* Department of Pathology and Department of Physiology, University of Edinburgh Medical School, Edinburgh, United
Kingdom EH8 9AG; § National Defense Medical Center and Tri-Service General Hospital, Taiwan 100; and
Rheumatic Diseases
Unit, Western General Hospital, Edinburgh, United Kingdom EH4 2XU
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Abstract |
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Chondrocyte function is regulated partly by
mechanical stimulation. Optimal mechanical stimulation maintains articular cartilage integrity, whereas abnormal mechanical stimulation results in development
and progression of osteoarthritis (OA). The responses of signal transduction pathways in human articular
chondrocytes (HAC) to mechanical stimuli remain unclear. Previous work has shown the involvement of integrins and integrin-associated signaling pathways in
activation of plasma membrane apamin-sensitive
Ca2+-activated K+ channels that results in membrane
hyperpolarization of HAC after 0.33 Hz cyclical mechanical stimulation. To further investigate mechanotransduction pathways in HAC and show that the hyperpolarization response to mechanical stimulation is a
result of an integrin-dependent release of a transferable
secreted factor, we used this response. Neutralizing
antibodies to interleukin 4 (IL-4) and IL-4 receptor inhibit mechanically induced membrane hyperpolarization and anti-IL-4 antibodies neutralize the hyperpolarizing activity of medium from mechanically stimulated cells. Antibodies to interleukin 1
(IL-1
) and
cytokine receptors, interleukin 1 receptor type I and the
common
chain/CD132 (
) have no effect on me-
chanically induced membrane hyperpolarization.
Chondrocytes from IL-4 knockout mice fail to show a
membrane hyperpolarization response to cyclical mechanical stimulation. Mechanically induced release of
the chondroprotective cytokine IL-4 from HAC with
subsequent autocrine/paracrine activity is likely to be
an important regulatory pathway in the maintenance of
articular cartilage structure and function. Finally, dysfunction of this pathway may be implicated in OA.
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Introduction |
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ARTICULAR cartilage covers the ends of long bones
within synovial joints and protects the underlying
bone against shearing and compressive forces.
Cartilage is composed of a proteoglycan and collagen-rich
extracellular matrix containing chondrocytes. Collagen
forms a meshwork that imparts tensile strength, and proteoglycans form large aggregates that provide resistance to
compression (Stockwell, 1991). The maintenance of cartilage matrix integrity is critically dependent on mechanical
stimulation and cartilage thickness reflects the total load
transmitted by the joint. Experiments on whole animals
with intact joints have shown that abnormal loading,
whether increased or decreased, influences cell metabolism and results in cellular and biochemical changes that
lead to cartilage breakdown and the development of osteoarthritis (OA1; Mow et al., 1992
). In vitro experiments
with chondrocytes in culture have demonstrated a variety
of physiological and biochemical responses to cyclical mechanical stimuli. These include changes in membrane potential, intracellular calcium concentration, and cAMP
levels, and inhibition or stimulation of glycosaminoglycan production (Veldhuijzen et al., 1979
; Urban, 1994
).
Cyclical and static mechanical stimulation are well recognized as having a variety of effects on a number of different cell types, including bone cells, chondrocytes, vascular
endothelium, and smooth muscle, from tissues normally
exposed to mechanical forces. Mechanical signals imparted by stretch, pressure, tension, fluid flow, or shear
stress rapidly lead to the activation of multiple intracellular signaling molecules and pathways, including opening of
stretch activated and calcium selective ion channels (Sachs, 1988), protein tyrosine phosphorylation (Yano et
al., 1996
), inositol lipid metabolism (Prasad et al., 1993
),
and activation of protein kinase C (PKC; Kimono et al.,
1996
). Activation of these and other signaling pathways in
turn leads to changes in gene expression and protein synthesis of important regulatory mechanisms controlling tissue structure and function, e.g., PDGF production by smooth
muscle cells (Wilson et al., 1993
); nitric oxide and prostaglandin production by endothelial cells (Davies, 1995
);
proteoglycan synthesis by chondrocytes (Veldhuijzen et al., 1979
); and bone matrix synthesis by bone cells (Harter et al., 1995
).
