Aquaporin water channels AQP1 and AQP3, are expressed in equine articular chondrocytes

8
Aquaporin water channels AQP1 and AQP3, are expressed in equine articular chondrocytes Ali Mobasheri a, * , Elisa Trujillo b , Susan Bell a , Stuart D. Carter a , Peter D. Clegg a , Pablo Mart ın-Vasallo c , David Marples d a Connective Tissue and Molecular Pathogenesis Research Groups, Faculty of Veterinary Science, University of Liverpool, Liverpool L69 7ZJ, UK b Servicio de Reumatologia, Hospital Universitario de Canarias, La Cuesta, Tenerife, Spain c Departamento de Bioqu ımica y Biolog ıa Molecular, Universidad de La Laguna, La Laguna, Tenerife, Spain d School of Biomedical Sciences, University of Leeds, Leeds, UK Accepted 1 August 2003 Abstract Chondrocytes exist in an unusual and highly variable ionic and osmotic environment in the extracellular matrix of articular cartilage. Alterations to the ionic and osmotic environment of chondrocytes influence the volume and ionic content of the cells, which, in turn, modifies the rate at which extracellular matrix macromolecules are synthesized and degraded. Thus, regulation of the water and solute content of chondrocytes will profoundly affect their anabolic and catabolic functions. The water content of cells is effectively influenced by the abundance of aquaporin (AQP) water channels. Recent studies have shown that several AQP water channel isoforms are expressed in chondrocytes from MeckelÕs cartilage, developing teeth and other orofacial tissues. The aim of the present investigation was to determine if chondrocytes from equine articular cartilage express AQP water channels. Polyclonal antibodies to AQP1, AQP2 and AQP3 were used in conjunction with immunohistochemistry, immunoblotting and quantitative flow cytometry to determine if AQP1, AQP2 and AQP3 are expressed in equine articular chondrocytes. Our studies show that AQP1 and AQP3 are expressed by chondrocytes in articular cartilage in situ and in isolated chondrocytes. We found no evidence for expression of AQP2, the vasopressin-regulated water channel in chondrocytes. AQP1 and AQP3 may be involved in the transport of water and small solutes and osmotically active metabolites across the chondrocyte plasma membrane during volume regulatory behaviour. AQP1 may be involved in transporting metabolic water. AQP3 may participate in the transport of glycerol and structurally related molecules. Ó 2003 Elsevier Ltd. All rights reserved. Keywords: Equine articular cartilage; Chondrocyte; AQP1; AQP3; Water permeability; Volume regulation; Immunohistochemistry; Flow cytometry 1. Introduction Articular chondrocytes are specialized cells responsi- ble for the synthesis and maintenance of cartilage matrix (Buckwalter and Mankin, 1998). The extracellular ma- trix of cartilage consists of water (up to 70% of total tissue weight), type II collagen, large aggregating pro- teoglycans, small proteoglycans and smaller non- collagenous matrix proteins. The cross-linked collagen forms a meshwork in which negatively charged proteo- glycans are trapped attracting cations (mainly Na þ ,K þ and Ca 2þ ) and osmotically obliged water which, in turn, results in swelling of the proteoglycans, increasing the tension within the collagen network. This swelling mechanism provides the extracellular matrix with the facility to resist tension and shear forces, thereby giving cartilage its ability to resist compression under static and dynamic mechanical load. Chondrocytes are excellent sensors of ionic and os- motic stimuli and respond to these signals in coordina- tion with other environmental, hormonal and genetic factors to regulate metabolic activity (Guilak, 2000; Mobasheri et al., 2002). Although this metabolic regu- lation is essential to the health, structural integrity and The Veterinary Journal 168 (2004) 143–150 The Veterinary Journal www.elsevier.com/locate/tvjl * Corresponding author. Tel.: +44-151-794-4284; fax: +44-151-794- 4243. E-mail address: [email protected] (A. Mobasheri). 1090-0233/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2003.08.001

Transcript of Aquaporin water channels AQP1 and AQP3, are expressed in equine articular chondrocytes

The

The Veterinary Journal 168 (2004) 143–150

Veterinary Journalwww.elsevier.com/locate/tvjl

Aquaporin water channels AQP1 and AQP3, are expressedin equine articular chondrocytes

Ali Mobasheri a,*, Elisa Trujillo b, Susan Bell a, Stuart D. Carter a,Peter D. Clegg a, Pablo Mart�ın-Vasallo c, David Marples d

a Connective Tissue and Molecular Pathogenesis Research Groups, Faculty of Veterinary Science, University of Liverpool, Liverpool L69 7ZJ, UKb Servicio de Reumatologia, Hospital Universitario de Canarias, La Cuesta, Tenerife, Spain

c Departamento de Bioqu�ımica y Biolog�ıa Molecular, Universidad de La Laguna, La Laguna, Tenerife, Spaind School of Biomedical Sciences, University of Leeds, Leeds, UK

