Epidermal-Specific Defect of GPI Anchor in Pig-a Null Mice Results in Harlequin Ichthyosis-Like Features
2004; Elsevier BV; Volume: 123; Issue: 3 Linguagem: Inglês
10.1111/j.0022-202x.2004.23227.x
ISSN1523-1747
AutoresMariko Hara‐Chikuma, Junji Takeda, Masahito Tarutani, Yoshikazu Uchida, Walter M. Holleran, Yoko Endo, Peter M. Elias, Shintaro Inoue,
Tópico(s)Dermatology and Skin Diseases
ResumoWe previously demonstrated that the epidermal-specific glycosylphosphatidylinositol (GPI)-anchor-deficient mice, generated by Pig-a gene disruption (Pig-a null mice), exhibited wrinkled and dry skin with hyperkeratosis and abnormal differentiation, and they died within a few days after birth. Here, we investigated the basis for the early demise of these animals, and the potential role of epidermal structural and biochemical abnormalities. The rapid demise of these animals was associated with both diminished epidermal permeability barrier function and decreased stratum corneum (SC) water content. The barrier abnormality could be attributed abnormal internal contents of lamellar bodies, with a downstream failure to generate normal extracellular lamellar bilayers in the SC. Moreover, processing profilaggrin to its monomeric form was impaired in Pig-a null mouse epidermis, while levels of the differentiation-specific proteins, involucrin, loricrin and profilaggrin were normal. Failure of filaggrin processing was accompanied by decreased activity of protein phosphatase 2A, an enzyme involved in profilaggrin to filaggrin processing. Thus, these studies demonstrate a critical role for GPI anchor and GPI-anchored proteins in divergent arms of epidermal terminal differentiation. While the permeability barrier abnormality can be attributed to defects in the lamellar body secretory system, the hydration abnormality is, in part, due to lack of availability of filaggrin-derived proteolytic products. Finally, since the dual abnormalities in the lamellar body secretory system and filaggrin processing resemble two key features of human Harlequin ichthyosis, Pig-a null mice could provide an appropriate analog for further studies of this disease. We previously demonstrated that the epidermal-specific glycosylphosphatidylinositol (GPI)-anchor-deficient mice, generated by Pig-a gene disruption (Pig-a null mice), exhibited wrinkled and dry skin with hyperkeratosis and abnormal differentiation, and they died within a few days after birth. Here, we investigated the basis for the early demise of these animals, and the potential role of epidermal structural and biochemical abnormalities. The rapid demise of these animals was associated with both diminished epidermal permeability barrier function and decreased stratum corneum (SC) water content. The barrier abnormality could be attributed abnormal internal contents of lamellar bodies, with a downstream failure to generate normal extracellular lamellar bilayers in the SC. Moreover, processing profilaggrin to its monomeric form was impaired in Pig-a null mouse epidermis, while levels of the differentiation-specific proteins, involucrin, loricrin and profilaggrin were normal. Failure of filaggrin processing was accompanied by decreased activity of protein phosphatase 2A, an enzyme involved in profilaggrin to filaggrin processing. Thus, these studies demonstrate a critical role for GPI anchor and GPI-anchored proteins in divergent arms of epidermal terminal differentiation. While the permeability barrier abnormality can be attributed to defects in the lamellar body secretory system, the hydration abnormality is, in part, due to lack of availability of filaggrin-derived proteolytic products. Finally, since the dual abnormalities in the lamellar body secretory system and filaggrin processing resemble two key features of human Harlequin ichthyosis, Pig-a null mice could provide an appropriate analog for further studies of this disease. ethylenediamine-N,N,N′,N′-tetraacetic acid glycosylphosphatidylinositol harlequin ichthyosis lamellar body okadaic acid phosphate-buffered