The ENaC Channel is Required for Normal Epidermal Differentiation
2002; Elsevier BV; Volume: 118; Issue: 4 Linguagem: Inglês
10.1046/j.1523-1747.2002.01721.x
ISSN1523-1747
AutoresTheodora M. Mauro, Martin J. Behne, Yuko Oda, Debbie Crumrine, László G. Kömüves, Ulrich Rassner, Peter M. Elias, Marjorie Guitard, Edith Hümmler,
Tópico(s)Ion Channels and Receptors
ResumoIonic fluxes are important for critical aspects of keratinocyte differentiation, including synthesis of differentiation-specific proteins, enzymatic catalysis of protein cross-linking, post-transcriptional processing of profilaggrin, and lipid secretion. The epithelial sodium channel is expressed in epidermis and the expression of its α and β subunits is enhanced as keratinocytes differentiate. In order to ascertain the role of the epithelial sodium channel in epidermal differentiation, we examined skin of mice in which the epithelial sodium channel α subunit had been deleted. Newborn –/– mice, in which the α subunit had been completely inactivated, demonstrated epithelial hyperplasia, abnormal nuclei, premature secretion of lipids, and abnormal keratohyaline granules. In addition, immunohistochemistry demonstrated that expression of the differentiation markers K1, K6, and involucrin were abnormal. These data suggest that the epithelial sodium channel modulates ionic signaling for specific aspects of epidermal differentiation, such as synthesis or processing of differentiation- specific proteins, and lipid secretion. Ionic fluxes are important for critical aspects of keratinocyte differentiation, including synthesis of differentiation-specific proteins, enzymatic catalysis of protein cross-linking, post-transcriptional processing of profilaggrin, and lipid secretion. The epithelial sodium channel is expressed in epidermis and the expression of its α and β subunits is enhanced as keratinocytes differentiate. In order to ascertain the role of the epithelial sodium channel in epidermal differentiation, we examined skin of mice in which the epithelial sodium channel α subunit had been deleted. Newborn –/– mice, in which the α subunit had been completely inactivated, demonstrated epithelial hyperplasia, abnormal nuclei, premature secretion of lipids, and abnormal keratohyaline granules. In addition, immunohistochemistry demonstrated that expression of the differentiation markers K1, K6, and involucrin were abnormal. These data suggest that the epithelial sodium channel modulates ionic signaling for specific aspects of epidermal differentiation, such as synthesis or processing of differentiation- specific proteins, and lipid secretion. epithelial Na+ channel The amiloride sensitive epithelial Na+ channel (ENaC) is an important modulator of Na+ homeostasis, and thereby plays a critical role in regulating blood pressure (reviewed byHummler and Horisberger, 1999Hummler E. Horisberger J.D. Genetic disorders of membrane transport V. The epithelial sodium channel and its implication in human diseases.Am J Physiol. 1999; 276: G567-G571PubMed Google Scholar), renal function (Shimkets, 1994Shimkets R.A. et al.Liddle's syndrome: heritable human hypertension caused by mutations in the β subunit of the epithelial sodium channel.Cell. 1994; 79: 407-414Abstract Full Text PDF PubMed Scopus (1141) Google Scholar;Snyder, 1995Snyder P.M. et al.Mechanism by which Liddle's syndrome mutations increase activity of a human epithelial Na+ channel.Cell. 1995; 83: 969-978Abstract Full Text PDF PubMed Scopus (380) Google Scholar;Chang, 1996Chang S.S. et al.Mutations in subunits of the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, pseudohypoaldosteronism type 1.Nat Genet. 1996; 12: 248-253Crossref PubMed Scopus (661) Google Scholar;Gründer, 1997Gründer S. et al.A mutation causing pseudohypoaldosteronism type 1 identifies a conserved glycine that is involved in the gating of the epithelial sodium channel.EMBO J. 1997; 16: 899-907Crossref PubMed Scopus (163) Google Scholar), and fluid balance in the lung (Stutts et al., 1995Stutts M.J. 