Epithelial Sodium Channels are Upregulated During Epidermal Differentiation
1999; Elsevier BV; Volume: 113; Issue: 5 Linguagem: Inglês
10.1046/j.1523-1747.1999.00742.x
ISSN1523-1747
AutoresYuko Oda, Ashkan Imanzahrai, Angela Kwong, László G. Kömüves, Peter M. Elias, Corey Largman, Theodora M. Mauro,
Tópico(s)Ion Transport and Channel Regulation
ResumoTerminal differentiation of epidermal keratinocytes is linked to transmembrane ion flux. Previously, we have shown that amiloride, an inhibitor of epithelial sodium channels, blocks synthesis of differentiation-specific proteins in normal human keratinocytes. Here, we have identified the specific subunits of amiloride-sensitive human epithelial sodium channels in relation to differentiation of cultured human keratinocytes, as well as to epidermal development. As assessed by northern hybridization, RNase protection assay, and reverse transcription–polymerase chain reaction, transcripts encoding functional α and regulatory β subunits of human epithelial sodium channels were expressed both in cultured keratinocytes and in epidermis at levels comparable with the kidney. The mRNA expression of both human epithelial sodium channel-α and -β increased during calcium-induced keratinocyte differentiation. Whereas the β subunit of human epithelial sodium channel was induced by elevated concentrations of calcium, the α subunit increased with duration of culture. The regulatory γ subunit was less abundant but also expressed in epidermis. Both human epithelial sodium channel-α and -β were localized throughout the nucleated layers of human adult epidermis, but these channels were not detected in early stages of fetal epidermal development. This co-ordinated expression of subunits suggests that epithelial sodium channels may play an important part in both epidermal differentiation and skin development, presumably by modulating ion transport required for epidermal terminal differentiation. Terminal differentiation of epidermal keratinocytes is linked to transmembrane ion flux. Previously, we have shown that amiloride, an inhibitor of epithelial sodium channels, blocks synthesis of differentiation-specific proteins in normal human keratinocytes. Here, we have identified the specific subunits of amiloride-sensitive human epithelial sodium channels in relation to differentiation of cultured human keratinocytes, as well as to epidermal development. As assessed by northern hybridization, RNase protection assay, and reverse transcription–polymerase chain reaction, transcripts encoding functional α and regulatory β subunits of human epithelial sodium channels were expressed both in cultured keratinocytes and in epidermis at levels comparable with the kidney. The mRNA expression of both human epithelial sodium channel-α and -β increased during calcium-induced keratinocyte differentiation. Whereas the β subunit of human epithelial sodium channel was induced by elevated concentrations of calcium, the α subunit increased with duration of culture. The regulatory γ subunit was less abundant but also expressed in epidermis. Both human epithelial sodium channel-α and -β were localized throughout the nucleated layers of human adult epidermis, but these channels were not detected in early stages of fetal epidermal development. This co-ordinated expression of subunits suggests that epithelial sodium channels may play an important part in both epidermal differentiation and skin development, presumably by modulating ion transport required for epidermal terminal differentiation. epithelial sodium channel glyceraldehyde-3-phosphate dehydrogenase Proliferation and differentiation of epidermal keratinocytes are linked to changes in ion concentration in the epidermis. Gradients for Ca2+ (Menon et al., 1985Menon G.K. Grayson S. Elias P. Ionic calcium reservoirs in mammalian epidermis: ultrastructural localization by ion-captured cytochemistry.J Invest Dermatol. 