Role for ELOVL3 and Fatty Acid Chain Length in Development of Hair and Skin Function
2004; Elsevier BV; Volume: 279; Issue: 7 Linguagem: Inglês
10.1074/jbc.m310529200
ISSN1083-351X
AutoresR.B. Westerberg, Petr Tvrdík, Anne-Birgitte Undén, Jan-Erik Månsson, Lars Norlén, Andreas Jakobsson, Walter H. Holleran, Peter M. Elias, Abolfazl Asadi, Per Flodby, Rune Toftgård, Mario R. Capecchi, Anders Jacobsson,
Tópico(s)Dermatology and Skin Diseases
ResumoVery little is known about the in vivo regulation of mammalian fatty acid chain elongation enzymes as well as the role of specific fatty acid chain length in cellular responses and developmental processes. Here, we report that the Elovl3 gene product, which belongs to a highly conserved family of microsomal enzymes involved in the formation of very long chain fatty acids, revealed a distinct expression in the skin that was restricted to the sebaceous glands and the epithelial cells of the hair follicles. By disruption of the Elovl3 gene by homologous recombination in mouse, we show that ELOVL3 participates in the formation of specific neutral lipids that are necessary for the function of the skin. The Elovl3-ablated mice displayed a sparse hair coat, the pilosebaceous system was hyperplastic, and the hair lipid content was disturbed with exceptionally high levels of eicosenoic acid (20:1). This was most prominent within the triglyceride fraction where fatty acids longer than 20 carbon atoms were almost undetectable. A functional consequence of this is that Elovl3-ablated mice exhibited a severe defect in water repulsion and increased trans-epidermal water loss. Very little is known about the in vivo regulation of mammalian fatty acid chain elongation enzymes as well as the role of specific fatty acid chain length in cellular responses and developmental processes. Here, we report that the Elovl3 gene product, which belongs to a highly conserved family of microsomal enzymes involved in the formation of very long chain fatty acids, revealed a distinct expression in the skin that was restricted to the sebaceous glands and the epithelial cells of the hair follicles. By disruption of the Elovl3 gene by homologous recombination in mouse, we show that ELOVL3 participates in the formation of specific neutral lipids that are necessary for the function of the skin. The Elovl3-ablated mice displayed a sparse hair coat, the pilosebaceous system was hyperplastic, and the hair lipid content was disturbed with exceptionally high levels of eicosenoic acid (20:1). This was most prominent within the triglyceride fraction where fatty acids longer than 20 carbon atoms were almost undetectable. A functional consequence of this is that Elovl3-ablated mice exhibited a severe defect in water repulsion and increased trans-epidermal water loss. Fatty acids consisting of up to 16 carbons are synthesized by the well studied fatty acid synthase complex (1Smith S. FASEB J. 1994; 8: 1248-1259Crossref PubMed Scopus (512) Google Scholar). However, a significant amount of the fatty acids produced by fatty acid synthase are further elongated into very long chain fatty acids (VLCFA). 1The abbreviations used are: VLCFAvery long chain fatty acidsTEWLtrans-epidermal water lossESembryonic stemHPTLChigh performance thin-layer chromatography.1The abbreviations used are: VLCFAvery long chain fatty acidsTEWLtrans-epidermal water lossESembryonic stemHPTLChigh performance thin-layer chromatography.VLCFA have been recognized as structural components in a variety of fat molecules such as glycerolipids and sphingolipids. They are found in virtually all cells and are major constituents of the brain, skin, and testis (2Stewart M.E. Downing D.T. Adv. Lipid Res. 1991; 24: 263-301Crossref PubMed Google Scholar, 3Coniglio J.G. Prog. Lipid Res. 1994; 4: 387-401Crossref Scopus (48) Google Scholar, 4Poulos A. Lipids. 