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 α inhibit mechanically induced membrane hyperpolarization and anti–IL-4 antibodies neutralize the hyperpolarizing activity of medium from mechanically stimulated cells. Antibodies to interleukin 1β (IL-1β) and cytokine receptors, interleukin 1 receptor type I and the common γ chain/CD132 (γ) 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.

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 (α and β 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 α5 integrin and β1 integrins, whereas the 0.104-Hz depolarization response is inhibited by antibodies to αVβ5 and not by anti-α5 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 1β (IL-1β), IL-4, and transforming growth factor β1 (TGF-β1) 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.

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 (β1, β5αV, α1, α3, and α5) 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 β-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 α (IL-4Rα; Idzerda et al., 1990), the common gamma chain (γc; Takeshita et al., 1992; Puck et al., 1993), and IL-13 receptor α (IL-13Rα; Aman et al., 1996) were used for the PCR reactions: IL-4 5′-TTTGAACAGCCTCACAGAGC-3′, 5′-TCCTTCACAGGACAGGAATT-3′; IL-4Rα 5′-CTTGTTCACCTTTGGACTGG-3′, 5′-CTTGAGCTCTGAGCATTGCC-3′; γc 5′-CTCCTTGCCTAGTGTGGATGG-3′, 5′-CACTGTAGTCTGGCTGCAGAC-3′; and IL-13Rα 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-4Rα, 2.5 mM; γc, 2 mM; and IL-13Rα, 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 MΩ 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-1β, IL-4, IL-4Rα, and γc were obtained from R&D Systems, Inc. Anti–β1 integrin (P4C10) and anti–αVβ5 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-1β, IL-4, TGF-β1, and interferon gamma (IFN-γ; 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.

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–β1 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–αVβ5 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-1β, IFN-γ, TGF-β1, 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-4Rα, γc, and IL-13Rα 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-4Rα/γc) and type II receptor (IL-4Rα/IL-13Rα).

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-1β had no effect (Fig. 3). Specific antibodies to IL-4Rα (10 μg/ml) prevented the hyperpolarization response of chondrocytes to mechanical stimulation, whereas inhibitory antibodies to the γc 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–β1 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).

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 α5β1 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-4Rα/IL-13Rα, 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.

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.