1985; 84: 508-512Crossref PubMed Scopus (367) Google Scholar;Mauro et al., 1998Mauro T. Bench G. Sidderas-Haddad E. Feingold K.R. Elias P.M. Cullander C. Acute barrier disruption abolishes the Ca2+ and K+ gradients in murine epidermis. quantitative measurement using PIXE.J Invest Dermatol. 1998; 111: 1198-1201Crossref PubMed Scopus (147) Google Scholar), Na+ (Warner et al., 1988aWarner R.R. Myers M.C. Taylor D.A. Electron probe analysis of human skin: element concentration profiles.J Invest Dermatol. 1988; 90: 78-85Abstract Full Text PDF PubMed Google Scholar), Cl (Warner et al., 1988aWarner R.R. Myers M.C. Taylor D.A. Electron probe analysis of human skin: element concentration profiles.J Invest Dermatol. 1988; 90: 78-85Abstract Full Text PDF PubMed Google Scholar;Mauro et al., 1998Mauro T. Bench G. Sidderas-Haddad E. Feingold K.R. Elias P.M. Cullander C. Acute barrier disruption abolishes the Ca2+ and K+ gradients in murine epidermis. quantitative measurement using PIXE.J Invest Dermatol. 1998; 111: 1198-1201Crossref PubMed Scopus (147) Google Scholar), and K+ (Warner et al., 1988aWarner R.R. Myers M.C. Taylor D.A. Electron probe analysis of human skin: element concentration profiles.J Invest Dermatol. 1988; 90: 78-85Abstract Full Text PDF PubMed Google Scholar;Mauro et al., 1998Mauro T. Bench G. Sidderas-Haddad E. Feingold K.R. Elias P.M. Cullander C. Acute barrier disruption abolishes the Ca2+ and K+ gradients in murine epidermis. quantitative measurement using PIXE.J Invest Dermatol. 1998; 111: 1198-1201Crossref PubMed Scopus (147) Google Scholar) are observed across the epidermis in vivo, with low levels in the basal and spinous layers. Ca2+ concentrations increase through the upper granular layers where the cells are differentiated whereas Na+ and K+ peak in the lower granular layers (Warner et al., 1988aWarner R.R. Myers M.C. Taylor D.A. Electron probe analysis of human skin: element concentration profiles.J Invest Dermatol. 1988; 90: 78-85Abstract Full Text PDF PubMed Google Scholar;Mauro et al., 1998Mauro T. Bench G. Sidderas-Haddad E. Feingold K.R. Elias P.M. Cullander C. Acute barrier disruption abolishes the Ca2+ and K+ gradients in murine epidermis. quantitative measurement using PIXE.J Invest Dermatol. 1998; 111: 1198-1201Crossref PubMed Scopus (147) Google Scholar), suggesting a role for these ions in the regulation of differentiation in vivo. Differentiation of keratinocytes in vitro also is induced by increasing extracellular Ca2+ concentrations (Hennings et al., 1980Hennings H. Michael D. Cheng C. Steinert P. Holbrook K. Yuspa S.H. Calcium regulation of growth and differentiation of mouse epidermal cells in culture.Cell. 1980; 19: 245-254Abstract Full Text PDF PubMed Scopus (1448) Google Scholar;Pillai et al., 1990Pillai S. Bikle D.D. Mancianti M.L. Cline P. Hincenbergs M. Calcium regulation of growth and differentiation of normal human keratinocytes: Modulation of differentiation competence by stages of growth and extracellular calcium.J Cell Physiol. 1990; 143: 294-302Crossref PubMed Scopus (193) Google Scholar;Bikle and Pillai, 1993Bikle D.D. Pillai S. Vitamin D, calcium, and epidermal differentiation.Endocrine Rev. 1993; 14: 3-19PubMed Google Scholar). Increases in intracellular Na+ and K+ also are observed during keratinocyte differentiation, and agents which deplete intracellular K+ block calcium-induced differentiation (Hennings et al., 1983Hennings H. Holbrook K.A. Yuspa S.H. Factors influencing calcium-induced terminal differentiation in cultured mouse epidermal cells.J Cell Physiol. 