Degradation of the organic phase of bone by osteoclasts: a secondary role for lysosomal acidification.
Osteoclasts degrade bone matrix by secretion of hydrochloric acid and proteases. We studied the processes involved in the degradation of the organic matrix of bone in detail and found that lysosomal acidification is involved in this process and that MMPs are capable of degrading the organic matrix in the absence of cathepsin K.


BACKGROUND
Osteoclasts resorb bone by secretion of acid by the vacuolar H+-adenosine triphosphatase (V-ATPase) and the chloride channel ClC-7, followed by degradation of the matrix, mainly collagen type I, by cathepsin K and possibly by matrix metalloproteinases (MMPs). However, the switch from acidification to proteolysis and the exact roles of both the ion transporters and the proteinases still remain to be studied.


METHODS
We isolated CD14+ monocytes from human peripheral blood from either controls or patients with autosomal dominant osteopetrosis type II (ADOII) caused by defective ClC-7 function and cultured them in the presence of RANKL and macrophage-colony stimulating factor (M-CSF) to generate osteoclasts. We decalcified cortical bovine bone slices and studied the osteoclasts with respect to morphology, markers, and degradation of the decalcified matrix in the presence of various inhibitors of osteoclast acidification and proteolysis, using normal calcified bone as a reference.


RESULTS
We found that ADOII osteoclasts not only have reduced resorption of the calcified matrix, but also 40% reduced degradation of the organic phase of bone. We found that both acidification inhibitors and cathepsin K inhibitors reduced degradation of the organic matrix by 40% in normal osteoclasts, but had no effect in the ADOII osteoclasts. Furthermore, we showed that inhibition of MMPs leads to a 70% reduction in the degradation of the organic bone matrix and that MMPs and cathepsin K have additive effects. Finally, we show that osteoclastic MMPs mediate release of the carboxyterminal telopeptide of type I collagen (ICTP) fragment in the absence of cathepsin K activity, and therefore, to some extent, are able to compensate for the loss of cathepsin K activity.


CONCLUSIONS
These data clearly show that osteoclastic acidification of the lysosomes plays a hitherto nonrecognized role in degradation of the organic matrix. Furthermore, these data shed light on the complicated interplay between acidification dependent and independent proteolytic processes, mediated by cathepsin K and the MMPs, respectively.
Henriksen
Kim
K
Pharmos Bioscience, Nordic Bioscience and Center for Clinical and Basic Research A/S, Herlev, Denmark. kh@nordicbioscience.com
Sørensen
Mette G
MG
Nielsen
Rasmus H
RH
Gram
Jeppe
J
Schaller
Sophie
S
Dziegiel
Morten H
MH
Everts
Vincent
V
Bollerslev
Jens
J
Karsdal
Morten A
MA
D000328:Adult; D000368:Aged; D001862:Bone Resorption / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D056657:Cathepsin K; D002403:Cathepsins / Q000378:metabolism; D002478:Cells, Cultured; D018118:Chloride Channels / Q000235:genetics / Q000378:metabolism*; D025063:Chromosome Disorders / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D017364:Collagenases / Q000378:metabolism; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D010010:Osteoclasts / Q000378:metabolism* / Q000473:pathology; D010024:Osteoporosis / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism*
D018118:Chloride Channels; D002403:Cathepsins; C534130:CTSK protein, human; D056657:Cathepsin K; D017364:Collagenases; D025262:Vacuolar Proton-Translocating ATPases