Accepted 1 August 2003

Abstract

Chondrocytes exist in an unusual and highly variable ionic and osmotic environment in the extracellular matrix of articular

cartilage. Alterations to the ionic and osmotic environment of chondrocytes influence the volume and ionic content of the cells,

which, in turn, modifies the rate at which extracellular matrix macromolecules are synthesized and degraded. Thus, regulation of the

water and solute content of chondrocytes will profoundly affect their anabolic and catabolic functions. The water content of cells is

effectively influenced by the abundance of aquaporin (AQP) water channels. Recent studies have shown that several AQP water

channel isoforms are expressed in chondrocytes from Meckel�s cartilage, developing teeth and other orofacial tissues. The aim of the

present investigation was to determine if chondrocytes from equine articular cartilage express AQP water channels. Polyclonal

antibodies to AQP1, AQP2 and AQP3 were used in conjunction with immunohistochemistry, immunoblotting and quantitative flow

cytometry to determine if AQP1, AQP2 and AQP3 are expressed in equine articular chondrocytes. Our studies show that AQP1 and

AQP3 are expressed by chondrocytes in articular cartilage in situ and in isolated chondrocytes. We found no evidence for expression

of AQP2, the vasopressin-regulated water channel in chondrocytes. AQP1 and AQP3 may be involved in the transport of water and

small solutes and osmotically active metabolites across the chondrocyte plasma membrane during volume regulatory behaviour.

AQP1 may be involved in transporting metabolic water. AQP3 may participate in the transport of glycerol and structurally related

molecules.

� 2003 Elsevier Ltd. All rights reserved.

Keywords: Equine articular cartilage; Chondrocyte; AQP1; AQP3; Water permeability; Volume regulation; Immunohistochemistry; Flow cytometry

1. Introduction

Articular chondrocytes are specialized cells responsi-

ble for the synthesis and maintenance of cartilage matrix

(Buckwalter and Mankin, 1998). The extracellular ma-trix of cartilage consists of water (up to 70% of total

tissue weight), type II collagen, large aggregating pro-

teoglycans, small proteoglycans and smaller non-

collagenous matrix proteins. The cross-linked collagen

forms a meshwork in which negatively charged proteo-

* Corresponding author. Tel.: +44-151-794-4284; fax: +44-151-794-

4243.

E-mail address: [email protected] (A. Mobasheri).

1090-0233/$ - see front matter � 2003 Elsevier Ltd. All rights reserved.

doi:10.1016/j.tvjl.2003.08.001

glycans are trapped attracting cations (mainly Naþ, Kþ

and Ca2þ) and osmotically obliged water which, in turn,

results in swelling of the proteoglycans, increasing the

tension within the collagen network. This swelling

mechanism provides the extracellular matrix with thefacility to resist tension and shear forces, thereby giving

cartilage its ability to resist compression under static and

dynamic mechanical load.

Chondrocytes are excellent sensors of ionic and os-

motic stimuli and respond to these signals in coordina-

tion with other environmental, hormonal and genetic

factors to regulate metabolic activity (Guilak, 2000;

Mobasheri et al., 2002). Although this metabolic regu-lation is essential to the health, structural integrity and

144 A. Mobasheri et al. / The Veterinary Journal 168 (2004) 143–150

functional performance of articulating joints (Buckw-alter and Mankin, 1998), the mechanisms by which ionic

and osmotic changes modulate chondrocyte metabolism

are poorly understood (Guilak et al., 2002). Adaptation

to ionic and osmotic stimuli involves changes in the

physical properties of the chondrocyte plasma mem-

brane and the activity of channels and transporters

(Guilak et al., 2002; Mobasheri et al., 1998, 2002).

Chondrocytes have the capacity to regulate their volumein response to hyper and hypo-osmotic stress (Guilak

et al., 1995). This volume regulatory activity involves

mobilization of osmotically active solutes (osmolytes)

and water across the chondrocyte plasma membrane.

However, the molecular identity of plasma membrane

systems responsible for osmolyte and water transport in

chondrocytes has remained unknown.