1983; 116: 265-281Crossref PubMed Scopus (90) Google Scholar). Some of the ion channels responsible for early calcium-induced differentiation have been characterized previously, including a nonselective cation channel (Mauro et al., 1993Mauro T.M. Isseroff R.R. Lasarow R. Pappone P.A. Ion channels are linked to differentiation in keratinocytes.J Membr Biol. 1993; 132: 201-209Crossref PubMed Scopus (58) Google Scholar), K+ channel (Mauro et al., 1997Mauro T. Dixon D.B. Komuves L. Hanley K. Pappone P.A. Keratinocyte K+ channels mediate Ca2+induced differentiation.J Invest Dermatol. 1997; 108: 864-870Crossref PubMed Scopus (49) Google Scholar), nicotinic channel (Grando et al., 1995Grando S. Horton R.M. Pereira E.F. Diethelm-Okita B.M. George P.M. Albaquerque E.X. Conti-Fine B.M. A nicotinic acetylcholine receptor regulating cell adhesion and motility is expressed in human keratinocytes.J Invest Dermatol. 1995; 105: 774-781Crossref PubMed Scopus (189) Google Scholar), and cyclic guanosine monophosphate gated channel (Oda et al., 1997Oda Y. Timpe L.C. McKenzie R.C. Sauder D.N. Largman C. Mauro T. Alternative spliced isoforms of the cGMP-gated channel in human keratinocyte.FEBS Lett. 1997; 414: 140-145Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). In addition, pharmacologic experiments have suggested that voltage-sensitive Ca2+ channels control lipid secretion in the last stages of differentiation (Lee et al., 1994Lee S.H. Elias P.M. Feingold K.R. Mauro T. A role for ions in barrier recovery after acute perturbation.J Invest Dermatol. 1994; 102: 976-979Abstract Full Text PDF PubMed Google Scholar). Amiloride block the formation of cornified envelopes, as well as the synthesis and activity of the calcium-dependent keratinocyte-specific isoform of transglutaminase, suggesting a key role for an amiloride-sensitive channel in epidermal differentiation (Mauro et al., 1995Mauro T. Dixon D.B. Hanley K. Isseroff R. Pappone P.A. Amiloride blocks a keratinocyte nonspecific cation channel and inhibits calcium-induced keratinocyte differentiation.J Invest Dermatol. 1995; 105: 203-208Crossref PubMed Scopus (25) Google Scholar). Amiloride-sensitive epithelial sodium channels (ENaC) (reviewed byMatolon et al., 1996Matolon S. Benos D.J. Jackson R.M. Biophysical and molecular properties of amiloride-inhibitable Na+ channels in alveolar epithelial cells.Am J Physiol. 1996; 271: L1-L22PubMed Google Scholar;Garty and Palmer, 1997Garty H. Palmer L.G. Epithelial sodium channels: Function, structure and regulation.Physiol Rev. 1997; 77: 359-396Crossref PubMed Scopus (994) Google Scholar;Fyfe et al., 1998Fyfe G.K. Quinn A. Canessa C.M. Structure and function of the Mec-ENaC family of ion channels.Semin Nephrol. 1998; 18: 138-151PubMed Google Scholar) first were shown to mediate active Na+ reabsorption through the apical membrane of absorbing epithelial cells, including those of the renal collecting duct, distal colon, and urinary bladder (Garty and Palmer, 1997Garty H. Palmer L.G. Epithelial sodium channels: Function, structure and regulation.Physiol Rev. 1997; 77: 359-396Crossref PubMed Scopus (994) Google Scholar). Moreover, this channel later was shown to be essential for transport of water and salt in normal lung (Hummler et al., 1996Hummler E. Barker P. Gatzy J. et al.Early death due to defective neonatal lung liquid clearance in a αENaC-deficient mice.Nature Genet. 1996; 12: 325-328Crossref PubMed Scopus (731) Google Scholar). ENaC-α-deficient mice die soon after birth because they cannot transport the water from their alveoli (Hummler et al., 1996Hummler E. Barker P. Gatzy J. et al.Early death due to defective neonatal lung liquid clearance in a αENaC-deficient mice.Nature Genet. 