Effects of Substance P on RANKL and OPG. mrna Expression in MG-63 Osteoblast-like Cells. Exposed to Polyethylene Particles

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1 Advanced Studies in Biology, Vol. 4, 2012, no. 7, Effects of Substance P on RANKL and OPG mrna Expression in MG-63 Osteoblast-like Cells Exposed to Polyethylene Particles Max Daniel Kauther Department of Trauma Surgery, University Hospital of Essen, Hufelandstraße 55, Essen, Germany Jie Xu Department of Orthopaedics, University Hospital of Essen, Hufelandstraße 55, Essen, Germany Julia Hartl Department of Orthopaedics, University Hospital of Essen, Hufelandstraße 55, Essen, Germany Sven Lendemans Department of Trauma Surgery, University Hospital of Essen, Hufelandstraße 55, Essen, Germany

2 318 Max Daniel Kauther et al Marcus Jäger Department of Orthopaedics, University Hospital of Essen, Hufelandstraße 55, Essen, Germany Christian Wedemeyer Department of Orthopaedics, University Hospital of Essen, Hufelandstraße 55, Essen, Germany Abstract Introduction: Particle induced osteolysis is the most frequent cause of aseptic loosening after total joint replacement. This in-vitro study of MG-63 osteoblast-like cells focused on the effect of the neurotransmitter substance P (SP) on receptor activator of nuclear factor-κb ligand (RANKL) and osteoprotegerin (OPG), two key regulators of particle-induced osteolysis. Materials and Methods: MG-63 cells were incubated with different concentration of SP (0.1, 1.0 and 10 ug/ml) and ultra-high molecular weight polyethylene particles (UHMWPE) in cell-particle ratios of 1:100 and 1:500. RNA was analyzed by quantitative SybrGreen RT-PCR. Results: SP treatment lead to a significantly decrease of RANKL mrna expression (p<0.05) after 48 and 72 hours in cell-particle ratios of 1:100 and 1:500. Osteoprotegerin expression in MG-63 cells was increased after 48 hours and 72hours incubation with SP (p<0.05) while the cell-particle ratio was 1:100. The up-regulation of OPG was almost neutralized with the increase of the cell-particle concentration (1:500). Conclusion: These results demonstrate that the neurotransmitter SP can inhibit the RANKL mrna expression and up-regulates the OPG gene transcripts of the particle-stimulated osteoblast-like cells. The neurotransmitter SP might be related to bone metabolism in conditions of particle-induced osteolysis. Keywords: Substance P, Particles, UHMWPE, aseptic loosening, particleinduced osteolysis

3 Effects of substance P on RANKL and OPG mrna expression 319 Introduction Periprosthetic osteolysis is known to be the major cause of aseptic loosening in joint arthroplasty. There is increasing evidence that wear debris in periprosthetic tissues has direct catabolic effects on osteoblasts and osteoblasts leading to osteolysis around the implant requiring challenging revision surgery [25]. Many studies have investigated particle-induced osteolysis from different points of view. An influence of the nervous system on particle-induced osteolysis has been discussed since the discovery of nerve fibers in the capsule of loosened arthroplasty [2]. Substance P (SP) receptors in untreated joint tissue [28] and the localization of SP-immunoreactive nerve fibers in the vicinity of bone cells in animals [8,13] and humans [21] suggest an influence on bone remodeling of the nervous system. SP is a neuropeptide of 11 amino acids that is release from sensory nerve endings by several stimuli [9,19]. Nerve fibers containing SP have been found not only in the nerve system but also in other locations including intestine, immune system, and skeletal system [10]. This neuropeptide transmits pain signals and regulates immune responses such as leukocyte phagocytosis, neutrophil chemotaxis, and antibody production from B cells [10,15,19,23]. SP stimulates vascular dilatation and increases blood vessel permeability [5,16], modulates the blood flow and induces angiogenesis in the bone marrow [3]. Our in-vivo study of particle induced osteolysis in SP-deficient mice suggested an osteoprotective influence of the neurotransmitter SP [27]. The osteoprotegerin/receptor activator of nuclear factor-κb ligand/receptor activator of nuclear factorκb (OPG/RANKL/RANK) system plays a key role in the cross-talk between osteoblasts and osteoclasts [14]. RANKL and OPG are members of a ligand-receptor system that directly regulates osteoclast differentiation and bone resorption, and both are produced and secreted by osteoblastic lineage cells. RANKL binds to its receptor RANK, which is expressed on osteoclast progenitors, and leads to osteoclast activation. OPG binds to RANKL and thereby inhibits osteoclast activation. The balance of OPG and RANKL is essential for bone remodeling [4]. This in-vitro study focused on the effect of the neurotransmitter SP on RANKL and OPG mrna expression in MG-63 osteoblast-like cells.