Aquaporins (AQPs) are a family of water channelproteins related to the major intrinsic protein (MIP or

AQP0). Mammalian AQP proteins form water-perme-

able channels that provide the plasma membranes of red

cells, kidney proximal and collecting tubules and other

tissues with high permeability to water, thereby per-

mitting water to move in the direction of an osmotic

gradient. AQPs are expressed in diverse tissues (Borgnia

et al., 1999; Marples, 2001). Some members of the AQPgene family are permeable, not only to water, but also,

to small solutes such as glycerol and urea. The mam-

malian aquaporin family consists of ten members that

fall into two categories: the traditional aquaporins are

water channels and include AQP0, AQP1, AQP2,

AQP4, AQP5, AQP6 and AQP8; the unorthodox,

multifunctional aquaglyceroporins may transport water

in addition to small solutes. This latter category includesAQP3, AQP7 and AQP9 (Borgnia et al., 1999).

AQP1, originally known as CHIP28 (Agre et al.,

1987), or AQP-CHIP, is a homotetramer with six bilayer

spanning domains and is expressed in most tissues in the

mammalian body (Agre et al., 1993). AQP1 is a possible

candidate for disorders involving imbalance in ocular

fluid movement. AQP2 is a vasopressin-regulated renal

water channel and is highly expressed in the cortex andmedulla of kidney (Fushimi et al., 1994). AQP3 is highly

expressed in the kidney, colon, liver and small intestine,

where it has been proposed to transport water and small

solutes such as glycerol and urea (Ishibashi et al., 1994.

Ma et al., 1994).

We have previously hypothesized that water and

osmolyte channels are involved in cell volume regulation

and possibly also mechanotransduction in chondrocytes(Mobasheri et al., 1998, 2002; Trujillo et al., 2000). The

aim of this study was to determine if aquaporin water

channels are expressed by articular chondrocytes.

Polyclonal antibodies to AQP1, AQP2 and AQP3 were

used in conjunction with immunohistochemistry, Wes-

tern blotting and quantitative flow cytometry to com-

pare the relative expression levels of these three

aquaporins in sections of equine cartilage and in isolatedequine chondrocytes.

2. Materials and methods

2.1. Chemicals

Unless otherwise stated, all chemicals used in thisstudy were molecular biology grade and purchased from

Sigma/Aldrich and Sigma Biosciences. Materials for

immunohistochemistry were purchased from Vector

Laboratories and Dako Cytomation.

2.2. Tissues

Equine articular cartilage was obtained from thefemoropatellar and metacarpophalangeal joints of

horses at the Equine Division at the Philip Leverhulme

Large Animal Hospital, University of Liverpool. The

horses used for cartilage samples were euthanased for

clinical reasons and not for the purposes of the present

investigation. Full depth sections (8 lm thickness) of

articular cartilage from all zones (superficial, middle,

deep and calcified) were provided by the histopathologyservice of the Department of Veterinary Pathology,

University of Liverpool. Formalin-fixed paraffin-

embedded ovine kidney mounted on 3-aminopropyl-

triethoxysilane (APES) treated slides were used as a

positive control for AQP1, AQP2 and AQP3 expression.

2.3. Antibodies

Immunohistochemical studies were conducted using a

panel of polyclonal isoform-specific antibodies raised in

rabbits against rat AQP1, AQP2 and AQP3 (Chemicon

International). Polyclonal antibodies to rat AQP1 and

AQP2 developed in the laboratory of Dr. D. Marples

were also used in this study.

2.4. Immunohistochemistry

The DAKO EnVisionTM+ System, HRP (Dako

Cytomation) was used for visualisation of the antibody

staining. This is a two-step immunohistochemical

staining technique. This system is based on a horserad-

ish peroxidase (HRP) labelled polymer which is conju-

gated with secondary antibodies specific for rabbit. The

labelled polymer does not contain avidin or biotin andconsequently, non-specific staining resulting from en-

dogenous avidin–biotin activity is eliminated. Briefly,

full depth equine cartilage explants, including sub-

chondral bone, were removed and fixed for 2 h in 4%

paraformaldehyde in phosphate-buffered saline (PBS).

Explants were decalcified overnight in 10% EDTA in

PBS before paraffin embedding and sectioning (7 lm)

