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• W2080254988 abstract "Liver X receptor-α and -β are members of the nuclear hormone receptor superfamily that heterodimerize with retinoid X receptor and are activated by oxysterols. In recent studies we found that treatment of cultured human keratinocytes with oxysterolstimulated differentiation, as demonstrated by increased expression of involucrin and transglutaminase, and inhibited proliferation. The aims of this study were to determine: (i) whether oxysterols applied topically to the skin of mice induce differentiation in normal epidermis; (ii) whether this effect is mediated via liver X receptor-α and/or liver X receptor-β; and (iii) whether oxysterols normalize epidermal morphology in an animal model of epidermal hyperplasia. Topical treatment of normal hairless mice with 22(R)-hydroxycholesterol or 24(S),25-epoxycholesterol resulted in a decrease in epidermal thickness and a decrease in keratinocyte proliferation assayed by proliferating cell nuclear antigen staining. Moreover, oxysterol treatment increased the levels of involucrin, loricrin, and profilaggrin protein and mRNA in the epidermis, indicating that oxysterols stimulate epidermal differentiation. Additionally, topical oxysterol pretreatment improved permeability barrier homeostasis. Whereas liver X receptor-α–/– mice revealed no alterations in epidermal differentiation, the epidermis was thinner in liver X receptor-β–/– mice than in wild-type mice, with a reduced number of proliferating cell nuclear antigen positive cells and a modest reduction in the expression of differentiation markers. Topical oxysterol treatment induced differentiation in liver X receptor-α–/– mice whereas in liver X receptor-β–/– mice there was no increase in the expression of differentiation markers. Whereas both liver X receptor-α and liver X receptor-β are expressed in cultured human keratinocytes and in fetal rat skin, only liver X receptor-β was observed on northern blotting in adult mouse epidermis. Finally, treatment of hyperproliferative epidermis with oxysterols restored epidermal homeostasis. These studies demonstrate that epidermal differentiation is regulated by liver X receptor-β and that oxysterols, acting via liver X receptor-β, can induce differentiation and inhibit proliferation in vivo. The ability of oxysterols to reverse epidermal hyperplasia suggests that these agents could be beneficial for the treatment of skin disorders associated with hyperproliferation and/or altered differentiation. Liver X receptor-α and -β are members of the nuclear hormone receptor superfamily that heterodimerize with retinoid X receptor and are activated by oxysterols. In recent studies we found that treatment of cultured human keratinocytes with oxysterolstimulated differentiation, as demonstrated by increased expression of involucrin and transglutaminase, and inhibited proliferation. The aims of this study were to determine: (i) whether oxysterols applied topically to the skin of mice induce differentiation in normal epidermis; (ii) whether this effect is mediated via liver X receptor-α and/or liver X receptor-β; and (iii) whether oxysterols normalize epidermal morphology in an animal model of epidermal hyperplasia. Topical treatment of normal hairless mice with 22(R)-hydroxycholesterol or 24(S),25-epoxycholesterol resulted in a decrease in epidermal thickness and a decrease in keratinocyte proliferation assayed by proliferating cell nuclear antigen staining. Moreover, oxysterol treatment increased the levels of involucrin, loricrin, and profilaggrin protein and mRNA in the epidermis, indicating that oxysterols stimulate epidermal differentiation. Additionally, topical oxysterol pretreatment improved permeability barrier homeostasis. Whereas