Purpose: Chondrocytes acquire a modified phenotype with ageing, resulting in increased risk of osteoarthritis (OA) due to alterations in the cartilage extracellular matrix (ECM). SnoRNAs direct chemical modification of RNA substrates and are involved in endoribonucleolytic pre-rRNA processing. The post-transcriptional 2'O-ribose methylation and pseudouridylation carried out by snoRNAs fine-tunes spliceosome and ribosome function, accommodating changing requirements for protein synthesis during health and disease. Control of snoRNA levels may be pivotal in regulating the transcriptional and translational capacity of high protein producing chondrocytes. This is interesting as in OA there is an imbalance between ECM protein anabolism and catabolism. To ensure continuous ECM deposition it is essential for a chondrocyte to control the number and quality of its ribosomes. We tested the hypothesis that the ribosome's translational capacity alters with age and disease due to dysregulation of expression and function of specific snoRNAs; contributing to the development of the OA chondrocyte phenotype. Methods: Total RNA was extracted from human OA knee cartilage of young (n=6; mean age±SD 22.7±4.1 years) normal and old n=6; (66.4±15.9 years) donors and hybridised onto Affymetrix miRNA 4.0 arrays. The probe set for Homo sapiens was used to determine differentially expressed snoRNAs. Relative ribosome number and chondrocyte marker gene expression was determined using qRT-PCR of 5.8, 18 and 28S rRNAs and of COL2A1, ACAN, SOX9, COL10A1, RUNX2, MMP13, ADAMTS5, COX-2, IL6, BAPX1 mRNAs. Total DNA content by SYBR Green detection and total protein was determined using BCA assay. Results: Normal samples correlated closely together, however OA samples clustered into three groups. When PCA was integrated with the Kellgren and Lawrence scores of OA donors the sub-populations were separated on OA severity. Analysis of the three subgroups identified 26 snoRNAs reduced in OA and 11 snoRNAs increased. These include 25 box C/D and 11 box H/ACA snoRNAs. To address the potential impact of aberrant snoRNAs expression on rRNA maturation, we determined the relative ribosome content in healthy and OA human articular chondorcytes (HAC). 18S and 5.8S rRNA (not 28S) levels were decreased in OA, together with a typical OA chondrocyte gene expression profile. Pre-rRNA levels were higher in OA, indicating aberrant pre-rRNA processing. In concert array results indicated that expression of U3 and U13 snoRNAs is deregulated in OA. In contrast to the majority of the snoRNAs these direct site-specific endoribonucleolytic cleavage of pre-rRNA. To address a potential involvement of the inflammatory compound of OA, healthy HACs were exposed to IL1β. Similar to OA chondrocytes 18S and 5.8S rRNA decreased on exposure and pre-rRNA increased. To address the question whether the chondrocyte phenotype responds in an OA-like fashion as a result of alterations in the cell's translation capacity we inhibited rRNA transcription using actinomycin D using normal HACs. 18S and 5.8S rRNA levels/cell were significantly downregulated whereas 28S rRNA levels remained unaffected. Due to ribosome depletion a reduction in total protein content/cell was observed, confirming functionally decreased translation capacity. The expression of RUNX2 and COL10A1 was upregulated, whereas COL2A1 expression was downregulated. Findings indicate that as a result of decreased chondrocyte ribosome content and translation capacity, chondrocytes phenotypically respond in an OA-like fashion. Conclusions:Since we found evidence for altered ribosome abundancy and auxiliary rRNA maturation machinery in ageing chondrocytes accompanied by differential OA cartilage-specific expression of snoRNAs, we believe that the translational capacity of the articular chondrocyte in OA is impaired, due to dysregulation of expression and function of specific snoRNAs, thereby contributing to the development of the OA chondrocyte phenotype.

In order to characterize a fibroblast cell line representing normal human skin fibroblasts in three-dimensional cultures, we compared the fibroblast line MSU-1.1, derived from human foreskin and immortalized by v-myc, to primary human dermal fibroblasts (NDF). Our results demonstrate that in contrast to NDF, all MSU-1.1 fibroblasts die within 3–4 d when cultured within three-dimensional contractile collagen matrices. Also, in contrast to NDF, MSU-1.1 cells die markedly in anchored collagen gels as well. Death is due to apoptosis and is attenuated by addition of antibodies against collagen-recognizing receptors α1β1 and α2β1. Apoptosis of NDF in collagen lattices was repressed by an inhibitor of caspase-1, which was ineffective on apoptosis of MSU-1.1. Further, apoptosis by MSU-1.1 fibroblasts was also observed in anchored, i.e., restrained collagen lattices, an environment that supports proliferation of NDF.

The mechanisms underlying the contraction-dependent apoptosis of primary fibroblasts are of prime importance in understanding anchorage-dependent survival/apoptosis of dermal fibroblasts. As integrins are essential extracellular matrix receptors in fibroblasts, their role in anchorage-dependent apoptosis/survival of fibroblasts was analyzed. Primary human fibroblasts displayed a marked reduction of apoptosis in mechanically relaxed collagen matrices in the presence of adhesion-blocking antibodies against alpha1beta1 or alpha2beta1. Anti-alphavbeta3 antibodies had a considerably weaker effect. In additional experiments RD cells, which lack alpha2 integrin, displayed no apoptosis in mechanically relaxed collagen matrices. Their susceptibility to apoptosis was restored after transfection with functional alpha2 integrin, and it could be blocked again by adhesion-blocking antibodies against alpha2beta1 integrin. Therefore we conclude that apoptosis of human primary fibroblasts in contractile collagen matrices is - at least in part - inhibited by adhesion-blocking anti-integrin antibodies, suggesting that the mode of apoptosis in this case is different from anoikis. Further, apoptosis in a mechanically relaxed collagen matrix could be abrogated by depolymerization of F-actin using cytochalasin D and also by disturbing actin-myosin interaction using 2,3-butanedione monoxime, indicating a possible dependence of apoptosis on mechanical forces and/or cell shape.

Apoptosis of primary fibroblasts was observed in vivo during wound healing in skin and is expected to occur in other organs as well; however, the environmental signal for induction of apoptosis in fibroblasts and the putative influence of cell-matrix interactions on the regulation of apoptosis remain to be identified. Here we provide evidence for the role of fibrillar collagen in this process, and demonstrate that normal human primary fibroblasts embedded in contractile collagen gels undergo apoptosis as shown by the appearance of cytoplasmatic histone-associated DNA fragments starting at day 1 of culture with a peak around days 2-4. This induction of apoptosis in primary fibroblasts seems to be specific for contractile collagen gels, because apoptosis of primary fibroblasts was neither observed in cells grown on culture dishes or on plastic dishes coated with collagen, nor observed in cells seeded in either anchored collagen gels or contractile fibrin gels. We therefore conclude that a distinct environment such as a contractile collagen matrix determines the susceptibility of normal primary fibroblasts to apoptosis.

A cDNA fragment coding for the sex-inducing glycoprotein of Volvox carteri f. nagariensis was expressed in a mammalian cell system (baby hamster kidney (BHK) cells). The transfection product exhibited a specific biological activity intermediate between the natural pheromone of the strains Volvox carteri f. nagariensis and Volvox carteri f. weismannia. Immunoblot analysis showed that the sex-inducing activity was expressed as a set of three iso-glycoproteins (35, 34 and 31 kDa).