A. Mobasheri et al. / The Veterinary Journal 168 (2004) 143–150 145

onto Vectabond coated slides (Vector Laboratories).The slides were de-waxed in xylene for 10 min at room

temperature (RT) before rehydration in a series of gra-

ded ethanol baths (100%, 70%, 50%). Wax boundaries

were drawn around the tissue sections using a paraffin

wax immunohistochemistry pen (DAKO). The endoge-

nous peroxidase activity in the cartilage sections was

quenched with by a one-hour incubation with 3% hy-

drogen peroxide solution in PBS (pH 7.4), containing 27mM KCl and 0.137 M NaCl. Sections were transferred

to a stainless steel humidified chamber and non-specific

binding blocked for 1 h using a 10% normal goat serum

(NGS) solution in PBS. The NGS was derived from a

donor herd of goats certified to have been pathogen free

at the time of bleeding and was essential for blocking

non-specific binding by the primary and secondary

antibodies.Full depth equine cartilage sections were incubated

overnight at 4 �C with the AQP1-3 antibodies diluted

1:200 in PBS containing 2% NGS. Subsequently, the

sections were rinsed 3� 15 min in PBS before incubation

with the secondary peroxidase-conjugated antibodies

(peroxidase-labelled polymer conjugated to goat anti-

rabbit immunoglobulins in Tris–HCl buffer containing

bovine serum albumin as a carrier protein) for a mini-mum of 1 h at RT. After rinsing in PBS (4� 15 min) the

peroxidase product was developed using DAKO Liquid

DAB+ Chromogen (3,30-diaminobenzidine chromogen

solution) which produces an insoluble brown coloured

product in the presence of the antibody–polymer com-

plex. Sections were counterstained with Mayer�s hae-

matoxylin, dehydrated in a series of ethanol baths

before being mounted in DPX and photographed usinga Nikon Microphot-FX microscope fitted with a Nikon

DXM1200 digital camera.

2.5. Equine chondrocyte isolation and FACS analysis

Equine chondrocytes were isolated by overnight di-

gestion of cartilage shavings in Dulbecco�s modified

Eagle�s medium (DMEM) supplemented with 5% fetalcalf serum, 1% antibiotic/antimycotic solution and

0.05% clostridial collagenase. Cell suspensions were

fixed for 10 min in 3.7% neutral buffered paraformal-

dehyde in PBS for 10 min at 37 �C. The fixed cells were

washed three times with PBS and incubated in 10%

NGS in PBS to quench non-specific binding sites.

Aliquots of 105 cells /100 lL PBS were placed into 1.5

mL Eppendorf tubes, spun down (2000 rpm, 5 min) andthe cell pellets were incubated with 100 lL aliquots of

AQP antibodies (AQP1, AQP2 and AQP3) at 4 �Covernight. Negative controls were incubated with 100

lL of a non-immune rabbit IgG (DAKO). Following

three washes with PBS, the cells were incubated with 100

lL FITC-conjugated goat anti-rabbit IgG diluted 1:100

(Sigma, UK) in PBS at 4 �C for 1 h in the dark. After

three further washes in PBS, the cells were resuspendedin 0.5 mL PBS and analyzed on a Becton–Dickinson

FACScan. Plot profiles and histograms were generated

using WinMDI flow cytometry software (http://facs.

scripps.edu/software.html). Statistical analysis was also

performed using WinMDI.

2.6. Western blotting with AQP1, AQP2 and AQP3

antibodies

Freshly isolated equine chondrocytes were washed

three times with serum-free DMEM and three times

with PBS. Total proteins were extracted with lysis buffer

(50 mM Tris–HCl, pH 7.2/150 mM NaCl/1% (v/v) Tri-

ton X-100/1 mM sodium orthovanadate/50 mM sodium

pyrophosphate/100 mM sodium fluoride/0.01% (v/v)

aprotinin/4 lg/mL pepstatin A/10 lg/mL leupeptin/1mM PMSF) on ice for 30 min. Insoluble material was

removed by centrifugation at 10 000g for 30 min. Ly-

sates were stored at )70 �C until use. For immuno-

blotting, equal amounts of total proteins were separated

on 12% polyacrylamide gels by SDS/PAGE under re-

ducing conditions. Proteins were transferred overnight

at 30 V onto nitrocellulose (Schleicher & Sch€ull) using a

mini transblot electrophoresis apparatus (Sigma).Membranes were blocked with 5% (w/v) skimmed-milk

powder in PBS/0.1% Tween 20 (blocking buffer) over-

night at 4 �C and incubated with primary antibodies to

AQP1 (1:500), AQP2 (1:500) and AQP3 (1:1000) diluted

in blocking buffer for 1 h at RT. After five washes in

blocking buffer, membranes were incubated with per-

oxidase-conjugated secondary antibody diluted in

blocking buffer for 30 min at RT. Membranes were fi-nally washed five times in blocking buffer, and devel-

oped using an Amersham Biosciences ECL enhanced

chemiluminescence kit.