4 320 Max Daniel Kauther et al Material and Methods Peptide SP (Sigma-Aldrich, Cat. No. S6883) was used in this study. The peptide was dissolved in an aqueous solution of acetic acid (0.001%, v/v) and stored at -20 before use. During cell seeding different doses of SP (0.1, 1.0, 10 ug/ml) were added to the MG-63 cells, while pure medium was added in the control groups. Preparation of wear particles The commercially pure ultra-high molecular weight polyethylene (UHMWPE) particles (Ceridust VP 3610, Clariant, Gersthofen, Germany) with a mean particle size (given as equivalent circle diameter) of 1.74 ± 1.43 μm (range ) were used in this study. For endotoxin removal, the particles were treated for 24 hours with 99% ethanol at room temperature and were afterwards dried in a desiccator. The efficacy of the method was checked using Limulus Amebocyte Lysate (LAL) Assay (Charles River, Kent, United Kingdom) with a sensitivity of 0.25 EU/ml according to the manufacturer s directions. The test was found to be negative. Subsequently, particles were re-suspended in 10% endotoxin-free fetal calf serum (FCS), vortexed and treated in a sonicating water bath. Flow cytometry was used to measure the number of particles per unit volume of solution. MG-63 cells The human osteoblast-like MG-63 cell line (CRL-1427 TM, ATCC) was obtained from the American Type Culture Collection. The cell line was cultured in RPMI 1640 medium (PAA, Pasching, Austria), supplemented with 100 U of penicillin G /ml (Gibco, BRL, Eggenstein, Germany), 100 µg of streptomycin /ml (Gibco), 2 mm L-glutamine (Gibco) and 10 % fetal calf serum (PCS) at 37 C in a humidified atmosphere (5% CO2 and 95% air). For the experiment, MG-63 cells were seeded into 6-well flat bottomed culture plates at the quantity of approximately cells per well. After 24 hours, an 80% confluence of the cells was reached. The supernatant was removed and a fresh medium containing

5 Effects of substance P on RANKL and OPG mrna expression 321 UHMWPE particles was added. During this procedure, different quantities of particles were added to form two different cell-particle ratios (1:100 and 1:500). Isolation of RNA and quantitative Real Time RT-PCR analysis Total RNA was isolated using Qiashraddle (Qiagen, Hilden, Germany) and purified using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Both procedures were performed according to the manufacturer s specification. RNA was analyzed by quantitative real time polymerase chain reaction (RT-PCR) in a Rotorgene Cycler (Corbett Research, Mortlake, Australia) using the QuantiFast SYBR Green RT-PCR kit (Qiagen, Hilden, Germany) according to the manufacturer s instructions. A conventional PCR was performed to obtain a product of amplification suitable for the construction of standard curves with the real-time PCR procedures. The incorporation of Sybr Green into the PCR products was monitored in real time after each PCR cycle, resulting in the calculation of the threshold cycle or Ct value that defines the PCR cycle number at which an exponential growth of PCR products begins. PCR cycle conditions were as follows: 10 minutes at 50 C, 5 minutes at 95 C, 35 to 40 cycles of 10 seconds at 95 C and 30 seconds at 60 C. Each PCR procedure included a negative control reaction without a template. To exclude residual DNA contamination of the RNA samples, RT-PCR was also performed without reverse transcriptase. For mrna amplification, the validated primers were obtained from Qiagen (Qiagen, Hilden, Germany): ß-actin (Cat. No. QT ), RANKL (Cat. No. QT ) and OPG (Cat. No. QT ). The PCR products were sequenced and found to be identical to the published sequences. The ß-actin housekeeping gene was used as reference for the relative quantification of the gene of interest, which was expressed as the ratio of concentration of the target to concentration of ß-actin. Statistical analysis Results from representative experiments are shown. They were expressed as mean ± standard deviation. A repeated measurement ANOVA for all continuous dependent variables determined if there was (a) a time-by-group interaction effect, (b) a time effect and (c) inter-group effect. When F-values corresponding to a time-by-group interaction effect for a given variable were found to be significant, simple effects testing was performed to determine a time effect within each experimental group. Subsequently, one-way ANOVA tests were used to determine the detectable change between the groups at each time point. One-way ANOVA