liver X receptor-α–/– mice revealed no alterations in epidermal differentiation, the epidermis was thinner in liver X receptor-β–/– mice than in wild-type mice, with a reduced number of proliferating cell nuclear antigen positive cells and a modest reduction in the expression of differentiation markers. Topical oxysterol treatment induced differentiation in liver X receptor-α–/– mice whereas in liver X receptor-β–/– mice there was no increase in the expression of differentiation markers. Whereas both liver X receptor-α and liver X receptor-β are expressed in cultured human keratinocytes and in fetal rat skin, only liver X receptor-β was observed on northern blotting in adult mouse epidermis. Finally, treatment of hyperproliferative epidermis with oxysterols restored epidermal homeostasis. These studies demonstrate that epidermal differentiation is regulated by liver X receptor-β and that oxysterols, acting via liver X receptor-β, can induce differentiation and inhibit proliferation in vivo. The ability of oxysterols to reverse epidermal hyperplasia suggests that these agents could be beneficial for the treatment of skin disorders associated with hyperproliferation and/or altered differentiation. 24(S),25-epoxycholesterol 22(R)-hydroxycholesterol differential interference–contrast proliferating cell nuclear antigen liver X receptor retinoid X receptor peroxisome proliferator-activated receptor transepidermal water loss Epidermal differentiation is required for the formation of the stratum corneum that provides both the mechanical and permeability barrier between the external environment and the internal milieu (Fuchs, 1990Fuchs E. Epidermal differentiation. The bare essentials.J Cell Biol. 1990; 111: 2807-2814Crossref PubMed Scopus (568) Google Scholar;Jackson et al., 1993Jackson S.M. Williams M.L. Feingold K.R. Elias P.M. Pathobiology of the stratum corneum.West J Med. 1993; 158: 279-285PubMed Google Scholar;Eckert et al., 1997Eckert R.L. Crish J.F. Robinson N.A. The epidermal keratinocyte as a model for the study of gene regulation and function.Physiol Rev. 1997; 77: 397-424Crossref PubMed Scopus (331) Google Scholar). The permeability barrier, which is essential for terrestrial life, is sustained by extracellular, lipid-enriched lamellar membranes in the stratum corneum. These extracellular lipid-enriched lamellar membranes inhibit water and aqueous solute movement (Elias and Menon, 1991Elias P.M. Menon G.M. Structural and biochemical correlates of the epidermal permeability barrier.Adv Lipid Res. 1991; 24: 1-26Crossref PubMed Google Scholar;Jackson et al., 1993Jackson S.M. Williams M.L. Feingold K.R. Elias P.M. Pathobiology of the stratum corneum.West J Med. 1993; 158: 279-285PubMed Google Scholar). Corneocytes in the stratum corneum are postmitotic, terminally differentiated, anuclear keratinocytes with a rigid cornified envelope that provides the extraordinary mechanical strength to the stratum corneum (Reichert et al., 1993Reichert U. Michel S. Schmidt R. The cornified cell envelope. A key structure of the terminally differentiated keratinocytes.in: Darmon M. Blumenberg M. Molecular Biology of the Skin. Academic Press, San Diego1993: 107-297Crossref Google Scholar;Steinert, 1995Steinert P.M. A model for the hierarchical structure of the human epidermal cornified cell envelope.Cell Death Differ. 