1996; 12: 325-328Crossref PubMed Scopus (731) Google Scholar,Hummler et al., 1997Hummler E. Barker P. Talbot C. et al.A mouse model for the renal salt–wasting syndrome pseudo-hypoaldosteronism.Proc Natl Acad Sci USA. 1997; 94: 11710-11715Crossref PubMed Scopus (144) Google Scholar). ENaC also might mediate mechanosensation (Awayda et al., 1995Awayda M.S. Ismailov I.I. Berdiev B.K. Benos D.J. A cloned renal epithelial Na+ channel protein displays stretch activation in planar lipid bilayers.Am J Physiol. 1995; 268: C1450-C1459PubMed Google Scholar;Kizer et al., 1997Kizer N. Guo X.L. Hruska K. Reconstitution of stretch-activated cation channels by expression of the α-subunit of the epithelial sodium channel cloned from osteoblasts.Proc Natl Acad Sci USA. 1997; 94: 1013-1018Crossref PubMed Scopus (138) Google Scholar) as suggested by their sequence homology with degenerins, a class of proteins that mediates touch sensitivity in the nematode, Caenorhabditis elegans (Corey and Garcia-Anoveros, 1996Corey D.P. Garcia-Anoveros J. Mechanosensation and the DEG/ENaC ion channels.Science. 1996; 273: 323-324Crossref PubMed Scopus (95) Google Scholar). ENaC channels are composed of different homologous proteins, denoted as the α, β, and γ subunits (Garty and Palmer, 1997Garty H. Palmer L.G. Epithelial sodium channels: Function, structure and regulation.Physiol Rev. 1997; 77: 359-396Crossref PubMed Scopus (994) Google Scholar). The activity of the functional α subunit is enhanced by coexpression of the β or γ regulatory subunits which alone are not active (Garty and Palmer, 1997Garty H. Palmer L.G. Epithelial sodium channels: Function, structure and regulation.Physiol Rev. 1997; 77: 359-396Crossref PubMed Scopus (994) Google Scholar). All of the ENaC channels appear to be highly conserved in vertebrates, because they already are expressed in amphibian skin (Puoti et al., 1995Puoti A. May A. Canessa C.M. Horisberger J.D. Schild L. Rossier B.C. The highly selective low-conductance epithelial Na channel of Xenopus laevis A6 kidney cells.Am J Physiol. 1995; 269: C188-C197PubMed Google Scholar), where Na+ is reabsorbed through the skin. The function of ENaC in mammalian epidermis, however, which is a nonabsorbing epithelium, is not clear. ENaC have been detected in adult rat skin, in hair follicles, interfollicular epidermis, and sweat glands, where they have been proposed to mediate Na+ absorption (Roudier-Pujol et al., 1996Roudier-Pujol C. Rochat A. Escoubet B. Eugene E. Barrandon Y. Bonvalet J.P. Farman N. Differential expression of epithelial sodium channel subunit mRNA in rat skin.J Cell Sci. 1996; 109: 379-385PubMed Google Scholar). In this report, we identified ENaC subunits in normal human keratinocytes and in epidermis, suggesting that ENaC play a part in keratinocyte differentiation and epidermal development. Furthermore, the potential regulatory roles of the three channel subunits were clarified by comparing their expression during keratinocyte differentiation induced by changes in either the concentration of extracellular calcium and/or length of culture (Bikle and Pillai, 1993Bikle D.D. Pillai S. Vitamin D, calcium, and epidermal differentiation.Endocrine Rev. 1993; 14: 3-19PubMed Google Scholar). The expression and localization of ENaC were also compared during embryonic development of epidermis. These findings, in conjunction with previous pharmacologic studies (Mauro et al., 1995Mauro T. Dixon D.B. Hanley K. Isseroff R. Pappone P.A. Amiloride blocks a keratinocyte nonspecific cation channel and inhibits calcium-induced keratinocyte differentiation.J Invest Dermatol. 