The routes by which a particular mechanical signal is
transduced into an intracellular response are being defined and evidence for a role for integrins is increasing
(Wang et al., 1993; Shyy and Chien, 1997
). Integrins are a
family of heterodimeric (
and
chain) transmembrane
glycoproteins that form specific receptors for extracellular
matrix (ECM) proteins (Hynes, 1992
). Many of the signal
transduction and gene expression events activated by mechanical stimuli are identical to those induced by integrin-mediated cell adhesion (Hynes, 1992
; Shyy and Chien,
1997
). Integrins associate with signaling molecules in the
focal adhesion complex that acts both as a signaling device
and a connection to the cytoskeleton through which they
can influence gene expression and control cell growth and
function. Experimental work has provided further evidence that integrins may act as mechanoreceptors in a variety of cell types. Integrins support a force-dependent
stiffening response in endothelial cells (Wang et al., 1993
)
and are involved in shear stress-dependent vasodilatation
of coronary arteries (Muller et al., 1997
) and transmitter
release from motor nerve terminals (Chen and Grinell,
1995). Also integrins were shown to be necessary for mechanically induced activation of ERK-2 and JNK-1 intracellular signaling pathways in cardiac fibroblasts (MacKenna et al., 1998
) and the membrane hyperpolarization
and depolarization responses of human articular chondrocytes (HAC) and bone cells to cyclical mechanical strain
(Salter et al., 1997
; Wright et al., 1997
).
We have developed a technique for applying controlled
forces to cultured cells allowing direct demonstration that
mechanical signals can be transmitted across ECM-cell
contacts (Wright et al., 1992, 1996
, 1997
). Using this technique we have stimulated mechanically sensitive cells including fibroblasts, human bone cells, and chondrocytes.
As a result, several electrophysiological, biochemical, and
molecular responses were affected, including changes in
cell membrane potential, protein-tyrosine phosphorylation (paxillin and FAK125), and c-fos activation, and (in
the case of chondrocytes) increased production of aggrecan mRNA and proteoglycan synthesis (Wright et al.,
1992
, 1996
, 1997
; Salter et al., 1997
). We have used the mechanically induced changes in membrane potential to dissect in detail molecules involved and pathways activated as a result of cyclical mechanical stimulation. The electrophysiological response to mechanical stimulation occurs
within 20 min, is dependent on the frequency of mechanical stimulation, and is also cell-type specific (Salter et al.,
1997
; Wright et al., 1997
). Stimulation at 0.33 Hz (2 s on/1 s
off) for 20 min at 37°C causes both human chondrocytes
and bone cells to undergo membrane hyperpolarization because small conductance Ca2+-dependent K+ channels
(SK) open. In contrast, stimulation at 0.104 Hz (2 s on/7.6 s
off) for 20 min with the same degree of microstrain results in membrane depolarization because the tetrodotoxin-sensitive Na+ channels are activated. Fibroblasts, on the
other hand, undergo membrane depolarization at 0.33 Hz
and hyperpolarization at 0.1 Hz (Wright et al., 1992
).
In this model system, signaling via integrins and integrin-associated signaling molecules (including actin cytoskeleton and tyrosine protein kinases) is necessary for
both the hyperpolarization and depolarization responses
to mechanical stimulation (Wright et al., 1996, 1997
; Salter
et al., 1997
). However, the 0.33-Hz hyperpolarization response is inhibited by antibodies to
5 integrin and
1 integrins, whereas the 0.104-Hz depolarization response is
inhibited by antibodies to
V
5 and not by anti-
5 integrin antibodies. This suggests specific roles of particular
integrins in the transduction of different forms of mechanical stimulation to cells (Salter et al., 1997
). Furthermore,
stretch sensitive ion channels, phospholipase C (PLC), the
inositol triphosphate (IP3) Ca2+-calmodulin pathway, and
PKC appear to be involved in the production of the hyperpolarization response only.