     
  • CM

    conditioned medium

  •  
  • γc

    common gamma chain

  •  
  • ECM

    extracellular matrix

  •  
  • HAC

    human articular chondrocytes

  •  
  • IFN-γ

    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-β1

    transforming growth factor β1

  •  
  • TIMP

    tissue inhibitor of matrix metalloproteinase

Akiama
T
,
Ishida
J
,
Nakagana
S
,
Ogawara
H
,
Watanabe
S
,
Itoh
N
,
Sibuya
M
,
Fukami
Y
Genistein, a specific inhibitor of tyrosine-specific protein kinases
J Biol Chem
1987
262
5592
5595
[PubMed]
Aman
MJ
,
Tayebi
N
,
Obiri
NI
,
Puri
RK
,
Modi
WS
,
Leonard
WJ
cDNA cloning and characterization of the human interleukin-13 receptor alpha chain
J Biol Chem
1996
271
29265
29270
[PubMed]
Arai
N
,
Nomura
D
,
Villaret
D
,
Malefijt
DR
,
Seiki
M
,
Yoshida
M
,
Minoshima
S
,
Fukuyama
R
,
Maekawa
M
,
Kudoh
J
et al
Complete nucleotide sequence of the chromosomal gene for human IL-4 and its expression
J Immunol
1989
142
274
282
[PubMed]
Ayajiki
K
,
Kindermann
M
,
Hecker
M
,
Fleming
I
,
Busse
R
Intracellular pH and tyrosine phosphorylation but not calcium determine shear stress-induced nitric oxide production in native endothelial cells
Circ Res
1996
78
750
758
[PubMed]
Blatz
AL
,
Magleby
KL
Single apamin-blocked Ca-activated K+channels of small conductance in cultured rat skeletal muscle
Nature
1986
323
718
720
[PubMed]
Chen
BM
,
Grinnell
AD
Integrins and modulation of transmitter release from motor nerve terminals by stretch
Science
1995
269
1578
1580
[PubMed]
Chevalier
X
,
Claudepierre
P
,
Groult
N
,
Zardi
L
,
Hornebeck
W
Presence of ED-A containing fibronectin in human articular cartilage from patients with osteoarthritis and rheumatoid arthritis
J Rheumatol
1996
23
1022
1030
[PubMed]
Cockcroft
S
,
Gomperts
BD
Role of guanine nucleotide binding protein in the activation of polyphosphoinositide phosphodiesterase
Nature
1985
314
534
536
[PubMed]
Davies
PF
Flow-mediated endothelial mechanotransduction
Physiol Rev
1995
75
519
560
[PubMed]
Davies
PF
,
Tripathi
SC
Mechanical stress mechanisms and the cell
Circ Res
1993
72
239
245
[PubMed]
Dean, D.D. 1991. Proteinase-mediated cartilage degradation in osteoarthritis. Semin. Arthritis Rheum. 20(Suppl. 2):2–11.
Friedlander
M
,
Brooks
PC
,
Sharfer
RW
,
Kincaid
CM
,
Varner
JA
,
Cheresh
DA
Definition of two angiogenic pathways by distinct aV integrins
Science
1995
270
1500
1502
[PubMed]
Glogauer
M
,
Arora
P
,
Yao
G
,
Sokholov
I
,
Ferrier
J
,
McCulloch
CAG
Calcium ions and tyrosine phosphorylation interact coordinately with actin to regulate cytoprotective responses to stretching
J Cell Sci
1997
110
11
21
[PubMed]
Harter
LV
,
Hruska
KA
,
Duncan
RL
Human osteoblast-like bone cells respond to mechanical strain with increased bone matrix protein production independent of hormonal regulation
Endocrinology
1995
136
528
535
[PubMed]
Hynes
RO
Integrins: versatility, modulation, and signaling in cell adhesion
Cell
1992
69
11
25
[PubMed]
Idzerda
RL
,
March
CJ
,
Mosley
B
,
Lyman
SD
,
Vanden
T
,
Bos
,
Gimpel
SD
,
Din
WS
,
Grabstein
KH
,
Widmer
MB
,
Park
LS
et al
Human interleukin 4 receptor confers biological responsiveness and defines a novel receptor superfamily
J Exp Med
1990
171
861
873
[PubMed]
Jobanputra
P
,
Lin
H
,
Jenkins
K
,
Bavington
C
,
Brennan
FR
,
Nuki
G
,
Salter
DM
,
Godolphin
JL
Modulation of human chondrocyte integrins by inflammatory synovial fluid
Arthritis Rheum
1996
39
1430
1432
[PubMed]
Kim
D
,
Lewis
DL
,
Graziadei
I
,
Neer
EJ
,
Bar-Sagi
D
,
Clapham
DE