1995; 105: 203-208Crossref PubMed Scopus (25) Google Scholar), suggest that the keratinocyte ENaC channels play an important part in epidermal differentiation. Human epidermis was isolated from normal newborn foreskin by dispase treatment, and primary keratinocyte cultures were prepared as described (Oda et al., 1998Oda Y. Tu C.L. Pillai S. Bikle D.D. The calcium sensing receptor and its alternatively spliced form in keratinocyte differentiation.J Biol Chem. 1998; 273: 23344-23352Crossref PubMed Scopus (130) Google Scholar). Keratinocyte suspensions were prepared from epidermal sheets by trypsinization. The cells were grown in serum-free keratinocyte growth medium (Clonetics, Walkersville, MD), supplemented with 0.07 mM Ca2+ for 2 d, then switched to the same medium containing either 0.03 mM, 0.1 mM, or 1.2 mM Ca2+ and cultured for different times before harvest. cDNA cloning and DNA sequencing was performed as previously described (Oda et al., 1997Oda Y. Timpe L.C. McKenzie R.C. Sauder D.N. Largman C. Mauro T. Alternative spliced isoforms of the cGMP-gated channel in human keratinocyte.FEBS Lett. 1997; 414: 140-145Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar;Oda et al., 1998Oda Y. Tu C.L. Pillai S. Bikle D.D. The calcium sensing receptor and its alternatively spliced form in keratinocyte differentiation.J Biol Chem. 1998; 273: 23344-23352Crossref PubMed Scopus (130) Google Scholar). The hENaC cDNA fragments were isolated from human epidermis by reverse transcription–PCR, and were subcloned into pCRII or pCR2.1 (Invitrogen, Carlsbad, CA). The α subunit of hENaC was cloned using a set of specific primers 5′-GAC AAG AAC AAC TCC AAC CTC-3′ and 5′-ACA CTC CTT GAT CAT GCT CTC-3′ designed from lung hENaC-α (Voilley et al., 1994Voilley N. Lingueglia E. Champigny G. Mattei M.G. Waldmann R. Lazdunski M. Barbry P. The lung amiloride-sensitive Na+ channel: Biophysical properties, pharmacology, ontogenesis, and molecular cloning.Proc Natl Acad Sci USA. 1994; 91: 247-251Crossref PubMed Scopus (203) Google Scholar). The β subunit was cloned using a set of degenerate primers derived from conserved regions of the rat, human and frog hENaC-β channels (Garty and Palmer, 1997Garty H. Palmer L.G. Epithelial sodium channels: Function, structure and regulation.Physiol Rev. 1997; 77: 359-396Crossref PubMed Scopus (994) Google Scholar) 5′-GG(A/G/C/T) AA(C/T) TG(C/T) TA(C/T) AT(A/C/T) TT(C/T) AA(C/T) TGG-3′ and 5′-(A/G)CA (A/G)TT (A/G)TG (A/G/T)AT CAT (A/G)TG (A/G)TC (C/T)TG (A/G)AA (G/A)CA-3′, which were a gift from Dr C. Lau, UCSF. A γ subunit was isolated using a set of degenerate primers designed from conserved amino acid sequences (G(S/N)CY(T/I/V/M)FN and C(Y/L) (R/H) SC(F/Y)Q). These regions were selected by alignment of 12 different ENaC-α, ENaC-β, and ENaC-γ sequences derived from bovine, rat, frog, and human ENaC (Garty and Palmer, 1997Garty H. Palmer L.G. Epithelial sodium channels: Function, structure and regulation.Physiol Rev. 1997; 77: 359-396Crossref PubMed Scopus (994) Google Scholar). The degenerate primers used were 5′-GGI AA(C/T) TG(C/T) TA(C/T) ACI TT(C/T) AA-3′ and 5′-TG(A/G)AA(A/G)CAI GA(A/G) TGI AG(A/G) CA-3′. The isolated cDNA clones were sequenced on both strands using vector-specific primers with dye termination cycle sequencing on an Applied Biosystems (Foster City, CA) 373A automated DNA sequencer. The DNA sequences were analyzed using the GCG DNA analysis package at the Computer Graphics Laboratory of the University of California, San Francisco. Poly(A)+ RNA was isolated from cultured keratinocytes or whole epidermis as described (Oda et al., 1997Oda Y. Timpe L.C. McKenzie R.C. Sauder D.N. Largman C. Mauro T. Alternative spliced isoforms of the cGMP-gated channel in human keratinocyte.FEBS Lett. 1997; 414: 140-145Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar,Oda et al., 1998Oda Y. Tu C.L. Pillai S. Bikle D.D. The calcium sensing receptor and its alternatively spliced form in keratinocyte differentiation.J Biol