6 322 Max Daniel Kauther et al tests, at each time point relative to the previous time point, determined if there were significant changes from each time-point. A p-value <0.05 was considered to indicate statistical significance. Results UHMWPE particles induced a dose-dependent increase of RANKL mrna in MG-63 cells A significant interaction between time and group for RANKL and OPG was found (F RANKL/ß-actin =25.44, p<0.05; F OPG/ß-actin =8.274, p<0.05). Simple effects testing verified an effect of time of releasing of RANKL on both particle groups with cell-particle ratios of 1:100 and 1:500 respectively. UHMWPE particles induced a significant increase of RANKL expression in concentrations of 1:100 and 1:500 after 72 hours compared to the untreated groups. SP down-regulated RANKL mrna expression in MG-63cells incubated with UHMWPE particles The depressive effect of SP on RANKL mrna expression was significant after 48 and 72 hours in both cell-particle concentrations (Figure 1, Figure 2). Simple effects testing verified that there was an effect of time on RANKL mrna expression inhibited by different concentration of SP (F=23.458, p<0.05). Considering particle concentration as an influencing factor, we compared the level of mrna-expression with the cell-particle concentration. No significant differences between cell-particle concentrations of 1:100 and 1:500 were found. Different doses of SP did not lead to a significant difference of RANKL mrna-expression. SP up-regulated RANKL mrna expression in MG-63cells incubated with UHMWPE particles In contrast to RANKL mrna expression, OPG mrna expression was stimulated after exposure to SP in cell-particle concentrations of 1:100 (Figure 3). After 48 hours, all SP-treated groups OPG mrna expression levels were

7 Effects of substance P on RANKL and OPG mrna expression 323 significantly higher compared to the untreated cells. After 72 hours incubation, the significant difference was only observed between the untreated group and 10 ug/ml-sp-incubated one (p<0.05). In cell-particle ratios of 1:500 significant up-regulation of OPG mrna-expression was only found after 72 hours (Figure 4). Using repeated measure ANOVA, a significant interaction between time and group was not confirmed for cells treated with different dose of SP (F=0.350, p=0.839). Discussion The role of UHMWPE wear debris in periprosthetic osteolysis, which ultimately results in total joint replacement s long-term failure, has been well established [7,22,25]. A linkage between neurotransmitters and particle-induced osteolysis has been found in several studies [12,26,27,29]. We believe that this is the first time that the effect of SP on MG-63 osteoblast like cells stimulated with UHMWPE particles has been examined in-vitro. UHMWPE particles caused a dose-dependent increase in RANKL mrna expression comparable to our further study of UHMWPE particle treated MG-63 cells [29]. These findings are complementary to a study by Itonaga et al. [11] that describes macrophage-osteoclast differentiation occurs in the presence of soluble RANKL, and that this process is inhibited by OPG. The OPG/RANKL/RANK system has been found to be one key regulator of osteoblastic regulation of osteoclastogenesis [11,14]. Recently, Ren et al. described the OPG/RANKL/RANK system to be essential for UHMWPE particle-induced bone resorption [20]. This study demonstrates an influence of UHMWPE particles and the neurotransmitter on the OPG/RANKL/RANK system that might be related to the process of particle induced osteolysis. We found that an increasing cell-particle ratio leads to an increase of RANKL and decrease of OPG mrna after 72 hours, corresponding to an increasing osteoclastic stimulation. In our study of MG-63 cells the neurotransmitter SP influenced the regulation of RANKL and OPG mrna expression. The depressive effect on RANKL mrna was found in all tested cell-particle concentrations. The up-regulation of OPG mrna after 72 hours further underlined the idea that SP