G-protein β gamma-subunits activate the cardiac muscarinic K+ channel via phospholipase A2
Nature
1989
337
557
560
[PubMed]
Kimono
I
,
Kudo
S
,
Yamakazi
T
,
Zou
Y
,
Shiojimi
I
,
Yazaki
Y
Mechanical stretch activates the stress activated protein kinases in cardiac myocytes
FASEB (Fed Am Soc Exp Biol) J
1996
10
631
636
[PubMed]
Kopf
M
,
Le Gros
G
,
Bachmann
M
,
Lamers
MC
,
Bluethmann
H
,
Kohler
G
Disruption of the murine IL-4 gene blocks Th2 cytokine responses
Nature
1993
362
245
248
[PubMed]
Lapadula
G
,
Iannone
F
,
Zuccaro
C
,
Grattagliano
V
,
Covelli
M
,
Patella
V
,
Lobianco
G
,
Pipitone
V
Integrin expression on chondrocytes: correlations with the degree of cartilage damage in human osteoarthritis
Clin Exp Rheumatol
1997
15
247
254
[PubMed]
Loeser
RF
,
Carlson
CS
,
McGee
MP
Expression of beta 1 integrins by cultured articular chondrocytes and in osteoarthritic cartilage
Exp Cell Res
1995
217
248
257
[PubMed]
MacKenna
DA
,
Dolf
F
,
Vuori
K
,
Ruoslahti
E
Extracellular signal-regulated kinase and c-jun NH2-terminal kinase activation by mechanical stretch is integrin-dependent and matrix-specific in rat cardiac fibroblasts
J Clin Invest
1998
101
301
310
[PubMed]
Maniotis
AJ
,
Chen
CS
,
Ingber
DE
Demonstration of mechanical connections between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure
Proc Natl Acad Sci USA
1997
94
849
854
[PubMed]
Miyake
S
,
Yagita
H
,
Maruyama
T
,
Hashimoto
H
,
Miyasaka
N
,
Okumura
K
β1 integrin-mediated interaction with extracellular matrix proteins regulates cytokine gene expression in synovial fluid cells of rheumatoid arthritis patients
J Exp Med
1993
177
863
868
[PubMed]
Mow
VC
,
Ratcliffe
A
,
Poole
AR
Cartilage and diarthrodial joints as paradigms for hierarchical materials and structures
Biomaterials
1992
13
67
97
[PubMed]
Muller
JM
,
Chilian
WM
,
Davis
MJ
Integrin signaling transduces shear stress-dependent vasodilation of coronary arterioles
Circ Res
1997
80
320
326
[PubMed]
Nemoto
O
,
Yamada
H
,
Kikuchi
T
,
Shimmei
M
,
Obata
K
,
Sato
H
,
Seiki
M
Suppression of matrix metalloproteinase-3 synthesis by interleukin-4 in human articular chondrocytes
J Rheumatol
1997
24
1774
1779
[PubMed]
Prasad
ARS
,
Logan
SA
,
Nerem
RM
,
Schwartz
CJ
,
Sprague
EA
Flow-related responses of intracellular inositol phosphate levels in cultured aortic endothelial cells
Circ Res
1993
72
827
836
[PubMed]
Puck
JM
,
Deschenes
SM
,
Porter
JC
,
Dutra
AS
,
Brown
CJ
,
Willard
HF
,
Henthorn
PS
The interleukin-2 receptor gamma chain maps to Xq13.1 and is mutated in X-linked severe combined immunodeficiency, SCIDX1
Hum Mol Genet
1993
2
1099
1104
[PubMed]
Ra
C
,
Yasuda
M
,
Yagita
H
,
Okumara
K
Fibronectin receptor integrins are involved in mast cell activation
J Allergy Clin Immunol
1994
94
625
628
[PubMed]
Sachs
F
Mechanical transduction in biological systems
Crit Rev Biomed Eng
1988
16
141
169
[PubMed]
Salter
DM
,
Hughes
DE
,
Simpson
R
,
Gardner
DL
Integrin expression by human articular chondrocytes
Br J Rheumatol
1992
31
231
234
[PubMed]
Salter
DM
Tenascin is increased in cartilage and synovium from arthritic knees
Br J Rheumatol
1993
32
780
786
[PubMed]
Salter
DM
,
Nuki
G
,
Wright
MO
Interleukin-4 expression in normal and osteoarthritic human articular cartilage
J Rheumatol
1996
23
1314
1315
[PubMed]
Salter
DM
,
Robb
JE
,
Wright
MO
Electrophysiological responses of human bone cells to mechanical stimulation: evidence for specific integrin function in mechanotransduction
J Bone Miner Res
1997
12
1133
1141
[PubMed]
Sanger
F
,
Nicklen
S
,
Coulson
AR
DNA sequencing with chain-terminating inhibitors
Proc Natl Acad Sci USA