saline phosphatidyl-inositolglycan class A phenylmethylsulfonylfluoride protein phosphatase 2A stratum corneum stratum granulosum transepidermal water loss A large number of eukaryotic membrane proteins are modified covalently by a glycosylphosphatidylinositol (GPI) moiety that serves as a membrane anchor. These GPI-anchored proteins exhibit diverse functions, including enzymes, cell adhesion molecules, cell surface antigens, and membrane-bound receptors (Kinoshita et al., 1995Kinoshita T. Inoue N. Takeda J. Defective glycosyl phosphatidylinositol anchor synthesis and paroxysmal nocturnal hemoglobinuria.Adv Immunol. 1995; 60: 57-103Crossref PubMed Google Scholar). In humans, a somatic mutation in hematopoietic stem cells of the X-linked Pig-a gene causes paroxysmal nocturnal hemoglobinuria, a hemolytic disease, due to deficiency of the GPI-anchored protein, CD 59 (Kinoshita et al., 1995Kinoshita T. Inoue N. Takeda J. Defective glycosyl phosphatidylinositol anchor synthesis and paroxysmal nocturnal hemoglobinuria.Adv Immunol. 1995; 60: 57-103Crossref PubMed Google Scholar). GPI anchor synthesis is a multistep process involving a series of gene products. All of the genes involved in GPI anchor biosynthesis are autosomal except Pig-a, which instead is located on the X chromosome (Kinoshita et al., 1997Kinoshita T. Ohishi K. Takeda J. GPI-anchor synthesis in mammalian cells: Genes, their products, and a deficiency.J Biochem (Tokyo). 1997; 122: 251-257Crossref PubMed Scopus (123) Google Scholar). Lack of the Pig-a gene, which encodes a class A phosphatidylinositol glycan, causes a variety of downstream deficiencies in the GPI anchor and in GPI-anchored proteins (Watanabe et al., 1998Watanabe R. Inoue N. Westfall B. Taron C.H. Orlean P. Takeda J. Kinoshita T. The first step of glycosylphosphatidylinositol biosynthesis is mediated by a complex of PIG-A, PIG-H, PIG-C and GPI1.EMBO J. 1998; 17: 877-885Crossref PubMed Scopus (126) Google Scholar). To investigate whether GPI anchor and GPI-anchored proteins are important in the epidermis, we generated a strain of epidermal-specific, Pig-a-gene-deficient mice (Pig-a null mice), utilizing a Cre-loxP recombinant system (Tarutani et al., 1997Tarutani M. Itami S. Okabe M. et al.Tissue-specific knockout of the mouse Pig-a gene reveals important roles for GPI-anchored proteins in skin development.Proc Natl Acad Sci USA. 1997; 94: 7400-7405Crossref PubMed Scopus (216) Google Scholar;Gao et al., 2002Gao X.H. Kondoh G. Tarutani M. et al.Rapid compensation for glycosylphosphatidylinositol anchor deficient keratinocytes after birth: Visualization of glycosylphosphatidylinositol-anchored proteins in situ.J Invest Dermatol. 2002; 118: 998-1002Crossref PubMed Scopus (2) Google Scholar). The skin of affected Pig-a null mice was wrinkled and dry, exhibiting prominent hyperkeratosis but no epidermal hyperplasia, and they died within a few days after birth. Although these studies demonstrated that GPI anchor and GPI-anchored proteins must be critical for epidermal structure and function, the basis for the early demise of the Pig-a-deficient animals remained unknown. The permeability barrier, which is required for life in a terrestrial environment, relies largely upon a lipid-enriched matrix localized within the extracellular domains of the outer layers of the epidermis, the stratum corneum (SC). A competent barrier requires organization of this hydrophobic matrix into mature lamellar membranes, which are formed from lipid contents secreted from epidermal lamellar bodies (LB) (Elias and Menon, 1991Elias P.M. Menon G.K. Structural and lipid biochemical correlates of the epidermal permeability barrier.Adv Lipid Res. 1991; 24: 1-26Crossref PubMed Google Scholar). LB appear first in the stratum spinosum layer, and their numbers increase in the stratum granulosum (SG) layer of the epidermis. In the outermost SG layer, LB migrate to the apical cytosol, where they fuse with the plasma membrane, releasing their contents into the extracellular domains at the