Recombinant human CX3CL1 and anti-human CX3CL1 were purchased from R&D Systems (Minneapolis, MN, USA). The following mAbs were used: anti-AKT1 (C-20), anti-ERK1/2 (C-16), anti-PCNA (PC-10) and anti-NFkbp65 (C-20) from Santa Cruz biotechnology (Santa Cruz, CA, USA), and anti-phospho AKT (Ser-473), anti-phospho p44/42 ERK (Thr-202/Tyr-204), anti-STAT3 and anti-phospho STAT3 (Ser727) from Cell Signaling Technology (Beverly, MA, USA).

Human multiple myeloma cell lines included RPMI-8226, KMS-12BM, KMS-12PE, L-363, OPM-2, KARPAS-620 and AMO-1 cells. All the cell lines were maintained in RPMI-1640 medium (Wako Pure Chemical Industries, Inc., Osaka, Japan) supplemented with 20% fetal bovine serum, 50 μM 2-mercaptoethanol (both from Invitrogen, Carlsbad, CA, USA), 100 u/ml penicillin and 100 μg/ml streptomycin (both from Meiji Seika Pharma, Tokyo, Japan). The cells were cultured at 37°C in an incubator with a humidified 5% CO₂ atmosphere.

RNA was extracted using an RNeasy Plus Mini kit (Qiagen, Hilden, Germany) and converted to cDNA using the SuperScript III First-Strand Synthesis System for RT-PCR (Life Technologies Corporation, Carlsbad, CA, USA). The expression of CX3CR1 was determined by RT-PCR. The PCR was performed for 30 cycles (denaturation, 98°C 5 sec; annealing, 60°C 5 sec; extension, 72°C 10 sec) using Sapphire Amp Fast PCR Master Mix (Takara Bio, Shiga, Japan) The PCR products were subjected to electrophoresis on a 1.5% agarose gel with SYBR-Green (Lonza Inc., Rockland, ME, USA) and then photographed under an ultraviolet transilluminator. GAPDH was used as a normalization control. The primers for GAPDH were as follows: sense, 5′-TGA AGG TCG GAG TCA ACG GAT TTG GT-3′ and antisense, 5′-CAT GTG GGC CAT GAG GTC CAC CAC-3′. The primers for CX3CR1 were as follows: sense, 5′-TGG CCT TGT CTG ATC TGC TGT TTG-3′ and antisense, 5′-ATG GCT TTG GCT TTC TTG TGG TTC-3′. The RNA from healthy donor plasma cell was purchased from AllCells (Alameda, CA, USA).

The western blot analysis was performed following incubation with or without 10 nM rCX3CL1. Antibodies to phosphorylated (p) or unphosphorylated JAK, Stat, Akt and Erk1/2 were used. Images of immunoblots were scanned and quantified with an ImageQuant LAS 4000 Lumino Image Analyzer from Fuji Film Corporation (Tokyo, Japan).

Triplicate wells of 96-well plates were coated with 1 μg fibronectin (Asahi Techno Glass, Co., Ltd., Tokyo, Japan) and vascular cell adhesion molecule-1 (VCAM-1) (R&D Systems). RPMI-8226 cells were stimulated by recombinant human CX3CL1 (10 nM) for 5 min. After stimulation, RPMI-8226 cells (1×10⁴ cells/100 μl in EMEM with 0.1% BSA) were seeded and incubated for 20 min at 37°C. Cells that adhered to the well were evaluated as previously described (20).

RPMI-8226 cells were cultured in the absence or presence of recombinant human CX3CL1 (10 nM) for 48 h, and then the conditioned cell culture medium was collected. Osteoclast precursors (RAW 264.7) were suspended in a-MEM supplemented with 10% FBS and cultured in a 24-well culture plates at 1x10⁶/well. After 48 h, the culture medium was replaced with 50% conditioned medium with or without 100 ng/ml of mouse recombinant soluble RANKL (Wako Pure Chemical Industries, Inc.). After 4 days, the cells were dehydrated with ethanol-acetone (1:1) for 1 min, dried and stained at room temperature with tartrate-resistant acid phosphatase (TRAP) staining solution. TRAP-positive cells appeared dark red. We counted TRAP-positive multi-nucleated cells containing three or more nuclei as osteoclasts.

