Inducible Heme Oxygenase 1 (HMOX1) Promotes Osteoblastogenesis, and Inhibits Osteoclastogenesis and Myeloma-Induced Bone Disease

Abstract 627

Cytotherapy with mesenchymal stem cells (MSCs) has been shown to promote bone formation, inhibit bone disease and reduce multiple myeloma (MM) growth in myelomatous bone (Yaccoby et al., 2006; Li et al., 2011). At the cellular level, the infused MSCs act as a bystander cells that activate endogenous osteoblasts and suppress osteoclast activity. To shed light on molecular mechanisms associated with the cytotherapeutic effects of MSCs we exploited the SCID-hu model engrafted with the Hg MM line. Hg cells are only maintained by passaging in SCID-hu/SCID-rab mice and produce severe bone disease. Human global gene expression profile (GEP) was performed in nonmyelomatous implanted bones (n=5), and in myelomatous implanted bones injected with human MSCs (1×10⁶ cells/bone) and analyzed immediately (control group, n=8) or 24 hours later (cytotherapy, n=6). Based on stringent criteria, (e.g. ≥2 folds, p<0.05, signal intensity ≥500 in upregulated or downregulated genes in cytotherapy and control groups, respectively), approximately 60 genes were found to be upregulated and 50 genes downregulated in myelomatous bones 24 hours after cytotherapy. Among the top upregulated genes we identified HMOX1 (5 folds, p<0.03), an antioxidant factor known to be involved in anti-inflammatory and wound healing processes. HMOX1 expression was 4 folds lower in whole myelomatous bones compared to nonmyelomatous bones (p<0.0004) and its expression in myelomatous bones was restored to normal level 24 hours after MSC cytotherapy. Clinical biopsies from newly diagnosed MM patients (n=369) also had lower HMOX1 expression than biopsies from healthy donors (n=20) or MM patients in remission (n=92, p<0.0001), supporting our experimental findings. The Hg MM line expressed very low level of HMOX1 while cultured MSCs expressed high level of HMOX1. In vitro, treatment with the HMOX1 inducer, hemin (50 μM), for 24 hours upregulated HMOX1 expression in MM cells (n=9) by 58±32 folds (p<0.009). Similar treatment with hemin induced HMOX1 expression in osteoblast precursors by 10±0.04 folds (p<0.001) and in osteoclast precursors by 1.6 folds (p<0.06). Hemin had modest inhibitory effect on growth of myeloma cell lines in culture but markedly inhibited formation of multinucleated osteoclasts by 49±2% (p<0.0001) and induced mineralization of osteoblasts analyzed by alizarin red staining. For testing hemin effects on MM growth and bone disease, hosts engrafted with Hg MM line were treated with vehicle or hemin (9 mice/group, 50 μM in 500 μl/mouse/injection) twice a week for 3 weeks. Hemin treatment had no effect on in vivo growth of Hg MM cells; however, in control hosts, bone mineral density (BMD) of the myelomatous bone was reduced by 17±3% from pretreatment levels whereas in hemin-treated hosts BMD of the myelomatous bone was reduced by 2±3% (p<0.005). The protective effect of hemin on bone disease was also visualized on x-rays. These data suggest that HMOX1 upregulation by MSC cytotherapy is involved in the therapeutic effects of this intervention and that induction of HMOX1 expression in myelomatous bone by hemin inhibits MM-induced bone disease.