| Summary: | Human articular cartilage is an avascular and aneural connective tissue covering the bone ends in the joints of the body. It functions in a hypoxic environment and cartilage cells, the chondrocytes, have adapted to use hypoxia to drive tissue-specific function. The maintenance of cartilage matrix homeostasis is an important topic in musculoskeletal research and is a subject of investigation in my laboratory. Previous work in the group has identified a non-coding RNA transcript (H19), which was highly expressed in human articular chondrocytes (to the levels of the most abundant matrix genes COL2A1 and AGC), and showed a pattern of expression strikingly similar to that of the known differentiated chondrocyte markers. Recently, H19 has been shown to give rise to a mature microRNA, namely miR-675. MicroRNAs have emerged as a new class of endogenous modulators of gene expression. Moreover, it was recently shown that microRNAs are essential for normal cartilage development in the mouse. Hence, the function of H19/miR-675 was investigated, in particular in terms of its importance in cartilage matrix gene regulation in normal, non-diseased, human articular chondrocytes. Overexpression of miR-675 increased type II collagen levels at the mRNA and secreted protein levels, whereas depletion of primary transcript H19 or inhibition of the mature microRNA (miR-675) caused a decrease in type II collagen message and protein levels. Finally, overexpression of miR-675 rescued type II collagen levels in H19-depleted or SOX9-depleted cells. Initial RNA interference experiments indicated that H19 is strongly regulated by the key cartilage transcription factor SOX9, as well as by another transcription factor important in chondrocytes, HIF-2α. These results were subsequently confirmed under endogenous conditions using chromatin immunoprecipitation assay (ChIP). The binding of these two transcription factors was found at specific sites downstream (SOX9) and upstream (HIF-2α) of the H19 transcription start site. Here we present a novel mechanism of COL2A1 regulation via miR-675 in human articular chondrocytes. We provide evidence that SOX9 directly targets H19, the primary transcript of miR- 675. This microRNA then positively regulates type II collagen in healthy human chondrocytes in an indirect manner. Additionally, HIF-2α directly binds both SOX9 and H19, thereby increasing levels of COL2A1 in hypoxia.
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