Tsubaki Masanobu, Kato Chisato, Manno Miyuki, Ogaki Mitsuhiko, Satou Takao, Itoh Tatsuki, Kusunoki Takashi, Tanimori Yoshihiro, Fujiwara Kimiko, Matsuoka Hiroshi, Nishida Shozo
Division of Pharmacotherapy, Kinki University School of Pharmacy, 3-4-1, Kowakae, Higashi-Osaka, Osaka 577-8502, Japan.
Mol Cell Biochem. 2007 Oct;304(1-2):53-60. doi: 10.1007/s11010-007-9485-7. Epub 2007 Jun 5.
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.
溶骨性病变在骨髓瘤晚期进展迅速，这些病变处出现的骨痛或骨折会显著降低患者的生活质量。关于这种骨质破坏的病因，最近有报道称，骨髓瘤患者大量产生的MIP-1α间接作用于破骨细胞前体细胞，并通过骨髓基质细胞或成骨细胞激活破骨细胞，尽管这一过程的细节仍不清楚。在本研究中，我们团队研究了MIP-1α诱导RANKL表达的机制以及MIP-1α对破骨细胞激活的影响。在ST2细胞和MC3T3-E1细胞中，RANKL mRNA和RANKL蛋白表达均以MIP-1α浓度依赖性方式增加。添加MIP-1α后1小时，RANKL mRNA表达开始增加；2小时时增加显著，随后观察到持续表达。添加MIP-1α后1、2和3天，ST2和MC3T3-E1细胞中RANKL蛋白表达的增加水平相似。添加MIP-1α后，与对照组相应量相比，磷酸化ERK1/2和Akt蛋白表达量增加。另一方面，添加MIP-1α后，磷酸化p38MAPK蛋白表达量与对照组相比有所下降。将U0126（一种MEK1/2抑制剂）或LY294002（一种PI3K抑制剂）添加到ST2和MC3T3-E1细胞中，发现其可抑制这些细胞中RANKL mRNA和RANKL蛋白表达。当添加p38MAPK抑制剂SB203580时，这些细胞中RANKL mRNA和RANKL蛋白表达增加。发现MIP-1α以MIP-1α浓度依赖性方式促进破骨细胞前体细胞系C7细胞的破骨细胞分化。与单独培养的C7细胞相比，与ST2和MC3T3-E1细胞共同培养的C7细胞中，MIP-1α更广泛地促进其分化为破骨细胞。这些结果表明，MIP-1α直接作用于破骨细胞前体细胞并诱导破骨细胞分化。该物质还通过促进骨髓基质细胞和成骨细胞中RANKL表达间接诱导破骨细胞分化。本研究结果表明，MEK/ERK和PI3K/Akt途径的激活以及p38MAPK途径的抑制参与了MIP-1α在骨髓基质细胞和成骨细胞中诱导的RANKL表达。这一发现可能有助于开发针对细胞内信号因子的破骨细胞抑制剂。
