MIP-1d Activates NFATc1 and Enhances Osteoclastogenesis: Involvement of Both PLCc2 and NFkB Signaling Kristy L. Weber, Michele Doucet, Adam Shaner, Nigel Hsu, David Huang, Jenna Fogel, Scott L. Kominsky* Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America Abstract Pathological bone resorption is a source of significant morbidity in diseases affecting the skeleton such as rheumatoid arthritis, periodontitis, and cancer metastasis to bone. Evidence indicates that elevated levels of inflammatory mediators such as IL-1, IL-6, and TNF-a play a role in this process by promoting the formation of bone-resorbing osteoclasts. Additionally, current studies have identified inflammatory chemokines of the macrophage inflammatory protein (MIP) family as potential mediators of pathological bone resorption, where both MIP-1a and -3a have been shown to enhance osteoclast (OCL) development. In this study we provide evidence that MIP-1d, whose expression is associated with renal cell carcinoma bone metastasis and rheumatoid arthritis, enhances OCL formation in vitro via a direct effect on OCL precursors. Consistent with this ability, exposure of OCL precursors to MIP-1d resulted in the activation of PLCc2 and NF-kB, two signaling pathways known to regulate OCL differentiation. Moreover, MIP-1d induced expression and nuclear translocation of NFATc1, a master regulator of osteoclastogenesis, which was dependent on activation of both the PLCc2 and NFkB signaling pathways. Lastly, consistent with in vitro studies, in vivo administration of MIP-1d significantly increased OCL number and resorption area as determined using a murine calvarial bone resorption model. Taken together, these data highlight the potential of MIP-1d as a mediator of pathological bone resorption and provide insight into the molecular mechanism through which MIP-1d enhances osteoclastogenesis. Citation: Weber KL, Doucet M, Shaner A, Hsu N, Huang D, et al. (2012) MIP-1d Activates NFATc1 and Enhances Osteoclastogenesis: Involvement of Both PLCc2 and NFkB Signaling. PLoS ONE 7(7): e40799. doi:10.1371/journal.pone.0040799 Editor: Paul Proost, University of Leuven, Rega Institute, Belgium Received February 23, 2012; Accepted June 13, 2012; Published July 9, 2012 Copyright: 2012 Weber et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Award Number R21AR056373 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Arthritis and Musculoskeletal and Skin Diseases or the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: kominsc@jhmi.edu Introduction Pathological bone resorption occurs in skeletal diseases such as rheumatoid arthritis, periodontitis, and cancer, resulting in substantial bone pain and loss of function. In the case of rheumatoid arthritis and periodontitis, bone loss occurs follow- ing chronic inflammation. Inflammatory mediators such as interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-a have been shown to elevate levels of the osteoclastogenic cytokine, receptor activator of nuclear factor kappa-B ligand (RANKL), enhancing the development of bone resorbing osteoclasts (OCL) [1], thereby disrupting the delicate balance of bone resorption and formation. Evidence also supports a role for inflammatory mediators (eg. IL-3, IL-6, and IL-8) in the OCL-mediated bone resorption observed in metastatic breast cancer and multiple myeloma [2,3,4]. Recent studies indicate that inflammatory chemokines of the macrophage inflammatory protein (MIP) family may also play a role in mediating pathological bone resorption. Currently, the MIP family consists of six members: MIP-1a, MIP-1b, MIP-1d, MIP-1c, MIP-3a, and MIP-3b. MIP-3a, whose expression is increased in bone biopsies from rheumatoid arthritis patients, has been shown to enhance OCL development by stimulating OCL precursor proliferation [5]. Similarly, it has also been detected in periodontitis where elevated expression was positively correlated with disease status [6,7,8]. Elevated levels of another family member, MIP-1a, were reported in bone marrow of multiple myeloma patients as compared to healthy adults [9]. Further studies indicate that MIP-1a is able to stimulate OCL development [10], while inhibition of MIP-1a significantly reduces bone destruction in a mouse model of multiple myeloma [11]. In line with these findings, we recently found levels of another MIP family member, MIP-1d (CCL15), to be signifi- cantly elevated in human renal cell carcinoma bone metastasis (RBM) tissues relative to bone marrow from healthy adults [12]. Further, consistent with the osteolytic nature of RBM, we provided in vitro evidence that MIP-1d stimulates chemotaxis of OCL progenitors and enhances OCL differentiation in response to RANKL. Here, we demonstrate the ability of MIP-1d to directly enhance the differentiation of OCL precursors in vitro, elucidate its effect on the signaling pathways and transcription factors regulating osteoclastogenesis, and provide the first evidence that MIP-1d can stimulate osteoclastogenesis and bone resorption in vivo, highlighting its potential as a mediator of pathological bone loss. PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e40799