Screening of protein kinase inhibitors identifies PKC inhibitors as inhibitors of osteoclastic acid secretion and bone resorption.
BACKGROUND
Bone resorption is initiated by osteoclastic acidification of the resorption lacunae. This process is mediated by secretion of protons through the V-ATPase and chloride through the chloride antiporter ClC-7. To shed light on the intracellular signalling controlling extracellular acidification, we screened a protein kinase inhibitor library in human osteoclasts.


METHODS
Human osteoclasts were generated from CD14+ monocytes. The effect of different kinase inhibitors on lysosomal acidification in human osteoclasts was investigated using acridine orange for different incubation times (45 minutes, 4 and 24 hours). The inhibitors were tested in an acid influx assay using microsomes isolated from human osteoclasts. Bone resorption by human osteoclasts on bone slices was measured by calcium release. Cell viability was measured using AlamarBlue.


RESULTS
Of the 51 compounds investigated only few inhibitors were positive in both acidification and resorption assays. Rottlerin, GF109203X, Hypericin and Ro31-8220 inhibited acid influx in microsomes and bone resorption, while Sphingosine and Palmitoyl-DL-carnitine-Cl showed low levels of inhibition. Rottlerin inhibited lysosomal acidification in human osteoclasts potently.


CONCLUSIONS
In conclusion, a group of inhibitors all indicated to inhibit PKC reduced acidification in human osteoclasts, and thereby bone resorption, indicating that acid secretion by osteoclasts may be specifically regulated by PKC in osteoclasts.
Sørensen
Mette G
MG
Nordic Bioscience A/S, Herlev, Denmark.
Karsdal
Morten A
MA
Dziegiel
Morten H
MH
Boutin
Jean A
JA
Nosjean
Olivier
O
Henriksen
Kim
K
D000143:Acids / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D000818:Animals; D001862:Bone Resorption / Q000201:enzymology / Q000378:metabolism / Q000517:prevention & control*; D002417:Cattle; D002478:Cells, Cultured; D006801:Humans; D010010:Osteoclasts / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000502:physiology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology* / Q000627:therapeutic use
D000143:Acids; D047428:Protein Kinase Inhibitors; D011493:Protein Kinase C