3. Results

Immunohistochemical studies revealed high levels ofAQP1 expression in equine articular cartilage, particu-

larly in the deep and calcified zones of articular cartilage

(Fig. 1). The vasopressin-regulated AQP2 was not de-

tected in any of the equine articular cartilage zones ex-

amined (Fig. 1E–H). AQP3 was moderately expressed in

equine articular cartilage, particularly in the deep and

calcified zones (Fig. 1I–L). Full depth sections of equine

articular cartilage revealed that in addition to articularchondrocytes AQP1 is abundantly expressed in cells

bearing the morphological characteristics of osteocytes

in the subchondral bone (Fig. 1D). AQP3 was also ex-

pressed in low quantities in cells resembling osteocytes

in the subchondral bone (Fig. 1L).

In order to demonstrate that the polyclonal anti-

bodies were specific to the aquaporin isoform against

Fig. 1. Immunohistochemical localization of AQP1 and AQP3 in all zones of normal equine articular cartilage including superficial, middle, deep and

calcified cartilage. The cartilage was obtained from the normal femoropatellar joint of a 5-year-old animal. Although AQP1 expression was evident in

chondrocytes of all zones (brown peroxidase staining in A–D) it was most abundantly expressed in cells of the deep zone (C). AQP2 expression was

not detected in any of the equine cartilage zones (E–H). AQP3 expression was expressed in lower quantities compared to AQP1 but it was present in

large quantities in deep zone chondrocytes (K). Moderate AQP1 and AQP3 expression was evident in bone cells in the calcified zone and subchondral

bone (D, L). Bars indicate 10 lm.

146 A. Mobasheri et al. / The Veterinary Journal 168 (2004) 143–150

which they were raised, we performed immunohisto-chemistry on formalin fixed, paraffin embedded sections

of normal ovine kidney. Due to the difficulties associ-

ated with obtaining fresh equine kidneys, we decided to

use fresh ovine kidneys as positive for AQP1, AQP2 and

AQP3 in the nephron. Fig. 2 shows expression of AQP1,

AQP2 and AQP3 in ovine renal cortex, medulla and

papilla. Abundant AQP1 immunostaining was observed

in the apical membranes of the proximal tubule and thedescending thin limb (as revealed by brown coloured

peroxidase staining in sites of AQP expression). AQP2

was expressed in the apical membrane domain of prin-

cipal cells in collecting ducts. AQP3 expression was

observed in lower quantities in basolateral membranes

of collecting ducts; labelling was mainly restricted to the

cortical, outer medullary, and inner medullary collecting

ducts.Quantitative FACS analysis demonstrated that iso-

lated equine chondrocytes express AQP1 and AQP3

(Fig. 3). Specific AQP1 fluorescence was detected

in 41.84% of the cells. AQP2 fluorescence was detected

in 1.13% of the cells (not shown). AQP3 was detected in

29.31% of the cells shown in the gated area (labelled R2

in Fig. 4A). These results confirm that antibodies to ratAQP1, AQP2 and AQP3 cross-react with equine AQPs.

Western blotting of freshly isolated equine articular

chondrocyte lysates was used to demonstrate the pres-

ence of AQP1 and AQP3 proteins. Fig. 4. shows the

Western blot data confirming AQP1 and AQP3 ex-

pression in equine chondrocytes. AQP2 expression was

not detected. We also confirmed the molecular weight of

AQP1 in equine chondrocytes; the main AQP1 bandwas approximately 28 kDa; the larger band in the AQP1

lane probably corresponds to the highly glycosylated

form of the AQP1 protein. AQP3 was detected as a

single protein migrating at around 28–30 kDa.

4. Discussion

Aquaporins belong to a large gene family of trans-

membrane proteins that form water permeable channels,

which may also be permeated by other small solutes.

Aquaporins are expressed in a variety of water trans-

porting epithelia and many other tissues where they fa-

cilitate water and solute transport across the cell

Fig. 2. Expression of AQP1, AQP2 and AQP3 in the ovine renal cortex, medulla and papilla. AQP1 was present in the apical (brush border)

membrane domain of proximal tubule segments (as indicated by brown peroxidase staining). AQP1 immunolabelling was also observed in the

squamous epithelium lining the descending thin limbs of the ovine loop of Henle which accounts for the high water permeability of this nephron

segment in the medulla (B) and to a lesser extent, the papilla (C). AQP1 expression was not detected in the thick ascending limb and medullary

collecting ducts. Expression of AQP2 was significantly lower than AQP1 and appeared to be particularly restricted to the apical membrane domain of

cells lining the distal nephron (cortical, medullary and papillary collecting ducts; D–F). AQP3 expression was more intense in the cortex and medulla

compared to the papilla. AQP3 expression was observed in basolateral membranes of the cortical and medullary collecting ducts (G, H).