1995; 2: 23-35PubMed Google Scholar). The cornified envelope is formed by extensive, covalent cross-linking of involucrin, loricrin, and other proteins (Steinert and Marenkov, 1995Steinert P.M. Marenkov L.N. The proteins elafin, filaggrin, keratin intermediate filaments, loricrin and SPRs are isopeptide crosslinked components of the human epidermal cornified cell envelope.J Biol Chem. 1995; 270: 17702-17711Crossref PubMed Scopus (464) Google Scholar,Steinert and Marenkov, 1997Steinert P.M. Marenkov L.N. Involucrin is an important early component in the assembly of the epidermal cornified cell envelope.J Biol Chem. 1997; 272: 2021-2030Crossref PubMed Scopus (193) Google Scholar). These proteins are expressed in a spatially and temporally regulated pattern as suprabasal keratinocytes undergo terminal differentiation during their upward migration towards the surface of the epidermis, and therefore, they serve as markers of the differentiation process (Fuchs, 1990Fuchs E. Epidermal differentiation. The bare essentials.J Cell Biol. 1990; 111: 2807-2814Crossref PubMed Scopus (568) Google Scholar;Eckert et al., 1997Eckert R.L. Crish J.F. Robinson N.A. The epidermal keratinocyte as a model for the study of gene regulation and function.Physiol Rev. 1997; 77: 397-424Crossref PubMed Scopus (331) Google Scholar). The cross-linking of these proteins is catalyzed by the keratinocyte-specific enzyme, transglutaminase I, which is also expressed late in keratinocyte differentiation (Fuchs, 1990Fuchs E. Epidermal differentiation. The bare essentials.J Cell Biol. 1990; 111: 2807-2814Crossref PubMed Scopus (568) Google Scholar;Steinert and Marenkov, 1995Steinert P.M. Marenkov L.N. The proteins elafin, filaggrin, keratin intermediate filaments, loricrin and SPRs are isopeptide crosslinked components of the human epidermal cornified cell envelope.J Biol Chem. 1995; 270: 17702-17711Crossref PubMed Scopus (464) Google Scholar,Steinert and Marenkov, 1997Steinert P.M. Marenkov L.N. Involucrin is an important early component in the assembly of the epidermal cornified cell envelope.J Biol Chem. 1997; 272: 2021-2030Crossref PubMed Scopus (193) Google Scholar;Eckert et al., 1997Eckert R.L. Crish J.F. Robinson N.A. The epidermal keratinocyte as a model for the study of gene regulation and function.Physiol Rev. 1997; 77: 397-424Crossref PubMed Scopus (331) Google Scholar). The viable precursors of corneocytes are the terminally differentiated keratinocytes of the stratum granulosum, which are characterized by the presence of keratohyalin granules, containing profilaggrin and loricrin as well as numerous lipid-containing lamellar bodies (Fuchs, 1990Fuchs E. Epidermal differentiation. The bare essentials.J Cell Biol. 1990; 111: 2807-2814Crossref PubMed Scopus (568) Google Scholar;Elias and Menon, 1991Elias P.M. Menon G.M. Structural and biochemical correlates of the epidermal permeability barrier.Adv Lipid Res. 1991; 24: 1-26Crossref PubMed Google Scholar;Jackson et al., 1993Jackson S.M. Williams M.L. Feingold K.R. Elias P.M. Pathobiology of the stratum corneum.West J Med. 1993; 158: 279-285PubMed Google Scholar;Eckert et al., 1997Eckert R.L. Crish J.F. Robinson N.A. The epidermal keratinocyte as a model for the study of gene regulation and function.Physiol Rev. 1997; 77: 397-424Crossref PubMed Scopus (331) Google Scholar). The proliferation rate of cells in the basal layer is balanced by the rates of apoptosis and corneocyte formation in the outer epidermis (Fuchs, 1990Fuchs E. Epidermal differentiation. The bare essentials.J Cell Biol. 1990; 111: 2807-2814Crossref PubMed Scopus (568) Google Scholar;Eckert et al., 1997Eckert R.L. Crish J.F. Robinson N.A. The epidermal keratinocyte as a model for the study of gene regulation and function.Physiol Rev. 1997; 77: 397-424Crossref PubMed Scopus (331) Google Scholar). Despite the importance of keratinocyte differentiation for epidermal function, the factors that regulate keratinocyte differentiation are not well understood (Fuchs, 1990Fuchs E. Epidermal differentiation. The bare essentials.J Cell Biol. 1990; 111: 2807-2814Crossref PubMed Scopus (568) Google Scholar;Eckert et al., 1997Eckert R.L. Crish J.F. Robinson N.A. The epidermal keratinocyte as a model for the study of gene regulation and function.Physiol Rev. 1997; 77: 397-424Crossref PubMed Scopus (331) Google Scholar). Nuclear hormone receptors are transcription factors that regulate many cellular functions, including cellular differentiation (Mangelsdorf et al., 1995Mangelsdorf D.J. Thummel C. Beato M. The nuclear receptor superfamily: The second decade.Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (5841) Google Scholar;Kliewer et al., 1999Kliewer S.A. Lehmann J.M. Willson T.M. Orphan nuclear receptors: Shifting endocrinology into reverse.Science. 1999; 284: 757-760Crossref PubMed Scopus (414) Google Scholar). The nuclear receptor superfamily has been divided into four major subgroups according to their dimerization and DNA binding properties (Mangelsdorf et al., 1995Mangelsdorf D.J. Thummel C. Beato M. The nuclear receptor superfamily: The second decade.Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (5841) Google Scholar). The class II subfamily consists of nuclear receptors that heterodimerize with retinoid X receptor (RXR). Members of this subfamily usually bind to direct repeats separated by a variable number of spacer nucleotides (Mangelsdorf et al., 1995Mangelsdorf D.J. Thummel C. Beato M. The nuclear receptor superfamily: The second decade.Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (5841) Google Scholar). Stimulation of several of the receptors in this subgroup, including the retinoic acid activated receptor (RAR), vitamin D receptor, and peroxisome proliferator activated receptor (PPAR)-α, regulates keratinocyte proliferation and differentiation (Bikle, 1996Bikle D.D. 1,25 OH2D3-modulated calcium induced keratinocyte differentiation.J Invest Dermatol Symp Proc. 1996; 1: 22-27PubMed Google Scholar;Eichner et al., 1996Eichner R. Gendimenico G.J. Kahn M. Mallon J.P. Capetola R.J. Mezick J.A. Effects of long-term retinoic acid treatment on epidermal differentiation in vivo, specific modification in the programme of terminal differentiation.Br J Dermatol. 1996; 135: 687-695Crossref PubMed Scopus (24) Google Scholar;Fisher and Voorhees, 1996Fisher G.J. Voorhees J.J. Molecular mechanisms of retinoid actions in skin.FASEB J. 1996; 10: 1002-1013Crossref PubMed Scopus (327) Google Scholar;Kang et al., 1996Kang S. Li X.-Y. Voorhees J.J. Pharmacology and molecular action of retinoids and vitamin D in skin.J Invest Dermatol Symp Proc. 1996; 1: 15-21Crossref PubMed Google Scholar;Hanley et al., 1998Hanley K. Jiang Y. He S.S. Keratinocyte differentiation is stimulated by activators of the nuclear hormone receptor PPAR-alpha.J Invest Dermatol. 1998; 110: 368-375Crossref PubMed Scopus (160) Google Scholar;Kömüves et al., 2000aKömüves L.G. Hanley K. Lefebvre A.M. Stimulation of peroxisome proliferator-activated receptor-α promotes epidermal keratinocyte differentiation in vivo.J Invest Dermatol. 2000; 115: 353-360https://doi.org/10.1046/j.1523-1747.2000.00073.xCrossref PubMed Scopus (137) Google Scholar,Kömüves et al., 2000bKömüves L.G. Hanley K. Man M.Q. Elias P.M. Williams M.L. Feingold K.R. Keratinocyte differentiation in hyperproliferative epidermis. Topical application of peroxisome proliferator-activated receptor-α activators restores tissue homeostasis.J Invest Dermatol. 2000; 115: 361-367https://doi.org/10.1046/j.1523-1747.2000.00076.xCrossref PubMed Scopus (91) Google Scholar). Moreover, keratinocyte differentiation is abnormal in transgenic mice that overexpress RAR or RXR dominant negative mutations in the epidermis, further showing the importance of nuclear hormone receptors that heterodimerize with RXR (Imakado et al., 1995Imakado S. Bickenbach J.R. Bundman D.S. Targeting expression of a dominant-negative retinoic acid receptor mutant in the epidermis of transgenic mice results in loss of barrier function.Genes Dev. 1995; 9: 317-329Crossref PubMed Scopus (121) Google Scholar;Saitou et al., 1995Saitou M. Sugai S. Tanaka T. Shimouchi K. Fuchs E. Narumiya S. Kakizuka A. Inhibition of skin development by targeted expression of a dominant-negative retinoic acid receptor.Nature. 