Protein kinase C affects microfilaments, bone resorption, and [Ca2+]o sensing in cultured osteoclasts.
The effects of protein kinase C (PKC) in the control of osteoclast activity are still unknown. We investigated the role of the enzyme in the control of microfilament organization, podosome assembly, bone resorption, and extracellular Ca2+ sensing in chicken and rabbit osteoclasts treated with agents known to affect PKC activity. Cells were treated for 20 min with a PKC activator [phorbol 12-myristate 13-acetate (PMA)], a PKC inhibitor (staurosporine), a protein kinase A (PKA) inhibitor (H-9), a guanosine 3',5'-cyclic monophosphate-dependent protein kinase-PKA-PKC inhibitor (H-7), or with the inactive phorbol, 4 alpha-phorbol, to examine microfilaments by decoration with rhodamine-phalloidin. In PMA-treated osteoclasts, the number of microfilament-containing adhesion structures (podosomes) per cell decreased. However, enlarged microfilamentous cores in podosomes and stress fiber-like filaments, otherwise absent in controls, appeared. Whereas H-7 induced increase of the number of podosomes, staurosporine, H-9, and 4 alpha-phorbol failed to change microfilament organization. Chicken osteoclasts received also long-term treatment with the agents in the presence of [3H]proline-prelabeled chicken or rat bone particles to measure bone resorption. PMA, as well as staurosporine and H-7, stimulated the resorbing activity, whereas cells were insensitive to H-9 and 4 alpha-phorbol. Measurement of cytosolic free calcium concentration in PMA-treated fura-2-loaded single osteoclasts demonstrated a synergistic effect of PKC activation on the inhibitory extracellular calcium concentration-sensing mechanism, which was, by contrast, blocked by H-7, staurosporine, and H-9 and was insensitive to 4 alpha-phorbol. These results indicate that PKC regulates osteoclast activity inducing both morphological and functional modifications.
Teti
A
A
Institute of Human Anatomy, School of Pharmacy, University of Bari, Italy.
Colucci
S
S
Grano
M
M
Argentino
L
L
Zambonin Zallone
A
A
D008841:Actin Cytoskeleton / Q000502:physiology*; D000818:Animals; D001862:Bone Resorption / Q000503:physiopathology*; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000648:ultrastructure; D009994:Osmolar Concentration; D010010:Osteoclasts / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D011493:Protein Kinase C / Q000502:physiology*
D011493:Protein Kinase C; D002118:Calcium