Chem. 1998; 273: 23344-23352Crossref PubMed Scopus (130) Google Scholar). The RNA were electrophoresed and hybridized with ENaC subunit specific 32P-labeled cDNA probes as previously described (Oda et al., 1997Oda Y. Timpe L.C. McKenzie R.C. Sauder D.N. Largman C. Mauro T. Alternative spliced isoforms of the cGMP-gated channel in human keratinocyte.FEBS Lett. 1997; 414: 140-145Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). A 376 bp DNA fragment for the α subunit, a 384 bp cDNA for the β subunit, and 375 bp cDNA for the γ subunit were prepared by digestion of the cloned cDNAs. The membranes were washed under stringent conditions (0.1 × sodium citrate/chloride buffer, 60°C). The same blot was re-probed with a 32P-labeled cDNA for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Clontech) as a control to estimate RNA loading. The message size was estimated by comparison with molecular weight markers (RNA ladder, 0.24–9.5 kb, Gibco BRL, Gaithersburg, MD). Anti-sense RNA probes were labeled with 32P[UTP] by transcription of linearized cDNA templates using MAXIscript (Ambion, Austin, TX). Probes were purified by gel electrophoresis, and hybridized with total RNA from cells or tissues. Probes were digested with RNase A + T1 and analyzed on urea/4% polyacrylamide gels (Oda et al., 1998Oda Y. Tu C.L. Pillai S. Bikle D.D. The calcium sensing receptor and its alternatively spliced form in keratinocyte differentiation.J Biol Chem. 1998; 273: 23344-23352Crossref PubMed Scopus (130) Google Scholar). Protected RNA band sizes were estimated using RNA molecular weight markers (RNA century marker, Ambion). Total RNA from normal human kidney, used as a control source of hENaC, was obtained from Clontech. hENaC subunits expression was determined by reverse transcription–PCR as described previously (Oda et al., 1997Oda Y. Timpe L.C. McKenzie R.C. Sauder D.N. Largman C. Mauro T. Alternative spliced isoforms of the cGMP-gated channel in human keratinocyte.FEBS Lett. 1997; 414: 140-145Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar,Oda et al., 1998Oda Y. Tu C.L. Pillai S. Bikle D.D. The calcium sensing receptor and its alternatively spliced form in keratinocyte differentiation.J Biol Chem. 1998; 273: 23344-23352Crossref PubMed Scopus (130) Google Scholar). The primer set for the hENaC-α subunit was as above. For the hENaC-β subunit primers: 5′-CTG GGA CAT CTT CAA CTG GG-3′ and 5′-CTT TGG AGA GGG CAC CAT AC-3′ were used. For the hENaC-γ subunit, the primer set of 5′-CAT GGG AAT TGC TAT ACT TTC-3′ and 5′-TGG AAG CAT GAA TGA AGG CA-3′ were used. Total RNA was isolated from cultured keratinocytes at various stages of differentiation or from normal neonatal foreskin epidermis. Total cDNA pools were prepared by reverse transcription using Superscript II (Gibco BRL). Each cDNA pool was also amplified using primers for human GAPDH (Clontech) as a control for cDNA loading in the PCR assay. Amplification was carried out using Taq polymerase PLUS (Stratagene, La Jolla, CA) for 25 cycles for both ENaC and GAPDH, which was shown to be within the linear range for both PCR reactions. The PCR products were electrophoresed on a 3% agarose gel, transblotted, and analyzed by southern hybridization using specific 32P-labeled cDNA probes for each hENaC subunit. Molecular weights of the PCR product bands were estimated using φX174/Hae III fragments (BRL) as standards. Plasma membranes were isolated from cultured keratinocytes and whole epidermis as described (Oda et al., 1997Oda Y. Timpe L.C. McKenzie R.C. Sauder D.N. Largman C. Mauro T. Alternative spliced isoforms of the cGMP-gated channel in human keratinocyte.FEBS Lett. 1997; 414: 140-145Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar,Oda et al., 1998Oda Y. Tu C.L. Pillai