1995; 374: 159-162Crossref PubMed Scopus (156) Google Scholar;Feng et al., 1997Feng X. Peng Z.H. Di W. Suprabasal expression of a dominant-negative RXR-alpha mutant in transgenic mouse epidermis impairs regulation of gene transcription and basal keratinocyte proliferation by RAR-selective retinoids.Genes Dev. 1997; 11: 59-71Crossref PubMed Scopus (57) Google Scholar). Additionally, in mice with a deficiency of RXR-α localized to the epidermis, epidermal hyperplasia occurs suggesting keratinocyte hyperproliferation, which is consistent with the inhibition of proliferation observed with vitamin D and PPAR-α ligands (Li et al., 2000Li M. Indra A.K. Warot X. et al.Skin abnormalities generated by temporally controlled RXRalpha mutations in mouse epidermis.Nature. 2000; 407: 633-636Crossref PubMed Scopus (263) Google Scholar). Recently, we have shown that liver X receptor (LXR) -α and LXR-β are expressed in cultured human keratinocytes and in fetal rat epidermis (Hanley et al., 1999Hanley K. Kömüves L.G. Bass N.M. Fetal epidermal differentiation and barrier development in vivo is accelerated by nuclear hormone receptor activators.J Invest Dermatol. 1999; 113: 788-795Crossref PubMed Scopus (83) Google Scholar,Hanley et al., 2000Hanley K. Ng D.C. He S.S. Oxysterols induce differentiation in human keratinocytes and increase AP-1 dependent involucrin transcription.J Invest Dermatol. 2000; 114: 545-553Crossref PubMed Scopus (89) Google Scholar). Moreover, we have demonstrated that certain oxysterol ligands for LXR increased cornified envelope formation, as well as the levels of involucrin and transglutaminase protein and mRNA in normal human keratinocytes, indicating that oxysterols stimulate keratinocyte differentiation in vitro (Hanley et al., 2000Hanley K. Ng D.C. He S.S. Oxysterols induce differentiation in human keratinocytes and increase AP-1 dependent involucrin transcription.J Invest Dermatol. 2000; 114: 545-553Crossref PubMed Scopus (89) Google Scholar). In contrast, oxysterols inhibited DNA synthesis in vitro by approximately 50% (Hanley et al., 2000Hanley K. Ng D.C. He S.S. Oxysterols induce differentiation in human keratinocytes and increase AP-1 dependent involucrin transcription.J Invest Dermatol. 2000; 114: 545-553Crossref PubMed Scopus (89) Google Scholar). Lastly, we have shown that oxysterols accelerate the development of the epidermis in fetal rats, accelerating the formation of the cutaneous permeability barrier (Hanley et al., 1999Hanley K. Kömüves L.G. Bass N.M. Fetal epidermal differentiation and barrier development in vivo is accelerated by nuclear hormone receptor activators.J Invest Dermatol. 1999; 113: 788-795Crossref PubMed Scopus (83) Google Scholar). The purpose of this study was to determine: (i) whether oxysterols, known to be ligands for LXR – 22(R)-hydroxycholesterol (22(R)OHCHOL) and 24(S),25-epoxycholesterol (24(S)25EPOCHOL) – when applied topically to the skin of mice induce differentiation in normal epidermis; (ii) whether this effect is mediated via LXR-α and/or LXR-β; and (iii) whether oxysterols normalize epidermal morphology in a hyperplastic animal disease model. Adult hairless mice 6–10 wk of age (Simonsen, Gilroy, CA or Charles River, Wilmington, MA) were treated topically twice a day for 4 d with 22(R)OHCHOL (Sigma, St Louis, MO) (500 nM, 200 μM, 500 μM dissolved in polypropylene glycol–ethanol 3:1 or in ethanol; 0.1 ml applied to 2 cm2 area on one flank). Moreover, 24(S)25EPOCHOL (a gift from Timothy Willson, Nuclear Receptor Discovery Research, GlaxoSmithKline, Research Triangle Park, NC) was applied to the flank in a similar fashion as 22(R)OHChol (500 nM in polypropylene glycol–ethanol 3 : 1). Control hairless mice were treated with vehicle alone in an identical fashion. The experiments were done in triplicate. LXR-α–/– (four adults), LXR-β–/– (four adults), and LXR-αβ–/– (three adults) deficient mice were produced as described previously (Peet et al., 1998Peet D.J. Turley S.D. Ma W. Janowski B.A. Lobaccaro J.