Epithelial Sodium Channels are Upregulated DuringEpidermal Differentiation

Yuko Oda, Ashkan Imanzahrai, Angela Kwong, Laszlo Komuves, Peter M. Elias, Corey Largman, andTheodora MauroDepartments of Medicine and Dermatology, University of California San Francisco, VA Medical Center San Francisco, San Francisco, California,U.S.A.

Terminal differentiation of epidermal keratinocytes is -β increased during calcium-induced keratinocyte dif-ferentiation. Whereas the β subunit of human epitheliallinked to transmembrane ion flux. Previously, we have

shown that amiloride, an inhibitor of epithelial sodium sodium channel was induced by elevated concentra-tions of calcium, the α subunit increased with durationchannels, blocks synthesis of differentiation-specific

proteins in normal human keratinocytes. Here, we of culture. The regulatory γ subunit was less abundantbut also expressed in epidermis. Both human epithelialhave identified the specific subunits of amiloride-

sensitive human epithelial sodium channels in relation sodium channel-α and -β were localized throughoutthe nucleated layers of human adult epidermis, butto differentiation of cultured human keratinocytes, as

well as to epidermal development. As assessed by these channels were not detected in early stages offetal epidermal development. This co-ordinatednorthern hybridization, RNase protection assay, and

reverse transcription–polymerase chain reaction, tran- expression of subunits suggests that epithelial sodiumchannels may play an important part in both epidermalscripts encoding functional α and regulatory β subunits

of human epithelial sodium channels were expressed differentiation and skin development, presumably bymodulating ion transport required for epidermal ter-both in cultured keratinocytes and in epidermis at

levels comparable with the kidney. The mRNA expres- minal differentiation. Key words: amiloride/epidermis/ionchannel/keratinocyte. J Invest Dermatol 113:796–801, 1999sion of both human epithelial sodium channel-α and

P channel (Grando et al, 1995), and cyclic guanosine monophosphategated channel (Oda et al, 1997). In addition, pharmacologicexperiments have suggested that voltage-sensitive Ca21 channelscontrol lipid secretion in the last stages of differentiation (Lee et al,1994). Amiloride block the formation of cornified envelopes,

roliferation and differentiation of epidermal ker-atinocytes are linked to changes in ion concentrationin the epidermis. Gradients for Ca21 (Menon et al,1985; Mauro et al, 1998), Na1 (Warner et al, 1988a),Cl– (Warner et al, 1988a; Mauro et al, 1998), and K1

(Warner et al, 1988a; Mauro et al, 1998) are observed across the as well as the synthesis and activity of the calcium-dependentepidermis in vivo, with low levels in the basal and spinous layers. keratinocyte-specific isoform of transglutaminase, suggesting a keyCa21 concentrations increase through the upper granular layers role for an amiloride-sensitive channel in epidermal differentiationwhere the cells are differentiated whereas Na1 and K1 peak in the (Mauro et al, 1995).lower granular layers (Warner et al, 1988a; Mauro et al, 1998), Amiloride-sensitive epithelial sodium channels (ENaC) (reviewedsuggesting a role for these ions in the regulation of differentiation by Matolon et al, 1996; Garty and Palmer, 1997; Fyfe et al, 1998)in vivo. Differentiation of keratinocytes in vitro also is induced by first were shown to mediate active Na1 reabsorption through theincreasing extracellular Ca21 concentrations (Hennings et al, 1980; apical membrane of absorbing epithelial cells, including those ofPillai et al, 1990; Bikle and Pillai, 1993). Increases in intracellular the renal collecting duct, distal colon, and urinary bladder (GartyNa1 and K1 also are observed during keratinocyte differentiation, and Palmer, 1997). Moreover, this channel later was shown to beand agents which deplete intracellular K1 block calcium-induced essential for transport of water and salt in normal lung (Hummlerdifferentiation (Hennings et al, 1983). Some of the ion channels et al, 1996). ENaC-α-deficient mice die soon after birth becauseresponsible for early calcium-induced differentiation have been they cannot transport the water from their alveoli (Hummler et al,characterized previously, including a nonselective cation channel 1996, 1997). ENaC also might mediate mechanosensation (Awayda(Mauro et al, 1993), K1 channel (Mauro et al, 1997), nicotinic et al, 1995; Kizer et al, 1997) as suggested by their sequence

homology with degenerins, a class of proteins that mediates touchsensitivity in the nematode, Caenorhabditis elegans (Corey andGarcia-Anoveros, 1996).

