Myostatin (also known as growth and differentiation factor 8) is a secreted member of the transforming growth factor-β (TGF-β) family that is mainly expressed in skeletal muscle, which is also its primary target tissue. Deletion of the myostatin gene (Mstn) in mice leads to muscle hypertrophy, and animal studies support the concept that myostatin is a negative regulator of muscle growth and regeneration. However, myostatin deficiency also increases bone formation, mainly through loading-associated effects on bone. Here we report a previously unknown direct role for myostatin in osteoclastogenesis and in the progressive loss of articular bone in rheumatoid arthritis (RA). We demonstrate that myostatin is highly expressed in the synovial tissues of RA subjects and of human tumor necrosis factor (TNF)-α transgenic (hTNFtg) mice, a model for human RA. Myostatin strongly accelerates receptor activator of nuclear factor κB ligand (RANKL)-mediated osteoclast formation in vitro through transcription factor SMAD2-dependent regulation of nuclear factor of activated T-cells (NFATC1). Myostatin deficiency or antibody-mediated inhibition leads to an amelioration of arthritis severity in hTNFtg mice, chiefly reflected by less bone destruction. Consistent with these effects in hTNFtg mice, the lack of myostatin leads to increased grip strength and less bone erosion in the K/BxN serum-induced arthritis model in mice. The results strongly suggest that myostatin is a potent therapeutic target for interfering with osteoclast formation and joint destruction in RA.

Vascular endothelial growth factor (VEGF), a multifunctional cytokine, potently stimulates angiogenesis including tumor neovascularization. Although well established in solid tumors, the role of VEGF in bone marrow neoangiogenesis and paracrine tumor-stromal cell interactions in lymphohematopoietic malignancies has not been fully elucidated. In multiple myeloma (MM), marrow neovascularization parallels disease progression. This parallel prompted us to investigate the expression and secretion of VEGF by myeloma cells and its potential effects in myeloma-marrow stroma interactions. The biologically active splice variants VEGF165 and VEGF121 were expressed and secreted by myeloma cell lines and plasma cells isolated from the marrow of patients with MM. As shown by immunocytochemistry or RT-PCR, myeloma cells did not express or weakly expressed the VEGF receptors FLT-1 and FLK-1/KDR, indicating that autocrine stimulation is unlikely. In contrast, FLK-1/KDR was abundantly expressed by marrow stromal cells. Therefore, we studied the effects of VEGF on marrow stroma, focusing on the secretion of interleukin-6 (IL-6), a potent growth factor for myeloma cells and an inhibitor of plasma cell apoptosis. Exposure of stromal and microvascular endothelial cells to recombinant human (rh) VEGF165 or VEGF121 induced a time- and dose-dependent increase in IL-6 secretion (14- to 27-fold at 50 ng/mL after 24 hours, P < .001). Conversely, rhIL-6 stimulated VEGF expression and secretion in myeloma cell lines (40%-60%; P < .05) and to a variable degree (up to 5.3-fold; P < .005) in plasma cells purified from the marrow of patients with MM. This mutual stimulation suggests paracrine interactions between myeloma and marrow stromal cells triggered by VEGF and IL-6.

Myeloma cells express basic fibroblast growth factor (bFGF), an angiogenic cytokine triggering marrow neovascularization in multiple myeloma (MM). In solid tumors and some lymphohematopoietic malignancies, angiogenic cytokines have also been shown to stimulate tumor growth via paracrine pathways. Since interleukin-6 (IL-6) is a potent growth and survival factor for myeloma cells, we have studied the effects of bFGF on IL-6 secretion by bone marrow stromal cells (BMSCs) and its potential reverse regulation in myeloma cells. Both myeloma-derived cell lines and myeloma cells isolated from the marrow of MM patients were shown to express and secrete bFGF. Cell-sorting studies identified myeloma cells as the predominant source of bFGF in MM marrow. BMSCs from MM patients and control subjects expressed high-affinity FGF receptors R1 through R4. Stimulation of BMSCs with bFGF induced a time- and dose-dependent increase in IL-6 secretion (median, 2-fold; P <.001), which was completely abrogated by anti-bFGF antibodies. Conversely, stimulation with IL-6 enhanced bFGF expression and secretion by myeloma cell lines (2-fold; P =.02) as well as MM patient cells (up to 3.6-fold; median, 1.5-fold; P =.002). This effect was inhibited by anti-IL-6 antibody. When myeloma cells were cocultured with BMSCs in a noncontact transwell system, both IL-6 and bFGF concentrations in coculture supernatants increased 2- to 3-fold over the sum of basal concentrations in the monoculture controls. The IL-6 increase was again partially, but significantly, inhibited by anti-bFGF. The data demonstrate a paracrine interaction between myeloma and marrow stromal cells triggered by mutual stimulation of bFGF and IL-6.

The small ubiquitin-like modifier (SUMO)-1 is an important posttranslational regulator of different signaling pathways and involved in the formation of promyelocytic leukemia (PML) protein nuclear bodies (NBs). Overexpression of SUMO-1 has been associated with alterations in apoptosis, but the underlying mechanisms and their relevance for human diseases are not clear. Here, we show that the increased expression of SUMO-1 in rheumatoid arthritis (RA) synovial fibroblasts (SFs) contributes to the resistance of these cells against Fas-induced apoptosis through increased SUMOylation of nuclear PML protein and increased recruitment of the transcriptional repressor DAXX to PML NBs. We also show that the nuclear SUMO-protease SENP1, which is found at lower levels in RA SFs, can revert the apoptosis-inhibiting effects of SUMO-1 by releasing DAXX from PML NBs. Our findings indicate that in RA SFs overexpression of SENP1 can alter the SUMO-1-mediated recruitment of DAXX to PML NBs, thus influencing the proapoptotic effects of DAXX. Accumulation of DAXX in PML NBs by SUMO-1 may, therefore, contribute to the pathogenesis of inflammatory disorders.inflammation ͉ autoimmunity ͉ DAXX ͉ SENP

Sclerostin, an inhibitor of the Wnt/β-catenin pathway, has anti-anabolic effects on bone formation by negatively regulating osteoblast differentiation. Mutations in the human sclerostin gene (SOST) lead to sclerosteosis with progressive skeletal overgrowth, whereas sclerostin-deficient (Sost(-/-)) mice exhibit increased bone mass and strength. Therefore, antibody-mediated inhibition of sclerostin is currently being clinically evaluated for the treatment of postmenopausal osteoporosis in humans. We report that in chronic TNFα (tumor necrosis factor α)-dependent arthritis, fibroblast-like synoviocytes constitute a major source of sclerostin and that either the lack of sclerostin or its antibody-mediated inhibition leads to an acceleration of rheumatoid arthritis (RA)-like disease in human TNFα transgenic (hTNFtg) mice with enhanced pannus formation and joint destruction. Inhibition of sclerostin also failed to improve clinical signs and joint destruction in the partially TNFα-dependent glucose-6-phosphate isomerase-induced arthritis mouse model, but ameliorated disease severity in K/BxN serum transfer-induced arthritis mouse model, which is independent of TNF receptor signaling, thus suggesting a specific role for sclerostin in TNFα signaling. Sclerostin effectively blocked TNFα- but not interleukin-1-induced activation of p38, a key step in arthritis development, pointing to a previously unrealized protective role of sclerostin in TNF-mediated chronic inflammation. The possibility of anti-sclerostin antibody treatment worsening clinical RA outcome under chronic TNFα-dependent inflammatory conditions in mice means that caution should be taken both when considering such treatment for inflammatory bone loss in RA and when using anti-sclerostin antibodies in patients with TNFα-dependent comorbidities.