Regulatory mechanism of 1α,25(OH)2D3-induced osteoclastogenesis by FGF (Fibroblast Growth Factor)-2

Abstract

[한글]

이 연구는 골세포와 연골세포의 기능에 있어 강력한 조절인자인 FGF-2의 작용을 규명하였다. 여러 종류의 FGF 중 FGF-2는 골세포에서 autocrine, 또는 paracrine 방식으로 작용하며, 이것이 골 대사, 골 형성과 흡수에 미치는 반응에 관해서는 논쟁의 여지가 있다.

따라서, 쥐의 두개골에서 분리한 조골세포와 골수세포와의 혼합배양에서 유도되는 파골세포 형성과정 중 FGF-2의 작용을 검토하였다. VitD3에 의한 파골세포 형성 유도시 다수의 TRAP 양성세포를 관찰할 수 있었으며, FGF-2 (0.1, 1,10 and 100 ng/ml) 첨가시 낮은 농도 (0.1, 1 ng/ml)에서는 VitD3에 의하여 유도된 파골세포 형성이 억제되었고 높은농도 (10, 100 ng/ml)에서는 변화를 보이지 않거나 촉진되었다. 또한, 실험에 사용된 FGF-2의 모든 농도에서 어떠한 세포독성도 나타내지 않았다. 따라서, 이는 파골세포 형성의 억제는 직접적인 독성작용에 의하지 않고 생리적 작용의 변화에 기인한 것임을 나타낸다.

FGF-2를 조골세포에 첨가하여 RANKL, OPG 및 M-CSF의 mRNA 발현을 측정한 결과 낮은 농도에서는 RANKL mRNA발현이 감소하였으나 OPG와 M-CSF는 큰 변화를 보이지 않았다. 그밖에도 FGF-2는 조골세포의 분화정도를 가늠하는 alkaline phosphatase와 세포외기질의 분해에 관련된 효소인 MMP13 (matrix metalloproteinase 13)의 mRNA발현을 감소시키고 파골세포 분화와 관련 있는 ADAM8 (A disintegrin and metalloprotease)의 발현에는 영향을 미치지 않는 것으로 나타났다. 이상의 결과로부터, FGF-2는 파골세포 형성과정에 있어 이중 효과 (dual effect)를 보이며, 특히 낮은 농도에서는 조골세포의 RANKL과 MMP13의 mRNA발현을 감소시킴으로써 파골세포의 형성을 억제한다

[영문]

This study investigated the action of FGF (fibroblast growth factor)-2, a potent regulator on the functions of bone and cartilage cells. Of FGFs, FGF-2 acts like an autocrine/paracrine factor for bone cells. With regards to the effects of FGF-2 on

bone cells, the response is still controversial in terms of bone metabolism, bone formation and bone resorption. To clarify the role of FGF-2, osteoclast formation was monitored in coculture system, mouse cavarial osteoblastic cells/bone marrow cells in the presence of 1α,25(OH)2D3 (VitD3). Numerous TRAP-positive cells (80~498) induced by VitD3 were observed in cocultures. When FGF-2 (0.1, 1, 10, and 100 ng/ml) was treated to the coculture system, VitD3-induced osteoclast formation was inhibited by FGF-2 at low concentrations (0.1, 1 ng/ml) while at high concentrations (10, 100 ng/ml) stimulated or not changed. It was also confirmed

that exposure of FGF-2 to cocultures did not show a toxic effect in the entire range of concentration (0.1, 1.0, 10, and 100 nM), suggesting that the effect of FGF-2 on osteoclast formation was caused by its changing the physiological activity, not by its direct toxic effect upon the cells. To see the effect of FGF-2

on the osteoblastic cells, expressions of RANKL, OPG and M-CSF mRNAs were monitored by RT-PCR after incubation for 96 hrs. The RANKL mRNA expression level was reduced at low concentration. However, FGF-2 did not make any remarkable change in OPG and

M-CSF mRNA expression. Consequently FGF-2, at low concentrations, resulted in the decrement of RANKL/OPG ratio. In addition, FGF-2 decreased the alkaline phosphatase activity which indicates the osteoblast differentiation. A disintegrin and metallo-proteinase (ADAM8), and matrix metallo-proteinase 13 (MMP13) which are responsible for osteoclast differentiation and degradation of extracellular matrix (ECM) respectively were examined by RT-PCR in the presence of FGF-2. FGF-2 did not exert any changes in ADAM8 while decreased the MMP13 mRNA expression at low concentration. Taken all together, these findings suggest that FGF-2 seems to have a dual effects on osteoclastogenesis, especially, FGF-2 at low concentration decreased osteoclast formation via the down-regulation of the RANKL and MMP13 mRNA

expressions in osteoblastic cells which in turn inhibits the osteoclast formation.