Manuscript received February 4, 1999; revised June 25, 1999; accepted ENaC channels are composed of different homologous proteins,for publication July 12, 1999. denoted as the α, β, and γ subunits (Garty and Palmer, 1997). TheReprint requests to: Dr. Yuko Oda, Department of Dermatology (190),

activity of the functional α subunit is enhanced by coexpression ofVeterans Affairs Medical Center, San Francisco, 4150 Clement Street, Santhe β or γ regulatory subunits which alone are not active (GartyFrancisco, CA 94121, U.S.A. Email: y2073@itsa.ucsf.eduand Palmer, 1997). All of the ENaC channels appear to be highlyAbbreviations: ENaC, epithelial sodium channel; GAPDH, glyceral-

dehyde-3-phosphate dehydrogenase. conserved in vertebrates, because they already are expressed in

0022-202X/99/$14.00 · Copyright © 1999 by The Society for Investigative Dermatology, Inc.

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molecular weight markers (RNA century marker, Ambion). Total RNAamphibian skin (Puoti et al, 1995), where Na1 is reabsorbedfrom normal human kidney, used as a control source of hENaC, wasthrough the skin. The function of ENaC in mammalian epidermis,obtained from Clontech.however, which is a nonabsorbing epithelium, is not clear. ENaC

have been detected in adult rat skin, in hair follicles, interfollicularReverse transcription–polymerase chain reaction (PCR)epidermis, and sweat glands, where they have been proposed toanalysis hENaC subunits expression was determined by reverse transcrip-mediate Na1 absorption (Roudier-Pujol et al, 1996). tion–PCR as described previously (Oda et al, 1997, 1998). The primer setIn this report, we identified ENaC subunits in normal human for the hENaC-α subunit was as above. For the hENaC-β subunit primers:

keratinocytes and in epidermis, suggesting that ENaC play a 59-CTG GGA CAT CTT CAA CTG GG-39 and 59-CTT TGG AGApart in keratinocyte differentiation and epidermal development. GGG CAC CAT AC-39 were used. For the hENaC-γ subunit, the primerFurthermore, the potential regulatory roles of the three channel set of 59-CAT GGG AAT TGC TAT ACT TTC-39 and 59-TGG AAG

CAT GAA TGA AGG CA-39 were used. Total RNA was isolated fromsubunits were clarified by comparing their expression duringcultured keratinocytes at various stages of differentiation or from normalkeratinocyte differentiation induced by changes in either theneonatal foreskin epidermis. Total cDNA pools were prepared by reverseconcentration of extracellular calcium and/or length of culturetranscription using Superscript II (Gibco BRL). Each cDNA pool was also(Bikle and Pillai, 1993). The expression and localization of ENaCamplified using primers for human GAPDH (Clontech) as a control forwere also compared during embryonic development of epidermis. cDNA loading in the PCR assay. Amplification was carried out using TaqThese findings, in conjunction with previous pharmacologic studies polymerase PLUS (Stratagene, La Jolla, CA) for 25 cycles for both ENaC

