ORCID
https://orcid.org/0000-0003-2073-9040
Date of Award
Summer 8-15-2016
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Osteoclasts (OCs), the specialized bone-resorbing cells, have high energy demands to facilitate mobility, protease secretion, and acidification of their resorptive lacunae. They are enriched with mitochondria to generate ATP for the membrane vATPases to acidify bone. Mitochondrial biogenesis (the generation of new mitochondrial DNA and proteins) and mitochondrial dynamics and turnover (degrading defective mitochondria and maintaining mitochondrial health) are two processes that have been well-studied in other mitochondria-dense cell types. Here, in two studies, we 1. investigate whether mitochondria biogenesis and OC differentiation are coupled processes and 2. assess the role of mitochondrial GTPase protein Mfn2 in regulating bone resorption. Both studies aim to shed light on how mitochondria biogenesis and health affect overall OC-mediated bone resorption.
In the first study we used mice with mutations in key alternative NF-κB pathway proteins, RelB and NIK, to dissect the complex relationship between mitochondrial biogenesis and osteoclastogenesis. In OC precursors lacking either NIK or RelB, RANKL was unable to increase mitochondrial DNA or OxPhos protein expression, and they exhibited lower oxygen consumption rates. Transgenic OC precursors expressing constitutively active NIK showed normal RANKL-induced mitochondrial biogenesis (OxPhos expression and mitochondria copy number) compared to controls, but larger mitochondrial dimensions and increased oxygen consumption rates, suggesting increased mitochondrial function. NIK- and RelB-deficient precursors failed to fully upregulate PGC-1β expression. Because PGC-1β has been reported to positively regulate both mitochondrial biogenesis and differentiation in OCs, we retrovirally overexpressed PGC-1β in RelB-/- cells, but surprisingly found that it did not affect differentiation, nor restore RANKL-induced mitochondrial biogenesis. To determine whether the blockade in osteoclastogenesis in RelB-deficient cells precludes mitochondrial biogenesis, we rescued RelB-/- differentiation via overexpression of NFATc1. Mitochondrial parameters in neither WT nor RelB-deficient cultures were affected by NFATc1 overexpression, and bone resorption in RelB -/- was not restored. Furthermore, NFATc1 co-overexpression with PGC-1β, while allowing OC differentiation, did not rescue mitochondrial biogenesis or bone resorption in RelB-/- OCs. This indicates that the alternative NF-κB pathway plays dual, but distinct roles in controlling the independent processes of OC differentiation and OC mitochondrial biogenesis.
In the second study, we aimed to understand the effect of targeting a protein regulating mitochondrial dynamics, Mfn2, to understand the link between mitochondrial health and turnover in OCs, and how that affects bone resorption. Mitophagy is an important process where damaged mitochondria are recycled via the autophagy pathway. A healthy mitochondrial network is also important to maintain integrity and function of mitochondria. Both mitophagy and tethering are dependent on the mitochondrial membrane GTPase Mitofusin 2 (Mfn2). To examine whether Mfn2 is important for osteoclast-mediated bone resorption, we conditionally deleted Mitofusin2 in OC-lineage cells using a LysozymeM-driven cre recombinase. In 16-20 week old males, there was no difference in trabecular bone mass between control (LysMcre/creMfn2+/+) and Mfn2 cKO (LysMcre/creMfn2fl/fl) groups. However, in females of the same age range, Mfn2 cKO mice had higher trabecular bone mass compared to controls. When the mice were aged to 40-50 weeks, neither sex showed differences in bone mass between cntrl and cKO groups. Neither group had significant differences in cortical bone mass at either age range. To study the OC-specific bone resorptive effect of Mfn2 deletion, we transplanted male control or cKO marrow into 9 week-old male WT recipients, and chimeras were analyzed 9.5 weeks later by microCT. Although there was no basal bone phenotype, super-physiological doses of RANKL injected over the calvarium of chimeric control or cKO mice showed Mfn2 deficiency protected mice from bone loss, by u-CT and histomorphometric analysis. Ex vivo, although 6-8 wk Mfn2 cKOs did not have any OC differentiation or mitochondrial biogenesis defects, 40-50 wk old cKOs exhibited both, and this was not due to a difference in viability. Lastly, Mfn2 cKOs showed fragmented mitochondrial morphology, decreased intracellular ATP production, and aberrant Ca2+ oscillation that could account for the failure of NFATc1 to rescue Mfn2 cKO’s OC differentiation defect. Although the exact mechanism of Mfn2 action in OC lineage cells remains to be elucidated, this molecule appears to be important in controlling both basal bone mass and pathological bone loss in mice.
Language
English (en)
Chair and Committee
Deborah Novack
Committee Members
Gerald Dorn, Roberta Faccio, Daniel Link, Deborah Novack, Eugene Oltz
Recommended Citation
Zeng, Rong, "Role of Mitochondria Biogenesis and Dynamics Regulating Osteoclast-mediated Bone Resorption" (2016). Arts & Sciences Electronic Theses and Dissertations. 908.
https://openscholarship.wustl.edu/art_sci_etds/908
Comments
Permanent URL: https://doi.org/doi:10.7936/K7BR8QK8