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Regulation of osteoclastic acid secretion by cGMP-dependent protein kinase
C Van Epps-Fung1, J P Williams, T L Cornwell
1Department of Pathology, University of Alabama at Birmingham 35294.
Abstract:
Nitric oxide (NO) down-regulates osteoclastic activity. The mechanism is unknown, although, in some cells NO acts by stimulating guanylate cyclase which activates cGMP-dependent proteins. We demonstrated cGMP-dependent protein kinase in osteoclasts by immunofluorescence microscopy. Specificity was confirmed by Western blot analysis showing a single 78 kDa band, the size of the Type I isoform, in isolated avian osteoclasts. Osteoclast function centers on HCl secretion at a specialized membrane organelle. We found that purified cGMP-dependent protein kinase inhibits ATP-dependent acid transport in reconstituted osteoclast membrane vesicles >90%, while cAMP-dependent kinase catalytic subunit, calmodulin kinase II, or cGMP alone were ineffective. This novel, direct modulation of acid transport by cGMP-dependent kinase and the occurrence of the enzyme in osteoclasts suggest that a mechanism of NO-regulation of bone turnover is via cGMP and cGMP-dependent protein kinase inhibition of HCl transport.
Insights
Nitric oxide (NO) regulates bone turnover by inhibiting osteoclast activity. This study shows NO works via cyclic guanosine monophosphate (cGMP)-dependent protein kinase to directly block acid secretion in osteoclasts.
Area of Science:
- Biochemistry
- Cell Biology
- Bone Physiology
Background:
- Nitric oxide (NO) is known to reduce osteoclast activity, a key process in bone remodeling.
- The precise molecular mechanisms underlying NO's effect on osteoclasts remain largely unelucidated.
- NO often signals through cyclic guanosine monophosphate (cGMP)-dependent pathways in various cell types.
Purpose of the Study:
- To investigate the presence and function of cGMP-dependent protein kinase (PKG) in osteoclasts.
- To determine if PKG plays a role in the NO-mediated regulation of osteoclast function, specifically HCl secretion.
Main Methods:
- Immunofluorescence microscopy was used to detect PKG in isolated avian osteoclasts.
- Western blot analysis confirmed the identity and size of PKG in osteoclasts.
- Functional assays using reconstituted osteoclast membrane vesicles assessed the effect of PKG on ATP-dependent acid transport.
Main Results:
- cGMP-dependent protein kinase (PKG) was identified in osteoclasts via immunofluorescence and confirmed as the Type I isoform by Western blot.
- Purified PKG significantly inhibited ATP-dependent acid transport in osteoclast membrane vesicles by over 90%.
- Other tested agents, including cAMP-dependent kinase, calmodulin kinase II, and cGMP alone, did not inhibit acid transport.
Conclusions:
- This study demonstrates that cGMP-dependent protein kinase (PKG) is present in osteoclasts and directly inhibits their acid transport function.
- These findings suggest a novel mechanism where nitric oxide (NO) regulates bone turnover by activating the cGMP-PKG pathway, leading to inhibition of osteoclast HCl secretion.