-M.A. Hammer R.E. Mangelsdorf D.J. Cholesterol and bile acid metabolism are impaired in mice lacking the nuclear oxysterol receptor LXR-alpha.Cell. 1998; 93: 693-704Abstract Full Text Full Text PDF PubMed Scopus (1190) Google Scholar). These “knockout” animals and age- and sex-matched controls (LXR-αβ+/+ adults) of the same genetic background were gently shaved and then topically treated twice a day for 4 d with either 22(R)OHCHOL in ethanol, or with ethanol alone. Epidermal hyperproliferation was induced in male hairless mice by repeated barrier abrogation with acetone, twice a day, for 5 d, as previously described (Denda et al., 1996Denda M. Wood L.C. Emami S. Calhoun C. Brown B.E. Elias P.M. Feingold K.R. The epidermal hyperplasia associated with repeated barrier disruption by acetone treatment or tape stripping cannot be attributed to increased water loss.Arch Dermatol Res. 1996; 288: 230-238Crossref PubMed Scopus (124) Google Scholar). For an additional 4 d these animals were treated topically with either 22(R)OHCHOL or vehicle as described above. Cutaneous permeability barrier function was determined by measuring transepidermal water loss (TEWL) with a Meeco electrolytic water analyzer, as described previously (Kömüves et al., 2000aKömüves L.G. Hanley K. Lefebvre A.M. Stimulation of peroxisome proliferator-activated receptor-α promotes epidermal keratinocyte differentiation in vivo.J Invest Dermatol. 2000; 115: 353-360https://doi.org/10.1046/j.1523-1747.2000.00073.xCrossref PubMed Scopus (137) Google Scholar,Kömüves et al., 2000bKömüves L.G. Hanley K. Man M.Q. Elias P.M. Williams M.L. Feingold K.R. Keratinocyte differentiation in hyperproliferative epidermis. Topical application of peroxisome proliferator-activated receptor-α activators restores tissue homeostasis.J Invest Dermatol. 2000; 115: 361-367https://doi.org/10.1046/j.1523-1747.2000.00076.xCrossref PubMed Scopus (91) Google Scholar). Barrier repair was measured by first disrupting the barrier by repetitive, gentle wipes with acetone or by repetitive tape stripping with cellophane tape until the TEWL levels were 3–7 mg per cm2 per h, and then determining TEWL at 3, 6, 16, and 24 h as an index of barrier homeostasis (Kömüves et al., 2000aKömüves L.G. Hanley K. Lefebvre A.M. Stimulation of peroxisome proliferator-activated receptor-α promotes epidermal keratinocyte differentiation in vivo.J Invest Dermatol. 2000; 115: 353-360https://doi.org/10.1046/j.1523-1747.2000.00073.xCrossref PubMed Scopus (137) Google Scholar,Kömüves et al., 2000bKömüves L.G. Hanley K. Man M.Q. Elias P.M. Williams M.L. Feingold K.R. Keratinocyte differentiation in hyperproliferative epidermis. Topical application of peroxisome proliferator-activated receptor-α activators restores tissue homeostasis.J Invest Dermatol. 