Osteoclasts are multinucleated bone-resorbing cells that use multiple pH regulation mechanisms to create an acidic pH in the resorption lacuna. Carbonic anhydrase II and vacuolar H(+)-ATPases produce and transport protons, while chloride channels provide a Cl(-) flux into the resorption site. These activities are required for inorganic matrix dissolution that precedes enzymatic removal of organic bone matrix. In other cell types it has become evident that carbonic anhydrase isoenzymes interact with AE proteins to form transport metabolons that regulate intracellular pH. Membrane-bound carbonic anhydrase isoenzymes may also compensate for the lack of cytoplasmic carbonic anhydrase II. Therefore, our goal was to explore the expression of membrane-bound carbonic anhydrase (CA) isoenzymes CA IV, CA IX, CA XII and CA XIV in bone-resorbing osteoclasts. Immunohistochemistry and confocal microscopy showed expression of CA IV, CA XII and CA XIV in cultured rat and human osteoclasts. To confirm these results, RT-PCR was used. Immunohistochemistry revealed distinct staining patterns for CA IV, CA XII and CA XIV in rat trabecular bone specimens. A plasma membrane staining was observed in bone lining cells with the CA XII antibody while osteoclast plasma membranes were stained with CA IV and CA XIV antibodies. Confocal microscopy of cultured human osteoclasts showed a punctated intracellular CA IV staining and a perinuclear CA XIV staining while no CA IX or CA XII staining was observed. To evaluate the physiological role of membrane-bound CAs in osteoclasts, we used PCS, a novel membrane-impermeable CA inhibitor. Increased osteoclast number and bone resorption activity was observed in rat osteoclast cultures exposed to a low concentration of PCS while higher concentrations affected cell survival. PCS treatment also disturbed intracellular acidification in osteoclasts, as determined by live cell microscopy. In conclusion, our data shows that membrane-bound carbonic anhydrase isoenzymes CA IV and CA XIV are expressed both at mRNA and protein levels in osteoclasts in vivo and in vitro. In addition, the inhibitor experiments provide novel evidence to support the hypothesis that intracellular pH regulation in osteoclasts may indeed involve transport metabolons.

Membrane-bound carbonic anhydrases in osteoclasts / R. Riihonen;C. T. Supuran;S. Parkkila;S. Pastorekova;H. K. Väänänen;T. Laitala-Leinonen. - In: BONE. - ISSN 8756-3282. - STAMPA. - 40:(2007), pp. 1021-1031. [10.1016/j.bone.2006.11.028]

Membrane-bound carbonic anhydrases in osteoclasts.

SUPURAN, CLAUDIU TRANDAFIR;
2007

Abstract

Osteoclasts are multinucleated bone-resorbing cells that use multiple pH regulation mechanisms to create an acidic pH in the resorption lacuna. Carbonic anhydrase II and vacuolar H(+)-ATPases produce and transport protons, while chloride channels provide a Cl(-) flux into the resorption site. These activities are required for inorganic matrix dissolution that precedes enzymatic removal of organic bone matrix. In other cell types it has become evident that carbonic anhydrase isoenzymes interact with AE proteins to form transport metabolons that regulate intracellular pH. Membrane-bound carbonic anhydrase isoenzymes may also compensate for the lack of cytoplasmic carbonic anhydrase II. Therefore, our goal was to explore the expression of membrane-bound carbonic anhydrase (CA) isoenzymes CA IV, CA IX, CA XII and CA XIV in bone-resorbing osteoclasts. Immunohistochemistry and confocal microscopy showed expression of CA IV, CA XII and CA XIV in cultured rat and human osteoclasts. To confirm these results, RT-PCR was used. Immunohistochemistry revealed distinct staining patterns for CA IV, CA XII and CA XIV in rat trabecular bone specimens. A plasma membrane staining was observed in bone lining cells with the CA XII antibody while osteoclast plasma membranes were stained with CA IV and CA XIV antibodies. Confocal microscopy of cultured human osteoclasts showed a punctated intracellular CA IV staining and a perinuclear CA XIV staining while no CA IX or CA XII staining was observed. To evaluate the physiological role of membrane-bound CAs in osteoclasts, we used PCS, a novel membrane-impermeable CA inhibitor. Increased osteoclast number and bone resorption activity was observed in rat osteoclast cultures exposed to a low concentration of PCS while higher concentrations affected cell survival. PCS treatment also disturbed intracellular acidification in osteoclasts, as determined by live cell microscopy. In conclusion, our data shows that membrane-bound carbonic anhydrase isoenzymes CA IV and CA XIV are expressed both at mRNA and protein levels in osteoclasts in vivo and in vitro. In addition, the inhibitor experiments provide novel evidence to support the hypothesis that intracellular pH regulation in osteoclasts may indeed involve transport metabolons.
2007
40
1021
1031
R. Riihonen;C. T. Supuran;S. Parkkila;S. Pastorekova;H. K. Väänänen;T. Laitala-Leinonen
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Utilizza questo identificatore per citare o creare un link a questa risorsa: https://hdl.handle.net/2158/775985
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