Naoyuki Tsutsumi

Effect of KCA-098 on the function of osteoblast-like cells and the formation of TRAP-positive multinucleated cells in a mouse bone marrow cell population.

KCA-098 (3,9-bis(N,N-dimethylcarbamoyloxy)-5H-benzofuro[3,2-c]quinol ine-6-one), an analogue of coumestrol (a naturally occurring weak phytoestrogen), dose-dependently increased alkaline phosphatase activity of osteoblastic ROS 17/2.8 cells and freshly-isolated osteoblasts from neonatal mouse calvaria, and reduced cell proliferation. In addition, KCA-098 increased the synthesis of collagenese-digestible protein (CDP) of ROS 17/2.8 cells. On the other hand, KCA-098 had no effect on the basal synthesis of osteocalcin but reduced the 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)(2)D3)-induced increase in osteocalcin synthesized by ROS 17/2.8 cells. Therefore, KCA-098 had a bidirectional effect on the differentiation of osteoblasts (i.e., stimulating both alkaline phosphatase activity and synthesis of CDP and inhibiting osteocalcin synthesis). However, as KCA-098 stimulated the mineralization of chick embryonic bone in organ culture and recovered the bone density reduced by ovariectomy of rats, it would serve overall to stimulate the differentiation of osteoblasts. On the other hand, KCA-098 inhibited the formation of tartrate-resistant, acid phosphate-positive multinucleated cells (TRAP(+)MNC) induced by 1 alpha,25(OH)(2)D(3), parathyroid hormone (PTH), and prostaglandin E2 (PGE2) in cultures of mouse bone marrow cells, showing that it inhibits the formation of osteoclast-like cells. Coumestrol and 17beta-estradiol had no effect on the proliferation and alkaline phosphatase activity of ROS 17/2.8 cells. They did, however, dose-dependently inhibit osteoclast-like cell formation as well as KCA-098 did, indicating that the main action of coumestrol and 17beta-estradiol on bone tissue is the inhibition of bone resorption.

In vitro effect of KCA-098, a derivative of coumestrol, on bone resorption of fetal rat femurs

The effects of 3,9-bis(N,N-dimethylcarbamoyloxy)-5H-benzofuro[3,2-c]quinoli ne-6-one (KCA-098), a derivative of coumestrol, on bone resorption was studied in organ cultures of 20-day fetal rat femora. KCA-098 increased the length, dry weight, and calcium and phosphorus contents of parathyroid hormone (PTH)-treated fetal rat femur. As PTH significantly reduced the calcium and phosphorus contents of the femora, probably by stimulating bone resorption, KCA-098 seems to inhibit bone resorption. In fact, KCA-098 inhibited the PTH-induced release of 45Ca from pre-labeled fetal rat femora into the medium in organ culture. Coumestrol also inhibited the release of 45Ca from bone into the medium. However, KCA-098 did not increase the uterine weight of ovariectomized rats, whereas coumestrol did so. Thus KCA-098 is a unique, new inhibitor of bone resorption that has no estrogenic activity.

Influence of epiphyseal cartilages on bone resorption of fetal rat femora

We developed a facile method for studying bone resorption using fetal rat femur by labelling the bone with 45Ca in vitro. We found that cartilages stimulated the bone resorption of a shaft which was obtained by cutting off both distal and proximal cartilages from the femur. When the shaft was co-cultured with the cartilages isolated by a 0.4-microns microporous membrane in the same Transwell, the bone resorption of the shaft was increased. This finding suggests that the stimulation of bone resorption by the cartilages is not a result of recruitment of osteoclasts or the precursor cells from the cartilages. Indomethacin (10(-6) M) failed to influence the bone resorbing activity of the cartilages. The bone resorbing activity in the supernatant obtained from the cartilage-culture was decreased by heating. The bone resorbing activity of the supernatant did not remain in the lipid-extract or the pronase-digested supernatant, but was present in a fraction whose molecular weight was greater than 50,000. These results collectively suggest that the cartilages produce a bone resorption-stimulating factor(s) which is water-soluble, is a non-prostanoid material, contains protein and has a molecular weight greater than 50,000.