Chan B Y, Gartland A, Wilson P J M, Buckley K A, Dillon J P, Fraser W D, Gallagher J A
Department of Clinical Biochemistry, Royal Liverpool University Hospital, The University of Liverpool, Liverpool, L69 3GA, UK.
Bone. 2007 Jan;40(1):149-59. doi: 10.1016/j.bone.2006.07.029. Epub 2006 Sep 28.
Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear steroid hormone superfamily and exist in three isoforms: PPARalpha, beta and gamma, each with specific functions. In this study, we have investigated the expression of PPARs by human osteoclast precursors and osteoclasts generated in vitro. In addition, the effects of fibrates and isoform-specific PPAR agonists on osteoclast formation and resorption in vitro were determined. Human peripheral blood mononuclear cells (PBMCs) were stimulated with human recombinant RANKL and M-CSF to generate osteoclasts. RNA was extracted at days 0, 7, 14 and 21 and RT-PCR for all three PPAR isoforms demonstrated their expression throughout this culture period. To determine the effect on osteoclast formation, PPAR agonists (10(-8) M to 10(-5) M) were added from the beginning of the culture until day 14 and the number of multinucleated osteoclasts counted. The effect of PPAR agonists on osteoclast function was similarly determined by treating mature, multinucleated osteoclasts cultured on dentine wafers with PPAR agonists (10(-8) M to 10(-5) M) for 7 days and quantifying resorption. Bezafibrate and fenofibrate, which non-discriminately activate all PPAR isoforms, significantly inhibited the formation of multinucleated osteoclasts from PBMC in vitro. Bezafibrate treatment of mature osteoclast resulted in 50% inhibition (at 10(-8) M and 10(-7) M) of resorption, yet fenofibrate had no significant effect. Activation of individual PPARs with isoform-specific agonist (GW9578, L165041 and ciglitizone which preferentially activate PPARalpha, beta and gamma respectively) resulted in significant dose-dependent inhibition of multinucleated osteoclast formation. Divergent effects on osteoclast resorption were observed; GW9578 had no significant effect on resorption, whereas ciglitizone and L165041 dose-dependently inhibited and stimulated resorption, respectively. These data show for the first time expression of all three PPAR isoforms throughout the development and maturation period of osteoclasts generated from human PBMCs. In addition, we demonstrate that isoform-specific PPAR agonists have strong effects on multinucleation and highly variable effects on bone resorption. In conclusion, this study highlights the potential of PPARs as therapeutic targets in diseases with accelerated osteoclast formation and resorption.
过氧化物酶体增殖物激活受体（PPARs）是核类固醇激素超家族的成员，有三种亚型：PPARα、β和γ，各有特定功能。在本研究中，我们调查了人破骨细胞前体及体外生成的破骨细胞中PPARs的表达情况。此外，还测定了贝特类药物及亚型特异性PPAR激动剂对体外破骨细胞形成和骨吸收的影响。用人重组RANKL和M-CSF刺激人外周血单核细胞（PBMCs）以生成破骨细胞。在第0、7、14和21天提取RNA，对所有三种PPAR亚型进行逆转录聚合酶链反应（RT-PCR），结果表明它们在整个培养期间均有表达。为确定对破骨细胞形成的影响，从培养开始至第14天添加PPAR激动剂（10⁻⁸ M至10⁻⁵ M），然后计数多核破骨细胞的数量。通过用PPAR激动剂（10⁻⁸ M至10⁻⁵ M）处理在牙本质薄片上培养的成熟多核破骨细胞7天，并对骨吸收进行定量分析，同样确定了PPAR激动剂对破骨细胞功能的影响。非选择性激活所有PPAR亚型的苯扎贝特和非诺贝特，在体外可显著抑制PBMCs中多核破骨细胞的形成。用苯扎贝特处理成熟破骨细胞，可导致骨吸收受到50%的抑制（在10⁻⁸ M和10⁻⁷ M时），而非诺贝特则无显著影响。用亚型特异性激动剂（分别优先激活PPARα、β和γ的GW9578、L165041和吡格列酮）激活单个PPARs，可导致多核破骨细胞形成受到显著的剂量依赖性抑制。在破骨细胞骨吸收方面观察到不同的作用；GW9578对骨吸收无显著影响，而吡格列酮和L165041分别剂量依赖性地抑制和刺激骨吸收。这些数据首次显示了在由人PBMCs生成的破骨细胞的整个发育和成熟过程中，所有三种PPAR亚型均有表达。此外，我们证明亚型特异性PPAR激动剂对多核化有强烈影响，而对骨吸收的影响则高度可变。总之，本研究突出了PPARs作为破骨细胞形成和骨吸收加速疾病治疗靶点的潜力。