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Agarwal M.K. Immunochemical analysis of the sodium channel in rodent and human eye.Exp Eye Res. 1999; 69: 21-32Crossref PubMed Scopus (64) Google Scholar) and has been found in a variety of epithelia, including epidermis, and in sweat glands (Roudier-Pujol et al., 1996Roudier-Pujol C. Rochat A. Escoubet B. Eugène E. Barrandon Y. Bonvalet J.P. Farman N. Differential expression of epithelial sodium channel subunit mRNAs in rat skin.J Cell Sci. 1996; 109: 379-385PubMed Google Scholar;Brouard et al., 1999Brouard M. Casado M. Djelidi S. Barrandon Y. Farman N. Epithelial sodium channel in human epidermal keratinocytes: expression of its subunits and relation to sodium transport and differentiation.J Cell Sci. 1999; 112: 3343-3352PubMed Google Scholar;Oda et al., 1999Oda Y. Imanzahrai A. Kwong A. Komuves L. Elias P. Largman C. Mauro T. Epithelial sodium channels are upregulated during epidermal differentiation.J Invest Dermatol. 1999; 113: 796-801https://doi.org/10.1046/j.1523-1747.1999.00742.xCrossref PubMed Scopus (26) Google Scholar). Expression of ENaC subunits is increased in more differentiated keratinocytes, and is found only in the later stages of fetal epidermal development (Oda et al., 1999Oda Y. Imanzahrai A. Kwong A. Komuves L. Elias P. Largman C. Mauro T. Epithelial sodium channels are upregulated during epidermal differentiation.J Invest Dermatol. 1999; 113: 796-801https://doi.org/10.1046/j.1523-1747.1999.00742.xCrossref PubMed Scopus (26) Google Scholar), suggesting that expression of this channel is linked to keratinocyte differentiation. Mammalian epidermis generally is not considered as a transporting epithelium, because transcutaneous movement of water and ions is thought to be regulated passively by an external permeability barrier composed of corneocytes embedded in a lipid-enriched intercellular matrix (Elias and Friend, 1975Elias P.M. Friend D.S. The permeability barrier in mammalian epidermis.J Cell Biol. 1975; 65: 180-191Crossref PubMed Scopus (541) Google Scholar). Yet, crucial aspects of keratinocyte differentiation, including the synthesis of cornified envelope and other differentiation-specific proteins (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;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 ([published erratum appears in J Membr Biol 134: 168, 1993]): 201-209Crossref PubMed Scopus (59) Google Scholar;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, conversion of profilaggrin to filaggrin (Resing et al., 1993Resing K.A. al-Alawi N. Blomquist C. Fleckman P. Dale B.A. Independent regulation of two cytoplasmic processing stages of the intermediate filament-associated protein filaggrin and role of Ca2+ in the second stage.J Biol Chem. 1993; 268: 25139-25145Abstract Full Text PDF PubMed Google Scholar), and secretion of stratum corneum lipid precursors (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), are controlled by transmembrane ionic fluxes, particularly Ca2+. Moreover, Na+ influx also modulates Ca2+-induced keratinocyte 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), presumably by modulations in membrane potential (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 ([published erratum appears in J Membr Biol 134: 168, 1993]): 201-209Crossref PubMed Scopus (59) Google Scholar;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). Further, application of amiloride, a known ENaC inhibitor, blocks Ca2+-induced differentiation in keratinocytes, although this result could be due to amiloride blockade of a nonspecific cation channel (Mauro et al., 1995Mauro T. Dixon D.B. Hanley K. Isseroff R.R. Pappone P.A. Amiloride blocks a keratinocyte nonspecific cation channel and inhibits Ca(++)-induced keratinocyte differentiation.J Invest Dermatol. 1995; 105: 203-208Crossref PubMed Scopus (25) Google Scholar). In order to determine the importance of ENaC for keratinocyte and epidermal differentiation, we studied keratinocytes and epidermis from mice in whom the ENaC α subunit had been deleted (Hummler, 1996Hummler E. et al.Early death due to defective neonatal lung liquid clearance in α-ENaC-deficient mice.Nat Genet. 