SG–SC interface, immediately prior to cornification (Elias et al., 1998Elias P.M. Cullander C. Mauro T. Rassner U. Komuves L. Brown B.E. Menon G.K. The secretory granular cell: The outermost granular cell as a specialized secretory cell.J Investig Dermatol Symp Proc. 1998; 3: 87-100Abstract Full Text PDF PubMed Scopus (122) Google Scholar). Cornified envelopes (CE), which consist of several cross-linked proteins; e.g., involucrin and loricrin, impart mechanical and chemical resistance to the corneocyte, as well as a scaffold for the organization of secreted LB contents into extracellular lamellar membranes. By linking adjacent keratin filaments, processed filaggrin also becomes a CE constituent, at least in the lower SC (Steven and Steinert, 1994Steven A.C. Steinert P.M. Protein composition of cornified cell envelopes of epidermal keratinocytes.J Cell Sci. 1994; 107: 693-700Crossref PubMed Google Scholar). Several proteases and phosphatases, including a protein phosphatase of the phosphatase 2A family, are believed to partake in profilaggrin to profilaggrin processing (Kam et al., 1993Kam E. Resing K.A. Lim S.K. Dale B.A. Identification of rat epidermal profilaggrin phosphatase as a member of the protein phosphatase 2A family.J Cell Sci. 1993; 106: 219-226PubMed Google Scholar;Pearton et al., 2001Pearton D.J. Nirunsuksiri W. Rehemtulla A. Lewis S.P. Presland R.B. Dale B.A. Proprotein convertase expression and localization in epidermis: Evidence for multiple roles and substrates.Exp Dermatol. 2001; 10: 193-203Crossref PubMed Scopus (80) Google Scholar). Filaggrin is a hydrophilic and cationic protein that aids both in the initial aggregation of keratin filaments within the corneocyte cytosol, and in attachment to keratin filaments to the CE (Ishida-Yamamoto et al., 1999Ishida-Yamamoto A. Tanaka H. Nakane H. Takahashi H. Hashimoto Y. Iizuka H. Programmed cell death in normal epidermis and loricrin keratoderma. Multiple functions of profilaggrin in keratinization.J Investig Dermatol Symp Proc. 1999; 4: 145-149Abstract Full Text PDF PubMed Scopus (35) Google Scholar). Higher in the SC, filaggrin is hydrolyzed to its constituent amino acids by an aspartate proteinase (Horikoshi et al., 1999Horikoshi T. Igarashi S. Uchiwa H. Brysk H. Brysk M.M. Role of endogenous cathepshin D-like and chymotrypsin-like proteolysis in human epidermal desquamation.Br J Dermatol. 1999; 141: 453-459Crossref PubMed Scopus (101) Google Scholar), and further deiminated to products that together comprise much of the natural humectants of the SC (Rawlings et al., 1994Rawlings A.V. Scott I.R. Harding C.R. Bowser P.A. Stratum corneum moisturization at the molecular level.J Invest Dermatol. 1994; 103: 731-741Abstract Full Text PDF PubMed Scopus (407) Google Scholar). Harlequin ichthyosis (HI) is a severe dermatological, autosomal recessive disorder (Dale et al., 1990Dale B.A. Holbrook K.A. Fleckman P. Kimball J.R. Brumbaugh S. Sybert V.P. Heterogeneity in harlequin ichthyosis, an inborn error of epidermal keratinization: Variable morphology and structural protein expression and a defect in lamellar granules.J Invest Dermatol. 1990; 94: 6-18Abstract Full Text PDF PubMed Google Scholar;Stewart et al., 2001Stewart H. Smith P.T. Gaunt L. et al.De novo deletion of chromosome 18q in a baby with harlequin ichthyosis.Am J Med Genet. 2001; 102: 342-345Crossref PubMed Scopus (15) Google Scholar), with affected infants often dying within days-to-weeks of birth, apparently due to massive hyperkeratosis and/or a severe barrier abnormality (Dale and Kam, 1993Dale B.A. Kam E. Harlequin ichthyosis. Variability in expression and hypothesis for disease mechanism.Arch Dermatol. 