Cell Biology International 1999, Vol. 23, No. 4, 237–240Article No. cbir.1999.0405, available online at http://www.idealibrary.com on

MINI-REVIEW

Epithelial sodium channels in skeletal cells; a role in mechanotransduction?

*To whom correspondence should be addressed: Dr A. Mobasheri,Senior Lecturer in Cellular & Molecular Biology, Department ofBiomedical Sciences, School of Biosciences, University ofWestminster, 115 New Cavendish Street, London W1M 8JS, U.K.;E-mail: mobasha@westminster.ac.uk

The amiloride sensitive epithelial sodium channel(ENaC) is a multimeric system consisting of threesubunits, �, � and � involved in apical Na+ uptakein a variety of epithelia (Canessa et al., 1993,1994). Epithelial sodium channels belong to theMEC/DEG/ENaC superfamily whose members areinvolved in diverse functions including acidsensing, maintenance of sodium homeostasis andtransduction of mechanical stimuli and nociceptivepain (Fyfe et al., 1998). In the epithelia of the renaltubule and the distal colon amiloride sensitiveepithelial sodium channels represent the rate-limiting step for sodium reabsorption (Rossieret al., 1994). ENaC is a major participant in thefine regulation of systemic sodium balance, bloodvolume and blood pressure and also plays a keyrole in lung fluid clearance in neonates (Hummleret al., 1996). Long before the identification andcloning of the ENaC superfamily gene members inmammalian species a number of genes required formechanosensitivity had already been identified inthe nematode worm genetic model Caenorhabditiselegans. Deletions in the mechanosensitive genesMEC-4 and MEC-10 and the degenerin geneDEG-1 rendered the invertebrates insensitive totouch and prone to neurodegeneration. The re-

1065–6995/99/040237+04 $30.00/0

markable structural similarity between mammalianENaC members and the C. elegans MEC/DEGgenes indicated that MEC and DEG encoded ionchannels and gave strong support to the hypoth-esis that mammalian ENaC genes may also beinvolved in the process of mechanotransduction(Tavernarakis and Driscoll, 1997).

The current information on ion channels inskeletal is relatively limited. However, it has beenestablished that skeletal cells such as osteoblastsand chondrocytes express a number of ion channelswith different biophysical and pharmacologicalproperties (for a review see Mobasheri et al., 1998and Duncan et al., 1998). In osteoblastic cells ingeneral, Ca2+ channels have been shown to playfundamental roles in cellular responses to externalstimuli including both mechanical forces andhormonal signals. Ca2+ channels are also proposedto modulate paracrine signalling between bone-forming osteoblasts and bone-resorbing osteoclastsat local sites of bone remodeling. Calcium signalsare characterized by transient increases in intracel-lular Ca2+ levels that are associated with activationof intracellular signaling pathways that control cellbehaviour and phenotype, including patterns ofgene expression. Development of Ca2+ signals is atightly regulated cellular process that involves theconcerted actions of plasma membrane and intra-cellular Ca2+ channels, along with Ca2+ pumpsand exchangers. Osteoblasts and chondrocyteshave been shown to be mechano-sensitive cells. Theprocess of anabolism and catabolism regulated bymechanical loading is a second-to-second, minute-to-minute, and hour-to-hour process that workstogether with local and systemic hormones to en-sure that the tissue can meet the demands of themechanical environment. Mechanical strain orloading acts as a mechanostimulant. Osteoblastsfor example respond to intermittent mechanicalstrain by increasing their mitotic rate and produc-tion of type I collagen extracellular matrix (Harteret al., 1994) and stretch-activated cation channelactivation (Duncan and Hruska, 1994). Similarlychondrocytes respond to mechanical loading by

Ali Mobasheri1* andPablo Martın-Vasallo2

1School of Biosciences,University of Westminster,London W1M 8JS, U.K.2Departamento de Bioquımicay Biologıa Molecular,Universidad de La Laguna,38206 La Laguna,Tenerife, Spain

� 1999 Academic Press

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Fig. 1. Expression of the � and � subunits of ENaC in human chondrocyte-like cells. The � subunit appears to be localized inthe plasma membrane whereas the expression of the � subunit is predominantly intracellular. Bars represent 10 �m (approx.).

decreasing production of extracellular matrixmacromolecules such as aggrecan (Buschmannet al., 1996). The metabolic response to mechanicalstrain led a number of groups to hypothesize thatfollowing physiological mechanostimulation theprocesses of mechanoreception and mechanotrans-duction may involve the cellular cytoskeleton(Wang et al., 1993; Ingber, 1997) and plasmamembrane ion channels (Cantiello, 1995a).

We have been interested in determining themolecular identity and subunit composition of ionchannels in skeletal cells. Recent studies performedby Killick and Richardson (1997) have establishedthat avian chondrocytes express �ENaC; Northernblot experiments clearly indicate that a 2.5-kbtranscript of ENaC is present in avian cartilage.Elegant studies by Kizer and colleagues (1997)have demonstrated expression of �ENaC at thetranscriptional and translational levels in an estab-lished rat osteoblast-like cell line (UMR-106) andprimary cultures of human bone marrow stromalcells. Using electrophysiological techniques theyalso showed that osteoblasts stably transfected withmammalian expression vector encoding �ENaCexpress a stretch activated, non-selective cationchannel that is only active when negative pressureis applied to cell-attached patches. The abovestudies prompted us to investigate the expression ofENaC subunits in human chondrocytes and osteo-blasts in vitro and in vivo. Chondrocytes are notonly mechano-sensitive, but also they are close

