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

VL - 378

SP - 804

EP - 809

JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

SN - 0006-291X

IS - 4

ER -