1993; 129: 1471-1477Crossref PubMed Scopus (44) Google Scholar). HI epidermis shows (1) absence or paucity of LB, (2) abnormal (decreased-to-absent) extracellular lamellar membranes structures, and (3) massive hyperkeratosis of the SC. Although the gene(s) that is (are) responsible for HI has (have) not yet been identified, HI has been divided into three subtypes (I–III), based in part on the status of profilaggrin and filaggrin processing (Dale et al., 1990Dale B.A. Holbrook K.A. Fleckman P. Kimball J.R. Brumbaugh S. Sybert V.P. Heterogeneity in harlequin ichthyosis, an inborn error of epidermal keratinization: Variable morphology and structural protein expression and a defect in lamellar granules.J Invest Dermatol. 1990; 94: 6-18Abstract Full Text PDF PubMed Google Scholar). A defect of conversion from profilaggrin to filaggrin has reported in HI types I and II (Dale and Kam, 1993Dale B.A. Kam E. Harlequin ichthyosis. Variability in expression and hypothesis for disease mechanism.Arch Dermatol. 1993; 129: 1471-1477Crossref PubMed Scopus (44) Google Scholar), while profilaggrin is lacking in HI type III. In addition, decreased protein phosphatase 2A (PP2A) activity is associated with HI type II (Kam et al., 1997Kam E. Nirunsuksiri W. Hager B. Fleckman P. Dale B.A. Protein phosphatase activity in human keratinocytes cultured from normal epidermis and epidermis from patients with harlequin ichthyosis.Br J Dermatol. 1997; 137: 874-882Crossref PubMed Scopus (18) Google Scholar). A relationship between HI pathogenesis and GPI-anchored protein(s) has not yet been reported. To investigate the role of GPI anchor and GPI-anchored proteins in epidermal function, we further analyzed epidermal-specific Pig-a null mice. We demonstrate: first, severe abnormalities of epidermal permeability barrier function; second, abnormal structure of both LB internal contents and their resultant extracellular membrane lamellae; and third, defects in the processing of profilaggrin to filaggrin, associated with a reduced PP2A activity. These studies show that GPI anchor and GPI-anchored proteins influence key epidermal functions, including epidermal permeability barrier, as well as SC hydration. Since the abnormalities in Pig-a null mice resemble key features of human HI, this model might allow new insights into the pathogenic mechanisms operative in HI. To determine whether GPI anchor and GPI-anchored proteins affect one or more critical epidermal functions, we first examined the epidermal permeability barrier function(s) and SC hydration. Transepidermal water loss (TEWL) across Pig-a null mouse skin explants were compared to rates for comparable samples from wild-type littermates (control) using a gravimetric technique (Nolte et al., 1993Nolte C.J. Oleson M.A. Bilbo P.R. Parenteau N.L. Development of a stratum corneum and barrier function in an organotypic skin culture.Arch Dermatol Res. 1993; 285: 466-474Crossref PubMed Scopus (68) Google Scholar). As shown in Figure 1a, TEWL values were markedly higher in the Pig-a null mice than in wild-type control (5.0-fold increase; p<0.01). Despite increased rates of water loss, high-frequency, skin-surface conductance, representative of SC water content, was significantly lower in Pig-a null mice than in control mice (p<0.01) (Figure 1b). Thus, two key epidermal functions: (1) epidermal permeability barrier function and (2) SC hydration, are compromised in epidermal-specific Pig-a null mice. To investigate the basis for the increased TEWL in the Pig-a null mice, we next performed an ultrastructural analysis of the outer epidermis by electron microscopy, utilizing both osmium tetroxide (OsO4) and ruthenium tetroxide (RuO4) post-fixed tissue samples (Figure 2). While normal extracellular lamellar structures were present in the SC of wild-type mice (Figure 2b), lamellar membrane structures in Pig-a null mouse SC were shortened, distorted, and replaced by incompletely processed, LB-derived materials (Figure 2a). Moreover, most of the LBs in the SG layer of Pig-a null epidermis also displayed abnormalities in size and/or internal contents, indicating an abnormality in LB formation, although the overall numbers of LB (=density) appeared to be normal in Pig-a null mice (Figure 2c). In contrast, the structure of corneocytes, including the cornified envelope (CE), appeared normal in Pig-a null mouse SC (Figure 