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- Materials and methods
Advanced chronic idiopathic myelofibrosis (IMF) with osteosclerosis and increase and thickening of bone trabeculae is typically contrasted by the absence or sparse presence of osteoclasts. Because osteoclast formation can be inhibited by osteoprotegerin (OPG) we investigated OPG expression in IMF with severe fibrosis and osteosclerosis, which expressed significantly higher (up to 71-fold) OPG mRNA levels when compared with prefibrotic cellular IMF and control cases. The receptor activator of nuclear factor kappaB ligand (RANKL), a positive regulator of osteoclast differentiation and putative antagonist of OPG was overexpressed by up to 34-fold exclusively in advanced IMF. Case-specific calculation of the RANKL/OPG ratio in advanced IMF showed a wide range without significant differences when compared with the prefibrotic IMF and non-neoplastic haematopoiesis. Immunohistochemical detection of OPG protein revealed strong labelling of endothelial cells within proliferating vessels in fibrotic IMF and heterogeneously labelled megakaryocytes, and fibroblasts. Osteosclerosis and impaired osteoclast function in IMF appears to be associated with upregulated endothelial OPG expression but concomitant reduction of the antagonist RANKL could not be demonstrated. We conclude that osteosclerosis in IMF is associated with increased endothelial OPG expression without concomitant RANKL downregulation.
Idiopathic myelofibrosis (IMF) belongs to the chronic myeloproliferative disorders (CMPD). Like the other CMPDs, it is caused by the clonal proliferation of a haematopoietic stem cell retaining the capacity to differentiate into all lineages (Tefferi, 2000). A feature that distinguishes IMF is the progression to bone marrow fibrosis and osteosclerosis whereby the proliferating fibroblasts are reactive and non-clonal in nature (Tefferi, 2000). In advanced stages with osteosclerosis there is an increase and thickening of bone trabeculae that characteristically lack rimming osteoblasts and osteoclasts (Ward & Block, 1971).
In normal bone homeostasis, osteoprotegerin (OPG) and the receptor activator of nuclear factor kappaB ligand (RANKL, also called TRANCE [tumour necrosis factor (TNF)-related, activation-induced cytokine)], participate in a cytokine axis that tightly controls the differentiation of osteoclasts from monocyte precursors. OPG, a member of the TNF-receptor family, acts as soluble decoy receptor for RANKL thereby limiting binding of RANKL to its functional receptor RANK (Hofbauer & Schoppet, 2004). RANKL is highly expressed in areas of trabecular bone remodelling and provides an important signal required for full osteoclast development, activation and survival (Hofbauer & Schoppet, 2004). Previous studies showed that, in patients with multiple myeloma and other diseases associated with bone destruction, an increase in RANKL expression along with a decrease in OPG expression triggered osteolysis by favouring osteoclast differentiation (Pearse et al, 2001; Grimaud et al, 2003). Accordingly, therapeutic studies using RANKL antagonists have been conducted in order to prove clinical benefit for patients with osteolysis and bone destruction (Sordillo & Pearse, 2003).
Recently, Chagraoui et al (2003a) demonstrated, in a murine model of IMF, that OPG induced inhibition of osteoclastogenesis might be responsible for the development of osteosclerosis by engrafting thrombopoietin-overexpressing haematopoietic cells into OPG-deficient recipients. In this animal model, stromal cells, but not haematopoietic cells, were identified as potential producers of OPG. Enhanced plasma levels of OPG were found in patients with manifest IMF (Wang et al, 2004). Expression of RANKL as well as the cellular source of enhanced OPG expression in IMF has not been studied to date. The aim of the present study was to (i) determine the expression level of RANKL and OPG mRNA in bone marrow cells derived from non-neoplastic haematopoiesis in comparison with IMF in the prefibrotic and advanced stage, (ii) demonstrate RANKL/OPG mRNA ratios in IMF and non-neoplastic haematopoiesis, and (iii) identify the cellular source of both RANKL and OPG protein in the bone marrow cells.
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- Materials and methods
Our study revealed increased OPG mRNA and protein expression in advanced IMF, which is in close agreement with a recent study demonstrating elevated OPG plasma levels in patients with IMF in comparison with normal volunteers (Wang et al, 2004). Initially identified as a novel member of the TNF receptor superfamily (Simonet et al, 1997), OPG represents a key molecule in the regulation of bone formation and turnover through inhibition of osteoclast differentiation. Recent data showed that OPG, upregulated in vivo in mice overexpressing TPO and TGFβ-1, led to severe osteosclerosis (Chagraoui et al, 2003a). In contrast, mice deficient for OPG developed extensive osteoporosis (Bucay et al, 1998).
