We analyzed the expression profile of proliferating RA-SF in comparison with that of nonproliferating RA-SF, and we observed that distinct sets of genes, including several novel genes that have not been associated with RA, were expressed differentially in proliferating RA-SF by using a PCR-based suppression subtractive hybridization technique. It is useful for development of novel therapeutic approaches to identify the expression profiles in disease-associated cells or conditions [12
], and there are some reports that identified genes specifically expressed in rheumatoid synovial cells by using a cDNA subtraction or a differential display method [14
]. Since we used RA-SF from the same patient, at the same passage and cultured with the same medium, we could exclude genetic polymorphism between individuals, and could identify specific genes dependent on proliferation or cell density.
In LD-specific subtracted cDNA, we isolated several interesting molecules that play an important role in cell proliferation. For example, we found upregulation of several ECM proteins. ECM proteins not only serve just as a mechanical structure, but also play an important role in many biological functions, such as cell migration, proliferation and differentiation [18
]. Indeed, it was reported that versican stimulates cell proliferation [19
], and EFEMP1 stimulates DNA synthesis when microinjected into fibroblasts [21
]. Fibronectin fragments play an important role in proliferation, adhesion and matrix metalloproteinase expression of RA-SF [22
]. The expression of ECM proteins in proliferating RA-SF, therefore, could stimulate their proliferation and differentiation in an autocrine and/or paracrine manner.
The expression of proliferation-related molecules such as AZI
was also observed in LD cells. AZI induces polyamine synthesis, which is essential for cell growth [23
], and it was reported that AZI is differentially expressed in tumor tissues rather than normal tissues [25
]. Although the function of Fbx3 has not been identified so far, F-box proteins are important for controlling the cell cycle [26
]. Simon et al.
] demonstrated that NAP1L1 is highly expressed in proliferating cells. On the other hand, unr
was also highly expressed in LD cells, and it was reported that unr interacts with apoptotic protease-activating factor-1 (Apaf-1) and could modulate a certain apoptotic pathway [28
]. We can hypothesize, therefore, that these molecules are not only essential for cell survival and proliferation in general, but could also play an important role in the proliferation of RA-SF in vitro
and in vivo
In HD-specific subtracted cDNA, we also obtained several interesting molecules. Indeed, we found that CD26
were specifically expressed in non-proliferating RA-SF. CD26 was originally identified as an adenosine deaminase complexing protein and T-cell activation molecule, and it was reported that activated T cells as well as fibroblasts in rheumatoid synovium expressed this molecule [29
]. Recently, it was shown that the malignant phenotype of melanoma cells is suppressed by the introduction of the exogenous CD26 gene [30
]. EPLIN is downregulated or lost in cancer cell lines and inhibits cell proliferation when overexpressed [31
]. On the other hand, RNASE 4 mediates the antiviral and antiproliferative roles of interferons and is induced during growth inhibition in murine cell lines [33
]. Recently, Taniguchi et al
] showed that cyclin-dependent kinase inhibitor p16INK4a
is induced in RA-SF by several growth-inhibited conditions, including irradiation, serum depletion and high cellular density. Surprisingly, they showed also that the gene transfer of p16 inhibits proliferation of synovial fibroblasts in vitro
and suppresses rat adjuvant arthritis in vivo
. In this context, EPLIN
genes might also be promising candidates for gene transfer as a novel approach to treating RA.