2a). Together, these findings suggest that the permeability barrier abnormality in Pig-a null mice results from defects in LB structure/contents, which in turn result in extracellular abnormalities in lamellar membrane structures. Since our prior study demonstrated hyperkeratosis in the Pig-a null mice (Tarutani et al., 1997Tarutani M. Itami S. Okabe M. et al.Tissue-specific knockout of the mouse Pig-a gene reveals important roles for GPI-anchored proteins in skin development.Proc Natl Acad Sci USA. 1997; 94: 7400-7405Crossref PubMed Scopus (216) Google Scholar), we next investigated whether alterations in either the levels of keratinocyte differentiation protein markers or their breakdown products could also contribute to the functional abnormalities in these animals. Western blot analysis revealed that the levels of profilaggrin, filaggrin dimer (Figure 3a), involucrin (Figure 3b) and loricrin (Figure 3c) were not altered in the Pig-a null versus normal control mice. The Pig-a null epidermis, however, exhibited a virtual absence of filaggrin monomer (Figure 3a). To determine whether the lack of filaggrin monomer in the Pig-a null mice reflects defective processing of profilaggrin to filaggrin or increased degradation of filaggrin to free amino acids, we next analyzed levels of free amino acids in the SC. Total free amino acid content was significantly reduced in the Pig-a null mice (2.9±0.5 vs 18.5±1.9 nmol per nmol, p<0.001). The distribution of amino acids was also altered in Pig-a null mouse SC in comparison to normal control mice (data not shown). Specially, the levels of arginine, glycine, histidine, and serine, which are the major components of filaggrin, were selectively decreased in Pig-a null mouse SC (data not shown). Moreover, a high correlation was found between the amino acid composition of wild type SC and that of murine filaggrin (Genebank accession #A31488) (r=0.99, p<0.01) (Figure 4a), while the same correlation between Pig-a null mice and murine filaggrin were low (r=0.56) (Figure 4b). In contrast, the composition of defective amino acids (i.e., the amino acid content of wild-type minus Pig-a null for each) correlated highly with the amino acid composition of normal murine filaggrin (r=0.92, p 400 kDa), is synthesized in the SG cell layer of the epidermis, where it localizes with in F-type keratohyalin granules (Presland et al., 2000Presland R.B. Boggess D. Lewis S.P. Hull C. Fleckman P. Sundberg J.P. Loss of normal profilaggrin and filaggrin in flaky tail (ft/ft) mice: An animal model for the filaggrin-deficient skin disease ichthyosis vulgaris.J Invest Dermatol. 2000; 115: 1072-1081Crossref PubMed Scopus (151) Google Scholar). During terminal differentiation, profilaggrin is processed to filaggrin dimer, which is followed by further dephosphorylation of filaggrin by PP2A, along with site-specific proteolysis yielding the filaggrin monomer (Presland et al., 1997Presland R.B. Kimball J.R. Kautsky M.B. Lewis S.P. Lo C.Y. Dale B.A. Evidence for specific proteolytic cleavage of the N-terminal domain of human profilaggrin during epidermal differentiation.J Invest Dermatol. 1997; 108: 170-178Crossref PubMed Scopus (73) Google Scholar). The observed decrease in PP2A activity correlates with, and likely explains, the lack of filaggrin monomer in the Pig-a null mouse epidermis. Yet, despite decreased enzyme activity, PP2A protein levels remained normal in Pig-a null mouse epidermis. Our results suggest further that GPI anchor and GPI-anchored proteins could be linked to the human disorder of cornification, HI, which bears clinical, structural, and biochemical resemblance to the skin phenotype of pig-a null mice (Table I). HI is a severe, autosomal recessive skin disorder (Dale et al., 1990Dale B.A. Holbrook K.A. Fleckman P. Kimball J.R. Brumbaugh S. Sybert V.P. Heterogeneity in harlequin ichthyosis, an inborn error of epidermal keratinization: Variable morphology and structural protein expression and a defect in lamellar granules.J Invest Dermatol. 