title: Macrophage inflammatory protein-1alpha (MIP-1alpha) enhances a receptor activator of nuclear factor kappaB ligand (RANKL) expression in mouse bone marrow stromal cells and osteoblasts through MAPK and PI3K/Akt pathways.
abstract: Osteolytic lesions are rapidly progressive during the terminal stages of myeloma, and the bone pain or bone fracture that occurs at these lesions decreases the patients' quality of life to a notable degree. In relation to the etiology of this bone destruction, it has been reported recently that MIP-1alpha, produced in large amounts in myeloma patients, acts indirectly on osteoclastic precursor cells, and activates osteoclasts by way of bone-marrow stromal cells or osteoblasts, although the details of this process remain obscure. In the present study, our group investigated the mechanism by which RANKL expression is induced by MIP-1alpha and the effects of MIP-1alpha on the activation of osteoclasts. RANKL mRNA and RANKL protein expressions increased in both ST2 cells and MC3T3-E1 cells in a MIP-1alpha concentration-dependent manner. RANKL mRNA expression began to increase at 1 h after the addition of MIP-1alpha; the increase became remarkable at 2 h, and continuous expression was observed subsequently. Both ST2 and MC3T3-E1 cells showed similar levels of increased RANKL protein expression at 1, 2, and 3 days after the addition of MIP-1alpha. After the addition of MIP-1alpha, the amount of phosphorylated ERK1/2 and Akt protein expressions showed an increase, as compared to the corresponding amount in the control group. On the other hand, the amount of phosphorylated p38MAPK protein expression showed a decrease from the amount in the control group after the addition of MIP-1alpha. U0126 (a MEK1/2 inhibitor) or LY294002 (a PI3K inhibitor) was added to ST2 and MC3T3-E1 cells, and was found to inhibit RANKL mRNA and RANKL protein expression in these cells. When SB203580, a p38MAPK inhibitor, was added, RANKL mRNA and RANKL protein expression were increased in these cells. MIP-1alpha was found to promote osteoclastic differentiation of C7 cells, an osteoclastic precursor cell line, in a MIP-1alpha concentration-dependent manner. MIP-1alpha promoted differentiation into osteoclasts more extensively in C7 cells incubated together with ST2 and MC3T3-E1 cells than in C7 cells incubated alone. These results suggested that MIP-1alpha directly acts on the osteoclastic precursor cells and induces osteoclastic differentiation. This substance also indirectly induces osteoclastic differentiation through the promotion of RANKL expression in bone-marrow stromal cells and osteoblasts. The findings of this investigation suggested that activation of the MEK/ERK and the PI3K/Akt pathways and inhibition of p38MAPK pathway were involved in RANKL expression induced by MIP-1alpha in bone-marrow stromal cells and osteoblasts. This finding may be useful in the development of an osteoclastic inhibitor that targets intracellular signaling factors.
authors.affiliation: Division of Pharmacotherapy, Kinki University School of Pharmacy, 3-4-1, Kowakae, Higashi-Osaka, Osaka 577-8502, Japan.
authors.forename: Masanobu
authors.initials: M
authors.lastname: Tsubaki
authors.forename: Chisato
authors.initials: C
authors.lastname: Kato
authors.forename: Miyuki
authors.initials: M
authors.lastname: Manno
authors.forename: Mitsuhiko
authors.initials: M
authors.lastname: Ogaki
authors.forename: Takao
authors.initials: T
authors.lastname: Satou
authors.forename: Tatsuki
authors.initials: T
authors.lastname: Itoh
authors.forename: Takashi
authors.initials: T
authors.lastname: Kusunoki
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Tanimori
authors.forename: Kimiko
authors.initials: K
authors.lastname: Fujiwara
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Matsuoka
authors.forename: Shozo
authors.initials: S
authors.lastname: Nishida
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000187:drug effects* / Q000378:metabolism; D002070:Butadienes / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D054405:Chemokine CCL3 / Q000494:pharmacology*; D002867:Chromones / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D007093:Imidazoles / Q000494:pharmacology; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D009570:Nitriles / Q000494:pharmacology; D051058:Oncogene Protein v-akt / Q000378:metabolism*; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011725:Pyridines / Q000494:pharmacology; D053245:RANK Ligand / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D017154:Stromal Cells / Q000187:drug effects / Q000378:metabolism
chemical_list: D002070:Butadienes; D054405:Chemokine CCL3; D002867:Chromones; D004791:Enzyme Inhibitors; D007093:Imidazoles; D009025:Morpholines; D009570:Nitriles; D011725:Pyridines; D053245:RANK Ligand; C505689:Tnfsf11 protein, mouse; C113580:U 0126; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D019869:Phosphatidylinositol 3-Kinases; D051058:Oncogene Protein v-akt; D048049:Extracellular Signal-Regulated MAP Kinases; C093642:SB 203580