2000; 115: 361-367https://doi.org/10.1046/j.1523-1747.2000.00076.xCrossref PubMed Scopus (91) Google Scholar). Skin samples were fixed overnight in 4% formaldehyde (freshly prepared from paraformaldehyde (Sigma) and embedded in paraffin (ParaplastPlus, Fisher Scientific, Pittsburgh, PA). Sections (5 µm for immunohistochemistry, 15 µm for in situ hybridization) were collected on SuperfrostPlus slides (Fisher Scientific), and processed as described below. Affinity-purified rabbit antibodies, specific for involucrin, profilaggrin/filaggrin, and loricrin (Covance/BabCo, Berkeley, CA), and a biotinylated monoclonal anti-proliferating cell nuclear antigen (anti-PCNA) antibody (CalTag Laboratories, Burlingame, CA) were used. Affinity-purified biotinylated goat anti-rabbit IgG, affinity-purified biotinylated goat anti-mouse IgG, and ABC-peroxidase (ABC-PO) were purchased from Vector (Burlingame, CA). Immunohistochemical localization of these proteins was performed as described previously (Kömüves et al., 1998Kömüves L.G. Hanley K. Jiang Y. Elias P.M. Wiliams M.L. Feingold K.R. Ligands and activators of nuclear hormone receptors regulate epidermal differentiation during fetal rat skin development.J Invest Dermatol. 1998; 111: 429-433Crossref PubMed Scopus (91) Google Scholar,Kömüves et al., 2000aKömüves L.G. Hanley K. Lefebvre A.M. Stimulation of peroxisome proliferator-activated receptor-α promotes epidermal keratinocyte differentiation in vivo.J Invest Dermatol. 2000; 115: 353-360https://doi.org/10.1046/j.1523-1747.2000.00073.xCrossref PubMed Scopus (137) Google Scholar,Kömüves et al., 2000bKömüves L.G. Hanley K. Man M.Q. Elias P.M. Williams M.L. Feingold K.R. Keratinocyte differentiation in hyperproliferative epidermis. Topical application of peroxisome proliferator-activated receptor-α activators restores tissue homeostasis.J Invest Dermatol. 2000; 115: 361-367https://doi.org/10.1046/j.1523-1747.2000.00076.xCrossref PubMed Scopus (91) Google Scholar), and peroxidase activity was revealed with diaminobenzidine (Vector). For the detection of PCNA proteins, antigen retrieval was performed with microwave treatment in 10 mM citrate buffer, pH = 6.0. Omission of the first antibodies, or incubation with the substrate solution resulted in no signal, showing that nonspecific binding of the second antibody and/or ABC-PO, or endogenous peroxidase activity were not contributing to the signals obtained. Biotin-labeled RNA probes to detect loricrin and profilaggrin mRNA were synthesized from linearized cDNA sequences (a gift from S. Yuspa, NIH), using reagents supplied by Roche Molecular Biochemicals (Indianapolis, IN). In situ hybridization was performed as described previously (Kömüves et al., 2000aKömüves L.G. Hanley K. Lefebvre A.M. Stimulation of peroxisome proliferator-activated receptor-α promotes epidermal keratinocyte differentiation in vivo.J Invest Dermatol. 2000; 115: 353-360https://doi.org/10.1046/j.1523-1747.2000.00073.xCrossref PubMed Scopus (137) Google Scholar), with the following modifications. The sections were hybridized at 45°C and the hybridization of the biotin-labeled probes to endogenous mRNA was detected with ABC-PO (Vector). Biotinylated tyramide (CSA kit, DAKO, Carpinteria, CA) was used for signal amplification, followed by incubation with ABC-alkaline phosphatase (Vector). Alkaline phosphatase activity was developed with VectorRed substrate (Vector). Hybridization with biotin-labeled, sense control probes resulted in no signal, indicating the specificity of hybridization with the anti-sense probes. Omitting the biotin-labeled anti-sense probes from the hybridization cocktail resulted in no signal, which demonstrated that only biotin-containing RNA hybrids were detected. Moreover, incubation with the BCIP/NTBT substrate reagents alone resulted in no staining, showing that endogenous alkaline phosphatase activity within the tissues did not contribute to the signal obtained. The tissue samples were analyzed with an Olympus BX50 microscope using 20 × UplanApo and 40 × UplanFl objectives with bright-field or differential interference–contrast (DIC) illumination. Images were acquired with a DEI 750 CCD camera (Optronics, Goleta, CA), controlled by BioQuant Nova imaging software (R&M