1990; 94: 6-18Abstract Full Text PDF PubMed Google Scholar;Stewart et al., 2001Stewart H. Smith P.T. Gaunt L. et al.De novo deletion of chromosome 18q in a baby with harlequin ichthyosis.Am J Med Genet. 2001; 102: 342-345Crossref PubMed Scopus (15) Google Scholar), whose genetic defect still remains unidentified. The most distinctive metabolic features of human HI are a paucity of LB and defective filaggrin, both of which are dominant features of the Pig-a null mouse. The defective processing of profilaggrin to filaggrin monomer has been demonstrated in HI types I and II (Dale et al., 1990Dale B.A. Holbrook K.A. Fleckman P. Kimball J.R. Brumbaugh S. Sybert V.P. Heterogeneity in harlequin ichthyosis, an inborn error of epidermal keratinization: Variable morphology and structural protein expression and a defect in lamellar granules.J Invest Dermatol. 1990; 94: 6-18Abstract Full Text PDF PubMed Google Scholar), which also was evident in Pig-a null epidermis. Moreover, as in the Pig-a null mouse, decreased PP2A activity, but not PP2Ac protein levels, are found in HI type II keratinocytes (Kam et al., 1997Kam E. Nirunsuksiri W. Hager B. Fleckman P. Dale B.A. Protein phosphatase activity in human keratinocytes cultured from normal epidermis and epidermis from patients with harlequin ichthyosis.Br J Dermatol. 1997; 137: 874-882Crossref PubMed Scopus (18) Google Scholar).Table IComparison of symptoms between Pig-a null mice and human HISymptomWild-typePig-a nullHIaHarlequin ichthyosis (HI) is subdivided into three subtype; type I, II and III (Dale and Kam, 1993).Morphology Hyperkeratosis-++ Lamellar bodyNormalAbnormalAbnormal SC lamellarPresentAbsentAbsentFilaggrin ProFG→FGProFG→FGProFG onlyProFG only or AbsentbOnly profilaggrin (ProFil) was expressed in type I and II HI. Both ProFil and filaggrin (Fil) are absent in type III HI case (Dale and Kam, 1993). PP2A activityNormalDecreaseDecreasecThe decrease in PP2A activity has been reported in type II HI case (Kam et al, 1997). Not reported in type I and III HI. PP2Ac protein+++cThe decrease in PP2A activity has been reported in type II HI case (Kam et al, 1997). Not reported in type I and III HI.ProFG, profilaggrin; FG, filaggrina Harlequin ichthyosis (HI) is subdivided into three subtype; type I, II and III (Dale and Kam, 1993Dale B.A. Kam E. Harlequin ichthyosis. Variability in expression and hypothesis for disease mechanism.Arch Dermatol. 1993; 129: 1471-1477Crossref PubMed Scopus (44) Google Scholar).b Only profilaggrin (ProFil) was expressed in type I and II HI. Both ProFil and filaggrin (Fil) are absent in type III HI case (Dale and Kam, 1993Dale B.A. Kam E. Harlequin ichthyosis. Variability in expression and hypothesis for disease mechanism.Arch Dermatol. 1993; 129: 1471-1477Crossref PubMed Scopus (44) Google Scholar).c The decrease in PP2A activity has been reported in type II HI case (Kam et al., 1997Kam E. Nirunsuksiri W. Hager B. Fleckman P. Dale B.A. Protein phosphatase activity in human keratinocytes cultured from normal epidermis and epidermis from patients with harlequin ichthyosis.Br J Dermatol. 1997; 137: 874-882Crossref PubMed Scopus (18) Google Scholar). Not reported in type I and III HI. Open table in a new tab ProFG, profilaggrin; FG, filaggrin An "HI-like mouse" has also been identified as a spontaneous mutation at Jackson Laboratory (Sundberg et al., 1997Sundberg J.P. Boggess D. Hogan M.E. et al.Harlequin ichthyosis (ichq): A juvenile lethal mouse mutation with ichthyosiform dermatitis.Am J Pathol. 1997; 151: 293-310PubMed Google Scholar). Although the prominent hyperkeratosis in these mice more clearly resembles human HI, recent studies have revealed the abnormality as due to mutations in cystatin M/E, a serine protease inhibitor (Zeeuwen et al., 2002Zeeuwen P.L. van Vlijmen-Willems I.M. Hendriks W. Merkx G.F. Schalkwijk J. A null mutation in the cystatin M/E gene of ichq mice causes juvenile lethality and defects in epidermal cornification.Hum Mol Genet. 