Immunology Letters 142 (2012) 34–40 Contents lists available at SciVerse ScienceDirect Immunology Letters j ourna l h o me page: www.elsevier.com/locate/immlet Inhibition of receptor activator of nuclear factor-B ligand (RANKL)-induced osteoclast formation by pyrroloquinoline quinine (PQQ) Erdenezaya Odkhuu, Naoki Koide, Abedul Haque, Bilegtsaikhan Tsolmongyn, Yoshikazu Naiki, Shoji Hashimoto, Takayuki Komatsu, Tomoaki Yoshida, Takashi Yokochi ∗ Department of Microbiology and Immunology, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan a r t i c l e i n f o Article history: Received 16 September 2011 Received in revised form 9 November 2011 Accepted 5 December 2011 Available online 13 December 2011 Keywords: Pyrroloquinoline quinine Receptor activator of nuclear factor-B ligand Osteoclast Nuclear factor of activated T cells c-Fos Type I interferon receptor a b s t r a c t The effect of pyrroloquinoline quinine (PQQ) on receptor activator of nuclear factor-B ligand (RANKL)- induced osteoclast formation was examined using RAW 264.7 macrophage-like cells. RANKL led to the formation of osteoclasts identified as tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in the culture of RAW 264.7 cells. However, PQQ inhibited the appearance of osteoclasts and pre- vented the decrease of F4/80 macrophage maturation marker on RANKL-stimulated cells, suggesting a preventive action of PQQ on RANKL-induced osteoclast differentiation. PQQ inhibited the activation of nuclear factor of activated T cells (NFATc1), a key transcription factor of osteoclastogenesis, in RANKL- stimulated cells. On the other hand, PQQ did not inhibit the signaling pathway from RANK/RANKL binding to NFATc1 activation, including NF-B and mitogen-activated protein kinases (MAPKs). PQQ augmented the expression of type I interferon receptor (IFNAR) and enhanced the IFN--mediated janus kinase (JAK1) and signal transducer and activator of transcription (STAT1) expression. Moreover, PQQ reduced the expression level of c-Fos leading to the activation of NFATc1. Taken together, PQQ was suggested to prevent RANKL-induced osteoclast formation via the inactivation of NFATc1 by reduced c-Fos expres- sion. The reduced c-Fos expression might be mediated by the enhanced IFN- signaling due to augmented IFNAR expression. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Osteoclasts are bone-resorbing multinuclear cells derived from hematopoietic stem cells [1]. The interaction between recep- tor activator of nuclear factor (NF)-B (RANK) and RANK ligand (RANKL) is essential for osteoclast differentiation and activation [2,3]. The binding of RANKL and RANK on osteoclast progenitor cells triggers the activation of tumor necrosis factor receptor-associated factor 6 (TRAF6) [4] and subsequently the activation of NF-B and mitogen-activated protein kinases (MAPKs), such as extracellu- lar signal-regulated kinase 1/2 (ERK1/2), p38 and stress-activated Abbreviations: AP1, activated protein 1; CREB, cAMP responsive element bind- ing protein; ERK, extracellular signal-regulated kinase; IFNAR, type I interferon receptor; IFN, interferon; iNOS, inducible type of nitric oxide synthase; JAK1, janus kinase 1; MAPK, mitogen-activated protein kinase; NFATc1, nuclear factor of acti- vated T cells 1; NF-B, nuclear factor-B; PQQ, pyrroloquinoline quinine; RANKL, receptor activator of nuclear factor-B ligand; SAPK/JNK, stress-activated protein kinase/c-Jun N-terminal kinase; SD, standard deviation; STAT1, signal transducer and activator of transcription 1; TRAF6, tumor necrosis factor receptor-associated factor 6; TRAP, tartrate-resistant acid phosphatase. ∗ Corresponding author. Tel.: +81 561 62 3311x2269; fax: +81 561 63 9187. E-mail address: yokochi@aichi-med-u.ac.jp (T. Yokochi). protein kinase/c-Jun N-terminal kinase (SAPK/JNK) [5,6]. Nuclear factor of activated T cells (NFATc1) is a downstream transcrip- tion factor in the RANKL/RANK signal pathway and plays a crucial role on the osteoclastogenesis [3,7]. NFATc1 as a key molecule of osteoclastogenesis induces a series of osteoclast-specific genes, including cathepsin K, tartrate-resistant acid phosphatase (TRAP), calcitonin receptor and osteoclast-associated receptor [7,8]. c-Fos is also an essential transcription factor for osteoclastogenesis [9] and positively regulates osteoclastogenesis via NFATc1 activation. On the other hand, c-Fos induces interferon (IFN)- [10] as a strong inhibitor of osteoclastogenesis and negatively regulates osteoclas- togenesis through type I interferon receptor (IFNAR) [11]. Pyrroloquinoline quinine (PQQ) possesses a variety of functions ranging from classical vitamin to anti- and pro-oxidant [12,13]. PQQ is widely dispersed in animals [13–15]. Dietary in vivo exper- iments reveal that PQQ deficiency exhibits growth impairment, compromised immune responsiveness and abnormal reproductive performance [16,17]. PQQ might be involved in bone metabolism via nitric oxide biosynthesis [12]. However, there is no report on the role of PQQ on RANKL-induced osteoclast formation. Recently, PQQ has been reported to regulate several intracellular signaling pathways, including Ras-related ERK1/2 activation [18], CREB- dependent mitochondriogenesis [19], and JAK/STAT activation [13]. 0165-2478/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2011.12.001