Histone acetylation regulated by class I histone deacetylases (HDACs) plays a pivotal role in matrix-specific gene transcription and cartilage development. While we previously demonstrated that microRNA (miR)-455-3p is upregulated during chondrogenesis and can enhance early chondrogenesis, the mechanism underlying this process remains largely unclear. In this study, we characterized the effect of miR-455-3p on histone H3 acetylation and its role during cartilage development and degeneration. We observed that miR-455-3p was highly expressed in proliferating and pre-hypertrophic chondrocytes, while HDAC2 and HDAC8 were primarily expressed in hypertrophic chondrocytes. Meanwhile, miR-455-3p suppressed the activity of reporter constructs containing the 3'-untranslated regions of HDAC2/8, inhibited HDAC2/8 expression and promoted histone H3 acetylation at the collagen 2 (COL2A1) promoter in human SW1353 chondrocyte-like cells. Treatment with the HDAC inhibitor trichostatin A (TSA) resulted in increased expression of cartilage-specific genes and promoted glycosaminoglycan deposition. Moreover, TSA inhibited matrix metalloproteinase 13 (Mmp13) expression and promoted nuclear translocation of SOX9 in interleukin-1-treated primary mouse chondrocytes. Lastly, knockdown of HDAC2/3/8 increased SRY (sex-determining region Y)-box 9 (SOX9) and decreased Runt-related transcription factor 2 (RUNX2) expression. Taken together, these findings suggest that miR-455-3p plays a critical role during chondrogenesis by directly targeting HDAC2/8 and promoting histone H3 acetylation, which raises possibilities of using miR-455-3p to influence chondrogenesis and cartilage degeneration.

Background/Aims: Long noncoding RNAs (lncRNAs) play important roles in stem cell differentiation. However, their role in osteogenesis of human adipose-derived stem cells (ASCs), a promising cell source for bone regeneration, remains unknown. Here, we investigated the expression profile and potential roles of lncRNAs in osteogenic differentiation of human ASCs. Methods: Human ASCs were induced to differentiate into osteoblasts in vitro, and the expression profiles of lncRNAs and mRNAs in undifferentiated and osteogenic differentiated ASCs were obtained by microarray. Bioinformatics analyses including subgroup analysis, gene ontology analysis, pathway analysis and co-expression network analysis were performed. The function of lncRNA H19 was determined by in vitro knockdown and overexpression. Quantitative reverse transcription polymerase chain reaction was utilized to examine the expression of selected genes. Results: We identified 1,460 upregulated and 1,112 downregulated lncRNAs in osteogenic differentiated human ASCs as compared with those of undifferentiated cells (Fold change ≥ 2.0, P < 0.05). Among these, 94 antisense lncRNAs, 85 enhancer-like lncRNAs and 160 lincRNAs were further recognized. We used 12 lncRNAs and 157 mRNAs to comprise a coding-non-coding gene expression network. Additionally, silencing of H19 caused a significantly increase in expression of osteogenesis-related genes, including ALPL and RUNX2, while a decrease was observed after H19 overexpression. Conclusion: This study revealed for the first time the global expression profile of lncRNAs involved in osteogenic differentiation of human ASCs and provided a foundation for future investigations of lncRNA regulation of human ASC osteogenesis.

Edited by Zhijie ChangKeywords: Chondrogenesis MiR-455-3p Runt-related transcription factor 2 a b s t r a c tThe expression of miR-455-3p has been shown to be up-regulated in chondrogenesis of mesenchymal stem cell, but its role in different stages during chondrogenesis remains unknown. Here, we show that miR-455-3p is increased in ATDC5 cells from 0 d to 21 d, but rapidly decreases at 28 d, and a similar expression kinetic is detected in the development of mouse embryos. We show that miR-455-3p functions as an activator for early chondrogenic differentiation, most likely by inhibiting the expression of Runt-related transcription factor 2 (Runx2) as indicated by luciferase reporter assays. In conclusion, miR-455-3p may activate early chondrogenesis by directly targeting Runx2.

Aim: The molecular pathways regulating cartilage degradation are unclear. miR-381 was identified as a putative regulator of chondrogenesis related genes. Here, we examined its role in chondrogenesis and osteoarthritic cartilage degeneration. Methods: miR-381 expression was assessed in vitro in response to IL-1β stimulation in primary human (PHC) and mouse (PMC) chondrocytes, and ATDC5 derived chondrocytes; and in vivo in mouse embryos and human osteoarthritic cartilage. The effects of miR-381 on chondrogenesis and NF-kB signaling were assessed using a synthetic RNA mimic or inhibitor and luciferase assay, respectively. Upstream regulators of miR381 were probed using siRNA or overexpression plasmids for Sox9 and Runx2. Results: miR-381 expression was elevated in chondrogenic and hypertrophic ATDC5 cells. miR-381 was induced in vitro by IL-1β in ATDC5 cells, PMCs, and PHCs, and was expressed in areas of cartilage degradation or absorption in vivo. Overexpression of Runx2 or Sox9 increased miR-381 expression in ATDC5 cells. miR-381 suppressed expression of collagen, type II, alpha 1, and enhanced expression of metalloproteinase-13 (MMP-13), but did not regulate NFKBIA and NKRF activity. Conclusion: miR-381 was highly expressed during chondrogenesis and in arthritic cartilage. It may contribute to absorption of the cartilage matrix by repressing type II collagen and inducing MMP-13.

Advanced glycation end products (AGEs) disturb bone remodeling during aging, and this process is accelerated in diabetes. However, their role in modulation of osteoclast-induced bone resorption is controversial, with some studies indicating that AGEs enhance bone resorption and others showing the opposite effect. We determined whether AGEs present at different stages of osteoclast differentiation affect bone resorption differently. Based on increased levels of tartrate-resistant acid phosphatase (TRAP) and cathepsin K (CTSK), we identified day 4 of induction as the dividing time of cell fusion stage and mature stage in RAW264.7 cell-derived osteoclast-like cells (OCLs). AGE-modified BSA (50-400 μg/ml) or control BSA (100 μg/ml) was then added at the beginning of each stage. Results showed that the presence of AGEs at the cell fusion stage reduced pit numbers, resorption area, and CTSK expression. Moreover, expression of receptor activator of nuclear factor-κB (RANK) as well as the number of TRAP-positive cells, nuclei per OCL, actin rings, and podosomes also decreased. However, the presence of AGEs at the mature stage enlarged the resorption area markedly and increased pit numbers slightly. Intriguingly, only the number of nuclei per OCL and podosomes increased. These data indicate that AGEs biphasically modulate bone resorption activity of OCLs in a differentiation stage-dependent manner. AGEs at the cell fusion stage reduce bone resorption dramatically, mainly via suppression of RANK expression in osteoclast precursors, whereas AGEs at the mature stage enhance bone resorption slightly, most likely by increasing the number of podosomes in mature OCLs.