(Mauro et al, 1995), suggest that the keratinocyte ENaC channels and GAPDH, which was shown to be within the linear range for bothplay an important part in epidermal differentiation. PCR reactions. The PCR products were electrophoresed on a 3% agarose

gel, transblotted, and analyzed by southern hybridization using specificMATERIALS AND METHODS 32P-labeled cDNA probes for each hENaC subunit. Molecular weights of

the PCR product bands were estimated using φX174/HaeIII fragmentsCell culture Human epidermis was isolated from normal newborn(BRL) as standards.foreskin by dispase treatment, and primary keratinocyte cultures were

prepared as described (Oda et al, 1998). Keratinocyte suspensions wereMembrane protein preparations from keratinocytes andprepared from epidermal sheets by trypsinization. The cells were grownepidermis Plasma membranes were isolated from cultured keratinocytesin serum-free keratinocyte growth medium (Clonetics, Walkersville, MD),and whole epidermis as described (Oda et al, 1997, 1998). The cells orsupplemented with 0.07 mM Ca21 for 2 d, then switched to the sametissues were sonicated in homogenization buffer (20 mM Tris–HCl, pH 7.4,medium containing either 0.03 mM, 0.1 mM, or 1.2 mM Ca21 andcontaining 0.25 M sucrose, 4 mM MgCl2, 5 mM ethyleneglycol-bis-(b-cultured for different times before harvest.aminoethylether)-N,N,N9,N9,-tetraacetic acid, and protease inhibitors).The homogenate was sedimented at 48 000 3 g for 30 min, and theIsolation of partial cDNA clones for hENaC cDNA cloning andsoluble cytoplasmic fraction was removed. The membrane fraction wasDNA sequencing was performed as previously described (Oda et al, 1997;extracted with 1% deoxycholate acid, 1% Triton X-100, 0.1% sodiumOda et al, 1998). The hENaC cDNA fragments were isolated from humandodecyl sulfate and separated from the insoluble cytoskeleton fraction byepidermis by reverse transcription–PCR, and were subcloned into pCRIIcentrifugation at 48 000 3 g for 30 min. The membrane protein fractionor pCR2.1 (Invitrogen, Carlsbad, CA). The α subunit of hENaC was(50 µg) was denatured, and then incubated at 30°C overnight with orcloned using a set of specific primers 59-GAC AAG AAC AAC TCCwithout 0.5 U of PNGase F (Boehringer Mannheim, Indianapolis, IN)AAC CTC-39 and 59-ACA CTC CTT GAT CAT GCT CTC-39 designed(Oda et al, 1998).from lung hENaC-α (Voilley et al, 1994). The β subunit was cloned using

a set of degenerate primers derived from conserved regions of the rat,Western analysis of hENaC proteins Membrane protein concentra-human and frog hENaC-β channels (Garty and Palmer, 1997) 59-GG(A/tions were determined using the BCA protein assay (Pierce, Rockford,G/C/T) AA(C/T) TG(C/T) TA(C/T) AT(A/C/T) TT(C/T) AA(C/T)IL). Membrane proteins (50 µg) were electrophoresed in duplicate onTGG-39 and 59-(A/G)CA (A/G)TT (A/G)TG (A/G/T)AT CAT (A/7.5% polyacrylamide gels and electroblotted on to polyvinylidene difluorideG)TG (A/G)TC (C/T)TG (A/G)AA (G/A)CA-39, which were a giftmembranes (0.2 µm, BioRad Laboratories, Hercules, CA), and blockedfrom Dr C. Lau, UCSF. A γ subunit was isolated using a set of degeneratewith 5% milk in TBST (10 mM Tris–Cl, pH 7.5, 150 mM NaCl, andprimers designed from conserved amino acid sequences (G(S/N)CY(T/I/0.1% Tween-20). The blots were incubated overnight at 4°C with eitherV/M)FN and C(Y/L) (R/H) SC(F/Y)Q). These regions were selected bythe purified IgG fraction (20 µg per ml) of a polyclonal antisera againstalignment of 12 different ENaC-α, ENaC-β, and ENaC-γ sequencesfull length fusion protein of bovine hENaC-α generated from the cDNAderived from bovine, rat, frog, and human ENaC (Garty and Palmer,clone (gift from Dr. D.J. Benos, University of Alabama) which cross react1997). The degenerate primers used were 59-GGI AA(C/T) TG(C/T)with human hENaC-α. The specificity and cross-reactivity of the antibodyTA(C/T) ACI TT(C/T) AA-39 and 59-TG(A/G)AA(A/G)CAI GA(A/G)was assessed using both immunofluorescence and western blotting (IsmailovTGI AG(A/G) CA-39. The isolated cDNA clones were sequenced onet al, 1996; Awayda et al, 1997). Subsequently, the blots were incubatedboth strands using vector-specific primers with dye termination cyclewith a sheep anti-mouse secondary antibody conjugated with horseradishsequencing on an Applied Biosystems (Foster City, CA) 373A automatedperoxidase (Amersham, Uppsala, Sweden) for 1 h at room temperature.DNA sequencer. The DNA sequences were analyzed using the GCGThe bound horseradish peroxidase was visualized using an enhancedDNA analysis package at the Computer Graphics Laboratory of thechemiluminescence system (Amersham). The antibody specificity wasUniversity of California, San Francisco.confirmed by incubating a replicate blot with same concentration ofpreimmune IgG which was provided together with the antibody byNorthern hybridization Poly(A)1 RNA was isolated from culturedDr. Benos.keratinocytes or whole epidermis as described (Oda et al, 1997, 1998). The

