Rheumatology 2004;43:1226–1231
Advance Access publication 6 July 2004
doi:10.1093/rheumatology/keh296
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
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-1b 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 RT–PCR 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-1b 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-1b-mediated signalling pathway.
KEY WORDS: Methotrexate, Integrin, Interleukin-1, Mechanical stimulation, Mechanotransduction, Gene expression.
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
(100–12 000 mg/m2) in the treatment of malignancy but in much
lower doses (5–25 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 [2–4]. Fractures, resulting from minimal
trauma, and pain, are increased in patients receiving long-term,
low-dose MTX treatment for RA [5–7].
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 [8–13]. 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 [15–19], 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.
Materials and methods
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,
Division of Pathology, University of Edinburgh Medical School, 1Department of Orthopaedics and 2Department of Oncology, Royal Hospital for Sick
Children, Edinburgh, UK.
Submitted 9 March 2004; revised version accepted 4 June 2004.
Correspondence to: D. M. Salter, Division of Pathology, Edinburgh University Medical School, Teviot Place, Edinburgh EH8 9AG, UK. E-mail:
[email protected]
1226
Rheumatology Vol. 43 No. 10 ß British Society for Rheumatology 2004; all rights reserved
Effects of MTX on HBC mechanotransduction
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 [20–23]. 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 strain on the base of the dish.
Electrophysiological recording
Membrane potentials of HBC were recorded using a single bridge
electrode and calibrator as previously described [20–23]. 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.
1227
buffer of 4 M guanidine thiocyanate, 0.75 M sodium citrate, 10%
(wt/vol) lauryl sarcosine and 7.2 l/ml -mercaptoethanol. The
quantity of RNA isolated was determined by spectrophotometry
using the absorbance reading at 260 nm.
Reverse transcriptase–PCR (RT–PCR)
Template cDNA was synthesized using 0.5–1.5 g 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,
50 -AAGATGGACTCAACGGTCTC-30 , 50 -AACCAGACATGC
CTCTTGTC-30 ; BMP-4, 50 -CAGCGGTCCAGGAAGAAGAA
TAAG-30 and 50 -TCTGCACAATGGCATGGTTG-30 .
A typical 20-l PCR reaction contained 16 mM ammonium
sulphate, 67 mM Tris–HCl, pH 8.8, 0.01 (vol/vol) Tween, 1 M
of each primer, 2 l cDNA, 100 M 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
Mann–Whitney U test, as appropriate, depending on whether the
F ratio of the variances of the two means reached significance.
Experimental protocol
Results
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).
Effect of MTX on HBC responses to 0.33 Hz mechanical
stimulation
RNA extraction
Total RNA was extracted from cultured bone cells as described for
the micro-RNA extraction kit (Stratagene) using a denaturing
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 M to 1 nM (Table 1). At
both 1 M 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 ¼ 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).
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).
K. J. Elliot et al.
1228
TABLE 1. Effect of methotrexate on human bone cell responses to mechanical stimulation
Membrane potential (mV): mean S.E.M.
Reagent
n
Resting cells
þMTX
After 0.33 Hz MS
% change
(resting – after MS)
Nil
MTX
1 M
50 nM
1 nM
5
32.6 1.29
n/a
54.4 2.87
þ67%*
5
5
5
46.2 1.96
41.4 2.48
38.0 3.17
45.0 3.99
43.2 5.26
37.0 2.30
42.0 3.89
39.6 2.69
50.4 4.94
–9% ns
–4% ns
þ33%*
Results shown are from a single experiment and are consistent between experiments and between cells from four donors.
MS, mechanical stimulation; ns, not significant.
*P < 0.05.
mRNA ratio (coll 1/GAPDH)
1.6
1.4
1.2
1
No MTX
0.8
MTX
0.6
0.4
0.2
0
NS
0
1
3
6
24
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 (Mann–Whitney U test). This trend is unaffected by the addition of 50 nM
MTX. Error bars are þ1 S.E.M. Pooled data for three donors, tests performed in duplicate (n ¼ 6).
mRNA ratio (BMP-4/GAPDH)
2.5
2.0
1.5
MTX
No MTX
1.0
0.5
0.0
NS
0
1
3
6
24
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, Mann–Whitney U test). The trend is unaffected by the addition of 50 nM MTX. Error bars are þ1 S.E.M. Pooled
data for three donors, tests performed in duplicate (n ¼ 6).