1995; 30: 1-14Crossref PubMed Scopus (150) Google Scholar). Depending on their chain length and degree of unsaturation, they contribute to membrane fluidity and other chemical properties of the cell.Formation of VLCFA is performed in the endoplasmic reticulum, in the early Golgi, and in mitochondria by membrane-bound enzymes, the former being more prominent (5Cinti D.L. Cook L. Nagi M.N. Suneja S.K. Prog. Lipid Res. 1992; 31: 1-51Crossref PubMed Scopus (190) Google Scholar, 6David D. Sundarababu S. Gerst J.E. J. Cell Biol. 1998; 143: 1167-1182Crossref PubMed Scopus (114) Google Scholar).Recently, five mammalian genes, Elovl1-5, 2In accordance with the Mouse and Human Nomenclature Committees the assigning symbols for the mouse genes Scc1, Ssc2, and Cig30 are changed to Elovl1, Elovl2, and Elovl3, respectively, and the human HELO1 gene is changed to ELOVL5.2In accordance with the Mouse and Human Nomenclature Committees the assigning symbols for the mouse genes Scc1, Ssc2, and Cig30 are changed to Elovl1, Elovl2, and Elovl3, respectively, and the human HELO1 gene is changed to ELOVL5. whose protein products belong to a highly conserved family of microsomal enzymes involved in the formation of VLCFA, have been identified (7Tvrdik P. Asadi A. Kozak L.P. Nedergaard J. Cannon B. Jacobsson A. J. Biol. Chem. 1997; 272: 31738-31746Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 8Leonard A.E. Bobik E.G. Dorado J. Kroeger P.E. Chuang L.-T. Thurmond J.M. Parker-Barnes J.M. Das T. Huang Y.-S. Mukerji P. Biochem. J. 2000; 350: 765-770Crossref PubMed Scopus (182) Google Scholar, 9Tvrdik P. Westerberg R. Silve S. Asadi A. Jakobsson A. Cannon B. Loison G. Jacobsson A. J. Cell Biol. 2000; 149: 707-717Crossref PubMed Scopus (180) Google Scholar, 10Zhang K. Kniazeva M. Han M. Li W. Yu Z. Yang Z. Li Y. Metzker M.L. Allikmets R. Zack D.J. Kakuk L.E. Lagali P.S. Wong P.W. MacDonald I.M. Sieving P.A. Figueroa D.J. Austin C.P. Gould R.J. Ayyagari R. Petrukhin K. Nat. Genet. 2001; 27: 89-93Crossref PubMed Scopus (374) Google Scholar). All five genes show a diverse tissue-specific expression pattern indicating a unique role for different VLCFA in different cell types.Although the general functions of the Elovl genes are partially understood (8Leonard A.E. Bobik E.G. Dorado J. Kroeger P.E. Chuang L.-T. Thurmond J.M. Parker-Barnes J.M. Das T. Huang Y.-S. Mukerji P. Biochem. J. 2000; 350: 765-770Crossref PubMed Scopus (182) Google Scholar, 9Tvrdik P. Westerberg R. Silve S. Asadi A. Jakobsson A. Cannon B. Loison G. Jacobsson A. J. Cell Biol. 2000; 149: 707-717Crossref PubMed Scopus (180) Google Scholar, 10Zhang K. Kniazeva M. Han M. Li W. Yu Z. Yang Z. Li Y. Metzker M.L. Allikmets R. Zack D.J. Kakuk L.E. Lagali P.S. Wong P.W. MacDonald I.M. Sieving P.A. Figueroa D.J. Austin C.P. Gould R.J. Ayyagari R. Petrukhin K. Nat. Genet. 2001; 27: 89-93Crossref PubMed Scopus (374) Google Scholar, 11Oh C.S. Toke D.A. Mandala S. Martin C.E. J. Biol. Chem. 1997; 272: 17376-17384Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar, 12Moon Y.A. Shah N.A. Mohapatra S. Warrington J.A. Horton J.D. J. Biol. Chem. 2001; 276: 45358-45366Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 13Matsuzaka T. Shimano H. Yahagi N. Yoshikawa T. Amemiya-Kudo M. Hasty A.H. Okazaki H. Tamura Y. Iizuka Y. Ohashi K. Osuga J. Takahashi A. Yato S. Sone H. Ishibashi S. Yamada N. J. Lipid Res. 2002; 43: 911-920Abstract Full Text Full Text PDF PubMed Google Scholar, 14Moon Y.A. Horton J.D. J. Biol. Chem. 2003; 278: 7335-7343Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar), very little is known about the role of specific fatty acid chain length in cellular responses and developmental processes. The ELOVL3 protein has been suggested to be involved in the formation of saturated and monounsaturated fatty acyl chains containing up to 24 carbon atoms (9Tvrdik P. Westerberg R. Silve S. Asadi A. Jakobsson A. Cannon B. Loison G. Jacobsson A. J. Cell Biol. 2000; 149: 707-717Crossref PubMed Scopus (180) Google Scholar). Elovl3 gene expression has only been detected in brown adipose tissue, liver, and skin (7Tvrdik P. Asadi A. Kozak L.P. Nedergaard J. Cannon B. Jacobsson A. J. Biol. Chem. 1997; 272: 31738-31746Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 9Tvrdik P. Westerberg R. Silve S. Asadi A. Jakobsson A. Cannon B. Loison G. Jacobsson A. J. Cell Biol. 2000; 149: 707-717Crossref PubMed Scopus (180) Google Scholar). To assess the in vivo role of ELOVL3, we disrupted the Elovl3 gene by homologous recombination in mouse. Here we describe the characterization of Elovl3 expression in skin and present evidence