Studies in osteoblasts and endothelial cells have demonstrated the production of soluble factors, such as prostaglandins and nitric oxide, in response to mechanical stimulation (Somjen et al., 1980; Ayajiki et al., 1996
). The
purpose of the study was to investigate whether soluble
mediators, in particular cytokines including interleukin 1
(IL-1
), IL-4, and transforming growth factor
1 (TGF-
1)
that are recognized as having important roles in regulation
of chondrocyte function via autocrine and paracrine signaling, were involved in the membrane hyperpolarization
response of HAC to mechanical stimulation.
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Materials and Methods |
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Isolation of Chondrocytes
Postmortem articular cartilage was aseptically removed from macroscopically normal femoral condyles and tibial plateaux of human knee joints.
Donors had died from a variety of diseases unrelated to the locomotor
system and were undergoing routine hospital autopsy. Cartilage was sampled from 8 males (mean age, 68 yr; range 58-83 yr) and 17 females (mean
age, 76 yr; range 37-93 yr). Cartilage from different anatomical regions of
the knee joint were pooled and chondrocytes were isolated by sequential enzyme digestion at 37°C in 95% air/5% CO2 with 0.25% trypsin (GIBCO
BRL) for 30 min and 3 mg/ml collagenase (type H; Sigma Chemical Co.)
for up to 24 h as described previously (Wright et al., 1996). Cells were
seeded in Ham's F12 medium supplemented with 10% FCS, 100 IU/ml
penicillin, and 100 µg/ml streptomycin to a final density of 5 × 105/ml in
55-mm plastic petri dishes (Nunc), and cultured in a 95% air/5% CO2 incubator at 37°C. Primary, nonconfluent, 5-10 d cultures of chondrocytes
were used in all experiments in an attempt to limit changes in gene expression (dedifferentiation). Morphologically, the cells studied were typically
flattened with a polygonal cell shape and did not show the fibroblastic appearance of dedifferentiated chondrocytes (Wright et al., 1997
). Immunological and molecular analyses confirmed production of similar ECM molecules (type II and VI collagen, fibronectin, and keratin sulphate) and
expression of identical integrin profile subunits (
1,
5
V,
1,
3, and
5)
to that of HAC in vivo (Salter et al., 1992
; Loeser et al., 1995
) and after
initial cell extraction (Jopanbutra et al., 1996).
In our experience these chondrocytes show a consistent and reproducible membrane hyperpolarization response to 0.33 Hz mechanical stimulation (Wright et al., 1992, 1996
, 1997
). We have assessed the electrophysiological response of chondrocytes from knee joint articular cartilage of >80
different individuals and observed no significant difference in the membrane response in cells with respect to gender, age, and cause of death as it
relates to patients without a history of locomotor system involvement (unpublished observations).
To investigate whether the membrane hyperpolarization response was
critically dependent on IL-4, chondrocytes from the joints of mice heterozygous for IL-4 or IL-4-deficient were studied. Chondrocytes were isolated by sequential enzymatic digestion and cultured as described above.
The IL-4 knockout mice used have been previously described (Kopf et al.,
1993). Mice were obtained from a colony maintained by Dr. M. Norval
(Department of Medical Microbiology, Edinburgh University, Edinburgh,
United Kingdom) with permission for use of these animals provided by
Professor Horst Bluethmann (Hoffmann-LaRoche AG, Basel, Switzerland).
RNA Extraction
Total RNA was extracted from cultured chondrocytes as described in the
micro RNA isolation kit (Stratagene), using a denaturing buffer of 4 M
guanidine thiocyanate, 0.75 M sodium citrate, 10% (wt/vol) lauroyl sarcosine, and 7.2 µl/ml -mercaptoethanol. The quantity of RNA isolated
was determined by spectrophotometry using the absorbance reading at
260 nm.
