The importance of TNF-α in the pathogenesis of RA has been well appreciated. Thus, anti-TNF-α antibodies and soluble TNF receptors have been demonstrated to have beneficial effects in the treatment of RA [4
]. On the other hand, increasing attention has been paid to the role of bone marrow abnormalities in the pathogenesis of RA. In this regard, we demonstrated that RA bone marrow CD34+ cells have abnormal capacities to respond to TNF-α and to differentiate into fibroblast-like cells producing MMP-1 [2
]. It should be noted that NFκB plays an important role in signal transduction and expression of a variety of genes, including MMP-1, under the influence of TNF-α [3
]. The results in the current study have demonstrated that the expression of mRNA for NFκB1 is increased in RA bone marrow CD34+ cells. Of note, the expression of NFκB1 mRNA was significantly correlated with that of NFκB1 protein. Moreover, the initial levels of NFκB1 mRNA in RA bone marrow CD34+ cells were correlated with their capacity to differentiate into fibroblast-like cells upon stimulation with TNF-α. The data suggest that the increased expression of NFκB1 mRNA might lead to constitutive overproduction of NFκB p50 molecules and thus result in abnormal responses to TNF-α of RA bone marrow CD34+ cells. Of note, bee venom and its major component melittin have been shown to display anti-arthritic effects through inactivation of NFκB [10
]. Since bee venom and melittin delay and reduce nuclear translocation of the p50 subunit of NFκB but not p65 (RelA) [10
], the importance of NFκB p50 rather than p65 in the pathogenesis of inflammatory arthritides has been underscored.
In the present study, significant numbers of RA patients were treated with MTX and oral steroids. However, there were no significant differences in the expression of NFκB1 mRNA in bone marrow CD34+ cells between RA patients receiving MTX or oral steroids and those who were not, although the expression of NFκB1 mRNA appeared to be lower in RA patients receiving these drugs. It is suggested, therefore, that administration of MTX and oral steroids might have made the differences in the expression of NFκB1 mRNA in bone marrow CD34+ cells between RA and OA less marked. On the other hand, the expression of NFκB1 mRNA in bone marrow CD34+ cells was not correlated with serum CRP levels in RA patients. The upregulation of NFκB1 mRNA in bone marrow CD34+ cells might not, therefore, be secondary to systemic inflammation, but may be a primary abnormality intrinsic to RA.
In the present study, the expression of mRNA for RelA (p65) appeared to be decreased in RA bone marrow CD34+ cells compared with that in OA bone marrow CD34+ cells, although this decrease did not reach statistical significance. Of note, a previous study demonstrated that embryonic fibroblasts from RelA-deficient mice are defective in the TNF-α mediated induction of mRNAs for IκBα [11
]. Moreover, in RelA deficient fibroblasts, IκBβ protein was absent, presumably due to the decreased stability of IκBβ mRNA [11
]. Since IκB plays an important role in inhibition of translocation of NFκB into the nucleus, the decrease in RelA mRNA might result in enhanced activation of NFκB related genes through upregulation of the translocation of NFκB. It is suggested, therefore, that the decreased expression of RelA mRNA in RA bone marrow CD34+ cells might also contribute to abnormal response to TNF-α.
It is possible that the upregulation of NFκB1 mRNA in bone marrow CD34+ cells might be secondary to the increased levels of TNF-α in the bone marrow. In fact, the treatment of bone marrow CD34+ cells from healthy individuals with TNF-α resulted in the increased expression of NFκB1 mRNA. However, TNF-α also enhanced the expression of mRNAs for NFκB2 and RelA in bone marrow CD34+ cells from healthy individuals. Of note, the expression of RelA mRNA appeared to be rather decreased in RA bone marrow CD34+ cells as mentioned above. Taken together, these data strongly suggest that the enhanced expression of NFκB1 mRNA might not be due simply to the increased levels of TNF-α in the bone marrow. Further studies to explore the mechanism of abnormal expression of NFκB1 mRNA in bone marrow CD34+ cells would be important for delineation of the pathogenesis of RA.
The role of the enhanced expression of NFκB1 mRNA in RA bone marrow CD34+ cells in their abnormal responses to TNF-α was further confirmed by the experiments of selective silencing of NFκB1 mRNA. Reduction of NFκB1 mRNA in RA bone marrow CD34+ cells by transfection of siRNA for NFκB1 markedly suppressed the generation of fibroblast-like cells as well as the production of MMP-1 and VEGF under the influence of TNF-α without affecting the viability or the capacity to produce β2MG. These results indicate that upregulation of NFκB1 mRNA expression leads to the enhanced responses of RA bone marrow CD34+ cells to TNF-α. Thus, the enhanced NFκB1 mRNA expression might be a critical defect in RA bone marrow CD34+ cells.
Autologous hematopoietic stem cell transplantation (HSCT) has been used to treat severe RA in limited case reports [12
]. However, a study with large numbers of patients has disclosed that recurrence of RA is frequent in patients who received autologous HSCT [14
]. Frequent recurrence after autologous HSCT for RA suggests that abnormalities in bone marrow stem cells might persist after the treatment [16
]. It is possible that the enhanced expression of NFκB1 mRNA might be closely related with such abnormalities in bone marrow stem cells, although further studies are required to confirm this point. It would also be important to explore whether there might be another transcription factor that could be inhibited without suppressing the differentiation of bone marrow CD34+ cells into fibroblast-like cells in order to confirm the importance of NFκB1 mRNA expression in the pathogenesis of RA.