that ELOVL3 participates in the formation of certain VLCFA and triglycerides in certain cells of the hair follicles and the sebaceous glands and that these lipid compounds are essential for function of the skin.MATERIALS AND METHODSElovl3 Gene Targeting Construct—A genomic liver DNA library from mouse strain 129/Sv cloned into the Lambda FIXII vector was used to isolate a DNA fragment containing the entire Elovl3 gene (15Tvrdik P. Asadi A. Kozak L.P. Nuglozeh E. Parente F. Nedergaard J. Jacobsson A. J. Biol. Chem. 1999; 274: 26387-26392Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). A 2.75-kb fragment between SacI and SalI upstream of the first exon was subcloned into the polylinker of the pBluescript SK plasmid in order to become the left arm (LA) in the Elovl3 gene targeting construct (Fig. 1A). The SacI site was blunt ended by T4 DNA polymerase (New England Biolab) and turned into a NotI site by ligation of NotI primers (BioSource International). A 1.2-kb fragment containing a neomycin resistance gene (neor) was inserted into the compatible SalI site of the plasmid containing the 2.75-kb left arm fragment. The ligation between SalI and XhoI ends consequently destroyed the recognition site for respective digestion enzymes. A 5.30-kb SalI/SalI fragment downstream of the second exon of the Elovl3 gene was ligated into the pBluescript SK plasmid and further digested with SalI and XhoI to receive a 2.77-kb fragment corresponding to the right arm (RA) in the Elovl3 gene-targeting construct. This fragment was subcloned into the XhoI site of the vector containing the 2.75-kb left arm fragment and the neor gene. The NotI/XhoI fragment from the resulting plasmid was ligated into a vector flanked by the thymidine kinase (tk) gene at the 3′-end.Gene-targeting in ES Cells and Blastocyst Injection—The Elovl3-targeted construct was linearized by NotI and electroporated into the R1 embryonic stem (ES) cells derived from male 129/Sv agouti mice (16Nagy A. Rossant J. Nagy R. Abramow-Newerly W. Roder J.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8424-8428Crossref PubMed Scopus (1960) Google Scholar). ES cells were cultured as earlier described and subjected to positive/negative selection using G418 and FIAU as previously described (17Thomas K.R. Capecchi M.R. Cell. 1987; 51: 503-512Abstract Full Text PDF PubMed Scopus (1799) Google Scholar, 18Mansour S.L. Thomas K.R. Capecchi M.R. Nature. 1988; 336: 348-352Crossref PubMed Scopus (1314) Google Scholar). ES cell DNA was prepared according to standard procedures (16Nagy A. Rossant J. Nagy R. Abramow-Newerly W. Roder J.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8424-8428Crossref PubMed Scopus (1960) Google Scholar). Probes as indicated in Fig. 1 and for the neor gene (not shown) were used to confirm ES cell clones heterozygous for the mutant Elovl3 allele.Elovl3-ablated ES cells were injected into C57BL/6J blastocysts and implanted into foster mothers (F1, CBAxC57BL6) according to standard procedures (19Gossler A. Doetschman T. Korn R. Serfling E. Kemler R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9065-9069Crossref PubMed Scopus (337) Google Scholar). The male offspring being the most chimeric, ∼80% agouti and 20% black in the coat color, were bred with C57BL/6J females to generate offspring heterozygous for the mutation. Genomic DNA was prepared from mouse tails by the simplified mammalian DNA isolation as described earlier (20Laird P.W. Zijderveld A. Linders K. Rudnicki M.A. Jaenisch R. Berns A. Nucleic Acids Res. 1991; 19: 4293Crossref PubMed Scopus (1297) Google Scholar). Tail biopsies were collected from 3-week-old mice and used directly for DNA isolation.The Elovl3-ablated mice were back-crossed with C57BL/6 (B&K Universal, Stockholm, Sweden) in the animal facility of the Institute. As control mice, we used heterozygote littermates or age-matched C57BL/6 mice that were bred under the same conditions as the Elovl3-ablated mice. Animals were fed ad libitum (rat and mouse standard diet No.1, BeeKay Feeds; B&K Universal, Stockholm, Sweden), had free access to water, and were kept on a 12:12-h light:dark cycle in single cages. Wild-type and Elovl3-ablated mice were bred in room temperature. Before exposing