MIP-1d Activates NFATc1 via PLCc2 and NFkB Figure 2. MIP-1d activates PLCc2 and NF-kB signaling pathways in OCL progenitors. BMM were treated with or without MIP-1d (0.1 pg/ml) and the effect on the ERK, JNK, PLCc2, and NF-kB signaling pathways was examined by Western analysis. (A) The effect of MIP-1d on the levels of total and phospho (p)-ERK, JNK, PLCc2, and IkBa was examined using equal amounts of protein from total cell lysates. (B) Nuclear translocation of p50, RelA, c-Rel, and (C) RelB following MIP-1d treatment was determined using equal amounts of protein from nuclear cell lysates. (C) Processing of p100 to p52 following MIP-1d treatment was determined using equal amounts of protein from total cell lysates. (D) Activation of NF-kB was determined 30 minutes following treatment with MIP-1d by non-radioactive EMSA analysis of nuclear cell lysates using NF-kB oligonucleotides. Oct-1 served as a loading control and 200-fold excess unlabeled oligonucleotide (200X-U.O.) served as a competitive control. Densitometry values are provided below respective lanes for ease of interpretation. Data are representative of at least two independent experiments. doi:10.1371/journal.pone.0040799.g002 resorption, underscoring its potential to contribute to pathological bone loss. While we have demonstrated the ability of MIP-1d to directly influence OCL precursors in vitro, it is possible that MIP-1d promotes OCL formation and bone resorption in vivo via additional mechanisms. Interestingly, expression of the receptors for MIP-1d, CCR1 and CCR3, is increased in mature OCL relative to OCL precursors [12]. Therefore it is conceivable that MIP-1d exerts effects on mature OCL in addition to OCL precursors, possibly influencing survival and activity. In addition to effects on bone-resorbing OCL, it is also possible that MIP-1d modulates the activity of bone-forming osteoblasts (OB). OB play a major role in OCL differentiation by producing RANKL, and its decoy receptor, osteoprotegerin (OPG). Since OB also express the receptors for MIP-d (S.L.K., unpublished observations/data), it may indirectly promote OCL formation and bone loss in vivo by influencing the production of RANKL and OPG by OB as well as by regulating OB survival and activity. Thus, in future studies it will be important to examine the effects of MIP-1d on mature OCL as well as pre and mature OB. Collectively, these data highlight the potential of MIP-1d to contribute to pathologic bone resorption associated with skeletal diseases such as rheumatoid arthritis and cancer metastasis to bone, where MIP-1d levels are elevated [12,25]. This work is timely as small molecule antagonists against CCRs are being developed and evaluated in clinical trials [26]. While this approach is promising, caution must be taken as many CCRs, including CCR1 and CCR3, are widely expressed in the body and serve as receptors for multiple ligands raising the potential for toxicity. Further investigation into the molecular mechanisms through which MIP-1d influences OCL formation and bone resorption in vivo are warranted and may lead to the discovery of more specific methods of inhibiting its effects. Materials and Methods Ethics Statement All experiments were carried out in accordance with the National Research Council’s ‘‘Guide to the Care and Use of Laboratory Animals’’. Animal use was approved by the Johns Hopkins Animal Care and Use Committee, animal welfare assurance #A3272-01, protocol #MO07M149. Generation of Bone Marrow Macrophages Bone marrow was isolated from the tibiae, femur, and humerus of 4–8 week old NIH Swiss mice. The ends of each bone were removed and the marrow collected by flushing the bone with aMEM containing 15% FBS and 1% penicillin and streptomycin PLoS ONE | www.plosone.org 4 July 2012 | Volume 7 | Issue 7 | e40799