RNA were electrophoresed and hybridized with ENaC subunit specific32P-labeled cDNA probes as previously described (Oda et al, 1997). A In situ hybridization Skin biopsies from adult and 10 wk old human376 bp DNA fragment for the α subunit, a 384 bp cDNA for the β fetal samples were fixed in 4% paraformaldehyde and embedded in paraffin.subunit, and 375 bp cDNA for the γ subunit were prepared by digestion Anti-sense and sense RNA probes were prepared from the same cDNAof the cloned cDNAs. The membranes were washed under stringent clones described above for northern hybridization, using pCRII dualconditions (0.1 3 sodium citrate/chloride buffer, 60°C). The same blot promoters. The hENaC cDNA clones were linearized and used as templateswas re-probed with a 32P-labeled cDNA for glyceraldehyde-3-phosphate to synthesize digoxigenin-labeled RNA probes, which were hybridizeddehydrogenase (GAPDH) (Clontech) as a control to estimate RNA loading. with the skin sections as previously described (Stelnicki et al, 1998). SignalsThe message size was estimated by comparison with molecular weight were visualized with a diaminobenzidine chromogen substrate. Sectionsmarkers (RNA ladder, 0.24–9.5 kb, Gibco BRL, Gaithersburg, MD). were counterstained with hematoxylin to visualize tissue morphology.

RESULTSRNase protection assay Anti-sense RNA probes were labeled with32P[UTP] by transcription of linearized cDNA templates using MAXIscript

All three hENaC subunits were expressed in human(Ambion, Austin, TX). Probes were purified by gel electrophoresis, andepidermis We first used reverse transcription–PCR to detect thehybridized with total RNA from cells or tissues. Probes were digestedhENaC subunits in human foreskin epidermis using specific orwith RNase A 1 T1 and analyzed on urea/4% polyacrylamide gels (Oda

et al, 1998). Protected RNA band sizes were estimated using RNA degenerate primers. The respective PCR fragments were subcloned

798 ODA ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

and sequenced. A 360 bp cDNA fragment for the α subunit wasobtained, using α-specific primers, whose sequence was identicalto human lung ENaC-α (930–1289) (Voilley et al, 1994). A 368 bpcDNA was isolated, using degenerate β-specific primers, whichwas identical to the human lung ENaC-β (940–1308) (Voilley et al,1995). Finally, reverse transcription–PCR was used with a set ofdegenerate primers, designed from conserved regions of 12 differentα, β, and γ sequences to isolate any related channels (see Materialsand Methods). A 359 bp PCR fragment cloned from this primer setwas identical to the γ subunit of kidney ENaC (865–1223)(McDonald et al, 1995; Voilley et al, 1995). These results demonstratethat human epidermis expresses all three ENaC subunits.

