Effects of methotrexate on human bone cell responses to mechanical stimulation
K. J. Elliot,
S. J. Millward-Sadler,
M. O. Wright,
J. E. Robb1,
W. H. B. Wallace2 and
D. M. Salter
Division of Pathology, University of Edinburgh Medical School, 1 Department of Orthopaedics and 2 Department of Oncology, Royal Hospital for Sick Children, Edinburgh, UK.
Correspondence to: D. M. Salter, Division of Pathology, Edinburgh University Medical School, Teviot Place, Edinburgh EH8 9AG, UK. E-mail: Donald.Salter{at}ed.ac.uk
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Abstract
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Objectives. Methotrexate (MTX), which is prescribed in the treatment of malignancy and autoimmune disease, has detrimental effects on a number of organ systems, including bone. At present, the exact mechanism of action of MTX on bone at the cellular level is unclear. Mechanical stimuli imparted by stretch, pressure, fluid flow and shear stress result in a variety of biochemical responses that are important in bone metabolism. Cyclical mechanical stimulation at 0.33 Hz induces rapid cell membrane hyperpolarization of human bone cells (HBC) via an integrin-mediated pathway which includes an IL-1ß autocrine/paracrine loop. This study was undertaken to investigate the effect of MTX on responses of HBC to 0.33 Hz mechanical stimulation.
Methods. Electrophysiological responses of HBC were measured before and after mechanical stimulation at 0.33 Hz in the presence or absence of MTX. Semiquantitative RTPCR was used to investigate effects of MTX on relative levels of type-1 collagen and bone morphogenetic protein-4 (BMP-4) following 0.33 Hz mechanical stimulation.
Results. MTX dose-dependently inhibited HBC hyperpolarization in response to 0.33 Hz mechanical stimulation. Production/release of IL-1ß was inhibited by MTX, whereas its effects on target cells were not. Mechanical stimulation of HBC at 0.33 Hz caused a significant decrease in relative levels of BMP-4 mRNA, whereas relative levels of type-1 collagen mRNA were consistently increased, although these increases did not reach statistical significance. These trends were unaffected by MTX.
Conclusions. These studies show that MTX affects HBC mechanotransduction by interfering with integrin-mediated signalling. The data also suggest that the mechanotransduction pathway responsible for the regulation of type-1 collagen and BMP-4 gene expression may be distinct from the IL-1ß-mediated signalling pathway.
KEY WORDS: Methotrexate, Integrin, Interleukin-1ß, Mechanical stimulation, Mechanotransduction, Gene expression
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Introduction
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Methotrexate (MTX) is prescribed widely, both in the treatment of cancers and in the treatment of inflammatory diseases such as rheumatoid arthritis (RA) [1]. It is used at high doses (10012 000 mg/m2) in the treatment of malignancy but in much lower doses (525 mg/week) in the treatment of RA. MTX has a number of side-effects in a variety of organ systems, including bone. High-dose MTX has detrimental effects on bone formation, and children who receive MTX therapy in the treatment of childhood malignancies often show growth retardation throughout the course of their treatment [24]. Fractures, resulting from minimal trauma, and pain, are increased in patients receiving long-term, low-dose MTX treatment for RA [57].
The mechanism of action of MTX on bone at the cellular level is not clear but may involve interaction with the function of a number of types of bone cells and their precursors. Several studies have reported that MTX has effects on osteoblasts in vitro and in vivo, including adverse influences on recruitment and differentiation of mesenchymal precursors, and inhibition of osteoblast function, including bone formation [813]. The routes by which MTX exerts these effects have yet to be defined and, although inhibition of folate metabolism is likely to be important in the prevention of proliferation-associated events, it is possible that MTX may influence the ability of osteoblasts to respond to environmental cues such as growth factors [14], cytokines and mechanical stimulation [1519], which are important in maintaining bone homeostasis.
The present study was performed to investigate whether MTX affects responses of human trabecular bone cells (HBC) to mechanical stimulation in vitro. The results suggest that MTX modulates at least some of the responses of HBC to mechanical stimulation.
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Materials and methods
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Human bone cell (HBC) culture
HBC cultures were established from trabecular bone explants as previously described [20, 21]. Bone was collected at surgery, with ethical approval and informed consent, from children undergoing corrective osteotomy, or from adults undergoing above- or below-knee amputation as a result of diseases unrelated to the osteoarticular system. The trabecular bone was cut into small pieces, washed in phosphate-buffered saline (pH 7.4) and cultured in minimal essential medium (MEM; Sigma, Poole, UK) containing 10% fetal calf serum (First Link), 2 mM glutamine and antibiotics (0.1 mg/ml streptomycin, 100 U/ml penicillin) in a humidified 5% CO2 atmosphere at 37°C. Initially, cultures were grown in tissue culture grade Petri dishes (Nunc, Paisley, UK) and, once established, were passaged into tissue culture flasks. The cells in the cultures show variable morphology, with both spindle cells and polygonal cells present. Cell populations grown from the explants showed osteoblast-like characteristics (type-1 collagen production, osteocalcin and osteopontin synthesis and alkaline phosphatase activity which was increased by 1,25-dihydroxycholecalciferol). Cells were not used beyond passage 5.