Effects of MTX on HBC mechanotransduction
1229
TABLE 2. Effect of conditioned medium (CM) from HBC mechanically stimulated for 20 min on the membrane potential of previously unstimulated HBC
Membrane potential (mV): mean 1 S.E.M.
Dish 1
Dish 2
Reagent
n
Resting
þMTX
After MS
(% change)
Resting
þMTX
With CM
(% change)
Nil (control)
MTX (50 nM)
(added to dish 1)
MTX (50 nM)
(added to dish 2)
5
5
27.6 1.7
27.6 1.8
n/a
29.4 1.6
43.0 3.3 (þ56%*)
26.2 2.2 (–5%, ns)
24.8 1.0
35.8 2.5
n/a
n/a
37.8 1.1 (þ52%**)
37.2 2.9 (þ4%, ns)
5
39.2 2.2
n/a
58.4 2.1 (þ49%*)
35.0 1.8
38.0 2.7
56.6 2.8 (þ49%***)
Results shown are from a single experiment and are consistent between experiments and between cells from three donors.
MS, mechanical stimulation; ns, not significant.
*P < 0.001; **P < 0.0005; ***P < 0.0005.
TABLE 3. Effect of MTX on the membrane hyperpolarization response of HBC treated with 10 pg/ml IL-1
Membrane potential (mV): mean S.E.M.
Stimulus
IL-1
IL-1
Reagent
n
Resting
þ MTX
IL-1
% change
(resting – after-IL-1)
Nil
50 nM MTX
5
5
29.4 1.3
31.2 2.0
n/a
29.4 2.8
51.4 2.4
51.4 2.8
74.8*
74.8**
Results shown are from a single experiment and are consistent between experiments and between cells from three donors.
*P < 0.0005; **P < 0.001.
Effects of MTX on IL1--dependent membrane
hyperpolarization
The membrane hyperpolarization response of HBC to 0.33 Hz
mechanical stimulation is the result of autocrine/paracrine IL-1
signalling following activation of 51 integrin. We have previously
demonstrated that neutralizing antibodies to IL-1 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 51 integrin function or IL-1 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-1, 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-1 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-1 (Table 3).
Discussion
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 apaminsensitive small conductance calcium-activated potassium (SK)
channels, follows activation of a signal cascade involving 51
integrin and initiation of an IL-1 autocrine/paracrine loop
[20, 21]. Attempts to quantify IL-1 concentrations using commercially available ELISA (enzyme-linked immunosorbent assay) kits
have proved unsuccessful. Possible reasons for this include (i) that
the concentration of IL-1 released is below the level of detection
from the assay, and (ii) that IL-1 release is a tightly regulated
process and that, upon its release, IL-1 is immediately taken up by
other HBC.
The results presented in this paper suggest that MTX modulates
the 51 integrin-mediated signal cascade by blocking the IL-1
release induced by mechanical stimulation rather than by interfering with downstream IL-1 responses, as shown by the inability
of MTX to influence the membrane hyperpolarization activity of
either exogenously added IL-1 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-1, with
evidence to support both effects on the production and the
subsequent secretion of IL-1. It has been shown that MTX
reduces IL-1 production in the early phase of antigen-induced
arthritis in rabbits, as assessed by intra-articular concentrations of
IL-1 [24], and is a potent inhibitor of lipopolysaccharide-induced
IL-1 release from rat peritoneal macrophages [25].
There is also, however, a substantial amount of evidence which
suggests that IL-1 synthesis is unaffected by MTX, but that
binding of IL-1 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
K. J. Elliot et al.
1230
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-1 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 stimulationinduced 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 [28–30]. 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-1
function. In the present study, however, it is not possible to
ascertain whether these gene expression responses are mediated
via 51 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 IL1, 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 61 integrin
expression by synovial fibroblast-like cells [32]. The hyperpolarization response of HBC following 0.33 Hz mechanical stimulation is
51 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 51
integrin-dependent pathway that results in IL-1 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 cell–cell
communication, in addition to potential effects on precursor cell
proliferation and differentiation.
Rheumatology
Key messages
MTX dose-dependently inhibits electrophysiological responses of human bone
cells by interfering with IL-1 synthesis/
release.
MTX does not affect mechanically
induced alterations in relative levels of
BMP-4 and type-1 collagen.
The authors have declared no conflicts of interest.
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