Reverse Transcriptase-PCR (RT-PCR)
Before cDNA synthesis, all RNA samples were incubated with DNase I
(Life Technologies) for 15 min in the presence of RNase inhibitor (Life
Technologies). Template cDNA was synthesized using 1-5 µg RNA, superscript II, and oligo dT(12-18; Life Technologies) according to the manufacturer's instructions. Primers specific for IL-4 (Arai et al., 1989), IL-4
receptor
(IL-4R
; Idzerda et al., 1990
), the common gamma chain (
c;
Takeshita et al., 1992
; Puck et al., 1993
), and IL-13 receptor
(IL-13R
;
Aman et al., 1996
) were used for the PCR reactions: IL-4 5'-TTTGAACAGCCTCACAGAGC-3', 5'-TCCTTCACAGGACAGGAATT-3';
IL-4R
5'-CTTGTTCACCTTTGGACTGG-3', 5'-CTTGAGCTCTGAGCATTGCC-3';
c 5'-CTCCTTGCCTAGTGTGGATGG-3', 5'-CACTGTAGTCTGGCTGCAGAC-3'; and IL-13R
5'-GTGAAACATGGAAGACCATC-3', 5'-GTGAAATAACTGGATCTGATAGGC-3'.
A typical 20-µl PCR reaction contained 16 mM ammonium sulphate,
67 mM Tris/HCl, pH 8.8, 0.01% (vol/vol) Tween 20, 1 µM of each primer,
2 µl cDNA, 100 µM dNTPs, 0.1% (wt/vol) BSA, and 0.25 U Taq polymerase (Bioline). The magnesium chloride concentrations for each primer
pair were: IL-4, 4 mM; IL-4R, 2.5 mM;
c, 2 mM; and IL-13R
, 1.5 mM.
The following program was used for all reactions: 94°C for 3 min; 35 cycles
of 94°C for 1 min, 60°C for 1 min, 72°C for 1 min 30 s; 72°C for 10 min.
PCR products were analyzed by electrophoresis using a 1% (wt/vol) agarose gel.
Cloning and Sequencing
PCR products were cloned into the TA cloning vector (Invitrogen Corp.)
as described in the manufacturer's protocol. Each insert was sequenced
using the Sanger dideoxy chain termination method (Sanger et al., 1977),
modified according to the protocol provided with the sequenase kit
(United States Biochemical Corp.).
Mechanical Stimulation of Chondrocytes and Electrophysiological Recording
The technique and apparatus used have been previously described in detail (Wright et al., 1996). For the induction of pressure-induced strain
(PIS), 55-mm diameter plastic petri dishes (Nunc) were placed in a sealed
pressure chamber with inlet and outlet ports. The chamber was pressurized using nitrogen 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) for 20 min,
37°C, at a pressure of 16 kPa above atmospheric pressure. This system was
shown to produce microstrain on the base of the culture dish (Wright et al.,
1996
). Membrane potentials of cells were recorded using a single electrode bridge circuit and calibrator, as previously described (Wright et al.,
1992
; Salter et al., 1997
). Microelectrodes with tip resistances of 40-60 M
and tip potentials of ~3 mV were used to impale the cells. Membrane potentials of isolated cells were measured and results were accepted if, on
cell impalement, there was a rapid change in voltage to the membrane potential level that remained constant for at least 60 s. Experiments were performed at 37°C. The membrane potentials of 5-10 cells were measured
before and after the period of PIS.
Anticytokine, antiintegrin, and anticytokine receptor antibodies were
added to chondrocytes 30 min before mechanical stimulation. Membrane
potentials were measured before and after addition of antibody and after
the period of mechanical stimulation. Antibodies had no effect on the
resting membrane potential. Antibodies remained in contact with cells
during cyclical PIS and when poststimulated membrane potentials were
measured. Antibodies against IL-1, IL-4, IL-4R
, and
c were obtained
from R&D Systems, Inc. Anti-
1 integrin (P4C10) and anti-
V
5 integrin (P1F6) were obtained from Life Technologies. For each condition
tested, at least three experiments were performed on different cells from
different donor knees on different days.