Method for inducing cyclical mechanical strain
The technique employed for inducing strain in the bone cells grown in monolayer was that described previously [2023]. Briefly, plastic 58 mm tissue culture grade dishes (Nunc) were placed in a sealed chamber containing inlet and outlet ports. The chamber was pressurized from below using helium gas from a cylinder, at a frequency determined by an electronic timer controlling the inlet and outlet valves. The standard stimulation regimen used was a frequency of 0.33 Hz (2 s on/1 s off) at a pressure of 30 mmHg for 20 min at 37°C. Cyclical pressurization of this system induces deformation of the base of the plastic culture dish and consequently on the attached cells. In the current series of experiments, a pressure of 30 mmHg (0.025 atmospheres) was used, which results in approximately 4000 µstrain on the base of the dish.
Electrophysiological recording
Membrane potentials of HBC were recorded using a single bridge electrode and calibrator as previously described [2023]. Microelectrodes having tip resistance between 30 and 60 M
and tip potential of approximately 3 mV were used to impale the cells. Cells selected for impalement showed polygonal morphology. Results were accepted if, upon impalement, there was a rapid change in voltage, which remained constant for at least 20 s.
Experimental protocol
Experiments were performed using subconfluent cultures of HBC. In each set of experiments, resting membrane potentials of five cells were measured in serum-free medium, and after addition of MTX for 30 min at 37°C, the membrane potential of a further five cells was assessed before mechanical stimulation. Finally, after the 20-min period of mechanical stimulation or the addition of recombinant cytokine for defined times, membrane potentials of a further five cells were recorded. Reagents being studied were in contact with cells during mechanical stimulation or stimulation with recombinant cytokines and also when post-stimulation membrane potentials were recorded. Different dishes were used for each reagent tested.
For the conditioned media (CM) transfer experiments, a dish of HBC (with or without 50 nM MTX) was stimulated at 0.33 Hz. Following mechanical stimulation, the membrane potentials of five cells were recorded and the medium was then transferred immediately to a dish of unstimulated HBC (with or without 50 nM MTX), membrane potential of the cells being assessed 20 min after transfer of the medium.
Each experiment was performed a minimum of three times, on different days, using cells from at least three donors. Assays for lactate dehydrogenase activity and acridine orange staining to assess apoptosis were undertaken, and showed no effect of MTX on cell viability over the time course of the experiments (results not shown).
RNA extraction
Total RNA was extracted from cultured bone cells as described for the micro-RNA extraction kit (Stratagene) using a denaturing buffer of 4 M guanidine thiocyanate, 0.75 M sodium citrate, 10% (wt/vol) lauryl sarcosine and 7.2 µl/ml ß-mercaptoethanol. The quantity of RNA isolated was determined by spectrophotometry using the absorbance reading at 260 nm.
Reverse transcriptasePCR (RTPCR)
Template cDNA was synthesized using 0.51.5 µg RNA, Superscript II and oligo dT (Life Technologies) according to the manufacturer's instructions. Primers specific for type-1 collagen (Coll 1) and BMP-4 were used for the PCR reactions: Coll 1, 5'-AAGATGGACTCAACGGTCTC-3', 5'-AACCAGACATGCCTCTTGTC-3'; BMP-4, 5'-CAGCGGTCCAGGAAGAAGAATAAG-3' and 5'-TCTGCACAATGGCATGGTTG-3'.
A typical 20-µl PCR reaction contained 16 mM ammonium sulphate, 67 mM TrisHCl, pH 8.8, 0.01 (vol/vol) Tween, 1 µM of each primer, 2 µl cDNA, 100 µM dNTPs, 0.01% (wt/vol) bovine serum albumin, 2.5 mM magnesium chloride and 0.25 U Taq polymerase (Biogene). The following programmes were used: type-1 collagen, 95°C for 3 min, 26 cycles of 95°C for 1 min, 60°C for 1 min, 72°C for 1 min 30 s; 72°C for 10 min; BMP-4, 95°C for 3 min, 26 cycles of 95°C for 35 s, 57°C for 35 s, 72°C for 1 min, 72°C for 10 min. PCR products were analysed by electrophoresis using a 1% (wt/vol) agarose gel and visualized by ethidium bromide staining. Semiquantitative analysis of PCR products was performed using the Enhanced Analysis System (EASY; Scotlab, Coatbridge, UK).