2002; 11: 2867-2875Crossref PubMed Google Scholar). Moreover, the LB secretory system is normal in these mice; and furthermore, recent studies exclude cystatin M/E mutations in HI patients (Zeeuwen et al., 2003Zeeuwen P.L. Dale B.A. de Jongh G.J. et al.The human cystatin M/E gene (CST6): Exclusion candidate gene for harlequin ichthyosis.J Invest Dermatol. 2003; 121: 65-68Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Not only do these "HI mice" display normal membrane and internal contents of LB, 2Elias PM, Crumrine D, Sunberg JA: Unpublished observations. there is no evidence of aberrant filaggrin processing. 3Hara M, Endo Y, Inoue S: Unpublished results. On the other hand, the Pig-a null mouse reveals close physiological, structural, and biochemical similarities to HI. Interestingly, Pig-a is an X-linked gene, while HI is an autosomal recessive disease. Therefore it is not likely that Pig-a mutation alone accounts for HI. Our results, however, suggest that another gene(s) involved in GPI-anchor biosynthesis, or a GPI-anchored protein, might be defective in HI. For example, GPI anchor or GPI-anchored protein(s) might be critical for cornification, such that alterations would affect both LB biosynthesis/secretion and filaggrin processing, as seen in both Pig-a-deficient mice and HI patients (type I and II). These results suggest that the Pig-a null mouse might be an appropriate model for future studies on the etiology and pathogenesis of HI. In summary, our results indicate that GPI anchor and GPI-anchored proteins appear to be important both for the development of a normal permeability barrier, and for the processing of profilaggrin to filaggrin monomers. Finally, our results show that defects of the GPI anchor and GPI-anchored proteins in the epidermis result in a HI-like disease with hyperkeratosis, abnormal both lamellar body secretory system and filaggrin processing, providing the first evidence that such defects could be etiological/pathogenic features of the human disorder. The generation of epidermal-specific GPI-anchor deficient mice through Pig-a gene disruption has been described previously (Tarutani et al., 1997Tarutani M. Itami S. Okabe M. et al.Tissue-specific knockout of the mouse Pig-a gene reveals important roles for GPI-anchored proteins in skin development.Proc Natl Acad Sci USA. 1997; 94: 7400-7405Crossref PubMed Scopus (216) Google Scholar). Newborn Pig-a null (-/-) mice were compared to wild-type (+/+) littermates. Genotypes were confirmed by PCR and Southern blot analysis (Tarutani et al., 1997Tarutani M. Itami S. Okabe M. et al.Tissue-specific knockout of the mouse Pig-a gene reveals important roles for GPI-anchored proteins in skin development.Proc Natl Acad Sci USA. 1997; 94: 7400-7405Crossref PubMed Scopus (216) Google Scholar). All animal experiments were approved by the Animal research Committee of Kanebo Basic Research laboratory in accordance with the National Research Council Guide (National Research Council: National Research Council (NRC) guide, 1996National Research Council: National Research Council (NRC) guide National Academy Press, Washington, DC1996Google Scholar). TEWL was measured gravimetrically from samples taken 6–8 h after birth, as described previously (Nolte et al., 1993Nolte C.J. Oleson M.A. Bilbo P.R. Parenteau N.L. Development of a stratum corneum and barrier function in an organotypic skin culture.Arch Dermatol Res. 1993; 285: 466-474Crossref PubMed Scopus (68) Google Scholar). Briefly, skin explants (1.8 cm2) were placed dermis-side down onto parafilm squares, then the lateral edges were sealed with petrolatum in order to prevent water loss from areas other than the epidermal surface. The skin samples were then weighed hourly at ambient temperature and humidity (25±2°C, 50%–55%) over a period of 6 h. SC hydration (water content) was measured by high-frequency surface electrical conductance using a Skicon-200 (IBS, Hamamatsu, Japan). Skin samples obtained 36 h after birth were minced to <0.5 mm3, fixed in modified Karnovsky's fixative overnight, and post-fixed in either 1.5% aqueous OsO4 containing 1.5% potassium ferrocyanide, or in 0.2% RuO4 in 0.1 M sodium cacodylate buffer, as previously described (Holleran et al., 1997Holleran W.M. Uchida Y. Halkier-Sorensen L. Haratake A. Hara M. Epstein J.H. Elias P.M. Structural and biochemical basis for the UVB-induced alterations in epidermal barrier function.Photodermatol Photoimmunol Photomed. 