1996; 12: 325-328Crossref PubMed Scopus (732) Google Scholar). These mice display a distinct phenotype, in which keratohyalin granule assembly or processing and lipid secretion appear to be altered. Epidermal differentiation also is retarded in these mice, and epidermal hyperplasia is present. These findings suggest that ENaC processes might modulate specific milestones in epidermal differentiation. Genotyping of mice was performed using polymerase chain reaction (PCR) on DNA isolated from tail tips and subjected to PCR-based analysis using primers as described previously (Hummler, 1997Hummler E. et al.A mouse model for the renal salt-wasting syndrome pseudohypoaldosteronism.Proc Natl Acad Sci USA. 1997; 94: 11710-11715Crossref PubMed Scopus (144) Google Scholar). Eight wild-type (+/+), 16 heterozygous mutant (+/–), and 12 homozygous mutant (–/–) newborn mice, and five –/– (–/–Tg) and four +/+ animals in which the α-ENaC subunit was selectively restored to lung tissue, enabling the mice to live beyond the perinatal period, were prepared as described previously (Hummler, 1997Hummler E. et al.A mouse model for the renal salt-wasting syndrome pseudohypoaldosteronism.Proc Natl Acad Sci USA. 1997; 94: 11710-11715Crossref PubMed Scopus (144) Google Scholar). Animal experiments followed approved institutional protocols at the University of Lausanne. Epidermal thickness was measured from the base of the stratum basale to the apex of the stratum granulosum in histologic sections of newborn α-ENaC +/+, +/–, and –/– mice. Sections were measured with the observer blinded to the genotype of the animals. Statistical significance was calculated using the ANOVA test. Full-thickness skin samples were obtained from euthanized animals, minced to < 0.5 mm3, and fixed in modified Karnovsky's fixative overnight. All samples were then divided and postfixed in the dark in either 1% ruthenium tetroxide or 1% aqueous osmium tetroxide containing 1.5% potassium ferrocyanide. Thin sections were examined, with or without further contrasting with lead citrate, in a Zeiss 10 A electron microscope operated at 60 kV. Keratinocytes from α-ENaC +/+, +/–, and –/– mice were isolated and cultured as described previously (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 (1450) Google Scholar;Hennings et al., 1994Hennings H. Mouse epidermal keratinocytes.in: Leigh I.M. Watt F.M. Keratinocyte Methods. Cambridge University Press, New York1994: 21-23Google Scholar). Total RNA (500 µg) was extracted from α-ENaC –/– and control keratinocytes, reverse transcribed, and amplified by PCR using the Titan One Tube RT-PCR System (Roche Laboratories). Amplified fragments were separated on a 2% agarose gel and visualized by staining with ethidium bromide. Primers were used as described previously (Hummler, 1996Hummler E. et al.Early death due to defective neonatal lung liquid clearance in α-ENaC-deficient mice.Nat Genet. 1996; 12: 325-328Crossref PubMed Scopus (732) Google Scholar). Skin samples from newborn mice were frozen in OCT compound. Frozen skin sections were fixed 10 min in paraformaldehyde 4%. For immunohistochemistry of differentiation markers, monospecific rabbit antisera (dilutions according to BAbCO Laboratories, Berkeley, CA) produced against mouse keratins 1, 6, and 14 (BAbCO Laboratories) were incubated 1 h at room temperature with frozen skin sections, followed by incubation for 45 min at room temperature with antirabbit IgY CY3 secondary antibody diluted at 1:100 (Jackson Immunoresearch Laboratories). Skin samples from newborn +/+ and –/– mice were fixed in 4% paraformaldehyde. Sections of skin were exposed to involucrin antibody (Covance, Richmond, CA) at a dilution of 1:500, followed by a biotinylated goat antirabbit secondary antibody and ABC peroxidase (both from Vector Laboratories, Burlingame, CA) to precipitate diaminobenzidine. Sections were counterstained with methylgreen. Biopsies were obtained from newborn –/– mice soon after birth, before respiratory distress had developed or hyperplasia might develop in response to a deficient barrier. Although pathologic skin changes were not apparent upon gross examination of newborn skin, light microscopy showed thickening of all suprabasal levels (Figure 1a) attributable to an increase in the average number of cell layers from three to four, six, seven. In addition, –/– epidermis demonstrated focal abnormalities in epidermal