IntroductionMultiple myeloma (MM) almost exclusively develops in the bone marrow and generates devastating bone destruction by osteoclasts (OCs) recruited around MM cells. A marked stimulation of osteoclastic bone resorption causes debilitating clinical symptoms, including intractable bone pain, disabling multiple fractures, and hypercalcemia. The severity of bone disease correlates with the tumor burden and is one of the major parameters in widely used Durie and Salmon clinical staging system. It is of note that the aggressive features of MM bone lesions have significantly contributed to its poor prognosis despite the recent development of intensive chemotherapeutic regimens. 1,2 Therefore, elucidation of the molecular mechanism of bone destruction and tumor progression is essential for the development of effective therapies to improve survival as well as quality of life of patients with MM.Interactions between receptor activator of nuclear factorkappaB (RANK) expressed on the surface of the OC lineage cells and RANK ligand expressed on stromal cells play a key role in the development and activation of OCs, whereas osteoprotegerin, a decoy receptor for RANK ligand secreted from stromal cells, inhibits RANK ligand-RANK signaling. 3-8 MM cells stimulate osteoclastogenesis by triggering a coordinated increase in RANK ligand and decrease in osteoprotegerin in bone marrow (BM) stromal cells. [9][10][11] We and others have demonstrated that osteoclastogenic CC chemokines macrophage inflammatory protein 1␣ (MIP-1␣) and MIP-1␤ are secreted from most of MM cells and play a critical role in the development of MM bone lesions. [12][13][14][15][16][17][18] These chemokines act on MM cells in an autocrine/paracrine fashion and enhance MM cell adhesion to stromal cells through activation of integrins, including very late antigen-4 (VLA-4). The interaction between MM and stromal cells then induces RANK ligand expression by stromal cells, leading to OC differentiation and activation. 12 Almost exclusive development of MM in the BM suggests that the BM microenvironment supports MM cell growth and survival. Among cell components in the BM, roles of stromal cells in MM cell growth and survival have been extensively studied. When cocultured with MM cells, stromal cells are stimulated to produce interleukin 6 (IL-6), which promotes proliferation of MM cells and prevents them from apoptosis induced by anticancer agents. [19][20][21] Other than stromal cells, OCs induced by MM cells are among major cellular components of the BM microenvironment. Administration of inhibitors of osteoclast activity, including bisphosphonates, RANK-Fc, and osteoprotegerin, not only prevented MMinduced bone destruction but also interfered with tumor progression in animal models of MM. 9,22-25 Repeated administration of bisphosphonates has also been reported to reduce the tumor burden without chemotherapy in a portion of patients with MM. 26 These observations raise a possibility that an interaction between OCs and MM Materials and methods ChemicalsThe f...

Mesenchymal stem cells (MSCs)3 have the potential to differentiate into different lineages, including osteoblasts, chondroblasts, and adipocytes (1-7). The osteoblast differentiation program of MSCs is characterized by cell recruitment, which is followed by timely expressed genes including Runx2, alkaline phosphatase (ALP), type I collagen (ColA1), and osteocalcin (OC), which is associated with extracellular matrix mineralization (8 -10). The program of MSC osteogenic differentiation can be induced by soluble molecules such as bone morphogenetic proteins (BMPs) or Wnt proteins that activate several signaling pathways to trigger osteoblast differentiation (11-15). Although various downstream signals are known to promote osteogenic differentiation (16 -20), the molecular mechanisms that control the early stages of MSC osteoblast differentiation are not fully elucidated.Fibroblast growth factors (FGFs) play an important major role in the control of cell proliferation, differentiation, and survival in several tissues including bone (21-24). Notably, FGF2 was found to promote cell growth and osteoblast differentiation in bone marrow-derived mesenchymal cells (25,26). Consistent with an important role of FGF signaling in the control of osteoprogenitor cells, deletion of FGF2 in mice results in decreased bone marrow stromal cell osteogenic differentiation and altered bone formation (27). The actions of FGFs are highly dependent on high affinity FGF receptors (FGFRs) (28). FGF binding to FGFRs leads to receptor dimerization and phosphorylation of intrinsic tyrosine residues, which leads to activation of several signal transduction pathways including phospholipase C␥ (PLC␥), mitogen-activated protein kinases (MAPK), and phosphatidylinositol 3-kinase (PI3K) (29,30). In bone, activation of extracellular-related kinase (ERK1/2) MAPK and protein kinase C (PKC) was found to enhance osteoblast gene expression (31, 32). The important role of FGFR signaling in bone formation is highlighted by the finding that gain-of-function mutations in FGFRs results in premature cranial osteogenesis (33, 34). FGFR1 was recently shown to be an important transducer of FGF signals in proliferating osteoblasts (35). In contrast, activated FGFR2 was shown to enhance osteoblast differentiation in Apert syndromic craniosynostosis (36 -41). However, the role of FGFR2 signaling in osteogenic differentiation of mesenchymal stem cells is yet to be elucidated.In the present study, we investigated the specific role of FGFR2 signaling on osteoblast commitment and differentiation