Effects of Cytokines on Chondrocyte Membrane Potential
Membrane potential of chondrocytes was measured before and 10 min after the addition of recombinant IL-1, IL-4, TGF-
1, and interferon
gamma (IFN-
; R&D Systems). To investigate signaling molecules involved in IL-4-induced hyperpolarization chondrocytes were treated, in
separate experiments, with a number of pharmacological inhibitors of cell
signaling for 30 min before addition of recombinant IL-4. The reagents
used (Sigma Chemical Co.) were: neomycin, an inhibitor of PLC (Cockcroft et al., 1985; Kim et al., 1989
); flunarizine, an inhibitor of IP3-mediated release of Ca2+ from the ER (Seiler et al., 1987
); genistein, a tyrosine
kinase inhibitor (Akiyama et al., 1987); apamin, a specific blocker of SK
channels (Blatz and Magleby, 1986
); and gadolinium, a blocker of stretch-activated ion channels (Yang and Sachs, 1989
).
Statistics
The mean, SD, and standard error of the mean were determined in each experiment. For statistical comparisons, when the F ratio of the two variances reached significance, the nonparametric Mann-Whitney test was used. When the ratio did not reach significance, the Student's t test was used.
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Results |
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A Transferable Factor Induces Membrane Hyperpolarization of HAC in Response to Mechanical Strain
HAC subjected to PIS at 0.33 Hz, 37°C for 20 min undergo
hyperpolarization of the plasma membrane by ~45% (Table I). Conditioned medium from mechanically stimulated
cells, when added to unstimulated chondrocytes, caused
membrane hyperpolarization of these cells similar to that
of the directly mechanical strained chondrocytes (Table I),
demonstrating the presence of a soluble, transferable factor secreted by the mechanically stimulated chondrocytes.
1 µg/ml P4C10, an anti-1 integrin antibody, when incubated with chondrocytes for 30 min at 37°C before stimulation, inhibited the hyperpolarization response to
mechanical stimulation. Medium from cells mechanically
stimulated in the presence 1 µg/ml P4C10, when transferred to unstimulated cells, did not significantly alter the
membrane potential of these cells (Table I). In contrast, 1 µg/ml P1F6, an anti-
V
5 integrin, had no effect on 0.33-Hz cyclical microstrain-induced hyperpolarization or production of a transferable factor that could induce membrane hyperpolarization of unstimulated chondrocytes.
|
Cytokines Induce Changes in Membrane Potential
When monolayer cultures of HAC were incubated in separate experiments with a panel of recombinant human cytokines (IL-1, IFN-
, TGF-
1, and IL-4), known to be involved in the regulation of chondrocyte metabolism and
potentially could function as autocrine/paracrine signaling
molecules, a change in membrane potential was seen (Fig. 1). Addition of IL-4 resulted in membrane hyperpolarization, whereas the other cytokines induced membrane depolarization. The effect of IL-4 on the membrane potential
of human chondrocytes was dose-dependent over a range
between 100 fg/ml and 10 ng/ml. A 17% hyperpolarization response was elicited at concentrations as low as 10 fg/ml
and a maximal response was obtained with 5-10 pg/ml (results not shown).