The three hENaC subunits are upregulated during ker-atinocyte differentiation In order to evaluate the potentialregulatory role of hENaC in epidermal differentiation, we nextused semiquantitative reverse transcription–PCR to examine theexpression of each of the three hENaC subunits during differenti-ation of cultured keratinoyctes. Well-defined changes in ker-atinocyte differentiation are induced both by culture confluencyand by progressively longer exposure to raised extracellular calcium(Younus and Bilchrest, 1992; Su et al, 1994; Gibson et al, 1996).Using the same conditions defined by Gibson et al (1996), wetested preconfluent (5 d of culture), near confluent (7 d), andpostconfluent (14 d) keratinocytes, cultured with low (0.03 mM),intermediate (0.1 mM), and high (1.2 mM) extracellular calcium.The α and γ subunits were expressed in undifferentiated ker-atinocytes and were upregulated by differentiation induced byextended confluent growth (Fig 1, upper and lower panels). Incontrast, the β subunit was not detected in undifferentiated cellsin keratinocytes, but appeared in fully differentiated cells byelevating calcium above 0.1 mM and under postconfluent condi-tions (Fig 1, middle panel, lanes 9 and 10). An independent

Figure 1. hENaC subunits of α, β, and γ were upregulated duringpreparation of keratinocytes showed reproducible results (data notkeratinocyte differentiation. Semi-quantitative reverse transcription–shown). Expression of the three hENaC subunits was also detectedPCR was used to analyze mRNA expression of hENaC. Normal humanin whole human epidermis (Fig 1, lane 11). The levels of control keratinocytes were grown in 0.07 mM Ca21 until 70% confluent (3 d)

message, GAPDH, detected by reverse transcription–PCR did not (lane 1) then switched to three different concentrations of Ca21 of 0.03 mMchange significantly among the samples used here (data not shown). (lanes 2, 5, and 8), 0.1 mM (lanes 3, 6, and 9), and 1.2 mM (lanes 4, 7,

We also evaluated the hENaC subunit expression levels quantitat- and 10). Total RNA was isolated on different confluency of the cells ofively by northern hybridization. Poly(A)1 RNAs were isolated from preconfluent (5 d), near confluent (7 d), and postconfluent (14 d) following

the calcium switch. The differentiation of these keratinocytes were definedundifferentiated keratinocytes (preconfluent, cultured in 0.03 mMby Gibson et al (1996). The protein expression of involucrin (INV) andCa21), differentiated keratinocytes (postconfluent, maintained intransglutaminase (TG) published were shown on the bottom. Epidermis1.2 mM Ca21), and from human epidermis. A 3.8 kb message forseparated from foreskin were also examined for hENaC expression (lanethe α subunit was detected in both keratinocytes and in epidermis11). Amplification products corresponding to the α (upper panel, 0.36 kb),(Fig 2A), and the expression was upregulated by differentiation β (middle panel, 1.2 kb), or γ (lower panel, 0.36 kb) subunits were