maturation, including a failure of suprabasal cells to flatten progressively, as well as nuclear atypia, and decreased amounts of keratohyalin. The epidermis of –/– mice was significantly thicker than that of +/– and +/+ mice, seen both in hematoxylin and eosin sections and confirmed by measurements of epidermal thickness (Figure 1b). Epidermal thickness of +/– and +/+ mice was not significantly different (Figure 1b), nor were defects in differentiation seen in +/– mice, suggesting that almost complete absence of the ENaC is required to produce defects in epidermal differentiation. Electron microscopy revealed additional defects. Keratohyaline granules were decreased in number (Figure 2a), and premature lipid secretion was noted in the mid-stratum granulosum in –/– mouse epidermis (Figure 3a). Mice in which transgenic expression of an α-ENaC cDNA rescues the perinatal lethality of α knockout mice (Hummler, 1997Hummler E. et al.A mouse model for the renal salt-wasting syndrome pseudohypoaldosteronism.Proc Natl Acad Sci USA. 1997; 94: 11710-11715Crossref PubMed Scopus (144) Google Scholar) were also examined. These animals retain approximately 15% of residual activity as estimated in lung explants (Hummler, 1996Hummler E. et al.Early death due to defective neonatal lung liquid clearance in α-ENaC-deficient mice.Nat Genet. 1996; 12: 325-328Crossref PubMed Scopus (732) Google Scholar). Skin samples from these –/– Tg mice retain the premature lipid secretion (Figure 3b), but demonstrate normal nuclei and keratohyaline granules and lack epidermal hyperplasia, suggesting that almost complete ablation of activity is necessary for disruption of keratinocyte differentiation (although not for control of lipid secretion) (Table I). Post-secretory processing of the lipids appeared to be normal, as assessed by ruthenium tetroxide staining (data not shown). Neither the β nor the γ subunits were upregulated in keratinocytes from α-ENaC –/– mice (Figure 4).Figure 3α ENaC –/– and –/– Tg (transgenic rescue) mice display premature lipid secretion. (A) Mid-stratum granulosum of –/– mice shows focal secretion of lamellar body contents into intercellular domains (arrows). (B) Upper and mid-stratum granulosum of –/– Tg rescue mice. Lipid is secreted normally in the upper stratum granulosum, but also is secreted prematurely in the mid-stratum granulosum (arrows). Scale bars: 0.5 µm.View Large Image Figure ViewerDownload (PPT)Table ISummary of skin changes in α-ENaC –/– miceFeatureKnockoutWild-typeRescueLipid secretionPrematureNormalPrematureHyperplasiaPresentNormalNormalMaturationAbnormalNormalNormalKeratohyalin granulesDecreasedNormalNormalProtein synthesisAbnormalNormalNot done Open table in a new tab Figure 4Detection of mRNA transcripts for α-, β-, and γ-ENaC in ENaC wild-type (α-ENaC +/+), heterozygous (+/–), and homozygous mutant (–/–) mouse keratinocytes. In α-ENaC –/– mice, no change in mRNA expression of the β- and γ-ENaC subunit was detected. The RT-PCR was controlled by detection of glyceraldehyde-3-phosphate dehydrogenase message. Positive control (+), α-ENaC cDNA; negative control (–), PCR reaction without RNA.View Large Image Figure ViewerDownload (PPT) Immunohistochemistry of murine epidermis demonstrated delayed expression of involucrin in the –/– newborn mice (Figure 5). Similarly, the expression of K1 was increased in –/– suprabasal layers compared to the +/+ mice (Figure 5). Expression of the basal marker K14 was similar in the interfollicular epidermis. The epidermis from α-ENaC knockout mice reacted positively with a mouse-specific K6 antibody, a marker for both the inner layer of the outer root sheath and nonspecific epidermal hyperplasia (Sundberg et al., 1997Sundberg J.P. Rourk M.H. Boggess D. Hogan M.E. Sundberg B.A. Bertolino A.P. Angora mouse mutation: altered hair cycle, follicular dystrophy, phenotypic maintenance of skin grafts, and changes in keratin expression.Vet Pathol. 