TY - JOUR
T1 - Characterization of acid flux in osteoclasts from patients harboring a G215R mutation in ClC-7
AU - Henriksen, Kim
AU - Gram, Jeppe
AU - Neutzsky-Wulff, Anita Vibsig
AU - Jensen, Vicki Kaiser
AU - Dziegiel, Morten H
AU - Bollerslev, Jens
AU - Karsdal, Morten A
N1 - Keywords: Acids; Arginine; Bone Resorption; Calcium; Chloride Channels; Genes, Dominant; Glycine; Humans; Hydrogen-Ion Concentration; Lysosomes; Mutation; Osteoclasts; Vacuolar Proton-Translocating ATPases
PY - 2008
Y1 - 2008
N2 - The chloride-proton antiporter ClC-7 has been speculated to be involved in acidification of the lysosomes and the resorption lacunae in osteoclasts; however, neither direct measurements of chloride transport nor acidification have been performed. Human osteoclasts harboring a dominant negative mutation in ClC-7 (G215R) were isolated, and used these to investigate bone resorption measured by CTX-I, calcium release and pit scoring. The actin cytoskeleton of the osteoclasts was also investigated. ClC-7 enriched membranes from the osteoclasts were isolated, and used to test acidification rates in the presence of a V-ATPase and a chloride channel inhibitor, using a H(+) and Cl(-) driven approach. Finally, acidification rates in ClC-7 enriched membranes from ADOII osteoclasts and their corresponding controls were compared. Resorption by the G215R osteoclasts was reduced by 60% when measured by both CTX-I, calcium release, and pit area when comparing to age and sex matched controls. In addition, the ADOII osteoclasts showed no differences in actin ring formation. Finally, V-ATPase and chloride channel inhibitors completely abrogated the H(+) and Cl(-) driven acidification. Finally, the acid influx was reduced by maximally 50% in the ClC-7 deficient membrane fractions when comparing to controls. These data demonstrate that ClC-7 is essential for bone resorption, via its role in acidification of the lysosomes and resorption lacunae in osteoclasts.
AB - The chloride-proton antiporter ClC-7 has been speculated to be involved in acidification of the lysosomes and the resorption lacunae in osteoclasts; however, neither direct measurements of chloride transport nor acidification have been performed. Human osteoclasts harboring a dominant negative mutation in ClC-7 (G215R) were isolated, and used these to investigate bone resorption measured by CTX-I, calcium release and pit scoring. The actin cytoskeleton of the osteoclasts was also investigated. ClC-7 enriched membranes from the osteoclasts were isolated, and used to test acidification rates in the presence of a V-ATPase and a chloride channel inhibitor, using a H(+) and Cl(-) driven approach. Finally, acidification rates in ClC-7 enriched membranes from ADOII osteoclasts and their corresponding controls were compared. Resorption by the G215R osteoclasts was reduced by 60% when measured by both CTX-I, calcium release, and pit area when comparing to age and sex matched controls. In addition, the ADOII osteoclasts showed no differences in actin ring formation. Finally, V-ATPase and chloride channel inhibitors completely abrogated the H(+) and Cl(-) driven acidification. Finally, the acid influx was reduced by maximally 50% in the ClC-7 deficient membrane fractions when comparing to controls. These data demonstrate that ClC-7 is essential for bone resorption, via its role in acidification of the lysosomes and resorption lacunae in osteoclasts.
U2 - 10.1016/j.bbrc.2008.11.145
DO - 10.1016/j.bbrc.2008.11.145
M3 - Journal article
C2 - 19070589
SN - 0006-291X
VL - 378
SP - 804
EP - 809
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 4
ER -