The 47S Pre-rRNA processing cascade of articular chondrocytes is impaired in an in vitro model of osteoarthritis

Abstract

Purpose: Osteoarthritis (OA) is the most common degenerative joint disorder, characterized by progressive loss and destruction of articular cartilage. Chondrocytes are highly specialized cells with primary function of producing and maintaining the extracellular matrix (ECM). This requires continuous translation of mRNAs by ribosomes. Mammalian ribosomes are large protein-RNA complexes containing 79 protein subunits and 4 ribosomal RNAs (18S, 5.8S, 28S, and 5 rRNAs). In the human system, three ribosomal RNAs, 18S, 5.8S, and 28S, arise from a single 47S pre-rRNA transcript. Maturation of these rRNAs takes place by a cascade of exo- and endo-ribonucleolytic cleavages of the primary 47S transcript. A limited number of small nucleolar RNAs (snoRNAs), such as RMRP are known to guide endoribonucleolytic cleavages of the 47S pre-rRNA transcript. Here, we hypothesized that 47S pre-rRNA processing is impaired in osteoarthritic articular chondrocytes, and contributes to OA progression. Methods: Primary human knee OA articular chondrocytes (pool of four donors, p2, seeding density 30,000 cells/cm2) were cultured in DMEM/F12, 10% FCS, 1% P/S, 1% NEAA and exposed to knee OA synovial fluid (OA-SF, 20% (v/v), pool of five donors) for 1-14 days. Expression levels of chondrocytes phenotypic marker genes (COL2A1, ACAN, COL10A1, COL1A1), selected 47S processing intermediates and RMRP snoRNA were measured by qPCR. Statistical significance was assessed by two-way ANOVA, Bonferroni post-tests, *p<0.05, **p<0.01, ***p<0.001 Results: To test our hypothesis, we set-up an OA-mimicking in vitro model using human primary articular chondrocytes exposed to osteoarthritic SF (OA-SF) for 1 to 14 days. The expression of chondrogenic marker genes COL2A1 (Figure 1A) and ACAN, as well as hypertrophic marker gene COL10A1, were significantly decreased immediately after the first day of treatment with OA-SF. The expression of COL1A1 (Figure. 1B), a marker of chondrocyte dedifferentiation, increased significantly upon OA-SF treatment in a time-dependent manner. To evaluate the 47S pre-rRNA processing cascade, primers covering selected cleavage sites (01, 2 and 4a) in the 47S transcript were designed (Figure 1C). Upon OA-SF treatment, we observed accelerated cleavage at site 01 within the 5’ ETS region, and site 2 within the ITS1. Cleavage at site 2 separates pre-18S and pre-5.8S-28S intermediates and it is guided by snoRNA RMRP, expression of which was increased upon OA-SF treatment. On the contrary, cleavage at site 4a within the ITS2 region, generating separated pre-5.8S and pre-28S precursors, was hindered in our model (Figure 1D). These results demonstrate that the dynamics of the 47S primary transcript processing is altered in our OA-mimicking in vitro model, showing accelerated processing towards mature 18S rRNA, and hindered processing towards mature 5.8S and 28S rRNAs. Conclusions: During OA, the chondrocyte’s environment changes dramatically, fueling a gradual change of the chondrocyte’s phenotype. In response to OA onset and progression, chondrocytes can adopt a variety of different OA-associated phenotypes ranging from proliferative, pre-hypertrophic, hypertrophic to a fibroblast-like. Upon OA-SF treatment we observed a progressive decrease in expression of COL2A1, ACAN, as well as COL10A1 genes, while expression of the dedifferentiation marker gene COL1A1 was significantly increased. Such an expression pattern is typical for dedifferentiating chondrocytes, and it indicates that chondrocytes treated with OA-SF are predominantly acquiring a fibroblast-like phenotype. Our results demonstrate alterations in the 47S pre-rRNA processing cascade, suggesting that 47S processing is vulnerable to changes in the chondrocyte’s extracellular environment. That can potentially lead to OA pathobiologically important alterations in ribosome biogenesis with consequences for the chondrocyte’s proteome. Future experiments are focussing on the validation of these findings by northern blotting and detailed unbiased mapping of the 47S processing cascade using RNA sequencing technology.