Detection of oncofetal h19 RNA in rheumatoid arthritis synovial tissue

Abstract

The expression of oncofetal H19 RNA and its localization/cellular source was analyzed in synovial tissue (ST) and isolated synovial macrophages (Mphi) or synovial fibroblasts (SFBs) by reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemistry. RT-PCR showed significantly higher H19 expression in ST from patients with rheumatoid arthritis (RA) (P = 0.000) and osteoarthritis (OA) (P = 0.009) than in normal/joint trauma controls (N/JT), but comparable levels in reactive arthritis. In situ hybridization demonstrated strong signals in all RA-ST samples (n = 8), with > or =85% positive cells in the lining layer, diffuse infiltrates, and stroma regions. In lymphoid aggregates and endothelial cells only 20% were positive. RA-ST contained a significantly higher percentage of strongly positive lining cells than OA-ST and N/JT-ST. H19 RNA was expressed in both Mphi and SFBs, as confirmed by RT-PCR in isolated RA Mphi and SFBs (n = 3). In RA-SFBs, low constitutive H19 RNA expression in culture (10% fetal calf serum) was strongly increased on starvation (3.5-fold, 1% fetal calf serum), with or without the addition of interleukin-1beta (10 to 100 U/ml), tumor necrosis factor-alpha (1 to 25 ng/ml), or platelet-derived growth factor-BB (2.5 to 10 U/ml). In OA-SFBs, this starvation-induced increase was lower (twofold), reaching significant differences compared with RA-SFBs after stimulation with interleukin-1beta and platelet-derived growth factor-BB. In both RA- and OA-SFBs, the MAP-kinase ERK-1/2 pathway and the phosphatidylinositol-3 kinase pathway influenced H19 RNA expression, as shown by inhibitor studies. Significant overexpression of H19 RNA and its increased sensitivity to starvation/cytokine regulation in RA suggests a pathogenetic role of this oncofetal gene, possibly reflecting embryonal dedifferentiation of the adult ST and/or ongoing inflammatory/oxidative stress.

Figure 1.

H19 RNA expression in ST (RT-PCR). Representative analysis of ST from patients with RA (n = 23), OA (n = 21), ReA (n = 2; lanes 1 and 2 in the two bottom gel panels), and normals/joint trauma (N/JT; n = 14; lanes 3 to 10 in the two bottom gel panels; A). Semiquantification and normalization to mRNA expression of GAPDH resulted in significantly higher H19 RNA expression in RA-ST versus N/JT-ST (***, P = 0.000) and in OA-ST versus N/JT-ST (**, P = 0.009; B). M, molecular size standard given in bp.

Figure 2.

Distribution and cellular sources of H19 RNA expression in ST (in situ hybridization). Six-μm cryostat sections from OA-ST (B, C, D) or RA-ST (A, E–I). The sense probe for H19 (negative control) did not bind to RA-ST (A) and OA-ST (B). The anti-sense probe for H19 showed a positive reaction in lining layer (arrowhead in C), diffuse infiltrates (arrow in C), stroma (# in C), and endothelial cells (* in C) of OA-ST (C) and RA-ST (E). Using double-in situ hybridization/IHC, H19 RNA was detected in CD68-positive Mφ and CD68-negative cells of OA-ST (D) and RA-ST (H and I, arrows); in serial sections, H19 expression in CD68-negative cells of RA-ST was predominantly in prolyl-4-hydroxylase-positive SFBs (F and G, arrowheads). Original magnifications: ×92 (A–E); ×184 (F–I).

Figure 3.

Distribution of H19 RNA expression in ST (semiquantitation of in situ hybridization). Strong signals for H19 RNA were obtained in all RA synovial specimens (n = 8). In lining layer, diffuse infiltrates, and stroma, ≥85% of the cells showed a positive signal, whereas lymphoid aggregates (only present in three of eight samples) and endothelial cells contained only ∼20% of positive cells. The H19 RNA expression in OA-ST (n = 5) was generally comparable to that in RA-ST. Significant differences between RA and OA (^§) were restricted to the lining layer. The percentage of H19-positive cells in lining layer and stroma of normal/joint trauma-ST (N/JT) was lower than in RA and OA, with significant differences in comparison with both RA and OA (*) in the lining layer. The percentage of H19-positive endothelial cells was comparable in RA-, OA-, and N/JT-ST.

Figure 4.

H19 RNA expression in synovial Mφ and SFBs isolated from RA (RT-PCR). A: Synovial Mφ and SFBs from patients with RA (n = 3) were analyzed by RT-PCR. B: Image analysis quantification and normalization to mRNA expression of GAPDH showed numerically higher H19 RNA expression in Mφ than in SFBs, but without statistical significance. M, molecular size standard given in bp.

Figure 5.

Regulation of H19 RNA expression in RA-SFBs/OA-SFBs on starvation (1% FCS) and stimulation with IL-1β, PDGF-BB, or TNF-α. A: Gel analysis of the H19 and β-actin PCR products from one RA patient. B: The means and standard errors of the mean (SEM) of the gel band quantification in four RA and five OA patients. Low constitutive expression of H19 RNA by RA-SFBs cultured in medium/10% FCS (A, B) was strongly (3.5-fold) induced after starvation for 96 hours in medium/1% FCS (B). This induction was significantly lower in OA-SFBs (approximately twofold). In neither RA- nor OA-SFBs, starvation-induced H19 expression was significantly influenced by the addition of IL-1β, PDGF, or TNF-α. M, molecular size standard given in bp. ^§, significant change.

Figure 6.

Influence of the blockade of MAP-kinase or PI3K signal transduction on the H19 RNA expression in RA-SFBs/OA-SFBs after stimulation with IL-1β or PDGF. A: Gel analysis of the H19 and β-actin PCR products from one RA patient. B: Means and standard errors of the mean (SEM) of the gel band quantification in three RA and three OA patients. In both RA- and OA-SFBs, the MAP-kinase ERK-1/2 and the phosphatidylinositol-3 kinase significantly influenced H19 RNA expression after IL-1β or PDGF-BB stimulation, as shown by inhibitor studies with U0126 and Wortmannin, respectively. M, molecular size standard given in bp.