Studies in a murine model of IMF combined with induced OPG deficiency suggested that not haematopoietic but stromal cells provide the relevant source for OPG production although megakaryocytes in mice have the potential to release OPG (Chagraoui et al, 2003b). The cellular origin of OPG in patients with IMF has not been elucidated. Therefore immunohistochemistry was performed to precisely delineate OPG overexpressing cells in the bone marrow. As shown in Fig. 5 the strongest labelling was observed in endothelial cells of extended and proliferating vessels. Besides endothelial cells, fibroblasts in areas with manifest fibrosis were also occasionally labelled for OPG. Megakaryocytes heterogeneously exhibited a rather dot-like and faint labelling (Fig. 5A and insert). Osteoblasts and osteoclasts in advanced IMF were infrequently demonstrated. Positive OPG labelling was detectable in both cell types (Fig. 2C, insert). With regard to endothelial cell labelling, reactive states and normal bone marrow endothelial cells also displayed OPG, but to a lesser degree, corresponding to the lower OPG mRNA level.
Endothelial cells have been identified as potential producers of OPG (Collin-Osdoby et al, 2001). Besides a role in bone homeostasis, in that endothelial cells may be involved (Simonet et al, 1997), OPG has also been suggested to be important for endothelial proliferation and survival (Malyankar et al, 2000). Neoangiogenesis is a prominent feature in progressed IMF (Mesa et al, 2003). Exaggerated production of OPG therefore might have a dual pathogenetic function in progressed IMF. Besides impairment of osteoclast formation, thereby inducing osteosclerosis, OPG might contribute to endothelial growth and neoangiogenesis.
Overexpression of OPG was not accompanied by a decreased expression of its putative antagonist RANKL. Indeed, RANKL mRNA was also overexpressed in advanced IMF. Case-specific RANKL/OPG ratios in advanced IMF revealed clearly increased and decreased, as well as balanced, subsets. Also, prefibrotic IMF and control haematopoiesis showed an almost similar pattern of ratios even though outliers could not be demonstrated to the same extent. Studies on osteolytic bone lesions suggested the principle of a shift towards an increase of the RANKL/OPG ratio (Grimaud et al, 2003). Considering this principle, the ratio should decrease inversely in a disease that displays bone apposition, such as IMF. Even though nearly half of the cases showed a RANKL/OPG ratio below 1, therefore favouring OPG, a straightforward decrease was not demonstrable. With regard to comparison of RANKL/OPG ratios in the total study group, both IMF in the prefibrotic phase as well as advanced stages in the median exhibited higher ratios when compared with non-neoplastic haematopoiesis.
As demonstrated for multiple myeloma the imbalance of increased RANKL and decreased OPG consecutively led to osteolysis and progression of the disease (Pearse et al, 2001). Accordingly, an opposite ratio that favours OPG might explain decreased bone turnover and bone apposition in advanced IMF. As shown, the RANKL/OPG ratios were rather higher for prefibrotic and advanced IMF when compared with the control cases even though this did not reach statistical significance. Indeed, variable intraindividual patterns for both factors under investigation led to a remarkable range of ratios. In IMF, deregulated bone remodelling therefore could not solely be explained by opposite expression patterns of RANKL and OPG. Both factors are generally synthesised and secreted by identical cell types, including those derived from stroma, bone matrix, and diverse haematopoietic progenitors. Recently, the important role of megakaryocytes in bone remodelling has been elegantly demonstrated in a co-culture system of osteoblasts and megakaryocytes (Bord et al, 2005). In this model, megakaryocytes increased type-1 collagen and OPG expression by osteoblasts along with a remarkable decrease of RANKL (Bord et al, 2005). The same group had previously demonstrated that megakaryocyte expression of OPG and RANKL is inversely modulated by oestrogens (Bord et al, 2004). It should be noted that in this study, the megakaryocytes under investigation were non-neoplastic. The complexity of mechanisms involved in bone remodelling has been recently demonstrated through the essential role of the dendritic cell-specific transmembrane protein (DC-STAMP) in osteoclastogenesis (Kukita et al, 2004). Indeed, RANKL induced DC-STAMP expression in osteoclast precursors and differentiation whereas small interfering RNAs and specific antibodies markedly suppressed formation of osteoclasts. In addition, the crucial role of TNF receptor-associated factors (TRAF), such as TRAF2 and TRAF6, in RANKL-induced osteoclast differentiation could be demonstrated (Kanazawa & Kudo, 2005; Lee et al, 2005). Accordingly, RANKL per se no longer appears to be the essential factor for osteoclastogenesis. IMF represents a neoplastic stem cell disease with atypia occurring in many cell lines. Hence, disturbance of the signal transduction in the monocyte lineage affecting the tight control of cellular differentiation cannot be excluded with certainty.
We conclude that OPG appears to be involved in reduced osteoclastogenesis in advanced IMF through overexpression by endothelial cells. In addition, an important role for OPG in endothelial cell survival and neoangiogenesis in advanced IMF must be considered. In contrast to other diseases showing lytic bone lesions because of RANKL upregulation, such as multiple myeloma, the RANKL/OPG ratio was not demonstrated to be notably shifted in IMF that showed decreased bone turnover. Future studies of other factors involved in bone remodelling downstream of RANKL, such as DC-STAMP, will shed light on the complex regulation of bone apposition and turnover in IMF.