In th e process of d evelop in g m ature osteoclasts, th e fu nction s of osteoblasts an d osteoclasts are intim ately related . Du rin g skeletal d evelop m ent an d bon e rem od elin g process, osteoblasts synth esize an d secrete sign al m olecu les th at control osteoclast differentiation (Ducy et al., 2000) an d cell-to-cell interaction s are n ecessary during th e w h ole procedure. This is a direct and crucial interaction th at has been w ell established in vivo (Bu rgess et al., 1999). Thu s osteoblasts p lay a cru cial role in differentiatin g th e m acroph age/ m onocyte to m ature osteoclasts. H ow ever, it has been only 4-5 year since clear m ech anism of m aturation of m acrop hages into osteoclasts has been id entified . There are tw o m olecules th at are essential and su fficient to prom ote osteoclastogen esis: M-CSF an d RAN KL (Teitelbau m , S., 2000).
M-CSF bin d s to its recep tor, c-fm s, on m acrop h age to provid e signals requ ired for their survival and proliferation . OPG is kn ow n to be a solu ble "d ecoy " receptor th at com p etes w ith RAN K for RAN KL. It h as been d escribed th at th e balance betw een th e expression of the stim ulator of osteoclastogenesis, RAN KL, an d of th e inhibitor, OPG, is the cru cial factor th at governs the am ou nt of bone resorbed .
In th e conju ction of m achin eries in osteoblast, it h as been rep orted th at N a/ K/ Cl cotran sp orter (N KCC1) is present in osteoblasts recently . We still d o n ot kn ow the role of N KCC1 in
osteoblast . N evertheless, it m ay be p ossible to hyp oth esize that th e N KCC1 is involved in th e bon e rem od eling p rocess, ju d gin g from th e facts that 1) N KCC1 cotran sp orter exists in osteoblasts, 2) N KCC1 tran sp orts the ion inw ard an d outw ard across th e ep ith elia and p lay an im p ortant role in regu latin g th e cell size an d volu m e, and 3) N KCC1 affects the d evelopm ent an d grow th of the cells (Ru ssell, J.M. et al., 2000). H ow ever, th ere is n o p lau sible exp lan ation for th e role of N KCC1 in osteoblast in concern w ith osteoclastogen esis (Fig. 4). Therefore, w e aim ed at revealing th e role of N KCC1 in osteoblast, inhibitin g th e N KCC1 activity u sing bu m etanid e.
Unexp ectedly, w e fou nd that bu m etanid e inhibited th e osteoclastogen esis in cocu ltu re system , osteoblast/ strom al cell.
This fact give u s th e new clu es th at N KCC1 is p robably resp on sible for the d ow n-regu lation of osteoclastogenesis.
Therefore, in this stu dy w e app lied osteoblast/ strom al cell cocu ltu re system to evalu ate 1) effect of bum etanid e on 1α,25(OH)2D3-indu ced osteoclast form ation, 2) d ecrease in cell volum e by bum etanid e, 3) chan ges in m RN A exp ression p rofile
bum etanid e did not ch ange 1α,25(OH)2D3-in duced osteoclast differentiation . Moreover, cell viability test sh ow ed th at bum etanid e did n ot sh ow toxic effect w hen ad d ed u p to 100 μ M to th e cocu ltu re (Fig. 8). It is clear th at bu m etanid e has som e effects on form ation of TRAP p ositive cell w ith no toxic effects.
So, cell volu m e w as m easu red to d eterm ine w heth er bu m etanid e h as any effect on cell volu m e since N KCC1 plays a m ajor role in controllin g th e volu m e of cells. As sh ow n in Fig. 9, 10 μM of bum etanid e redu ced th e relative cell volum e to th e half level.
Osteoclastic bon e resorp tion con sists of m u ltip le step s such as th e differentiation of osteoclast p recu rsors into m on onuclear p refu sion osteoclasts (pOC), the fu sion of p OCs to form m u ltinucleated osteoclasts, an d the activation of osteoclasts to resorb bone. Several factors related in th e differentiation, m aturation and activation of osteoclasts h ave been rep orted, w hich inclu d e RAN KL, OPG and M-CSF. Since osteoclast differentiation h as been rep orted to be m ediated by th ese factors, m RN A exp ression s w ere investigated . Th e exp ression of RAN KL m RN A in duced by 1α,25(OH)2D3 w as d ow n-regulated w ith the increase of bu m etanid e concentration . Moreover, the expression of OPG m RN A, a n ovel TN F receptor fam ily m em ber w as increased . These findings in dicate th at bu m etanid e inhibits osteoclast differentiation via inhibiting th e exp ression of RAN KL m RN A an d enh ancin g the expression of OPG m RN A.
On the oth er h and, as an inhibitor of N KCC1, bum etanid e
inhibits cellu lar u ptake of N a+, K+, and Cl- ion s, w hich resu lts in th e cellular volum e ch an ge. O'Donnell et. al. d em onstrated th at p h osp hatase inhibition results in th e elevation of en d othelial cell volum e, thu s su ggestin g th at restin g en d oth elial cell volum e is also d eterm in ed by th e integrated activities of kin ases an d p h osp hatases. The stim ulation of co-tran sp orter by cell shrinkage w as n ot enhanced by ph osp h atase inhibition, in dicating th at the effects of shrinkage an d p h osp hatase inhibition w ere n ot ad ditive, su ggestin g th at shrinkage inhibits th e p hosph atase. In this stu dy, bum etanid e did not h ave any effect on the expression of JN K.
Total am ou nt of JN K (t-JN K) w as rem ained con stant in th e ran ge of bu m etanid e concentration to be u sed bu t th e activated form of JN K, ph osp horylated JN K (p -JN K), w as d ecreased accordin g to th e increase of bu m etanid e concentration . JN K is on e of the m itogen activated p rotein kinase (MARK) fam ily . It h as been kn ow n to exhibit activation by hyp er-osm olarity and cellu lar volum e ch an ge. Th e d ecrease of p -JN K in dicates th at th e cell volum e ch an ge of osteoclasts are du e to activation of JN K in osteoclasts.
From th ese resu lts, it m ight be inferred that cellu lar shrinkage of osteoblastic cell in du ced by th e ad dition of bu m etanid e, w hich resu lts the inhibition of N KCC1 activity, is an im p ortant m odu lator in regulating osteoclastogen esis.
Fig . 15. Prop osed m od el for th e alteration of osteoclastogen esis via N KCC1 m odu lation . Th e osm otic shock on osteoblastic cell resu lts in m odu lation of N KCC1. It is p roved that th e shirinkage in osteoblasts resu lt in d ecrease of osteoclast activity.