Characterization of the Role of Peroxisome Proliferator Activator Receptor (PPAR)-Delta in Osteoclast Biology

Joao P. Marcal Fidalgo (2011). Characterization of the Role of Peroxisome Proliferator Activator Receptor (PPAR)-Delta in Osteoclast Biology PhD Thesis, Institute for Molecular Bioscience, The University of Queensland.

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Author Joao P. Marcal Fidalgo
Thesis Title Characterization of the Role of Peroxisome Proliferator Activator Receptor (PPAR)-Delta in Osteoclast Biology
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
Publication date 2011-06
Thesis type PhD Thesis
Supervisor Dr. Alan Ian Cassady
Dr. Dmitry Ovchinnikov
Prof. George Muscat
Total pages 195
Total colour pages 25
Total black and white pages 170
Subjects 06 Biological Sciences
Abstract/Summary Osteoclasts are the cells responsible for the resorption of bone and their activity is tightly controlled to maintain bone structure and calcium homeostasis. The elucidation of the function of known genes and the identification of novel genes involved with osteoclast biology are both critically important in the development of novel treatments for bone diseases. The objective of this project was to investigate the involvement of PPAR-delta in osteoclast formation and/or function using both in vitro and in vivo models through gene inactivation and gene overexpression/activation approaches. In the RAW/C4 osteoclast cell model, osteoclastogenic treatment with RANKL and CSF-1 induces upregulation of expression of PPAR-delta whereas expression of PPAR-alpha and PPAR-gamma subtypes is downregulated. The pattern of PPAR-delta expression during osteoclast differentiation is similar to typical osteoclast markers that normally show maximal expression levels at about day 5 of osteoclastogenesis. Treatment of cells with GW501516, a powerful and highly specific synthetic PPAR-delta agonist results in an increase in expression of all three osteoclast expression markers that tested: TRAP, NFATc1 and CTR. Over-expression of PPAR-delta in RANKL/CSF-1-stimulated RAW/C4 osteoclast cells markedly up-regulates the expression of osteoclast markers. NFATc1 is an early osteoclast differentiation marker whereas both TRAP and CTR are functional markers expressed at later stages so the impact of PPAR-delta over-expression is observed at both early and later stages of osteoclast differentiation. A moderate increase in the formation of giant multinucleated osteoclasts was detected in PPAR-delta over-expressing cells and also a further increase with GW501516 treatment. Even though PPAR-delta over-expression does not result in a significant increase in osteoclast numbers, an interesting observation was that it markedly induced TRAP enzyme activity in single nucleated cells that did not fuse to form the mature multinucleated osteoclasts, a potential indication of direct regulation by PPAR-delta. Using the Cre/LoxP recombination system, the expression of PPAR-delta in bone-marrow derived osteoclasts was knocked-down by 60% of normal values. Expression profiling of osteoclasts with reduced levels of PPAR-delta transcript revealed that TRAP, CTR and NFATc1 were downregulated in these cells. The PPAR-delta deficient osteoclasts also show reduced potential to differentiate into fully mature osteoclasts under suboptimal RANKL conditions. A mouse model was developed with a conditional knock-out of PPAR-delta specifically in osteoclasts and the resulting bone phenotype was characterized using histology, immunohistochemistry, X-ray, bone densitometry (DXA) and bone tomography (microCT) analysis. There were no skeletal differences in the PPAR-delta deficient animals when compared to normal control mice, and indication that PPAR-delta might not be essential for osteoclast differentiation/function under physiological conditions. The relative importance of the two PPAR-gamma isoforms in osteoclasts was investigated, by over-expressing them in RAW/C4 cells and assessing the effect on osteoclastogenic potential. PPAR-gamma1 expression is markedly upregulated during RANKL/CSF-1-stimulated differentiation whereas PPAR-gamma2 follows an opposite trend being markedly downregulated. Interestingly, a completely opposite expression profile for these two isoforms is obtained in adipocyte differentiation where PPAR-gamma2 showed to be the main molecular switch in that pathway whereas PPAR-gamma1 does not seem to be involved in that process. Over-expression of PPAR-gamma1, but not PPAR-gamma2, also stimulates TRAP expression and activity. Consistent with these results, PPAR-gamma1 overexpression clearly stimulates osteoclast formation, whereas PPAR-gamma2 does not induce any major changes in this process. Furthermore, PPAR-gamma1 overexpression not only significantly increases the total osteoclast number but also markedly increases the number of giant osteoclasts, cells with 25 or more nuclei. The results obtained in this study show that PPAR-delta has a role in in vitro osteoclast biology, possibly by regulating NFATc1. However, the moderate effect of over-expression on the number of osteoclast formed suggests that whilst PPAR-delta is involved in osteoclast differentiation and/or function, parallel pathways to PPAR-delta may be more decisive in osteoclast formation. The over-expression of PPAR-delta in RAW/C4 cells does not seem to take precedence over these pathways by having a potent effect on osteoclast differentiation. The lack of bone phenotype obtained in the PPAR-delta conditional knock-out mice indicates that PPAR-delta might not be essential for osteoclastogenesis under physiological conditions. Nevertheless, the investigation of the importance of the two PPAR-gamma isoforms in osteoclasts allows to speculate that PPAR-delta deficiency can be compensated by PPAR-gamma1 in these animals.
Keyword PPAR-delta, osteoclast, bone, osteoclast differentiation, PPAR-gamma
Additional Notes 25, 32, 33, 38, 51, 61, 71, 96, 100, 109, 111, 119, 125, 127, 130, 131, 133, 134, 136, 149, 151, 153, 191, 192, 193

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Created: Thu, 02 Jun 2011, 14:38:32 EST by Mr Joao Marcal Fidalgo on behalf of Library - Information Access Service