A. Mobasheri et al. / The Veterinary Journal 168 (2004) 143–150 147

membrane (Borgnia et al., 1999; Fushimi and Marumo,

1995; Nielsen and Agre, 1995). It is generally accepted

that AQP1 is the ubiquitously expressed, archetypal

water channel (Agre et al., 1993; Denker et al., 1998;

Nielsen et al., 1993; Sabol�ıc et al., 1992). AQP2 is avasopressin-regulated water channel expressed in renal

medullary collecting ducts (Fushimi et al., 1994; Nielsen

et al., 1993; Sabol�ıc et al., 1995). Unlike AQP1, which is

constitutively present in the plasma membrane, AQP2

abundance is regulated by vasopressin through short-

term exocytosis to the membrane and protein recycling

(Marples, 2001). AQP3 is a multifunctional channel

protein permeable to water, glycerol, urea and othersolutes (Ishibashi et al., 1994; Ma et al., 1994).

Although a great deal is known about the expression,

regulation and physiological function of aquaporins in

absorptive and secretory epithelia, thus far, there are

only three indirect reports of aquaporin expression in

skeletal cells. Early developmental studies in the rat have

demonstrated abundant AQP1 mRNA in the mesen-

chyme surrounding developing calcified bone (Bondy

et al., 1993). AQP1 has been found in the periosteum of

the guinea-pig inner ear (Stankovic et al., 1995). In ad-dition AQP1 and AQP3 have been identified in devel-

oping human teeth and selected orofacial tissues (Wang

et al., 2003). There are no reports of aquaporins in

articular chondrocytes from articular cartilage of load-

bearing joints despite the potential importance of aqu-

aporins in volume regulatory chondrocyte responses

load-induced to ionic and osmotic changes in their ex-

tracellular environment.Chondrocytes have been shown to be particularly

sensitive to their physicochemical environment (Guilak,

2000, 1995; Guilak et al., 2002). In response to osmotic

stresses that are often secondary to mechanical com-

pression of cartilage, chondrocytes re-organize their

Fig. 3. FACS analysis of isolated equine chondrocytes probed with polyclonal antibodies to AQP1, AQP3 and secondary FITC-conjugated anti-

rabbit IgG. (A) Scatter plot of starting chondrocyte population based on cell size and granularity. (B) Background fluorescence from control

chondrocytes (incubated with FITC–conjugated secondary IgG only). (C) Scatter plot of chondrocytes probed with AQP1 antibodies. (D) Specific

fluorescence from cells incubated with antibodies to AQP1. (E) Scatter plot of chondrocytes probed with AQP3 antibodies. (F) Specific fluorescence

from cells incubated with antibodies to AQP3. AQP1 and AQP3 were expressed in 41.84% and 29.31% of the cells, respectively. The peaks shown

under the M1 bar in D and F represents cells exhibiting a specific fluorescence signal above the pre-set threshold limit shown under the M1 bar in B.

Antibodies to AQP2 did not result in any specific staining or fluorescence signal (results not shown).

148 A. Mobasheri et al. / The Veterinary Journal 168 (2004) 143–150

cytoskeleton and activate plasma membrane transport

mechanisms to regulate normal cell volume. This regu-lation is essential for the maintenance of the extracel-

lular matrix since the metabolic activity of chondrocytes

is influenced by alterations in the osmotic environment

of cartilage tissue (Guilak et al., 2002). The transport of

water across the chondrocyte plasma membrane con-

stitutes an important component of volume regulatory

activities during recovery from swelling and shrinkage.

In view of this potentially important function and the

apparent lack of published information, in this study,

we examined the expression of three aquaporins (AQP1,AQP2 and AQP3) in sections of articular cartilage and

freshly isolated chondrocytes and showed that AQP1

and AQP3 are present in chondrocytes.

The immunohistochemical, FACS and Western blot

analyses presented in this paper strongly indicate that

AQP1 and AQP3 are expressed in equine articular car-

tilage and isolated equine chondrocytes. The immuno-

histochemistry data presented clearly show plasma

Fig. 4. Western blot analysis of AQP1 and AQP3 expression in equine

articular chondrocytes. The Western blot data confirm the presence of

AQP1 and AQP3 but reveal the absence of AQP2 expression in equine

chondrocytes. The molecular weight of AQP1 in equine chondrocytes

was confirmed to be approximately 28 kDa; larger bands in the AQP1

lane may correspond to the highly glycosylated form of the AQP1

protein. The AQP3 protein was detected as a single protein of 28–30

kDa.