(lane 2). In contrast, the 2.6 kb transcript of the β subunit was not shown by southern hybridization using 32P-labeled cDNA probes. Controldetected in undifferentiated keratinocytes (Fig 2B, lane 4), but was amplifications on epidermis without reverse transcription did not showstrongly induced with differentiation (lane 5), confirming the reverse any DNA band as demonstrated by no reverse transcription control (lane 12).transcription–PCR data. The β subunits also was detected inepidermis (lane 6). In contrast to the signals obtained by semiquantit- corresponding to the γ subunit also was detected in whole epidermisative reverse transcription–PCR, the γ subunit was detected only (data not shown).as a faint band in differentiated keratinocytes as a 3.5 kb mRNA Our data demonstrate that both the hENaC-α and hENaC-β(data not shown). The sizes of all three subunits were comparable mRNA are expressed at high levels in normal epidermis, whereaswith those reported in human kidney (McDonald et al, 1995). the γ subunit was at a lower level. All of the transcripts increasedFinally, we used the RNase protection assay to compare mRNA with keratinocyte differentiation, although the β subunit appearedlevels of epidermal and kidney hENaC. The expected 360 nt to require more complete differentiation to be induced.protected fragment corresponding to the α subunit was detectedin undifferentiated keratinocytes (Fig 3A, lane 3). The hENaC-α hENaC-α protein is expressed in both cultured keratinocytessignal was increased 2-fold following differentiation (lane 4) and and epidermis hENaC-α protein expression was examined byalso was detected in whole epidermis (lane 5), at a lower level than Western blotting. The membrane protein fraction from undifferenti-kidney (lane 11) but comparable with the expression in kidney after ated keratinocytes, two different stages of differentiated ker-normalization using GAPDH expression. ENaC-β was detected as atinocytes, and whole epidermis were used. The hENaC-α specifica 380 nt protected fragment in epidermis (Fig 3B, lane 12) at levels antibody identified a µ130 kDa protein band and multiple bandscomparable with the kidney (lane 11). The β subunit was not about µ90 kDa in undifferentiated cultured keratinocytes, whichdetected in total RNA from undifferentiated keratinocytes (lane increased in differentiated cells (Fig 4). Strong expression of the13) but was expressed in differentiated cells (lane 14). When µ90 kDa protein was observed in epidermis. After deglycosylationpoly(A)1 RNA was used instead of total RNA for hybridization with PNGase F, the µ90 kDa band was shifted to µ82 kDa, whichto increase sensitivity, a weak signal for hENaC-β was detected in corresponds to the approximate molecular weight of the kidneyundifferentiated keratinocytes (lane 15) which increased seven times hENaC-α (McDonald et al, 1994). A control replicate blot incub-

ated with IgG from preimmune serum did not detect these bandsfollowing differentiation (lane 16). A very faint 359 nt band

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Figure 3. hENaC-α and hENaC-β transcripts were expressed inboth cultured keratinocytes and in epidermis, which are comparablelevels with the kidney. The expression levels of hENaC were examinedby RNase protection assay using 32P-labeled anti-sense RNA probe ofhENaC-α (A, lane 1) and hENaC-β (B, lane 10). Total RNA (10 µg)from undifferentiated preconfluent keratinocytes cultured in 0.03 mMcalcium (lanes 3 and 13), from postconfluent differentiated cells maintainedin 1.2 mM calcium for 10 d (lanes 4 and 14), from epidermis (lanes 5 and12), and from normal kidney (lanes 2 and 11) were hybridized withprobes and digested by RNase. The poly(A)1 RNA (1 µg) from sameundifferentiated (lane 15) and differentiated keratinocytes (lane 16) werealso used instead of total RNA. The molecular weight of protected bands

Figure 2. Changes in hENaC-α and hENaC-β transcripts in cultured were estimated by RNA molecular weight marker (lanes 7 and 8). Nokeratinocytes, and their expression in epidermis. The expression of band was detected in tRNA control (lanes 6 and 9). The expressions ofhENaC were detected by northern hybridization. Poly(A)1 RNAs isolated hENaC were normalized by RNA loading control of GAPDH shown onfrom undifferentiated preconfluent keratinocytes cultured in 0.03 mM Ca21 the bottom of blot.(lanes 1 and 4), postconfluent differentiated keratinocytes cultured in1.2 mM Ca21 for 10 d (lanes 2 and 5), and human epidermis separated following epidermal development in vivo. These data are consistentfrom foreskin (lanes 3 and 6) were hybridized with 32P-labeled probes of with an important role for this channel in the regulation of epidermalhENaC-α (A) and hENaC-β (B). A GAPDH control probe was used to differentiation and development. The induction of hENaC-α,demonstrate RNA loading on the same gels.