1997; 34: 171-179Crossref PubMed Scopus (52) Google Scholar) (Figure 6), confirming the epidermal hyperplasia observed with hematoxylin and eosin studies. The pathologic findings of normal versus knockout mice are summarized in Table I.Figure 6K6 immunostaining confirms epidermal hyperplasia in skin from α-ENaC –/– mice. Skin sections of newborn –/– pups are positively stained for K6.View Large Image Figure ViewerDownload (PPT) These data suggest that the ENaC α-subunit controls selective aspects of epidermal differentiation, including synthesis of markers of differentiation, keratohyalin granule formation or processing, and lipid secretion. Moreover, control of lipid secretion seems to require a greater degree of channel activity, whereas the contribution of the ENaC to epidermal differentiation can be maintained by relatively low levels of expression. Our initial pharmacologic experiments, which demonstrated that an amiloride-sensitive conductance controls Ca2+-induced keratinocyte differentiation (Mauro et al., 1995Mauro T. Dixon D.B. Hanley K. Isseroff R.R. Pappone P.A. Amiloride blocks a keratinocyte nonspecific cation channel and inhibits Ca(++)-induced keratinocyte differentiation.J Invest Dermatol. 1995; 105: 203-208Crossref PubMed Scopus (25) Google Scholar), led us next to probe keratinocytes and epidermis for the amiloride-sensitive ENaC. We and others demonstrated ENaC subunit expression in both keratinocytes and epidermis (Roudier-Pujol et al., 1996Roudier-Pujol C. Rochat A. Escoubet B. Eugène E. Barrandon Y. Bonvalet J.P. Farman N. Differential expression of epithelial sodium channel subunit mRNAs in rat skin.J Cell Sci. 1996; 109: 379-385PubMed Google Scholar; Brouard etal, 1999;Oda et al., 1999Oda Y. Imanzahrai A. Kwong A. Komuves L. Elias P. Largman C. Mauro T. Epithelial sodium channels are upregulated during epidermal differentiation.J Invest Dermatol. 1999; 113: 796-801https://doi.org/10.1046/j.1523-1747.1999.00742.xCrossref PubMed Scopus (26) Google Scholar). ENaC expression, particularly of the β subunit, increases in more differentiated keratinocytes (Oda et al., 1999Oda Y. Imanzahrai A. Kwong A. Komuves L. Elias P. Largman C. Mauro T. Epithelial sodium channels are upregulated during epidermal differentiation.J Invest Dermatol. 1999; 113: 796-801https://doi.org/10.1046/j.1523-1747.1999.00742.xCrossref PubMed Scopus (26) Google Scholar). We now report that ENaC is important for specific processes of epidermal maturation and differentiation, based upon our observations in skin in which the α-ENaC subunit has been deleted. The α-ENaC –/– defects in keratohyalin formation/processing, premature lipid secretion, and abnormal expression of involucrin, K1, and K6 parallel the known increase in ENaC expression that occurs with differentiation (Oda et al., 1999Oda Y. Imanzahrai A. Kwong A. Komuves L. Elias P. Largman C. Mauro T. Epithelial sodium channels are upregulated during epidermal differentiation.J Invest Dermatol. 1999; 113: 796-801https://doi.org/10.1046/j.1523-1747.1999.00742.xCrossref PubMed Scopus (26) Google Scholar). Differentiation in keratinocytes also appears to be modulated by other plasma membrane ion channels, including a nonselective cation channel (Mauro et al., 1995Mauro T. Dixon D.B. Hanley K. Isseroff R.R. Pappone P.A. Amiloride blocks a keratinocyte nonspecific cation channel and inhibits Ca(++)-induced keratinocyte differentiation.J Invest Dermatol. 1995; 105: 203-208Crossref PubMed Scopus (25) Google Scholar), nicotinic ACh channel (Grando, 1995Grando S.A. et al.Keratinocyte muscarinic acetylcholine receptors: immunolocalization and partial characterization.J Invest Dermatol. 1995; 104: 95-100Crossref PubMed Scopus (74) 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), and Cl– 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 ([published erratum appears in J Membr Biol 134: 168, 1993]): 201-209Crossref PubMed Scopus (59) Google Scholar;Wohlrab and Markwardt, 1999Wohlrab D. Markwardt F. Influence of ion channel blockers on proliferation and free intracellular Ca2+ concentration of human keratinocytes.Skin Pharmacol Appl Skin Physiol. 