A. Mobasheri et al. / The Veterinary Journal 168 (2004) 143–150 149

membrane AQP1 and AQP3 expression in chondro-

cytes. Chondrocytes in the deep zone of articular carti-

lage exhibited the strongest AQP1 and AQP3 expression

levels. These studies confirm our preliminary observa-

tions in human and porcine articular cartilage (Trujillo

et al., 2000) and support a role for an aquaporin (AQP1)and an aquaglyceroporin (AQP3) in water and small

osmolyte transport across the plasma membranes of

these cells, respectively. AQP1 may be responsible for

conferring water permeability to chondrocytes and may

play an important role in allowing chondrocytes to re-

spond to changes in their ionic and osmotic environ-

ment. AQP3 may be involved in the transport of water,

glycerol and structurally related small osmolytes acrossthe chondrocyte membrane. Recent studies in peritoneal

mesothelial cells indicate AQP1 expression is up-regu-

lated by osmotic agents such as glucose (Lai et al., 2001).

In addition, studies in cultured human keratinocytes

show that osmotic stress up-regulates AQP3 gene ex-

pression (Sugiyama et al., 2001). Thus, AQP1, AQP3

and other AQPs may enable chrondrocytes to respond

to changes in their osmotic environment by regulatingcell volume.

In conclusion, we have shown that AQP1 and AQP3

are expressed in equine articular chrondrocytes. These

water channel proteins are likely to play a critical role in

the regulation of intracellular homeostasis in chron-

drocytes and may be important in allowing chondro-

cytes to respond to changes in their ionic and osmotic

environment by volume regulatory behaviour. Thepresence of AQP1 in equine chondrocytes supports a

role for AQP1 in water transport in across the chon-

drocyte membrane. The water being transported across

the chondrocyte membrane is likely to be metabolic or

extracellular matrix water. AQP3 may be involved in

water and solute transport in equine chondrocytes.

Based on the data presented, we propose a novel hy-

pothesis that extracellular matrix properties may influ-ence the chondrocyte�s internal environment and vice

versa and expression of sodium and water transporters

is a manifestation of such physiological adaptation. It is

possible that the expression of AQPs is altered when

chondrocytes are removed from their surrounding ex-tracellular matrix. It is also possible that mechanical and

osmotic pressure and consequent effects on the osmotic

environment of the cells will result in alterations in

chondrocyte AQPs. Future studies will focus on the

expression of AQP1, AQP3 and other members of the

AQP gene family in pathologies of articular cartilage

and their sensitivity to mercurial and stilbene derived

compounds capable of interfering with AQP function.

Acknowledgements

Supported by grants from the University of Liverpool

Research Development Fund (AM), the Medical Re-

search Council (DM) and Programa Sectorial de Pro-

mocion General del Conocimiento, Ministerio deEducacion y Cultura, Spain (ET, P M-V). The authors

wish to thank Dr. Alan Verkman (Cardiovascular Re-

search Institute, University of California at San Fran-

cisco) for helpful suggestions.

References

Agre, P., Saboori, A.M., Asimos, A., Smith, B.L., 1987. Purification

and partial characterization of the Mr 30,000 integral membrane

protein associated with the erythrocyte Rh(D) antigen. Journal of

Biological Chemistry 262, 17497–17503.

Agre, P., Preston, G.M., Smith, B.L., Jung, J.S., Raina, S., Moon, C.,

Guggino, W.B., Nielsen, S., 1993. Aquaporin-CHIP: the archetypal

molecular water channel. American Journal of Physiology 265,

F463–F476.

Bondy, C., Chin, E., Smith, B.L., Preston, G.M., Agre, P., 1993.

Developmental gene expression and tissue distribution of aquapo-

rin-CHIP. Proceedings of the National Academy of Sciences of the

United States of America 90, 4500–4504.

Borgnia, M., Nielsen, S., Engel, A., Agre, P., 1999. Cellular and

molecular biology of the aquaporin water channels. Annual

Review of Biochemistry 68, 425–458.

Buckwalter, J.A., Mankin, H.J., 1998. Articular cartilage: tissue design

and chondrocyte–matrix interactions. Instructional Course Lec-

tures 47, 477–486.

Denker, B.M., Smith, B.L., Kuhajda, F.P., Agre, P., 1998. Identifi-

cation, purification, and partial characterization of a novel Mr

28,000 integral membrane protein from erythrocytes and renal

tubules. Journal of Biological Chemistry 263, 15634–15642.

Fushimi, K., Sasaki, S., Yamamoto, T., Hayashi, M., Furukawa, T.,

Uchida, S., Kuwahara, M., Ishibashi, K., Kawasaki, M., Kihara, I,

et al., 1994. Functional characterization and cell immunolocaliza-

tion of AQP-CD water channel in kidney collecting duct. American

Journal of Physiology 267, F573–F582.