|
Human Chondrocytes Express IL-4 and IL-4 Receptors
Using immunohistochemical techniques we have shown
IL-4 to be present in HAC (Salter et al., 1996). However,
its production by these cells and the expression of IL-4 receptors were not previously described. RT-PCR on total
RNA isolated from primary cultured chondrocytes using
primers specific for IL-4 resulted in amplification of a 269-bp region of DNA (Fig. 2). This DNA region, when cloned and sequenced, displayed 100% identity to the published
sequence of human lymphocyte IL-4 mRNA (Arai et al.,
1989
). RT-PCR reactions using primers to IL-4R
,
c, and
IL-13R
revealed DNA products of 465, 356, and 450 bp,
respectively (Fig. 2), corresponding to the components of
both the type I IL-4 receptor (IL-4R
/
c) and type II receptor (IL-4R
/IL-13R
).
|
IL-4 Is Necessary for the Membrane Hyperpolarization Response to Mechanical Stimulation
Neutralizing antibodies to IL-4 abolished the hyperpolarization response to cyclical strain, whereas neutralizing
antibodies to IL-1 had no effect (Fig. 3). Specific antibodies to IL-4R
(10 µg/ml) prevented the hyperpolarization response of chondrocytes to mechanical stimulation,
whereas inhibitory antibodies to the
c subunit had no effect on the response (Fig. 3). Anti-IL-4 antibodies (1 µg/ml),
added to medium after mechanical stimulation but before transfer of that medium to unstimulated cells, prevented
subsequent hyperpolarization of unstained cells (Table II).
Chondrocytes isolated from the articular cartilage of knee
joints from IL-4 knockout mice did not show a significant
change in membrane potential after 20 min of mechanical
stimulation at 0.33 Hz (Fig. 4). In contrast, chondrocytes
isolated from knee joints of heterozygous mice showed a
similar hyperpolarization response (Fig. 4) to mechanical stimulation as that seen with HAC.
|
|
|
IL-4-mediated Membrane Hyperpolarization Involves PLC and IP3
The hyperpolarization response of HAC to recombinant
human IL-4 (10 pg/ml) was unaffected by P4C10 (anti-1
integrin), genistein (a tyrosine kinase inhibitor), and gadolinium (a blocker of mechanosensitive ion channels; Table
III), although these agents were shown previously to inhibit the hyperpolarization response of HAC to mechanical stimulation (Wright et al., 1996
, 1997
). Neomycin (an
inhibitor of PLC), flunarizine (an inhibitor of IP3-mediated release of Ca2+ from the ER), and apamin (an SK
channel blocker) each inhibited the chondrocyte hyperpolarization response to IL-4 (Table III).
|
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Discussion |
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This study has shown for the first time that IL-4 and its receptor are expressed by HAC. Furthermore this study also has shown that the cytokine receptor pair are involved in the integrin-dependent signaling pathway activated by 0.33-Hz cyclical strain that leads to the opening of SK channels and membrane hyperpolarization.
Close associations between integrin and growth factor-
mediated signaling in regulation of cell function are being
identified. Cell adhesion-dependent activation of the Ras/
MAPK pathway may involve tyrosine phosphorylation of
PDGF receptors (Sundberg and Rubin, 1996). Angiogenic
effects of a number of growth factors including basic fibroblast growth factor and vascular endothelial growth factor
are integrin-regulated (Friedlander et al., 1995
). Integrin-mediated cell adherence also has been shown to be important in cytokine gene expression in synovial fluid cells
from patients with rheumatoid arthritis (Miyake et al.,
1993
) and by mast cells after Ig E receptor aggregation
(Ra et al., 1994
). Wilson et al. (1993)
have demonstrated
previously that mechanical strain induces growth of vascular smooth muscle cells via an autocrine action of PDGF.
However, the growth-promoting effect required 36-48 h of
mechanical stimulation and was associated with increased
levels of PDGF mRNA, suggesting slow production and
release of the cytokine rather than the rapid release of a
preformed mediator after mechanical stimulation, as demonstrated in our system.