Data were analyzed for statistical significance using the Student’s t-test. P<0.05 was considered to indicate a statistically significant result. The mean and SD were calculated for all variables.

We first compared the expression of CX3CR1 in seven human multiple myeloma cell lines and plasma cells (Cd138-positive cell fraction) by RT-PCR (Fig. 1). CX3CR1 expression was detected in three (RPMI-8226, OPM-2 and KARPAS-620) of the seven human multiple myelomas, even though CX3CR1 was not expressed in the plasma cells that derived from healthy donors. CX3CR1 expression was apparently induced during the process of malignant transformation of normal plasma cells to multiple myeloma.

The observation of the CX3CR1 expression in human multiple myeloma cells prompted us to examine the biological responses of these cells to CX3CL1. The mechanisms underlying the differences in CX3CL1-induced progression and cell survival were investigated by analyzing signal transduction. Seven multiple myeloma cell lines were incubated with or without recombinant CX3CL1 and were subjected to western blotting using antibodies against unphosphorylated and phosphorylated Akt, Erk1, Erk2 and STAT3. Akt, Erk1 and Erk2, but not STAT3, were constitutively phosphorylated in the two cell lines (Fig. 2A). Incubation of RPMI-8226 cells with CX3CL1-induced p-Erk1/2 expression after 1 min; this expression peaked from 2 to 5 min, while p-Akt expression peaked after 2 min (Fig. 2B). Next, the RPMI-8226 cells were stimulated with the antibody for 2 min. We also observed that CX3CL1-induced activation of Akt and ERK in RPMI-8226 cells was inhibited selectively by an anti-CX3CL1 antibody (Fig. 3). These results indicate a rapid CX3CL1-driven signaling for progression and cell survival after stimulation of CX3CL1 in multiple myeloma.

We next examined whether CX3CL1 regulates cell adhesion of human multiple myeloma. The number of RPMI-8226 cells adhering to fibronectin and VCAM-1 increased by ~17- and 3-fold respectively, in response to pretreatment with recombinant CX3CL1 (Fig. 4). The adhesion of multiple myeloma cells to fibronectin and VCAM-1, which are mainly expressed in bone marrow stromal cells, activates many pathways and results in the upregulation of the cell cycle regulating proteins and anti-apoptotic proteins (9,21). These results suggest that CX3CL1-induced the progression of multiple myeloma in bone microenvironments.

We previously reported that CX3CL1 expressed by osteoblasts plays an important role in osteoclast differentiation, possibly acting through its dual functions as a chemotactic factor and adhesion molecule for osteoclast precursors expressing CX3CR1 (22,23).

Given the apparent expression of CX3CR1 by multiple myeloma cells, along with the inducible effect of its ligand CX3CL1 on multiple myeloma cell adhesion to bone microenvironment ECM. We also investigated the possible synergy between multiple myeloma and osteoclast precursors in osteoclast differentiation via CX3CL1. Osteoclast precursors, RAW 264.7 were differentiated by RANKL in conditioned medium collected from multiple myeloma cells stimulated by CX3CL1. After 4 days, TRAP-positive multinuclear osteoclasts were counted (Fig. 5). The conditioned medium increased the number of TRAP-positive multinuclear osteoclasts, while treatment with rat anti-CX3CL1 mAb suppressed the induction of TRAP-positive multinuclear osteoclasts. These results suggest that CX3CL1 indirectly induces osteoclast differentiation by promoting the secretion of a factor from multiple myeloma in bone microenvironments.