Statistics
Differences between means were tested for statistical significance using either a pooled-variance t-test or the non-parametric MannWhitney U test, as appropriate, depending on whether the F ratio of the variances of the two means reached significance.
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Results
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Effect of MTX on HBC responses to 0.33 Hz mechanical stimulation
Membrane potential
Following 20 min mechanical stimulation at 0.33 Hz, HBC showed a reproducible membrane hyperpolarization consistent with previous studies (Table 1). MTX dose-dependently inhibited HBC hyperpolarization in response to mechanical strain over a range of 1 µM to 1 nM (Table 1). At both 1 µM and 50 nM, MTX completely inhibited the hyperpolarization response. A significant hyperpolarization response was seen when cells were incubated with 1 nM but the response was less than that of cells stimulated in the absence of MTX.
Type-1 collagen mRNA expression
Mechanical stimulation of HBC for 20 min at 0.33 Hz resulted in a time-dependent increase in relative levels of type-1 collagen mRNA and appeared to reach a maximum 6 h after stimulation. Although this increase was consistent between experiments, the results did not quite reach statistical significance (P = 0.095). MTX at 50 nM, a concentration which abolished the electrophysiological response, had no effect on the mechanical stimulation induced elevation of type-1 collagen mRNA (Fig. 1).

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FIG 1. Effects of MTX on type-1 collagen mRNA expression following mechanical stimulation. Increased type-1 collagen levels are observed in cells subjected to RNA extraction 1, 3, 6 and 24 h after mechanical stimulation, when compared with non-stimulated (NS) cells, but these increases do not reach statistical significance (MannWhitney U test). This trend is unaffected by the addition of 50 nM MTX. Error bars are +1 S.E.M. Pooled data for three donors, tests performed in duplicate (n = 6).
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BMP-4 mRNA expression
Following 20 min of mechanical stimulation at 0.33 Hz, HBC showed a decrease in relative levels of BMP-4 mRNA, when compared with non-stimulated cells. A significant reduction was seen by 3 h after stimulation (P < 0.01) and maintained 24 h after stimulation (P < 0.01). A similar pattern was seen when HBC were mechanically stimulated in the presence of 50 nM MTX (Fig. 2).

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FIG 2. Effects of MTX on BMP-4 RNA expression following mechanical stimulation. There are statistically significant decreases in BMP-4 levels in cells subjected to RNA extraction 3, 6 and 24 h after mechanical stimulation, when compared with non-stimulated (NS) cells (P < 0.01, MannWhitney U test). The trend is unaffected by the addition of 50 nM MTX. Error bars are +1 S.E.M. Pooled data for three donors, tests performed in duplicate (n = 6).
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Effects of MTX on IL1-ß-dependent membrane hyperpolarization
The membrane hyperpolarization response of HBC to 0.33 Hz mechanical stimulation is the result of autocrine/paracrine IL-1ß signalling following activation of
5ß1 integrin. We have previously demonstrated that neutralizing antibodies to IL-1ß abolish HBC hyperpolarization following cyclical mechanical stimulation (results not shown) [21]. A series of experiments were undertaken to ascertain whether the effects of MTX were due to interference with either
5ß1 integrin function or IL-1ß signalling in this transduction pathway. CM taken from cells which had been mechanically stimulated for 20 min at 0.33 Hz, when added to unstimulated bone cells for 20 min, induced membrane hyperpolarization of these cells (Table 2). In contrast, medium from HBC that had been mechanically stimulated in the presence of 50 nM MTX did not cause membrane hyperpolarization of unstimulated cells (Table 2). This suggests either that the production of the transferable active agent, IL-1ß, by mechanical stimulation is inhibited or the ability of this agent to induce membrane hyperpolarization is affected. To address this, media from cells that had been mechanically stimulated for 20 min at 0.33 Hz was transferred to unstimulated cells that had been preincubated for 30 min with 50 nM MTX. Transfer of media to these cells resulted in cell membrane hyperpolarization of these cells (Table 2). Addition of recombinant IL-1ß to resting cells for 10 min caused hyperpolarization of these cells. This hyperpolarization response was not affected by incubation with 50 nM MTX for 30 min before addition of IL-1ß (Table 3).
