L. Johansson et al., A peptidyl derivative structurally based on the inhibitory center of cystatin C inhibits bone resorption in vitro, BONE, 26(5), 2000, pp. 451-459
Human cystatin C is a cysteine proteinase inhibitor belonging to the cystat
in superfamily, which previously has been shown to inhibit bone resorption
in bone organ culture, The aminoterminal segment, Arg(8)-Leu(9)-Va(10)-Gly(
11) (RLVG), of the single polypeptide chain of cystatin C constitutes an es
sential part of its inhibitory center, In the present study, the effect of
benzyloxycarbonyl-Arg(8)-Leu(9)-Val(10)-Gly(11)-diazomethane (Z- RLVG-CHN2)
on bone resorption in vitro was compared with the effects of cystatin C an
d calcitonin, Bone resorption was assessed by the release of Ca-45 and H-3
from mouse calvarial bones prelabeled with [Ca-45]CaCl2 and [H-3]-proline,
respectively. Z-RLVG-CHN2 concentration-dependently inhibited the release o
f Ca-45 and H-3 in bones stimulated by parathyroid hormone (PTH), with half
-maximal inhibition obtained at 1 mu mol/L. The inhibitory actions of Z-RLV
G-CHN2 and cystatin C were persistent, whereas action induced initially by
calcitonin was lost with time. The inhibition caused by Z-RLVG-CHN2 and cys
tatin C on PTH-stimulated Ca-45 release was observed after 6 h, whereas inh
ibition by calcitonin was seen already after 2 h, In contrast, the inhibito
ry effects of Z-RLVG-CHN2 and cystatin C, as well as that of calcitonin, on
H-3 release was seen already after 2 h, Z-RLVG-CHN2, in which the reactive
carboxyterminal diazomethane was substituted by nonreactive groups [-OH, -
NH2, or -N(CH3)(2)], resulted in peptidyl derivatives, which, in contrast t
o Z-RLVG-CHN2 and cystatin C, inhibited neither cysteine proteinases nor bo
ne resorption, In contrast to wild-type cystatin C, recombinant human cysta
tin C with Gly substitutions for residues Arg(8), Leu(9), Val(10), and Trp(
106), and with low or nonexistent affinity for cysteine proteinases, did no
t display any inhibitory effect on bone resorption, These data strongly ind
icate that Z-RLVG-CHN2 inhibits bone resorption in vitro by a mechanism tha
t seems primarily to be due to an inhibition of bone matrix degradation via
cysteine proteinases. The data also corroborate the hypothesis that cystat
in C inhibits bone resorption by virtue of its cysteine proteinase inhibito
ry capacity. (C) 2000 by Elsevier Science Inc, All rights reserved.