1977
74
5436
5467
Schmidt
C
,
Pommerenke
H
,
Durr
F
,
Nebe
B
,
Rychly
J
Mechanical stressing of integrin receptors induces enhanced tyrosine phosphorylation of cytoskeletally anchored proteins
J Biol Chem
1998
273
5081
5085
[PubMed]
Seiler
SM
,
Arnold
AJ
,
Stanton
HC
Inhibitors of inositol triphosphate induced Ca2+release from isolated platelet membrane vesicles
Biochem Pharmacol
1987
36
3331
3337
[PubMed]
Shingu
M
,
Miyauchi
S
,
Nagai
Y
,
Yasutake
C
,
Horie
K
The role of IL4 and IL6 in IL1-dependent cartilage matrix degradation
Br J Rheumatol
1995
34
101
106
[PubMed]
Shyy
JY-J
,
Chien
S
Role of integrins in cellular responses to mechanical stress and adhesion
Curr Opin Cell Biol
1997
9
707
713
[PubMed]
Somjen
D
,
Binderman
I
,
Berger
E
,
Harell
A
Bone remodelling induced by physical stress is prostaglandin E2 mediated
Biochim Biophys Acta
1980
627
91
100
[PubMed]
Stockwell
RA
Cartilage failure in osteoarthritis: relevance of normal structure and function. A review
Clin Anat
1991
4
161
191
Sundberg
C
,
Rubin
K
Stimulation of β1 integrins on fibroblasts induces PDGF independent tyrosine phosphorylation of PDGF β receptors
J Cell Biol
1996
132
741
752
[PubMed]
Takeshita
T
,
Asao
H
,
Ohtani
K
,
Ishii
N
,
Kumaki
S
,
Tanaka
N
,
Munakata
H
,
Nakamura
M
,
Sugamura
K
Cloning of the gamma chain of the human IL-2 receptor
Science
1992
257
379
382
[PubMed]
Urban
JP
The chondrocyte: a cell under pressure
Br J Rheumatol
1994
33
901
908
[PubMed]
Veldhuijzen
JP
,
Bourret
LA
,
Rodan
GA
In vitro studies of the effect of intermittent compressive forces on cartilage cell proliferation
J Cell Physiol
1979
98
299
306
[PubMed]
Wang
N
,
Butler
JP
,
Ingber
DE
Mechanotransduction across the cell surface and through the cytoskeleton
Science
1993
260
1124
1127
[PubMed]
Wilson
E
,
Mai
Q
,
Sudhir
K
,
Weiss
RH
,
Ives
HE
Mechanical strain induces growth of vascular smooth muscle cells via autocrine action of PDGF
J Cell Biol
1993
123
741
747
[PubMed]
Wright
MO
,
Stockwell
RA
,
Nuki
G
Response of plasma membrane to applied hydrostatic pressure in chondrocytes and fibroblasts
Connect Tissue Res
1992
28
49
70
[PubMed]
Wright
MO
,
Jobanputra
P
,
Bavington
C
,
Salter
DM
,
Nuki
G
Intracellular signal transduction pathways in human chondrocytes stimulated by intermittent pressurization
Trans Orth Res Soc
1995
29
88
Wright
MO
,
Jobanputra
P
,
Bavington
C
,
Salter
DM
,
Nuki
G
The effects of intermittent pressurization on the electrophysiology of cultured human chondrocytes: evidence for the presence of stretch activated membrane ion channels
Clin Sci
1996
90
61
71
[PubMed]
Wright
MO
,
Nishida
K
,
Bavington
C
,
Godolphin
JL
,
Dunne
E
,
Walmsley
S
,
Jobanputra
P
,
Nuki
G
,
Salter
DM
Hyperpolarization of cultured human chondrocytes follows cyclical pressure-induced strain: evidence of a role for α5β1 integrin as a chondrocyte mechanoreceptor
J Orthop Res
1997
15
742
747
[PubMed]
Wright, M.O., S.J. Millward-Sadler, G. Nuki, and D.M. Salter. 1998. Aberrant membrane depolarization in cultured chondrocytes from osteoarthritic cartilage following cyclical mechanical strain and exposure to IL4. Br. J. Rheumatol. 37(Suppl. 1):154.
Yang
XC
,
Sachs
F
Block of stretch-activated ion channels in Xenopusoocytes by gadolinium and calcium ions
Science
1989
243
1068
1071
[PubMed]
Yano
Y
,
Geibel
J
,
Sumpio
BE
Tyrosine phosphorylation of pp125FAK and paxillin in aortic endothelial cells induced by mechanical strain
J Cell Physiol
1996
271
c635
c649

Author notes

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@ed.ac.uk