It is unclear how integrin-mediated signaling causes IL-4
release. Rapid release of neurotransmitter from frog muscle motor nerve terminals after stretch is integrin-dependent and requires both intra and extracellular calcium
(Chen and Grinnell, 1995). The data from our studies suggest that mechanical stimulation induced release of IL-4
by human chondrocytes after recognition and transduction
of the mechanical signal by
5
1 integrin. Furthermore, activation of a signaling pathway involving tyrosine kinases, stretch-activated ion channels, and the actin cytoskeleton is consistent with other models of integrin-mediated mechanotransduction (Glogauer et al., 1997
;
Maniotis et al., 1997
; Muller et al., 1997
, Schmidt et al.,
1998
). IL-4 in turn binds to the chondrocyte IL-4 receptor
heterodimer, IL-4R
/IL-13R
, initiating a signal cascade
involving PLC and IP3-mediated Ca2+ release and subsequent activation of SK channels, leading to K+ efflux and
membrane hyperpolarization.
Coordinated activations of integrin and IL-4-associated
signaling pathways in chondrocytes are of potential importance in regulating the structure and function of normal
and diseased articular cartilage. Regulation occurs by mediating other biochemical responses to mechanical strain,
e.g., proteoglycan synthesis (Veldhuijzen et al., 1979), or
altering the expression of other ECM proteins, matrix
metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs) involved in the pathogenesis of
OA (Dean, 1991
). Studies of cytokine effects on chondrocytes in vitro suggest that IL-4 alters the ratio of MMPs
and TIMPs in favor of TIMPs by suppressing IL-1-stimulated MMP3 production (Shingu et al., 1995
; Nemoto et al.,
1997
). Integrin-regulated production of IL-4, as a result of
optimal mechanical stimulation in normal articular cartilage in vivo, would be chondroprotective by inhibiting cartilage degradation and promoting matrix synthesis in normal articular cartilage. In contrast, in joint diseases such as
OA, normal mechanotransduction pathways may be disrupted following changes in integrin expression by chondrocytes (Lapadula et al., 1997
) or neo-expression of adhesive and antiadhesive molecules such as fibronectin
(Chevalier et al., 1996
) and tenascin (Salter, 1993
) in the
pericellular matrix, resulting in abnormal chondrocyte activity. Indeed, preliminary data from our laboratory indicate that chondrocytes from OA cartilage show an abnormal electrophysiological response to both mechanical stimulation and direct application of IL-4 (Wright et al.,
1998
). Further elucidation of the signaling events activated
by mechanical stimuli in HAC from normal and diseased
cartilage should lead to a better understanding of how cartilage is maintained by mechanical stimuli in health and
disease. These studies suggest that better understanding of
the signaling molecules involved in mechanotransduction in chondrocytes may also lead to the identification of
novel targets for therapy in OA.
![]() |
Footnotes |
---|
Address correspondence to Dr. D.M. Salter, Department of Pathology, University of Edinburgh Medical School, Teviot Place, Edinburgh, United Kingdom EH8 9AG. Tel.: 44-31-650-2946. Fax: 44-31-650-6528. E-mail: Donald.Salter{at}ed.ac.uk
Received for publication 21 July 1998 and in revised form 1 March 1999.
We thank Dr. M. Norval and Dr. A. El-Ghorr for providing us with the heterozygous and IL-4 knockout mice, Professor Horst Bluethmann for his kind permission to use these animals, and Mr. M. Lawson for his advice on PCR.
This work was funded by a grant from the Arthritis Research Campaign.
![]() |
Abbreviations used in this paper |
---|
CM, conditioned medium;
c, common
gamma chain;
ECM, extracellular matrix;
HAC, human articular chondrocytes;
IFN-
, interferon gamma;
IL, interleukin;
IP3, inositol triphosphate;
MMP, matrix metalloproteinase;
OA, osteoarthritis;
PIS, pressure-induced strain;
PLC, phospholipase C;
PKC, protein kinase C;
RT-PCR, reverse transcriptase-PCR;
SK, small conductance Ca2+-dependent K+
channels;
TGF-
1, transforming growth factor
1;
TIMP, tissue inhibitor
of matrix metalloproteinase.
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