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A peptidyl derivative structurally based on the inhibitory center of cystatin C inhibits bone resorption in vitro

Johansson, L; Grubb, Anders LU ; Abrahamson, Magnus LU ; Kasprzykowski, F; Kasprzykowska, R; Grzonka, Z and Lerner, UH (2000) In Bone 26(5). p.451-459
Abstract
Human cystatin C is a cysteine proteinase inhibitor belonging to the cystatin superfamily, which previously has been shown to inhibit bone resorption in bone organ culture. The aminoterminal segment, Arg8-Leu9-Val10-Gly11 (RLVG), of the single polypeptide chain of cystatin C constitutes an essential part of its inhibitory center. In the present study, the effect of benzyloxycarbonyl-Arg8-Leu9-Val10-Gly11-diazomethane (Z-RLVG-CHN2) on bone resorption in vitro was compared with the effects of cystatin C and calcitonin. Bone resorption was assessed by the release of 45Ca and 3H from mouse calvarial bones prelabeled with [45Ca]CaCl2 and [3H]-proline, respectively. Z-RLVG-CHN2 concentration-dependently inhibited the release of 45Ca and 3H in... (More)
Human cystatin C is a cysteine proteinase inhibitor belonging to the cystatin superfamily, which previously has been shown to inhibit bone resorption in bone organ culture. The aminoterminal segment, Arg8-Leu9-Val10-Gly11 (RLVG), of the single polypeptide chain of cystatin C constitutes an essential part of its inhibitory center. In the present study, the effect of benzyloxycarbonyl-Arg8-Leu9-Val10-Gly11-diazomethane (Z-RLVG-CHN2) on bone resorption in vitro was compared with the effects of cystatin C and calcitonin. Bone resorption was assessed by the release of 45Ca and 3H from mouse calvarial bones prelabeled with [45Ca]CaCl2 and [3H]-proline, respectively. Z-RLVG-CHN2 concentration-dependently inhibited the release of 45Ca and 3H in bones stimulated by parathyroid hormone (PTH), with half-maximal inhibition obtained at 1 μmol/L. The inhibitory actions of Z-RLVG-CHN2 and cystatin C were persistent, whereas action induced initially by calcitonin was lost with time. The inhibition caused by Z-RLVG-CHN2 and cystatin C on PTH-stimulated 45Ca release was observed after 6 h, whereas inhibition by calcitonin was seen already after 2 h. In contrast, the inhibitory effects of Z-RLVG-CHN2 and cystatin C, as well as that of calcitonin, on 3H 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 to Z-RLVG-CHN2 and cystatin C, inhibited neither cysteine proteinases nor bone resorption. In contrast to wild-type cystatin C, recombinant human cystatin C with Gly substitutions for residues Arg8, Leu9, Val10, and Trp106, and with low or nonexistent affinity for cysteine proteinases, did not display any inhibitory effect on bone resorption. These data strongly indicate that Z-RLVG-CHN2 inhibits bone resorption in vitro by a mechanism that seems primarily to be due to an inhibition of bone matrix degradation via cysteine proteinases. The data also corroborate the hypothesis that cystatin C inhibits bone resorption by virtue of its cysteine proteinase inhibitory capacity. (Less)
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organization
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type
Contribution to journal
publication status
published
subject
in
Bone
volume
26
issue
5
pages
451 - 459
publisher
Elsevier
external identifiers
  • scopus:0034032289
ISSN
1873-2763
DOI
10.1016/S8756-3282(00)00261-1
language
English
LU publication?
yes
id
8b5ad791-2f70-4841-9563-7e9b87b76f1a (old id 1118347)
date added to LUP
2008-06-17 13:57:37
date last changed
2017-05-21 04:42:15
@article{8b5ad791-2f70-4841-9563-7e9b87b76f1a,
  abstract     = {Human cystatin C is a cysteine proteinase inhibitor belonging to the cystatin superfamily, which previously has been shown to inhibit bone resorption in bone organ culture. The aminoterminal segment, Arg8-Leu9-Val10-Gly11 (RLVG), of the single polypeptide chain of cystatin C constitutes an essential part of its inhibitory center. In the present study, the effect of benzyloxycarbonyl-Arg8-Leu9-Val10-Gly11-diazomethane (Z-RLVG-CHN2) on bone resorption in vitro was compared with the effects of cystatin C and calcitonin. Bone resorption was assessed by the release of 45Ca and 3H from mouse calvarial bones prelabeled with [45Ca]CaCl2 and [3H]-proline, respectively. Z-RLVG-CHN2 concentration-dependently inhibited the release of 45Ca and 3H in bones stimulated by parathyroid hormone (PTH), with half-maximal inhibition obtained at 1 μmol/L. The inhibitory actions of Z-RLVG-CHN2 and cystatin C were persistent, whereas action induced initially by calcitonin was lost with time. The inhibition caused by Z-RLVG-CHN2 and cystatin C on PTH-stimulated 45Ca release was observed after 6 h, whereas inhibition by calcitonin was seen already after 2 h. In contrast, the inhibitory effects of Z-RLVG-CHN2 and cystatin C, as well as that of calcitonin, on 3H 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 to Z-RLVG-CHN2 and cystatin C, inhibited neither cysteine proteinases nor bone resorption. In contrast to wild-type cystatin C, recombinant human cystatin C with Gly substitutions for residues Arg8, Leu9, Val10, and Trp106, and with low or nonexistent affinity for cysteine proteinases, did not display any inhibitory effect on bone resorption. These data strongly indicate that Z-RLVG-CHN2 inhibits bone resorption in vitro by a mechanism that seems primarily to be due to an inhibition of bone matrix degradation via cysteine proteinases. The data also corroborate the hypothesis that cystatin C inhibits bone resorption by virtue of its cysteine proteinase inhibitory capacity.},
  author       = {Johansson, L and Grubb, Anders and Abrahamson, Magnus and Kasprzykowski, F and Kasprzykowska, R and Grzonka, Z and Lerner, UH},
  issn         = {1873-2763},
  language     = {eng},
  number       = {5},
  pages        = {451--459},
  publisher    = {Elsevier},
  series       = {Bone},
  title        = {A peptidyl derivative structurally based on the inhibitory center of cystatin C inhibits bone resorption in vitro},
  url          = {http://dx.doi.org/10.1016/S8756-3282(00)00261-1},
  volume       = {26},
  year         = {2000},
}