Elevated pro-inflammatory signalling coupled with catabolic metalloproteinase expression is a common feature of arthritis, leading to cartilage damage, deterioration of the joint architecture and the associated pain and immobility. Countering these processes, histone deacetylase inhibitors (HDACi) have been shown to suppress matrix metalloproteinase (MMP) expression, block cytokine-induced signalling and reduce the cartilage degradation in animal models of the arthritis. In order to establish which specific HDACs account for these chondro-protective effects an HDAC1-11 RNAi screen was performed. HDAC6 was required for both the interleukin (IL)-1 induction of MMP expression and pro-inflammatory interleukin expression in chondrocytes, implicating an effect on NF-κB signalling. Depletion of HDAC6 post-transcriptionally up-regulated inhibitor of κB (IκB), prevented the nuclear translocation of NF-κB subunits and down-regulated NF-κB reporter activation. The pharmacological inhibition of HDAC6 reduced MMP expression in chondrocytes and cartilage collagen release. This work highlights the important role of HDAC6 in pro-inflammatory signalling and metalloproteinase gene expression, and identifies a part for HDAC6 in the NF-κB signalling pathway. By confirming the protection of cartilage this work supports the inhibition of HDAC6 as a possible therapeutic strategy in arthritis.

MicroRNAs are non-coding RNAs that act to downregulate the expression of target genes by translational repression and degradation of messenger RNA molecules. Individual microRNAs have the ability to specifically target a wide array of gene transcripts, therefore allowing each microRNA to play key roles in multiple biological pathways. miR-324 is a microRNA predicted to target thousands of RNA transcripts and is expressed far more highly in the brain than in any other tissue, suggesting that it may play a role in one or multiple neurological pathways. Here we present data from the first global miR-324-null mice, in which increased excitability and interictal discharges were identified in vitro in the hippocampus. RNA sequencing was used to identify differentially expressed genes in miR-324-null mice which may contribute to this increased hippocampal excitability, and 3'UTR luciferase assays and western blotting revealed that two of these, Suox and Cd300lf, are novel direct targets of miR-324. Characterisation of microRNAs that produce an effect on neurological activity, such as miR-324, and identification of the pathways they regulate will allow a better understanding of the processes involved in normal neurological function and in turn may present novel pharmaceutical targets in treating neurological disease.

miR-140 is selectively expressed in cartilage. Deletion of the entire Mir140 locus in mice results in growth retardation and early-onset osteoarthritis-like pathology; however, the relative contribution of miR-140-5p or miR-140-3p to the phenotype remains to be determined. An unbiased small RNA sequencing approach identified miR-140-3p as significantly more abundant (>10-fold) than miR-140-5p in human cartilage. Analysis of these data identified multiple miR-140-3p isomiRs differing from the miRBase annotation at both the 5' and 3' end, with >99% having one of two seed sequences (5' bases 2-8). Canonical (miR-140-3p.2) and shifted (miR-140-3p.1) seed isomiRs were overexpressed in chondrocytes and transcriptomics performed to identify targets. miR-140-3p.1 and miR-140-3p.2 significantly down-regulated 694 and 238 genes, respectively, of which only 162 genes were commonly down-regulated. IsomiR targets were validated using 3'UTR luciferase assays. miR-140-3p.1 targets were enriched within up-regulated genes in rib chondrocytes of Mir140-null mice and within down-regulated genes during human chondrogenesis. Finally, through imputing the expression of miR-140 from the expression of the host gene WWP2 in 124 previously published data sets, an inverse correlation with miR-140-3p.1 predicted targets was identified. Together these data suggest the novel seed containing isomiR miR-140-3p.1 is more functional than original consensus miR-140-3p seed containing isomiR.