Purpose: Similar to osteoclastogenesis, angiogenesis is enhanced in the bone marrow in myeloma in parallel with tumor progression.We showed previously that myeloma cells and osteoclasts are mutually stimulated to form a vicious cycle to lead to enhance both osteoclastogenesis and tumor growth. The present study was undertaken to clarify whether myeloma cell-osteoclast interaction enhances angiogenesis and whether there is any mutual stimulation between osteoclastogenesis and angiogenesis. Experimental Design: Myeloma cells and monocyte-derived osteoclasts were cocultured, and angiogenic activity produced by the cocultures was assessed with in vitro vascular tubule formation assays and human umbilical vascular endothelial cell (HUVEC) migration and survival. Osteoclastogenic activity was determined with rabbit bone cell cultures on dentine slices. Results: Myeloma cells and osteoclasts constitutively secrete proangiogenic factors, vascular endothelial growth factor (VEGF) and osteopontin, respectively. A cell-to-cell interaction between myeloma cells and osteoclasts potently enhanced vascular tubule formation. Blockade of both VEGF and osteopontin actions almost completely abrogated such vascular tubule formation as well as migration and survival of HUVECs enhanced by conditioned medium from cocultures of myeloma cells and osteoclasts. Furthermore, these factors in combination triggered the production of osteoclastogenic activity by HUVEC. Conclusions: Osteoclast-derived osteopontin and VEGF from myeloma cells cooperatively enhance angiogenesis and also induce osteoclastogenic activity by vascular endothelial cells. These observations suggest the presence of a close link between myeloma cells, osteoclasts, and vascular endothelial cells to form a vicious cycle between bone destruction, angiogenesis, and myeloma expansion.

Caveolin-3, the muscle-specific isoform of caveolins, plays important roles in signal transduction. Dominant-negative mutations of the caveolin-3 gene cause autosomal dominant limb-girdle muscular dystrophy 1C (LGMD1C) with loss of caveolin-3. However, identification of the precise molecular mechanism leading to muscular atrophy in caveolin-3-deficient muscle has remained elusive. Myostatin, a member of the muscle-specific TGF-β superfamily, negatively regulates skeletal muscle volume. Here we report that caveolin-3 inhibited myostatin signaling by suppressing activation of its type I receptor; this was followed by hypophosphorylation of an intracellular effector, Mad homolog 2 (Smad2), and decreased downstream transcriptional activity. Loss of caveolin-3 in P104L mutant caveolin-3 transgenic mice caused muscular atrophy with increase in phosphorylated Smad2 (p-Smad2) as well as p21 (also known as Cdkn1a), a myostatin target gene. Introduction of the myostatin prodomain, an inhibitor of myostatin, by genetic crossing or intraperitoneal administration of the soluble type II myostatin receptor, another inhibitor, ameliorated muscular atrophy of the mutant caveolin-3 transgenic mice with suppression of p-Smad2 and p21 levels. These findings suggest that caveolin-3 normally suppresses the myostatin-mediated signal, thereby preventing muscular atrophy, and that hyperactivation of myostatin signaling participates in the pathogenesis of muscular atrophy in a mouse model of LGMD1C. Myostatin inhibition may be a promising therapy for LGMD1C patients.