Fushimi, K., Marumo, F., 1995. Water channels. Current Opinion in

Nephrology and Hypertension 4, 392–397.

Guilak, F., 2000. The deformation behavior and viscoelastic properties

of chondrocytes in articular cartilage. Biorheology 37, 27–44.

Guilak, F., Ratcliffe, A., Mow, V.C., 1995. Chondrocyte deformation

and local tissue strain in articular cartilage: a confocal microscopy

study. Journal of Orthopaedic Research 13, 410–421.

Guilak, F., Erickson, G.R., Ting-Beall, H.P., 2002. The effects of

osmotic stress on the viscoelastic and physical properties of

articular chondrocytes. Biophysical Journal 82, 720–727.

150 A. Mobasheri et al. / The Veterinary Journal 168 (2004) 143–150

Ishibashi, K., Sasaki, S., Fushimi, K., Uchida, S., Kuwahara, M.,

Saito, H., Furukawa, T., Nakajima, K., Yamaguchi, Y., Gojobori,

T., et al., 1994. Molecular cloning and expression of a member of

the aquaporin family with permeability to glycerol and urea in

addition to water expressed at the basolateral membrane of kidney

collecting duct cells. Proceedings of the National Academy of

Sciences of the United States of America 91, 6269–6273.

Lai, K.N., Li, F.K., Lan, H.Y., Tang, S., Tsang, A.W., Chan, D.T.,

Leung, J.C., 2001. Expression of aquaporin-1 in human peritoneal

mesothelial cells and its upregulation by glucose in vitro. Journal of

the American Society for Nephrology 12, 1036–1045.

Ma, T., Frigeri, A., Hasegawa, H., Verkman, A.S., 1994. Cloning of a

water channel homolog expressed in brain meningeal cells and

kidney collecting duct that functions as a stilbene-sensitive glycerol

transporter. Journal of Biological Chemistry 269, 21845–21849.

Marples, D., 2001. Aquaporins: roles in renal function and peritoneal

dialysis. Peritoneal Dialysis International 21, 212–218.

Mobasheri, A., Mobasheri, R., Francis, M.J., Trujillo, E., Alvarez De

La Rosa, D., Martin-Vasallo, P., 1998. Ion transport in chondro-

cytes: membrane transporters involved in intracellular ion homeo-

stasis and the regulation of cell volume, free [Ca2þ] and pH.

Histology and Histopathology 13, 893–910.

Mobasheri, A., Carter, S.D., Mart�ın-Vasallo, P., Shakibaei, M., 2002.

Integrins and stretch activated ion channels: putative components

of functional cell surface mechanoreceptors in articular chondro-

cytes. Cell Biology International 26, 1–18.

Nielsen, S., Agre, P., 1995. The aquaporin family of water channels in

kidney. Kidney International 48, 1057–1068.

Nielsen, S., Smith, B.L., Christensen, E.L., Agre, P., 1993. Distri-

bution of the aquaporin CHIP in secretory and resorptive

epithelia and capillary endothelia. Proceedings of the National

Academy of Sciences of the United States of America 90, 7275–

7279.

Sabol�ıc, I., Valenti, G., Verbavatz, J.M., Van Hoek, A.N., Verkman,

A.S., Ausiello, D.A., Brown, D., 1992. Localization of the CHIP28

water channel in rat kidney. American Journal of Physiology 263,

C1225–C1233.

Sabol�ıc, I., Katsura, T., Verbavatz, J.M., Brown, D., 1995. The AQP2

water channel: effect of vasopressin treatment, microtubule disrup-

tion, and distribution in neonatal rats. Journal of Membrane

Biology 143, 165–175.

Stankovic, K.M., Adams, J.C., Brown, D., 1995. Immunolocalization

of aquaporin CHIP in the guinea pig inner ear. American Journal

of Physiology 269, C1450–C1456.

Sugiyama, Y., Ota, Y., Hara, M., Inoue, S., 2001. Osmotic stress up-

regulates aquaporin-3 gene expression in cultured human kerati-

nocytes. Biochimica et Biophysica Acta 1522, 82–88.

Trujillo, E., Marin, R., Masrani, B., Marples, D., Martin-Vasallo, P.,

Mobasheri, A., 2000. Aquaporin water channels in porcine and

human articular cartilage; differential expression in pathologies of

human cartilage. International Journal of Experimental Pathology

81, A37 (abstract).

Wang, W., Hart, P.S., Piesco, N.P., Lu, X., Gorry, M.C., Hart, T.C.,

2003. Aquaporin expression in developing human teeth and

selected orofacial tissues. Calcified Tissue International 72,

222–227.