8 324 Max Daniel Kauther et al might have and osteoprotective influence on aseptic loosening [6]. These findings contradict the opinion of a catabolic influence of SP on bone metabolism [17]. In our previous study of SP knock-out mice we found less particle-induced osteolysis compared to the wild-type [28]. One explanation might be that this in-vitro study only illuminates the effect on osteoblastic cells. The influence of SP on osteoclasts might overrule the depressive effect on RANKL. Adamus MA et al. [1] found that SP (10-10 to 10-8 M) increased the incorporation of [ 3 H] thymidine and [ 3 H] praline, but slightly decreased alkaline phosphatase specific activity and calcium deposition in osteogenic cells derived from rat marrow and suggested that SP affects the proliferation and differentiation of osteoblastic cells. Furthermore, Shih C [24] found a SP dose-dependent increase in the number of osteoblasts, which was caused by stem cell mitosis. Thereby the total amount of RANKL mrna in the periprosthetic gap might even be increased. Beside for the osteoblasts, RANKL mrna expression of other cells of the joint cavity could be further be influenced by SP and induce the proliferation of osteoclasts. Increased expression of RANKL mrna in synovial fibroblastic cells after the addition of SP was demonstrated by Matayoshi [18]. His results suggested that SP may induct an increased osteoclast formation through increased RANKL mrna expression in synovial fibroblastic cells. Another mechanism of SP on bone remodeling could partly be mediated through its action on blood vessels, thereby regulating local blood flow [5,16]. Conclusion All in all, this study shows that the neurotransmitter SP might have an influence on aseptic prosthetic loosening via the OPG/RANKL/RANK system. Further animal models of particle-induced osteolysis with SP stimulation would further help to understand the influence of SP in osteoblast-osteoclast interactions. Acknowledgement The authors thank Kaye Schreyer for editorial assistance with the manuscript. The study was supported by Deutsche Forschungsgemeinschaft grand WE 3634/1-1 and WE 3634/1-2 (Christian Wedemeyer).

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12 328 Max Daniel Kauther et al [29] J. Xu, M.D. Kauther, J. Hartl, C. Wedemeyer, Effects of alpha-calcitonin gene-related peptide on osteoprotegerin and receptor activator of nuclear factor-κb ligand expression in MG-63 osteoblast-like cells exposed to polyethylene particles, J Orthop Surg Res 5 (2010), 83. Figures: Figure 1: Time courses of RANKL mrna levels in MG-63 cells after UHMWPE particle and SP treatment in cell-particle concentrations of 1:100. Significant differences are marked. (*P<0.05)

13 Effects of substance P on RANKL and OPG mrna expression 329 Figure 2: Time courses of RANKL mrna levels in MG-63 cells after UHMWPE particle and SP treatment in cell-particle concentrations of 1:100. Significant differences are marked. (*P<0.05)

14 330 Max Daniel Kauther et al Figure 3: Time courses of OPG mrna levels in MG-63 cells after UHMWPE particle and SP treatment in cell-particle concentrations of 1:100. Significant differences are marked. (*P<0.05)

15 Effects of substance P on RANKL and OPG mrna expression 331 Figure 4: Time courses of OPG mrna levels in MG-63 cells after UHMWPE particle and SP treatment in cell-particle concentrations of 1:500. Significant differences are marked. (*P<0.05) Received: April, 2012