S. Bikle D.D. The calcium sensing receptor and its alternatively spliced form in keratinocyte differentiation.J Biol Chem. 1998; 273: 23344-23352Crossref PubMed Scopus (130) Google Scholar). The cells or tissues were sonicated in homogenization buffer (20 mM Tris–HCl, pH 7.4, containing 0.25 M sucrose, 4 mM MgCl2, 5 mM ethyleneglycol-bis-(b-aminoethylether)-N,N,N′,N′,-tetraacetic acid, and protease inhibitors). The homogenate was sedimented at 48 000 × g for 30 min, and the soluble cytoplasmic fraction was removed. The membrane fraction was extracted with 1% deoxycholate acid, 1% Triton X-100, 0.1% sodium dodecyl sulfate and separated from the insoluble cytoskeleton fraction by centrifugation at 48 000 × g for 30 min. The membrane protein fraction (50 μg) was denatured, and then incubated at 30°C overnight with or without 0.5 U of PNGase F (Boehringer Mannheim, Indianapolis, IN) (Oda et al., 1998Oda Y. Tu C.L. Pillai S. Bikle D.D. The calcium sensing receptor and its alternatively spliced form in keratinocyte differentiation.J Biol Chem. 1998; 273: 23344-23352Crossref PubMed Scopus (130) Google Scholar). Membrane protein concentrations were determined using the BCA protein assay (Pierce, Rockford, IL). Membrane proteins (50 μg) were electrophoresed in duplicate on 7.5% polyacrylamide gels and electroblotted on to polyvinylidene difluoride membranes (0.2 μm, BioRad Laboratories, Hercules, CA), and blocked with 5% milk in TBST (10 mM Tris–Cl, pH 7.5, 150 mM NaCl, and 0.1% Tween-20). The blots were incubated overnight at 4°C with either the purified IgG fraction (20 μg per ml) of a polyclonal antisera against full length fusion protein of bovine hENaC-α generated from the cDNA clone (gift from Dr. D.J. Benos, University of Alabama) which cross react with human hENaC-α. The specificity and cross-reactivity of the antibody was assessed using both immunofluorescence and western blotting (Ismailov et al., 1996Ismailov I.I. Berdiev B.K. Bradform A.L. Awayda M.S. Fuller C.M. Benos D.J. Associated proteins and renal epithelial sodium channel.J Membr Biol. 1996; 149: 123-132Crossref PubMed Scopus (37) Google Scholar;Awayda et al., 1997Awayda M.S. Tousson A. Benos. D.J. Regulation of a cloned epithelial Na+ channel by its β- and γ-subunits.Am J Physiol. 1997; 273: C1889-C1899PubMed Google Scholar). Subsequently, the blots were incubated with a sheep anti-mouse secondary antibody conjugated with horseradish peroxidase (Amersham, Uppsala, Sweden) for 1 h at room temperature. The bound horseradish peroxidase was visualized using an enhanced chemiluminescence system (Amersham). The antibody specificity was confirmed by incubating a replicate blot with same concentration of preimmune IgG which was provided together with the antibody by Dr. Benos. Skin biopsies from adult and 10 wk old human fetal samples were fixed in 4% paraformaldehyde and embedded in paraffin. Anti-sense and sense RNA probes were prepared from the same cDNA clones described above for northern hybridization, using pCRII dual promoters. The hENaC cDNA clones were linearized and used as templates to synthesize digoxigenin-labeled RNA probes, which were hybridized with the skin sections as previously described (Stelnicki et al., 1998Stelnicki E.J. Komuves L. Kwang A.O. et al.HOX homeobox genes exhibit spatial and temporal changes in expression during human skin development.J Invest Dermatol. 