MicroRNAs (miRNA) are short RNA molecules regulating the expression of specific mRNAs. We investigated the expression pattern and potential targets of mouse miR-140 and found that miR-140 is specifically expressed in cartilage tissues of mouse embryos during both long and flat bone development. MiR-140 expression was detected in the limbs of E11.5 embryos in the primorida of future bones both in the fore and hindlimb and across autopod, zeugopod and stylopod. All digits of E14.5 fore-and hindlimbs showed accumulation of miR-140, except the first digit of the hindlimb. MiR-140 expression was also detected in the cartilagenous base of E17.5 skulls and in the sternum, the proximal rib heads and the developing vertebral column of E15.5 embryos. A potential target of miR-140, histone deacetylase 4, was validated experimentally and the possible role of miR-140 in long bone development is discussed.

SummaryReplicative senescence occurs when normal somatic cells stop dividing. Senescent cells remain viable, but show alterations in phenotype, e.g. altered expression of matrix metalloproteinases ( MMPs ); these enzymes are known to be involved in cartilage destruction. It is assumed that cells deplete their replicative potential during aging, and age is a major risk factor for osteoarthritis (OA). Therefore, we hypothesized that chondrocytes in aging or diseased cartilage become senescent with associated phenotypic changes contributing to development or progression of OA.Articular cartilage was obtained from OA patients undergoing arthroplasty, with 'normal' cartilage from trauma surgery for hip fracture. Senescent cells were identified using the senescence-associated β β β β -galactosidase (SA-β β β β -gal) marker. Telomere length was assessed using Southern blot. MMP expression was measured at the mRNA level using Taqman® RT-PCR.No SA-β β β β -gal staining was observed in control cartilage regardless of patient age. In contrast, SA-β β β β -gal staining was observed in damaged OA cartilage adjacent to the lesion. Cultured chondrocytes isolated from sites near a lesion contained a greater percentage of SA-β β β β -gal positive cells than cultures isolated from distal sites or normal cartilage. Mean telomere length was shorter in cells near the lesion compared to distal sites in the same joint; thus the former population has undergone cell division. The expression of collagenases MMP-1 , -8 and -13 and tissue inhibitor of metalloproteinases (TIMP)-1 was altered in OA cartilage, but no difference was detected between lesion and distal sites in the same joint (i.e. no correlation was found between senescent cells and proteinase/ inhibitor expression).

The balance between matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), is pivotal in the remodeling of extracellular matrix. TGF-␤ has profound effects on extracellular matrix homeostasis, in part via its ability to alter this balance at the level of gene expression.

Cartilage destruction in osteoarthritis (OA) is thought to be mediated by two main enzyme families; the matrix metalloproteinases (MMPs) are responsible for cartilage collagen breakdown, whereas enzymes from the 'a disintegrin and metalloproteinase domain with thrombospondin motifs' (ADAMTS) family mediate cartilage aggrecan loss. Tissue inhibitors of metalloproteinases (TIMPs) regulate the activity of these enzymes. Although cartilage destruction in OA might be driven by the chondrocyte, low-grade synovitis is reported in patients with all grades of this disease.Our earlier work profiling these gene families in cartilage identified a number of genes that are regulated in OA, which are hence implicated in the disease process. Because the synovium might contribute to cartilage-matrix destruction in OA, we have extended the screening in the current study. We have profiled MMP, ADAMTS and TIMP genes in both cartilage and synovium from patients with either OA of the hip or a fracture to the neck of femur (NOF), giving a more complete picture of proteolysis in this disease.The four most significantly upregulated genes (P < 0.0001) in OA synovium compared to the fractured NOF are MMP28, ADAMTS16, ADAMTS17 and TIMP2. For MMP9, MMP10, MMP12, MMP17, MMP23, MMP28, ADAMTS4, and ADAMTS9, there is a significant correlation between expression levels in the synovium and cartilage, suggesting similar mechanisms of regulation. Additionally, we have shown that in cartilage the median level of steady-state mRNA for MMP13 is approximately 20-fold higher than MMP28 and approximately 1,500-fold higher than ADAMTS16, with expression of this latter gene approximately 150-fold higher in synovium than cartilage.This study is the most comprehensive analysis of the metzincin family of proteinases in the joint to date and has identified several proteinase genes not previously reported to be expressed or regulated in synovium.

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