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TABLE 2. Effect of conditioned medium (CM) from HBC mechanically stimulated for 20 min on the membrane potential of previously unstimulated HBC
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Discussion
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In the present study we show that MTX inhibits the membrane hyperpolarization response of HBC subjected to 20 min of mechanical stimulation at 0.33 Hz, but has no effect on mechanically induced changes in type-1 collagen or BMP-4 gene expression. The hyperpolarization response, a result of activation of apamin-sensitive small conductance calcium-activated potassium (SK) channels, follows activation of a signal cascade involving
5ß1 integrin and initiation of an IL-1ß autocrine/paracrine loop [20, 21]. Attempts to quantify IL-1ß concentrations using commercially available ELISA (enzyme-linked immunosorbent assay) kits have proved unsuccessful. Possible reasons for this include (i) that the concentration of IL-1ß released is below the level of detection from the assay, and (ii) that IL-1ß release is a tightly regulated process and that, upon its release, IL-1ß is immediately taken up by other HBC.
The results presented in this paper suggest that MTX modulates the
5ß1 integrin-mediated signal cascade by blocking the IL-1ß release induced by mechanical stimulation rather than by interfering with downstream IL-1ß responses, as shown by the inability of MTX to influence the membrane hyperpolarization activity of either exogenously added IL-1ß or that released by HBC as a result of mechanical stimulation.
There is growing evidence that the immunosuppressive activity of MTX is, at least in part, a result of modulation of IL-1ß, with evidence to support both effects on the production and the subsequent secretion of IL-1ß. It has been shown that MTX reduces IL-1ß production in the early phase of antigen-induced arthritis in rabbits, as assessed by intra-articular concentrations of IL-1ß [24], and is a potent inhibitor of lipopolysaccharide-induced IL-1ß release from rat peritoneal macrophages [25].
There is also, however, a substantial amount of evidence which suggests that IL-1ß synthesis is unaffected by MTX, but that binding of IL-1ß to its receptor and subsequent signalling is inhibited. It has been shown that MTX has no effect on the production of IL-1 in murine splenic macrophages or human peripheral blood mononuclear cells [26], but that the activity of IL-1, as assessed by two assays that measure different activities of IL-1, is inhibited by MTX. This work did not address whether the effect of MTX on IL-1 activity was direct or indirect, although suggestions were made that there may be a physical or chemical interaction between MTX and IL-1, that MTX may down-regulate IL-1 receptors, or that there may be post-receptor inhibition of intracellular mechanisms requiring IL-1. A separate study has demonstrated that MTX blocks the binding of IL-1ß to the IL-1 receptor on human lymphocytes, monocytes and granulocytes, thus inhibiting the cytokine cascade [27]. This study also demonstrated that the integrity of the IL-1 receptor is unaffected.
In addition to the effect of MTX on the mechanical stimulation-induced membrane hyperpolarization response, we also studied its effect on mechanical stimulation-induced gene expression. Effects of mechanical stimulation on gene expression vary depending on the type of bone cells being studied and the frequency and magnitude of the mechanical stimulus [2830]. In our system, mechanical stimulation of HBC resulted in a decrease in the relative levels of BMP-4 mRNA, whereas relative levels of type-1 collagen mRNA were consistently increased, although these increases failed to reach statistical significance. These changes suggest that the mechanical stimulus that is being provided in the current model induces a differentiation/anabolic response, driving the cells towards a more mature active bone cell phenotype. Although MTX had no effect on the mechanical response, relative levels of type-1 collagen were consistently decreased in the presence of MTX, raising the possibility of effects on gene expression through different routes. These results may also indicate that the mechanotransduction pathway by which type-1 collagen and BMP-4 gene expression is regulated is not reliant on IL-1ß function. In the present study, however, it is not possible to ascertain whether these gene expression responses are mediated via
5ß1 integrin or depend on other mechanically responsive membrane components, such as stretch-activated ion channels.
We have not investigated in detail the mechanisms by which MTX may modulate mechanical stimulation-induced release of IL-1ß, but a number of possibilities exist. MTX has been shown to interfere with integrin expression and integrin-dependent adhesion of leucocytes [31], and additionally down-regulates
6ß1 integrin expression by synovial fibroblast-like cells [32]. The hyperpolarization response of HBC following 0.33 Hz mechanical stimulation is
5ß1 integrin-dependent [20], and the effects of MTX on expression of this integrin or adhesion to its extracellular matrix ligand would be likely to interfere with bone cell mechanotransduction.
In conclusion, the present study suggests that MTX has effects on human bone cell mechanotransduction by disrupting the
5ß1 integrin-dependent pathway that results in IL-1ß secretion and membrane hyperpolarization. MTX has no effect on mechanical stimulation-induced changes in type-1 collagen or BMP-4 mRNA expression. The effects of MTX on bone metabolism are likely to be complex and involve both the regulation of mechanical responses of cells directly and through the modulation of cellcell communication, in addition to potential effects on precursor cell proliferation and differentiation.
The authors have declared no conflicts of interest.
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Submitted 9 March 2004;
revised version accepted 4 June 2004.