA. Results Functionally, Ech enhanced the viability of rat chondrocytes, repressed the inflammatory response and ECM degradation of rat chondrocytes induced by IL-1β with restrained oxidative stress. Mechanically, Ech repressed IL-1β-induced chondrocyte injury by activating the Nrf2/HO-1 signaling pathway. Meanwhile, Ech alleviated the degree of articular cartilage injury in rats and exerted protective effects on the rat model of OA in vivo. Discussion and conclusions Ech alleviated OA in rats by activating the Nrf2-HO-1 signaling pathway.

Objective Osteoarthritis (OA) is a degenerative disease of the joints characterized by inflammation and cartilage degeneration. Zinc finger E-box binding homeobox 2 (ZEB2) contains various function domains that interact with multiple transcription factors involved in various cellular functions. However, the function of ZEB2 in OA has not been clearly illustrated. Method Interleukin-1β (IL-1β) was used to establish an OA model in vitro. We quantified the ZEB2 expression in cartilage tissues from OA patients and IL-1β-induced chondrocytes through reverse transcription–quantitative polymerase chain reaction and Western blot. We then used functional assays to explore the function of ZEB2 during OA progression. Results ZEB2 expression was increased in OA cartilage tissues and chondrocytes. The silencing of ZEB2 increased aggrecan and collagen II levels, and reduced the content of matrix metalloproteinase-3 (MMP-3), MMP-9, and MMP-13. ZEB2 knockdown inhibited the effects of IL-1β on the production of nitric oxide and prostaglandin E2, and the expression of inducible nitric oxide synthase and cyclooxygenase-2. ZEB2 inhibition also suppressed the levels of IL-6 and tumour necrosis factor-α, and increased the IL-10 level in IL-1β-treated cells. Mechanically, ZEB2 knockdown blocked the activation of the Wnt/β-catenin pathway in chondrocytes. Conclusion Knockdown of ZEB2 alleviated IL-1β-induced cartilage degradation and the inflammatory response through the Wnt/β-catenin pathway in chondrocytes.

Dapagliflozin suppress endoplasmic reticulum stress mediated apoptosis of chondrocytes by activating Sirt1

ObjectivesOsteoarthritis (OA) patients with a neuropathic pain (NP) component may represent a specific phenotype. This study compares joint damage, pain and functional disability between knee OA patients with a likely...

AAV Vector-mediated Reversal of Hypoglycemia in Canine and Murine Glycogen Storage Disease Type Ia

A4.2 IL4–10 synerkine for the treatment of osteoarthritis

The mammalian target of rapamycin (mTOR) interacts with raptor to form the protein complex mTORC1 (mTOR complex 1), which plays a central role in the regulation of cell growth in response to environmental cues. Given that glucose is a primary fuel source and a biosynthetic precursor, how mTORC1 signaling is coordinated with glucose metabolism has been an important question. Here, we found that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) binds Rheb and inhibits mTORC1 signaling. Under low-glucose conditions, GAPDH prevents Rheb from binding to mTOR and thereby inhibits mTORC1 signaling. High glycolytic flux suppresses the interaction between GAPDH and Rheb and thus allows Rheb to activate mTORC1. Silencing of GAPDH or blocking of the Rheb-GAPDH interaction desensitizes mTORC1 signaling to changes in the level of glucose. The GAPDH-dependent regulation of mTORC1 in response to glucose availability occurred even in TSC1-deficient cells and AMPK-silenced cells, supporting the idea that the GAPDH-Rheb pathway functions independently of the AMPK axis. Furthermore, we show that glyceraldehyde-3-phosphate, a glycolytic intermediate that binds GAPDH, destabilizes the Rheb-GAPDH interaction even under low-glucose conditions, explaining how high-glucose flux suppresses the interaction and activates mTORC1 signaling. Taken together, our results suggest that the glycolytic flux regulates mTOR's access to Rheb by regulating the Rheb-GAPDH interaction, thereby allowing mTORC1 to coordinate cell growth with glucose availability.