hENaC-β, and hENaC-γ parallels the increased keratinocyte differ-entiation shown by involucrin (Warhol et al, 1985; Gibson et al,(data not shown). Taken together, these data suggest that the1996), loricrin (Hohl et al, 1991) (precursors for the cornifiedµ90 kDa band represents the glycosylated form of the hENaC-α.envelope), filaggrin (Yuspa et al, 1989) (an intermediate filament-The nature of the µ130 kDa band is not clear. These resultsassociated protein), keratins K1 and K10 (Yuspa et al, 1989), andshowed that hENaC-α protein is also expressed in both culturedtransglutaminase (Gibson et al, 1996), which cross-links thesekeratinocytes and epidermis.constitutive proteins in a calcium-dependent manner into the

hENaC-α and hENaC-β expression increases during epi- cornified envelope (Thacher and Rice, 1985). In particular, hENaC-dermal development. The localization of hENaC-α and β, but not hENaC-α, appears coincident with protein kinase CαhENaC-β mRNA were examined in relation to epidermal develop- (Ng and Bikle, personal communication), which plays a criticalment using in situ hybridization. Anti-sense and sense RNA probes part in the calcium-induced signal transduction of keratinocytefor hENaC-α and hENaC-β were hybridized with sections of differentiation (Cenning et al, 1995). As the ENaC-β subunit has10 wk old fetal and adult skin. In 10 wk old human fetal epidermis, been shown to be phosphorylated by protein kinase C (Shimketsonly background levels of staining, which were comparable with et al, 1998), the activity of ENaC may be modulated by theirsense control, were seen (Fig 5A, B). The hENaC-α and hENaC-β phosphorylation in epidermal differentiation.transcripts were abundantly expressed throughout the nucleated The three channel subunits appear to be differentially expressedlayers of adult epidermis as shown by brown staining (Fig 5C, D). in keratinocytes. Although the α and β subunits were expressed atNo staining was detected in sense controls on adult skin (Fig 5E, comparable level with the kidney, γ subunit was less abundant.F). The differential staining through basal to suprabasal cells could The dominant α and less abundant γ subunits were expressed innot be observed because whole epidermis was heavily stained, and undifferentiated keratinocytes. In contrast, the β subunit appearedskin pigmentation overlapped with labeling of the basal layer. only in fully differentiated cells. ENaC channels reconstituted with

These results demonstrate that ENaC-α and ENaC-β mRNAs α and γ subunits in vitro exhibited different single channel kineticsare abundantly localized throughout the nucleated layers of adult in comparison with the α and β combination (Fyfe and Canessa,epidermis, but are absent in early fetal epidermis at which time the 1998). As ENaC are known to form oligomeric assemblies (Chengepidermis is still undifferentiated, suggesting their part in epidermal et al, 1998), their activity likewise might be regulated in ker-development. atinocytes by different combinations of the α, β, and γ subunits.

This expression pattern, having equal α and β but less γ, is similarDISCUSSION to that in human lung and bladder (Burch et al, 1995; Smith et al,Our results demonstrate that the hENaC were induced in calcium 1998), in contrast to the approximately equal expression of the

three subunits in kidney and colon (Garty and Palmer, 1997).stimulated keratinocyte differentiation in culture, and increased

800 ODA ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

The observed differential expression of the three subunits mightreflect differences in gene regulation. The ENaC-α mRNA wasnot increased by calcium, but it was upregulated by cell confluency.In contrast, the regulatory β subunit more clearly responded tocalcium, increasing with elevated levels of extracellular calcium.These differential responses to calcium are reminiscent of thehormone sensitivity of these subunits. For example, the β and γsubunits in the distal colon increase 10-fold, whereas the α subunitdoes not change in response to aldosterone (Lingueglia et al, 1994).Further, the γ subunit preferentially is induced compared with theα and β subunits in cultured lung epithelial cells with dexamethasonetreatment (Venkatesh et al, 1997). In contrast, progesterone andestradiol increase the α and γ, but not the β subunit in fetal lung(Sweezey et al, 1998).