1997; 13: 117-128Crossref PubMed Scopus (113) Google Scholar). Ultrathin sections were examined by an electron microscope (Zeiss 10A, Carl Zeiss, Thornwood, New York) operated at 60 kV, with or without further contrasting with lead citrate. Immunoblotting was performed as described previously (Sakai et al., 2003Sakai S. Endo Y. Ozawa N. et al.Characteristics of the epidermis and stratum corneum of hairless mice with experimentally induced diabetes mellitus.J Invest Dermatol. 2003; 120: 79-85Crossref PubMed Scopus (62) Google Scholar) using polyclonal antibodies to mouse filaggrin (AF 111, BabCo, Berkeley, California), involucrin (BabCo) and loricrin (AF 62, BabCo), and monoclonal antibodies to keratin 6 (LHK6B, NeoMarkers, Fremont, California) and PP2Ac (Upstate Biotechnology, Lake Placid, New York). Epidermal sheets were separated from the dermis by incubation in PBS solution containing 10 mM EDTA at 37°C for 60 min, and homogenized as previously described (Kam et al., 1997Kam E. Nirunsuksiri W. Hager B. Fleckman P. Dale B.A. Protein phosphatase activity in human keratinocytes cultured from normal epidermis and epidermis from patients with harlequin ichthyosis.Br J Dermatol. 1997; 137: 874-882Crossref PubMed Scopus (18) Google Scholar). Protein phosphatase (PP) assay was performed as previously described (Kam et al., 1997Kam E. Nirunsuksiri W. Hager B. Fleckman P. Dale B.A. Protein phosphatase activity in human keratinocytes cultured from normal epidermis and epidermis from patients with harlequin ichthyosis.Br J Dermatol. 1997; 137: 874-882Crossref PubMed Scopus (18) Google Scholar). Phosphorylated glycogen phophorylase (Phos A) was prepared from purified glycogen phosphorylase (Phos B, Sigma, St Louis, Missouri) and [32P]-γ-ATP (Amersham, Piscataway, New Jersey, 110 GBq per mmol) by incubation with phosphorylase kinase and used as the substrate for the PP kinetic assay. PP activity was calculated as radioactivity (c.p.m.) from the dephosphorylation of the substrate per minute per microgram of protein. Okadaic acid (OA), a potent PP inhibitor of PP2A rather than PP1, was added to the assay mixture, and then the reaction was started by adding substrate. Protein was determined using the Bio-Rad DC protein assay kit (Bio-Rad Laboratories, Hercules, California). SC samples were removed from the skin by tape stripping and incubated in 10 mM HCl to extract the water-soluble fractions. Amino acids were analyzed using a Hitachi Amino Acid Analyzer L-8800 as described previously (Sakai et al., 2003Sakai S. Endo Y. Ozawa N. et al.Characteristics of the epidermis and stratum corneum of hairless mice with experimentally induced diabetes mellitus.J Invest Dermatol. 2003; 120: 79-85Crossref PubMed Scopus (62) Google Scholar). The total amino acid content derived from the SC proteins was assayed as described previously (Sakai et al., 2000Sakai S. Sasai S. Endo Y. Matue K. Tagami H. Inoue S. Characterization of the physical properties of the stratum corneum by a new tactile sensor.Skin Res Technol. 2000; 6: 128-134Crossref PubMed Scopus (25) Google Scholar). The free amino acid content per total amino acid content from the SC proteins (nmol/nmol) was calculated as described previously (Sakai et al., 2000Sakai S. Sasai S. Endo Y. Matue K. Tagami H. Inoue S. Characterization of the physical properties of the stratum corneum by a new tactile sensor.Skin Res Technol. 2000; 6: 128-134Crossref PubMed Scopus (25) Google Scholar). Statistical significance was assessed using SAS analysis system (SAS Institute Japan, Tokyo, Japan). The authors thank Debbie Crumrine for expert technical assistance.
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