1999; 12: 257-265Crossref PubMed Scopus (13) Google Scholar). The mechanism(s) by which ENaC controls keratohyalin granule formation/dispersal and lipid secretion is (are) unknown, but probably relates to its control of Na+ and/or Ca2+ fluxes. Whereas the delayed expression of differentiation markers might simply reflect delayed skin maturation, α-ENaC –/– mice also exhibit premature lipid secretion that is not seen with a simple delay in skin maturation. We have reproduced these lipid secretion abnormalities by short-term treatment of adult hairless mouse skin with 1 µM amiloride, 1Behne MJ, Meyer J, Hanson KM, et al: Functional role of the sodium-hydrogen antiporter, NHE1, in the epidermis: pharmacologic and NHE1 null-allele mouse studies. J Invest Dermatol 114:797 (277), 2000. Full paper in preparation. a concentration that pharmacologically blocks the ENaC. Whereas amiloride also blocks the NHE1 antiporter at this concentration, lipid secretion abnormalities were not seen in mice treated with a selective NHE inhibitor, HOE-694, nor were lipid secretory abnormalities seen in NHE1 –/– mice, suggesting that lipid secretion is controlled by the ENaC. 1Behne MJ, Meyer J, Hanson KM, et al: Functional role of the sodium-hydrogen antiporter, NHE1, in the epidermis: pharmacologic and NHE1 null-allele mouse studies. J Invest Dermatol 114:797 (277), 2000. Full paper in preparation. Finally, when skin from newborn α-ENaC –/– mice was grafted onto athymic recipient mice, 2Guitard M, Mauro TM, Behne MJ, Hummler E: Implication for epithelial sodium channel (ENaC) in epidermal differentiation and hair follicle development. J Invest Dermatol 114:409, 2001. Full paper in preparation. epidermal hyperplasia persisted for the duration of the grafts, i.e., up to 8 wk, whereas +/+ skin, grafted in parallel, appeared normal. Currents through the ENaC could direct differentiation by signaling a number of different pathways. In fact, it is likely that the mechanism of action varies in spinous versus granular layer keratinocytes, and in the ENaC modulation of differentiation versus lipid secretion, because different types of ion channels are found in undifferentiated (basal) versus differentiated (granular layer) keratinocytes. Although the Ca2+-permeable channels in the granular layer keratinocytes have not yet been characterized by molecular or electrophysiologic methods, pharmacologic and ion-substitution experiments suggest that these keratinocytes express T- or L-type voltage-sensitive Ca2+ channels (Lee et al., 1992Lee S.H. Elias P.M. Proksch E. Menon G.K. Mao-Quiang M. Feingold K.R. Calcium and potassium are important regulators of barrier homeostasis in murine epidermis.J Clin Invest. 1992; 89: 530-538Crossref PubMed Scopus (177) Google Scholar,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;Resing et al., 1993Resing K.A. al-Alawi N. Blomquist C. Fleckman P. Dale B.A. Independent regulation of two cytoplasmic processing stages of the intermediate filament-associated protein filaggrin and role of Ca2+ in the second stage.J Biol Chem. 1993; 268: 25139-25145Abstract Full Text PDF PubMed Google Scholar;Grando et al., 1996Grando S.A. Horton R.M. Mauro T.M. Kist D.A. Lee T.X. Dahl M.V. Activation of keratinocyte nicotinic cholinergic receptors stimulates calcium influx and enhances cell differentiation.J Invest Dermatol. 1996; 107: 412-418Crossref PubMed Scopus (148) Google Scholar). Na+ influx through the ENaC could depolarize the plasma membrane, thus increasing Ca2+ influx through voltage-sensitive channels (Lee et al., 1992Lee S.H. Elias P.M. Proksch E. Menon G.K. Mao-Quiang M. Feingold K.R. Calcium and potassium are important regulators of barrier homeostasis in murine epidermis.J Clin Invest. 1992; 89: 530-538Crossref PubMed Scopus (177) Google Scholar;Mauro et al., 1995Mauro T. Dixon D.B. Hanley K. Isseroff R.R. Pappone P.A. Amiloride blocks a keratinocyte nonspecific cation channel and inhibits Ca(++)-induced keratinocyte differentiation.J Invest Dermatol. 1995; 105: 203-208Crossref PubMed Scopus (25) Google Scholar). Moreover, raised intracellular Ca2+ blocks lipid secretion in a manner that is reversible by L-type channel blockers (Lee et al., 1992Lee S.H. Elias P.M. Proksch E. Menon G.K. Mao-Quiang M. Feingold K.R. Calcium and potassium are important regulators of barrier homeostasis in murine epidermis.J Clin Invest. 