We also detected immunoreactive µ130 kDa and µ90 kDaENaC-α proteins in cultured keratinocyte and epidermal membranefractions. The µ90 kDa band appears to be a glycosylated form ofENaC-α, because it could be converted to a smaller µ82 kDaprotein by PNGaseF, which corresponds to the previously estimatedmolecular weight of the ENaC protein (McDonald et al, 1994).Our data showed that ENaC was glycosylated with N-linkedcarbohydrates in epidermis. The glycosylation of ENaC wasreported in reconstituted channels in HEK 293 cells (Cheng et al,1998), but not in other tissues. It is not clear whether thisglycosylation is unique in epidermis. In contrast, the µ130 kDa

Figure 4. hENaC-α protein is expressed in cultured keratinocytes species was not altered by glycosidase treatment, and may representand epidermis. Membrane protein fractions (50 µg) were isolated from multimeric or aggregated ENaC-α protein.preconfluent undifferentiated keratinocytes cultured with 0.03 mM calcium Our studies demonstrating the upregulation of ENaC in ker-(lane 1), differentiated keratinocytes maintained with 1.2 mM calcium for atinocyte differentiation and in epidermal development suggest that4 d (lane 2) or 7 d (lane 3), and from whole epidermis (lanes 4 and 5). these channels play an important part in keratinocyte differentiationThese proteins were electrophoresed and analyzed using polyclonal IgG

or epidermal salt and water homeostasis. ENaC might maintain anagainst hENaC-α. The multiple bands about µ90 kDa and µ130 kDaappropriate level of hydration in epidermis by absorbing Na1,were observed. The µ90 kDa band (lane 4) shifted to µ82 kDa by removaltransporting ions and water from the extracellular fluid. The waterof N-linked oligosaccharide by PNGaseF (lane 5).content of the epidermis is decreased in the differentiated outer

Figure 5. hENaC-α and hENaC-β mRNAs wereinduced during epidermal development. Thelocalization of hENaC were examined on adult (C,D) and fetal skin (A, B) using in situ hybridization.hENaC-α and hENaC-β mRNA were observedthroughout adult human epidermis as shown by thebrown signal (C) compared with sense controls whichshow only green counterstaining (E), but not in fetalepidermis (A). The same expression pattern wasobserved for hENaC-β in adult (D compared withF) and fetal (B) skin. Pigmentation was observedin the basal layer of the adult skins (C–F). Finalmagnification of all pictures 5 3200.

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amiloride-inhibitable Na1 channels in alveolar epithelial cells. Am J Physiollayer (Warner et al, 1988b; Zglinicki et al, 1993) and keratinocyte271:L1–L22, 1996size increases as the cells differentiate (Bergstresser et al, 1978; Watt Mauro TM, Isseroff RR, Lasarow R, Pappone PA: Ion channels are linked to

and Green, 1982). differentiation in keratinocytes. J Membr Biol 132:201–209, 1993Mauro T, Dixon DB, Hanley K, Isseroff R, Pappone PA: Amiloride blocks a

keratinocyte nonspecific cation channel and inhibits calcium-inducedkeratinocyte differentiation. J Invest Dermatol 105:203–208, 1995

Mauro T, Dixon DB, Komuves L, Hanley K, Pappone PA: Keratinocyte K1 channelsThis work was supported in part by the research service of the Department ofmediate Ca21induced differentiation. J Invest Dermatol 108:864–870, 1997Veterans Affairs and by grant AR44341 from NIH. We appreciate Dr. Cris Lau Mauro T, Bench G, Sidderas-Haddad E, Feingold KR, Elias PM, Cullander C:

and Dr. Les Timpe for providing degenerate primers. We thank Dr. Sophia Acute barrier disruption abolishes the Ca21 and K1 gradients in murineepidermis. quantitative measurement using PIXE. J Invest Dermatol 111:1198–Rosenfeld for support in several of the molecular biologic studies and Ms. Juniper1201, 1998Pennypacker for technical assistance. We also are grateful to Dr. Ann Bradford and

McDonald FJ, Synder PM, Mccray PB, Welsh MJ: Cloning, expression, and tissueDr. D.J. Benos for providing the antibody against ENaC-α. distribution of a human α amiloride-sensitive Na1 channel. Am J Physiol

266:L728–L734, 1994McDonald FJ, Price MP, Snyder PM, Welsh MJ: Cloning and expression of the β-

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