relatives of osteoblasts and are also derived frommesenchymal progenitor cells (Grigoriadis et al.,1988). Using polyclonal antibodies raised againstthe �, � and � subunits of ENaC (Duc et al., 1994)we have obtained strong evidence for the presenceof � and � ENaC subunits in situ in humanchondrocytes (Trujillo et al., 1998, 1999) and in ahuman chondrocyte cell line (Mobasheri et al.,1999; see Fig. 1). Using a similar approach we haveidentified � and � ENaC in human femoral bonederived osteoblasts (Mobasheri et al., unpublishedobservations). ENaC expression is therefore notstrictly limited to epithelia. Indeed, ENaC expres-sion has also been demonstrated in epidermalkeratinocytes surrounding touch sensitive hair fol-licles (Roudier-Pujol et al., 1996). All this evidencepoints to the presence of epithelial sodium channelsin the plasma membranes of mechanosensitive cellsand leads us to hypothesize that ENaC or itshomologues may mediate mechanotransduction inskeletal cells. Further support for this hypothesiscomes from cloning studies of unc105, another C.elegans homologue that is believed to interact withtype IV collagen, providing evidence that mech-anostimulation of the extracellular matrix may betransduced into electrochemical signals within thecell. The extracellular domain of the proteinencoded by unc-105 probably interacts with colla-gen on the extracellular face and mechanical forcesexerted on the collagen network could open thechannel (Liu et al., 1996).

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Remarkably, the expression and distribution ofepithelial sodium channels appears to vary inpathologies of human articular cartilage. Althoughchondrocytes from normal cartilage express � and� ENaC, in osteoarthritis, ENaC seems to beabsent altogether. In contrast, in rheumatoidcartilage ENaC expression is upregulated (Trujilloet al., 1999). The significance of these findings isthat the altered extracellular matrix and/or theinflammatory response in degenerate cartilageappear to have a direct influence on the expressionand abundance of stretch-activated ENaC. Thus, interms of cellular pathophysiology our results sug-gest that the expression and abundance of epithelialsodium channels is altered in pathologies ofarticular cartilage.

The � subunit of ENaC shows sequence homol-ogy with a group of actin-binding proteins knownas dystrophins. The � and � dystrophins are impli-cated in the ability of muscle cells to contract.Interestingly dystrophins are also members of anactin-binding family including spectrin and fodrin(Ervasti and Campbell, 1993). Studies on renalepithelial cells have demonstrated that the epi-thelial sodium channel is linked to the cytoskeleton(Smith et al., 1991). Thus, if ENaC was alsodemonstrated to be physically and functionallylinked to the cellular cytoskeleton in skeletal cells itwould perfectly fulfill the criteria for a mechano-sensitive ion channel. This hypothesis of cyto-skeletal regulation is partially supported by at leastthree other ion transport proteins. The catalytic �subunit of the Na+, K+-ATPase is connected to thecytoskeleton via attachments to ankyrin and fodrin(Morrow et al., 1989) and the enzyme itself hasbeen shown to bind actin in a way that its ATPhydrolytic activity is enhanced (Cantiello, 1995b).Stimulation of the Na+/H+ exchanger by serum isregulated by F-actin (Watson et al., 1992) and theband-3 anion exchanger AE is attached to thecytoskeleton via ankyrin and spectrin in the ratkidney and cultured epithelial cells (Drenckhahnet al., 1985).

In terms of ion channel cell biology the findingsdescribed raise a number of important and funda-mental questions: for example, does ENaC func-tion as a stretch-activated ion channel in vivo as itdoes in vitro? Given that Na+ is the most abundantcation in the extracellular matrix of articular carti-lage is ENaC specific for Na+ or is it unable todiscriminate cations? Is ENaC attached to thecytoskeleton in skeletal cells? Does the cytoskeletonor the extracellular matrix regulate ENaC? Is itpossible to interfere with ENaC function by usingcompounds that disassemble the cellular cyto-

skeleton? Does ENaC contribute to the expressionof a mechanosensitive channel complex in skeletalcells? Are there any other members of the ENaCsuperfamily present in skeletal cells? If so, do theyplay a role in normal growth and skeletal develop-ment? Whether ENaC functions as a stretch-activated channel or contributes to the expressionof a mechanosensitive complex in skeletal cells is achallenging question and remains to be determined.

In summary, we feel that the discovery ofepithelial sodium channels in osteoblasts andchondrocytes has reinvigorated interest in skeletalmechanotransduction. Although the direct involve-ment of ENaC in transducing mechanical signals inthe Xenopus laevis oocyte or planar lipid bilayersremains controversial (Rossier, 1998) studies inmechanically responsive cells such as osteoblastsand chondrocytes should provide a more suitablephysiological model for studying mechanotrans-duction. Furthermore, we regard skeletal cellssuperior to cultured epithelial cell systems forstudies of mechanostimulation, mechanotransduc-tion and for elucidating the detailed molecularanatomy of interactions between ion channels,the cytoskeleton and extracellular matrix macro-molecules.

ACKNOWLEDGEMENTS

We wish to thank our collaborator Dr CeciliaCanessa (Molecular & Cellular Physiology, YaleUniversity School of Medicine) for making thiswork possible. We also thank our colleague DrMartin Francis (Nuffield Orthopaedic Centre,Oxford University) for critically reading the manu-script before submission. Our work is supportedby FIS, Spain (P. M.-V.), Intersep Limited, ThePhysiological Society, U.K. and the TeachingCompany Directorate, Department of Trade &Industry, U.K. (A.M.).

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