1998; 110: 110-115Crossref PubMed Scopus (103) Google Scholar). Signals were visualized with a diaminobenzidine chromogen substrate. Sections were counterstained with hematoxylin to visualize tissue morphology. We first used reverse transcription–PCR to detect the hENaC subunits in human foreskin epidermis using specific or degenerate primers. The respective PCR fragments were subcloned and sequenced. A 360 bp cDNA fragment for the α subunit was obtained, using α-specific primers, whose sequence was identical to human lung ENaC-α (930–1289) (Voilley et al., 1994Voilley N. Lingueglia E. Champigny G. Mattei M.G. Waldmann R. Lazdunski M. Barbry P. The lung amiloride-sensitive Na+ channel: Biophysical properties, pharmacology, ontogenesis, and molecular cloning.Proc Natl Acad Sci USA. 1994; 91: 247-251Crossref PubMed Scopus (203) Google Scholar). A 368 bp cDNA was isolated, using degenerate β-specific primers, which was identical to the human lung ENaC-β (940–1308) (Voilley et al., 1995Voilley N. Bassilana F. Mignon C. et al.Cloning, chromosomal localization, and physical linkage of the β and γ subunits (SCNN1B and SCNN1G) of the human epithelial amiloride-sensitive sodium channel.Genomics. 1995; 28: 560-565Crossref PubMed Scopus (82) Google Scholar). Finally, reverse transcription–PCR was used with a set of degenerate primers, designed from conserved regions of 12 different α, β, and γ sequences to isolate any related channels (see Materials and Methods). A 359 bp PCR fragment cloned from this primer set was identical to the γ subunit of kidney ENaC (865–1223) (McDonald et al., 1995McDonald F.J. Price M.P. Snyder P.M. Welsh M.J. Cloning and expression of the β- and γ-subunits of the human epithelial sodium channel.Am J Physiol. 1995; 268: C1157-C1163PubMed Google Scholar;Voilley et al., 1995Voilley N. Bassilana F. Mignon C. et al.Cloning, chromosomal localization, and physical linkage of the β and γ subunits (SCNN1B and SCNN1G) of the human epithelial amiloride-sensitive sodium channel.Genomics. 1995; 28: 560-565Crossref PubMed Scopus (82) Google Scholar). These results demonstrate that human epidermis expresses all three ENaC subunits. In order to evaluate the potential regulatory role of hENaC in epidermal differentiation, we next used semiquantitative reverse transcription–PCR to examine the expression of each of the three hENaC subunits during differentiation of cultured keratinoyctes. Well-defined changes in keratinocyte differentiation are induced both by culture confluency and by progressively longer exposure to raised extracellular calcium (Younus and Bilchrest, 1992Younus J. Bilchrest B. Modulation of mRNA levels during human keratinocyte differentiation.J Cell Physiol. 1992; 152: 232-239Crossref PubMed Scopus (46) Google Scholar;Su et al., 1994Su M.J. Bikle D.D. Mancianti M.L. Pillai S. 1,25-Dihydroxyvitamin D3 potentiated the keratinocyte response to calcium.J Biol Chem. 1994; 269: 14723-14729Abstract Full Text PDF PubMed Google Scholar;Gibson et al., 1996Gibson F.C. Ratman A.V. Bikle D.D. Evidence for separate control mechanism at the message, protein, and enzyme activation levels for transglutaminase during calcium-induced differentiation of normal and transformed human keratinocytes.J Invest Dermatol. 1996; 106: 154-161Crossref PubMed Scopus (62) Google Scholar). Using the same conditions defined byGibson et al., 1996Gibson F.C. Ratman A.V. Bikle D.D. Evidence for separate control mechanism at the message, protein, and enzyme activation levels for transglutaminase during calcium-induced differentiation of normal and transformed human keratinocytes.J Invest Dermatol. 1996; 106: 154-161Crossref PubMed Scopus (62) Google Scholar, we tested preconfluent (5 d of culture), near confluent (7 d), and postconfluent (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 keratinocytes and were upregulated by differentiation induced by extended confluent growth (Figure 1, upper and lower panels). In contrast, the β subunit was not detected in undifferentiated cells in keratinocytes, but appeared in fully differentiated cells
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