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Annals of the Rheumatic Diseases logoLink to Annals of the Rheumatic Diseases
. 2004 Oct;63(10):1205–1211. doi: 10.1136/ard.2003.006312

Discrepancy between mRNA and protein expression of tumour suppressor maspin in synovial tissue may contribute to synovial hyperplasia in rheumatoid arthritis

J Schedel 1, O Distler 1, M Woenckhaus 1, R Gay 1, B Simmen 1, B Michel 1, U Muller-Ladner 1, S Gay 1
PMCID: PMC1754744  PMID: 15361372

Abstract

Objective: To investigate the expression of maspin in RA synovial tissue and compare it with the expression in osteoarthritis (OA) and normal synovial tissue (NS).

Methods: Using specific primers for maspin, a 237 bp fragment was amplified from cDNA obtained from cultured RA, OA, and normal synovial fibroblasts (SF) by RT-PCR. Additionally, mRNA expression levels were determined quantitatively by real time PCR. mRNA expression of maspin was investigated on snap frozen and paraffin embedded synovial tissue sections by in situ hybridisation. Immunohistochemistry was used to identify the cell type expressing maspin. SDS-PAGE and western blotting were performed to evaluate the protein expression in cultured SF. To confirm protein synthesis in situ, immunohistochemistry with specific anti-maspin antibodies was performed in synovial tissue sections of patients with RA.

Results: RT-PCR showed expression of maspin in all cDNA samples from cultured SF. Maspin mRNA was found to be decreased in RA SF twofold and 70-fold compared with OA SF and NS SF, respectively. Maspin mRNA was expressed in RA, OA, and normal synovial tissue. Importantly, maspin transcripts were also found at sites of invasion into cartilage and bone. At the protein level, maspin could be detected in RA and, less prominently, OA SF. In RA synovial tissue, maspin protein was detected in only a few synovial lining cells.

Conclusion: Maspin is expressed intensively in RA SF at the mRNA level, but only slightly at the protein level, possibly owing to down regulation of maspin; this may contribute to the hyperplasia of synovial tissue in RA.

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Figure 1.

Figure 1

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 RT-PCR using maspin-specific primers spanning a 237 bp fragment showed expression of maspin in all synovial fibroblast RNA samples examined (representative RA, OA, normal synovial tissue samples).

Figure 2.

Figure 2

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 SF from seven patients with RA, four with OA, and one trauma patient were examined by real time PCR with maspin-specific primers and probe (TaqMan system, PE Biosystems). Less maspin was expressed in RA samples than in OA and normal synovial fibroblast samples (twofold and 70-fold lower in comparison with OA and NS fibroblast samples, respectively).

Figure 3.

Figure 3

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 In situ hybridisation on paraffin embedded synovial tissue sections showed maspin mRNA expression in RA, OA, and normal synovial tissue. Whereas the expression of maspin was restricted to SF in lining and sublining in normal and OA synovial tissue (B and C), in RA, maspin mRNA was additionally seen within perivascular infiltrates (D). Prostate tissue was used as positive control showing staining in epithelial cells (A). In situ hybridisation using the sense probe remained negative (negative control; E). Higher magnification disclosed maspin expression in mononuclear cells around vessels (F) as well as in multinucleated cells resembling osteoclasts at sites of invasion of the synovial tissue into cartilage and bone (G). Cultured RA SF on chamber slides also showed a positive signal for maspin (H). Original magnification x100 (E), x200 (B, C, D), x400 (A, F), x630 (G, H).

Figure 4.

Figure 4

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 To further characterise the cell type expressing maspin, double labelling with anti-CD68 (A, C) and anti-CD3 antibodies (B, D) was performed after in situ hybridisation with maspin probes on paraffin embedded RA synovial tissue samples. Predominantly, maspin was detected in CD68 and CD3 negative SF. Blue-black colour: maspin transcripts, red-brown colour: anti-CD68 and anti-CD3 positive cells, respectively. Original magnificationx200 (A, C), x400 (B, D).

Figure 5.

Figure 5

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 Maspin mRNA expression was additionally investigated in RA synovial tissue at sites of cartilage and bone destruction. Maspin positive cells with both fibroblast-like (A) and macrophage-like as well as osteoclast-like morphology (C) were found. Double labelling with anti-CD68 antibodies showed both maspin positive, CD68 negative (figs 5B and 3G) as well as maspin positive, CD68 positive cells (D). Original magnification of all figures x400.

Figure 6.

Figure 6

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 To examine the protein expression of maspin in RA SF, SDS-PAGE and western blot with anti-maspin antibodies was performed in SF of seven patients with RA and four with OA. A positive band of the correct size (42 kDa) was detected in all RA SF and OA SF examined. In normal SF, a discrete band could also be detected (A). To confirm these results in situ, immunohistochemistry using anti-maspin antibodies was performed in synovial tissue samples of four patients with RA. Prostate tissue was used as positive control (result not shown). Maspin could be detected only in a few single cells in the synovial lining as well as single cells in the sublining (B, arrows). Original magnification x200.

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