Biometrics, Nashville, TN). The digitized images were resized to 300 dpi resolution and assembled using Photoshop 5.5 (Adobe Systems, Mountain View, CA). Apart from adjusting the background intensities (using the Level tool in Photoshop), and cropping to size, no other digital tools were applied to the images. The figures were printed with Epson Stylus Photo 870 inkjet printer. Epidermal thickness was determined using a computer-generated micrometer (Carl Zeiss Vision, Munich, Germany). Epidermal thickness was defined as the distance between the basement lamina and the stratum granulosum–stratum corneum interface. Epidermal proliferation was determined by quantitating PCNA positive nuclei in the basal layer of the epidermis per unit length of basement membrane. To separate whole epidermis from full thickness skin, skin was placed in 10 mM ethylenediamine tetraacetic acid in calcium-free and magnesium-free phosphate-buffered saline, pH 7.4, for 35 min at 37°C. The epidermis was removed by scraping with a scalpel blade. RNA from epidermis and liver was prepared by a variation of the guanidinium thiocyanate method, as described previously (Jackson et al., 1992Jackson S.M. Wood L.C. Lauer S. Taylor J.M. Cooper A.D. Elias P.M. Feingold K.R. Effect of cutaneous permeability barrier disruption on HMG CoA reductase, LDL receptor and apoprotein E mRNA levels in the epidermis of hairless mice.J Lipid Res. 1992; 33: 1307-1314Abstract Full Text PDF PubMed Google Scholar). Total RNA was purified and added to oligo(dT)-cellulose to obtain poly(A)+ RNA. Northern blots were prepared as described previously (Jackson et al., 1992Jackson S.M. Wood L.C. Lauer S. Taylor J.M. Cooper A.D. Elias P.M. Feingold K.R. Effect of cutaneous permeability barrier disruption on HMG CoA reductase, LDL receptor and apoprotein E mRNA levels in the epidermis of hairless mice.J Lipid Res. 1992; 33: 1307-1314Abstract Full Text PDF PubMed Google Scholar). We first asked whether oxysterols induce keratinocyte differentiation in intact mice, as previously shown in cultured keratinocytes (Hanley et al., 2000Hanley K. Ng D.C. He S.S. Oxysterols induce differentiation in human keratinocytes and increase AP-1 dependent involucrin transcription.J Invest Dermatol. 2000; 114: 545-553Crossref PubMed Scopus (89) Google Scholar). Topical applications of several concentrations (500 nm, 200 µM, 500 µM) of 22(R)OHCHOL (Figure 1), or 500 nm 24(S)25EPOCHOL (not shown) for 4 d resulted in a decrease in epidermal thickness as compared with vehicle-treated controls (Figure 1, oxysterol 10.97 ± 0.69 µm, one to two epidermal cell layers vs vehicle 20.19 ± 0.49 µm, p < 0.001, three to four epidermal cell layers). Moreover, these treatments also resulted in a decrease in the proliferative pool of epidermal keratinocytes, as assayed by PCNA immunostaining (oxysterol 13.2% PCNA positive nuclei per unit length of basement membrane vs vehicle 32.8%, p < 0.001). The occasional suprabasal, PCNA-positive cells that occur in vehicle-treated epidermis (Figure 1c) were completely eliminated by oxysterol treatments (Figure 1d). On the other hand, involucrin, filaggrin, and loricrin protein levels all increased in oxysterol-treated epidermis (Figure 2). Whereas we observed increased labeling with the respective antibodies, staining remained restricted to the granular and upper spinous epidermal cell layers. The changes were especially pronounced for involucrin (Figure 2a vs Fig" @default.
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• W2080254988 date "2002-01-01" @default.
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• W2080254988 title "Oxysterol Stimulation of Epidermal Differentiation is Mediated by Liver X Receptor-β in Murine Epidermis" @default.
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