Inhibitory effect of menaquinone-7 (vitamin K_2) on osteoclast-like cell formation and osteoclastic bone resorption in rat bone tissues in vitro

摘要

The effect of menaquinone-7 (MK-7; vitamin K_2) on osteoclast-like cell formation and osteoclastic bone resorption in rat femoral tissues in vitro was investigated. The bone marrow cells were cultured for 7 days in a α-minimal essential medium (α-MEM) containing a well-known bone resorbing agent [parathyroid hormone (1-34) (PTH) or prostaglandin E_2 (PGE_2)] with an effective concentration. Osteoclast-like cells were estimated by staining for tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The presence of PTH (10~(-8) M) or PGE_2 (10~(-6) M) induced a remarkable increase in osteoclast-like multinucleated cells. These increases were significantly inhibited by MK-7 (10~(-8)-10~(-5) M). MK-7 (10~(-7) and 10~(-6) M) significantly inhibited phorbol 12-myristate 13-acetate-induced osteoclast-like cell formation, whereas MK-7 did not inhibit dibutyryl cyclic adenosine monophosphate (DcAMP) (10~(-5) M)-induced osteoclast-like cell formation. These results suggest that the inhibitory action of MK-7 is partly involved in protein kinase C signaling. The bone cells isolated from rat femoral tissues were cultured for 48 h in an α-MEM containing either vehicle or MK-7 (10~(-8)-10~(-5) M). The presence of MK-7 (10~(-6) and 10~(-5) M) caused a significant decrease in the number of mature osteoclasts. Such a decrease was also seen in the presence of calcitonin (10~(-10)-10~(-8) M), DcAMP (10~(-6) and 10~(-5) M), or calcium chloride (10~(-4) and 10~(-3) M). The effect of MK-7 (10~(-6) M) in decreasing the number of osteoclasts was not further enhanced in the presence of calcitonin (10~(-8) M), DcAMP (10~(-5) M), or calcium chloride (10~(-3) M), and was completely abolished by the presence of dibucaine (10~(-6) M) or staurosporine (10~(-7) M), which are inhibitors of Ca~(2+)-dependent protein kinases. These results suggested that MK-7 has a suppressive effect on osteoclasts. Moreover, the femoral-metaphyseal tissues obtained from rats were cultured for 48 h in Dulbecco's modified Eagle's medium containing either vehicle, PTH (10~(-7) M), or PGE_2 (10~(-5) M) in the absence or presence of MK-7 (10~(-7)-10~(-5) M). The presence of PTH or PGE_2 induced a significant decrease in bone calcium content. These decreases were significantly blocked by MK-7 (10~(-7)-10~(-5) M). This study demonstrates that MK-7 has an inhibitory effect on osteoclastic bone resorption in vitro.