Molecular profile of synovial fibroblasts in rheumatoid arthritis depends on the stage of proliferation

Abstract

The aim of this study was to explore the molecular profile of proliferating rheumatoid arthritis synovial fibroblasts (RA-SF). Total RNA was extracted from two cultures of RA-SF (low-density [LD] proliferating cells and high-density [HD] nonproliferating cells) and suppression subtractive hybridization was performed to compare differential gene expression of these two cultures. Subtracted cDNA was subcloned, and nucleotide sequences were analyzed to identify each clone. Differential expression of distinct clones was confirmed by semiquantitative RT-PCR. The expression of certain genes in synovial tissues was examined by in situ hybridization. In both LD and HD cells, 44 clones were upregulated. Of the 88 total clones, 46 were identical to sequences that have previously been characterized. Twenty-nine clones were identical to cDNAs that have been identified, but with unknown functions so far, and 13 clones did not show any significant homology to sequences in GenBank (NCBI). Differential expression of distinct clones was confirmed by RT-PCR. In situ hybridization showed that certain genes, such as S100A4, NFAT5, unr and Fbx3, were also expressed predominantly in synovial tissues from patients with RA but not from normal individuals. The expression of distinct genes in proliferating RA-SF could also be found in RA synovium, suggesting that these molecules are involved in synovial activation in RA. Most importantly, the data indicate that the expression of certain genes in RA-SF depends on the stage of proliferation; therefore, the stage needs to be considered in any analysis of differential gene expression in SF.

Figure 1

The list of clones obtained by forward (LD-specific) subtraction. We divided these clones into three categories. (a) This category consists of already identified and characterized genes. (b) This category includes the genes with sequences previously identified, but with unknown functions so far. The name of each clone is shown. (c) List of novel gene fragments that did not show any significant homology to sequences in GenBank (NCBI). We mention just the name of clones in our experiments.

Figure 2

The list of clones obtained by reverse (HD-specific) subtraction. We divided these clones into the same three categories as described in Figure 1.

Figure 3

The differential expression of distinct genes. It was confirmed by semiquantitative RT-PCR. (a) LD-specific genes such as EFEMP1, unr and Fbx3, and (b) HD-specific genes such as CD26, 13kdap and RNASE4 were upregulated in LD and HD cells, respectively. (c) The results of actin-β and GAPDH indicated that we compared equal amounts of template cDNA in these experiments. We analyzed three independent RA-SF and show the representative results.

Figure 4

The expression of S100A4 mRNA in sections from patients with RA by in situ hybridization. This figure shows antisense (a,b) and sense (c) staining, respectively. We also performed HE staining (d) in serial sections. The original magnifications are 200× (a,c,d) and 400× (b), respectively.

Figure 5

The expression of NFAT5 mRNA in sections from patients with RA by in situ hybridization. This figure shows antisense (a,b) and sense (c) staining, respectively. We also performed HE staining (d) in serial sections. The original magnifications are 100× (a,c,d) and 200× (b), respectively.

Figure 6

The expression of NFAT5 mRNA in sections from patients with RA by in situ hybridization. This figure shows antisense (a,b) and sense (c) staining, respectively. The positive signal was observed not only in fibroblast-like cells (arrowheads) but also in osteoclast-like cells (arrows). We also performed HE staining (d) in serial sections. The original magnifications are 200× (a,c,d) and 400× (b), respectively.

Supplementary Figure 1

The strategy of our experiments. SF from one patient with RA (passage6) were cultured in 75 cm² flasks until reaching confluence. Then, these cells were collected and seeded in 12-well culture plates (1.0 × 10⁵ cells/well). At 1, 2, 4, 7, 10 and 14 days after seeding, the number of cells was counted by the trypan blue exclusion method. At 4 days, the cells at low density (LD) proliferated exponentially, and at 14 days, the cells at high density (HD) almost ceased to proliferate as a result of contact inhibition. The inserts show the HE staining of SF on chamber slides at 4 and 14 days after seeding, respectively.

Supplementary Figure 2

The expression of unr mRNA in sections from patients with RA as shown by in situ hybridization. This figure shows antisense (a,b) and sense (c) staining, respectively. We also performed HE staining (d) in serial sections. The original magnifications are 200× (a,c,d) and 400× (b), respectively.

Supplementary Figure 3

The expression of Fbx3 mRNA in sections from patients with RA as shown by in situ hybridization. This figure shows antisense (a,b) and sense (c) staining, respectively. We also performed HE staining (d) in serial sections. The original magnifications are 100× (a,c,d) and 400× (b), respectively.

Supplementary Figure 4

The expression of Fbx3 mRNA in sections from patients with RA by in situ hybridization. This figure shows antisense (a,b) and sense (c) staining, respectively. The positive signal was observed not only in fibroblast-like cells (arrowheads) but also in osteoclast-like cells (arrow). We also performed HE staining (d) in serial sections. The original magnifications are 200× (a,c,d) and 400× (b), respectively.

Supplementary Figure 5

Summary of the expression of LD-specific genes in synovial tissues from patients with RA (●) and from normal individuals (○). In each experiment, we counted the number of positive cells in the lining of synovial tissues, and the levels of the mRNA expression were calculated as a percentage of positive cells. Then, we divided these data into three groups; namely, below 10% (Low), from 10 to 50% (Moderate) and more than 50% (High).