HE Jin Cheng Showa Univ., School of Dentistry Assistant, 歯学部, 助手 (10205049)
TAKAHASHI Naoyuki Showa Univ., School of Dentistry Lecturer, 歯学部, 講師 (90119222)
SUDA Tatsuo Showa Univ., School of Dentistry Professor, 歯学部, 教授 (90014034)
TANAKA Hirofumi Showa Univ., School of Dentistry Assistant (30146899)
宮浦 千里 昭和大学, 歯学部・生化学教室, 助手 (20138382)
|Budget Amount *help
¥6,500,000 (Direct Cost : ¥6,500,000)
Fiscal Year 1990 : ¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1989 : ¥4,500,000 (Direct Cost : ¥4,500,000)
Experiment 1 : It is known that most bone-resorbing agents including 1alpha, 25 (OH) _2D_3, interleukin 1 (IL-1) and tumor necrosis factor (TNF) first act on osteoblasts to induce osteoclastic bone resorption. We there for examined novel proteins produced by osteoblasts in response to those bone resorbing agents. We also examined the mechanism of regulation of trascription and translation of inter-leukin 6 (IL-6) and leukemia inhibitory factor (LIF) in osteoblasts in response to bone-absorphon agents.
[Methods] A mouse osteoblastic cell line (E1) and mouse primary osteoblasts were cultured for 3 days with various bone-resorbing agents. After incubation, conditioned media and cells were collected for determining protein contents and mRNA levels of IL-6 and LIF. To explore novel proteins produced by osteoblasts in response to bone-resorbing agents, E1 cells were cultured for 3 days with [^<35>S] -methio- nine in the presence of 1alpha, 25 (OH) _2D_3 and conditioned media were analyzed by
[Results] IL-1, TNF and lypopolysaccharides (LPS) greatly increased mRNA levels of IL-6 and LIF both in E1 cells and primary osteoblasts and increased production of IL-6 by E1 cells. Both recombinant IL-6 and LIF stimulated bone-resorbing activity at higher doses. Simultaneous addition of the two cytokines showed similar bone-resorbing activity at much lower doses. 1alpha, 25 (OH) _2D_3 induced 68 kDa and 116 kDa proteins under a reducing condition in E1 cells. The 68 kDa protein appeared to be identical with osteopontin, since the molecular weight and the regulation pattern by 1alpha, 25 (OH) _2D_3 were similar. Purification of the 116 kDa protein and analysis of the N-terminal amino acid sequence are in progress.
Experiment 2 : We have reported that 1alpha, 25 (OH) _2D_3, interleukin 4 (IL-4) and a macrophage fusion factor (MFF) present in conditioned medium of mouse spleen cells treated with concanavalin A similarly induce fusion of mouse alveolar macrophages. In this experiment, we examined the mechanism of fusion induced by 1alhpa, 25 (OH) _2D_3 and identified MFF.
[Methods] Mouse alveolar macrophages were treated with 1alpha, 25 (OH) _2D_3 for appropriate times in various concentrations of medium calcium to determine the condition for inducing macrophage fusion. After incubation with 1alpha, 25 (OH) _2D_3, a calcium-dependent protein, transglutaminase, was detected by western blot analysis. To determine the nature of MFF, the medium content of various cytokines was measured and neutralized by adding various antibodies.
[Results] 1alpha, 25 (OH) _2D_3 induced macrophage fusion at medium calcium concentration higher than 2 mM. The vitamin also induced transglutaminase time-dependently. It was concluded by the neutralization experiments with the antibodies that MFFs in the conditioned emdium of spleen cells were IL-4 and GM-CSF. Less