1992; 89: 530-538Crossref PubMed Scopus (177) Google Scholar;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. Thus, abolishing Na+ influx through the ENaC would hyperpolarize the membrane and decrease Ca2+ influx, thereby allowing unregulated lipid secretion. Decreased Ca2+ influx also inhibits the post-translational processing of profilaggrin to filaggrin (Resing et al., 1993Resing K.A. al-Alawi N. Blomquist C. Fleckman P. Dale B.A. Independent regulation of two cytoplasmic processing stages of the intermediate filament-associated protein filaggrin and role of Ca2+ in the second stage.J Biol Chem. 1993; 268: 25139-25145Abstract Full Text PDF PubMed Google Scholar), which could explain the presence of abnormal keratohyalin granules in the –/– granular cells (Presland et al., 1995Presland R.B. Bassuk J.A. Kimball J.R. Dale B.A. Characterization of two distinct calcium-binding sites in the amino-terminus of human profilaggrin.J Invest Dermatol. 1995; 104: 218-223Crossref PubMed Scopus (51) Google Scholar). In humans, mutations in all ENaC subunits have been reported, resulting in hypoactivity of the ENaC that results in pseudohypoaldosteronism, or PHA-1 (Gründer, 1997Gründer S. et al.A mutation causing pseudohypoaldosteronism type 1 identifies a conserved glycine that is involved in the gating of the epithelial sodium channel.EMBO J. 1997; 16: 899-907Crossref PubMed Scopus (163) Google Scholar). No specific dermatologic findings are reported for this condition. All mutations tested so far retain significant rest activity of ENaC, however, which might explain the absence of severe pulmonary pathology in PHA-1 patients, whereas inactivation of the α-ENaC subunit is rapidly fatal due to respiratory failure (Hummler, 1996Hummler E. et al.Early death due to defective neonatal lung liquid clearance in α-ENaC-deficient mice.Nat Genet. 1996; 12: 325-328Crossref PubMed Scopus (732) Google Scholar;Bonny et al., 1999Bonny O. Chraibi A. Loffing J. Jaeger N.F. Gründer S. Horisberger J.D. Rossier B.C. Functional expression of a pseudohypoaldosteronism type I mutated epithelial Na+ channel lacking the pore-forming region of its α subunit.J Clin Invest. 1999; 104: 967-974Crossref PubMed Scopus (91) Google Scholar). The knockouts for the β- and γ-ENaC subunit are available, but were not analyzed for a potential skin phenotype (Barker et al., 1998Barker P.M. Nguyen M.S. Gatzy J.T. et al.Role of γENaC subunit in lung liquid clearance and electrolyte balance in newborn mice. Insights into perinatal adaptation and pseudohypoaldosteronism.J Clin Invest. 1998; 102: 1634-1640Crossref PubMed Scopus (213) Google Scholar;McDonald, 1999McDonald F.J. et al.Disruption of the β subunit of the epithelial Na+ channel in mice: hyperkalemia and neonatal death associated with a pseudohypoaldosteronism phenotype.Proc Natl Acad Sci USA. 1999; 96: 1727-1731Crossref PubMed Scopus (205) Google Scholar). Rest activity of the ENaC has been demonstrated in γ-ENaC –/– mice (Barker et al., 1998Barker P.M. Nguyen M.S. Gatzy J.T. et al.Role of γENaC subunit in lung liquid clearance and electrolyte balance in newborn mice. Insights into perinatal adaptation and pseudohypoaldosteronism.J Clin Invest. 1998; 102: 1634-1640Crossref PubMed Scopus (213) Google Scholar). These findings are consistent with our experimental observations, reported above, that almost complete ablation of the α-ENaC subunit is required to produce defects in epidermal differentiation, although a higher level of ENaC expression is required for normal lipid secretion. To a large extent, the histologic and ultrastructural findings in ENaC –/– mice resemble features of psoriasis and other psoriasiform dermatoses. Although we have not examined these disease states for alterations in the expression or function of the ENaC, the similar histologic presentation suggests that the ENaC channel could modulate, or even underlie, some of the defects in keratinocyte differentiation that are seen in these conditions. Graft experiments and skin-specific gene targeting of α-ENaC might help to further identify the role of this sodium channel in epidermis. This work was supported by NIH grant PO AR 39448-11A1 (TM) and the Medical Research Service, San Francisco Veteran's Affairs, and the Swiss National Science Foundation #31–063801.0O/1 (EH).
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