Cysteine‐rich with epidermal growth factor (EGF)‐like domains 2 (CRELD2) is an endoplasmic reticulum (ER)‐resident chaperone highly activated under ER stress in conditions such as chondrodysplasias; however, its role in healthy skeletal development is unknown. We show for the first time that cartilage‐specific deletion of Creld2 results in disrupted endochondral ossification and short limbed dwarfism, whereas deletion of Creld2 in bone results in osteopenia, with a low bone density and altered trabecular architecture. Our study provides the first evidence that CRELD2 promotes the differentiation and maturation of skeletal cells by modulating noncanonical WNT4 signaling regulated by p38 MAPK. Furthermore, we show that CRELD2 is a novel chaperone for the receptor low‐density lipoprotein receptor‐related protein 1 (LRP1), promoting its transport to the cell surface, and that LRP1 directly regulates WNT4 expression in chondrocytes through TGF‐β1 signaling. Therefore, our data provide a novel link between an ER‐resident chaperone and the essential WNT signaling pathways active during skeletal differentiation that could be applicable in other WNT‐responsive tissues. © 2020 American Society for Bone and Mineral Research. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research..

Purpose: Histone deacetylases (HDACs) regulate the pattern of the post-translational modification acetylation, especially on chromatin, to control gene expression, determine cell differentiation and mammalian development. Previous studies reported HDACs as potential regulators of the expression of major collagenases in articular cartilage and gene silencing studies identified class I HDACs, specifically HDAC3, as a key regulator of their expression. Moreover, the chondroprotective role of HDAC inhibitors is supported in in vivo models of osteoarthritis. The aim of this project is to examine the role of HDAC3 in catabolic gene expression in osteoarthritis and to determine the role of HDAC3 loss in chondrocyte differentiation in vivo and in vitro. Methods: Chemical inhibitors against all HDACs (e.g. TSA) and specifically against HDAC3 (Apicidin) were used in conjunction with HDAC3 RNAi in human SW1353 chondrocytes (stimulated with IL-1) and in a mesenchymal stem cell (MSC) chondrogenic differentiation model. Gene expression was quantified using real-time PCR and protein by western blotting, immunohistochemistry and biochemical methods. RNA expression microarray analysis (Illumina HT12) was also performed, following RNA interference or Apicidin treatment, across a time-course of IL-1 stimulation in chondrocytes to identify critical regulators of catabolic gene expression. Subsequently the role of E2F-1 transcription factor was studied with gain or loss of function experiments. Transcription factor activity was assessed by reporter gene activation and interaction with HDAC3 determined by immunoprecipitation. Furthermore we have generated an HDAC3 conditional knockout mouse model (HDAC3ΔCartilage) to reveal the role of HDAC3 in cartilage development. A systematic analysis of HDAC3Fl/Fl; Cre/+ versus HDAC3Fl/Fl; Cre0 using x-ray analysis and femur length measurements will be performed. Also whole mount skeletal staining of new-born skeletons stained with alizarin red and alcian blue and growth plate sections analysis following Goldner's trichrome staining of six-week old animals will be used. Results: HDAC3 is essential for IL-1-induced collagenase expression in chondrocytes and for NF-κB transcriptional activity. Additionally HDAC3 regulates anabolic gene expression, including COL2A1 and ACAN, during MSC chondrogenesis, establishing it as a critical regulator of both catabolic and anabolic gene expression in chondrocytes. Microarray analysis suggests the E2F-1 transcription factor may mediate the effect of HDAC3 on regulating gene expression. Accordingly, modulation of E2F1 levels alters IL-1-induced collagenase expression and HDAC3 directly interacts with E2F1 and effect on gene expression. Ultimately, with the use of our HDAC3ΔCartilage mouse we are establishing whether these in vitro findings are replicated in vivo. Conclusions: We propose a novel role for HDACs and specifically HDAC3 in the regulation of the expression of genes implicated in cartilage degradation in osteoarthritis. This effect could be mediated through direct interaction of HDAC3 with E2F1 transcription factor and binding of this protein complex to the promoters or enhancers of target genes. Much evidence that the expression of chondrogenic genes is mediated by HDAC-dependent mechanisms has been provided by our MSC differentiation model and the HDAC3 conditional knockout mouse model.