Transcriptome-wide analysis of messenger RNA decay in normal and osteoarthritic human articular chondrocytes

Abstract

Objective: Messenger RNA (mRNA) decay rates control not only gene expression levels, but also responsiveness to altered transcriptional input. We undertook this study to examine transcriptome-wide posttranscriptional regulation in both normal and osteoarthritic (OA) human articular chondrocytes.

Methods: Human articular chondrocytes were isolated from normal or OA tissue. Equine articular chondrocytes were isolated from young or old horses at a commercial abattoir. RNA decay was measured across the transcriptome in human cells by microarray analysis following an actinomycin D chase. Messenger RNA levels in samples were confirmed using quantitative reverse transcription-polymerase chain reaction.

Results: Examination of total mRNA expression levels demonstrated significant differences in the expression of transcripts between normal and OA chondrocytes. Interestingly, almost no difference was observed in total mRNA expression between chondrocytes from intact OA cartilage and those from fibrillated OA cartilage. Decay analysis revealed a set of rapidly turned over transcripts associated with transcriptional control and programmed cell death that were common to all chondrocytes and contained binding sites for abundant cartilage microRNAs. Many transcripts exhibited altered mRNA half-lives in human OA chondrocytes compared to normal cells. Specific transcripts whose decay rates were altered were generally less stable in these pathologic cells. Examination of selected genes in chondrocytes from young and old healthy horses did not identify any change in mRNA turnover.

Conclusion: This is the first investigation into the "posttranscriptome" of the chondrocyte. It identifies a set of short-lived chondrocyte mRNAs likely to be highly responsive to altered transcriptional input as well as mRNAs whose decay rates are affected in OA chondrocytes.

Figure 1

A, Scatterplots demonstrating the relationships between log₂ gene expression levels between normal chondrocytes, intact osteoarthritic (OA) chondrocytes, and fibrillated OA chondrocytes (green and red dots represent genes that exhibit change between groups with P < 0.01). B, Pie charts representing the occurrence of major ontological annotations associated with genes significantly up-regulated and down-regulated in OA chondrocytes, either in location (top) or in process (bottom) categories of the Panther Classification System. Exploded regions on charts emphasize the number of extracellular-associated genes differentially regulated. C, Heatmap representing expression levels of the top 25 up-regulated and the top 25 down-regulated genes in OA chondrocytes. Both the gene and sample lists were subjected to hierarchical clustering. Color key indicates the normalized log₂ hybridization values. Average histologic scores of samples at the bottom are shown in Table1. ECM = extracellular matrix.

Figure 2

Profile of mRNA half-life in normal and osteoarthritic (OA) chondrocytes and in OA chondrocytes stratified into those isolated from intact OA and fibrillated OA tissue. The analysis caps longer half-lives at 24 hours, which accounts for the high peak at the far right in each panel (which extends beyond the limit of the y-axis in each case). Inserts show the same data on zoomed y-axis.

Figure 3

Effect of osteoarthritis (OA) on mRNA decay. A, Scatterplot relating the observed fold change in total mRNA levels between normal and OA cells to the fold change in mRNA half-life. The vertical line represents a normal:OA total mRNA ratio of 1:1, such that all points to the left of the line represent genes with increased expression in OA. The horizontal line represents a 2-fold change in the normal:OA mRNA half-life ratio, such that genes that are above this line are less stable in OA cells. B, Pie charts representing the occurrence of major ontological annotations associated with genes significantly up-regulated and down-regulated in OA chondrocytes, either in location (top) or in process (bottom) categories of the Panther Classification System. Exploded regions on charts emphasize the number of extracellular-associated genes differentially regulated. C, Real-time reverse transcription–polymerase chain reaction analysis of mRNA decay, confirming alterations between normal and intact OA chondrocytes in mRNA decay of selected genes. Values are mean ± SEM fold change in mRNA levels compared to 0 hours of the decay curve (data from 5 normal donors and 5 OA donors; see Table1). Data are presented for 6 genes in which the false discovery rate (FDR) was <0.05 in microarray analysis and for 3 genes in which the average half-life was altered but the FDR was >0.05. ECM = extracellular matrix.

Figure 4

Effect of age on mRNA decay of candidate genes in equine articular chondrocytes. Plots show mRNA half-life measured in hours from freshly isolated cells, plotted against the age in years of the donor animal. Data are from 7 animals ranging in age from 3 years to 20 years. Symbols represent individual samples.