the animals to the cold, the mice were housed at 30 °C (thermoneutrality) for 10 days. Mice were placed in the cold (4 °C) for the times indicated.Southern Blot Analysis of DNA from ES Cells and Mouse Tails—The DNA for the probes were purified according to the Jetsorb gel extraction kit (Genomed Inc.) and were labeled with [α-32P]dCTP using a random primed labeling kit (Roche Applied Science). Ten μg digested DNA from mouse tails and ES cells was separated on 0.8% agarose gel and transferred to Hybond-N membrane (Amersham Biosciences) in 20× SSC. The membrane was prehybridized with a solution containing 5× SSC, 5× Denhardt's, 0.5% SDS, 50 mm sodium phosphate, 50% formamide, and 100 mg/ml degraded DNA from herring sperm (Sigma) at 45 °C. After prehybridization, the membrane was transferred to a similar solution containing the denatured probe. The hybridization was carried out overnight at 55 °C. Hybond-N membrane was washed twice in 2× SSC, 0.2% SDS at 30 °C for 20 min each and then once in 0.1× SSC, 0.2% SDS at 55 °C for 30 min. The filters were analyzed by phosphorimaging (Molecular Dynamics) and quantified with the ImageQuant program.RNA Analysis—Total RNA was isolated using Ultraspec (Biotech Lab) from 50-100 mg (w/w) of each tissue as described earlier (7Tvrdik P. Asadi A. Kozak L.P. Nedergaard J. Cannon B. Jacobsson A. J. Biol. Chem. 1997; 272: 31738-31746Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). For Northern blot analysis, 20 μg total RNA was separated on a 1.2% (w/v) formaldehyde agarose gel and blotted onto Hybond-N membrane (Amersham Biosciences) in 20× SSC. The hybridization procedure was identical to that for Southern blot analysis, except that hybridization was carried out overnight at 45 °C. The membrane was then washed twice in 2× SSC, 0.2% SDS at 30 °C for 20-30 min each and then twice in 0.1× SSC, 0.2% SDS at 50 °C for 45 min. A corresponding cDNA fragment of Elovl3 (895-bp open reading frame) was used as a probe as previously described (7Tvrdik P. Asadi A. Kozak L.P. Nedergaard J. Cannon B. Jacobsson A. J. Biol. Chem. 1997; 272: 31738-31746Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 9Tvrdik P. Westerberg R. Silve S. Asadi A. Jakobsson A. Cannon B. Loison G. Jacobsson A. J. Cell Biol. 2000; 149: 707-717Crossref PubMed Scopus (180) Google Scholar). The filters were analyzed by phosphorimaging as for Southern analysis.Histology—Skin biopsies of age- and sex-matched animals were taken from similar body sites. Skin samples were fixed overnight at 4 °C in a phosphate-buffered, pH 7.4, 4% formaldehyde solution. Semithin sections were stained with hematoxylin and eosin and examined by light microscopy.For electron microscopy analysis, skin samples were taken at autopsy of 10-week-old mice and processed for electron microscopy. Samples were fixed overnight in Karnovsky's fixative, washed two times with 0.1 m cacodylate buffer, and post-fixed in either 1% osmium tetroxide (OsO4) in 0.1 m cacodylate buffer, pH 7.3, or in ruthenium tetroxide (RuO4) as described previously (21Madison K.C. Swartzendruber D.C. Wertz P.W. Downing D.T. J. Investig. Dermatol. 1987; 88: 714-718Abstract Full Text PDF PubMed Scopus (331) Google Scholar, 22Hou S.Y. Mitra A.K. White S.H. Menon G.K. Ghadially R. Elias P.M. J. Investig. Dermatol. 1991; 96: 215-223Abstract Full Text PDF PubMed Scopus (265) Google Scholar). After fixation, samples were dehydrated in graded ethanol solutions and embedded in an Epon/epoxy mixture. Ultrathin sections were examined both with and without further contrasting with lead citrate in an electron microscope (Zeiss 1A; Carl Zewiss, Thornwood, NY) operated at 60 kV.In Situ Hybridization—RNA probes were prepared from Elovl3 open reading frame mouse cDNA. In situ hybridization was performed as described previously (23Nilsson M. Unden A.B. Krause D. Malmqwist U. Raza K. Zaphiropoulos P.G. Toftgård R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3438-3443Crossref PubMed Scopus (355) Google Scholar). Briefly, a 895-bp fragment corresponding to nucleotides 162-1056 in the Elovl3 cDNA was cloned into a pCI-neo vector (Promega), appropriately linearized, and in vitro transcribed to obtain antisense and sense probes. Sections were treated with proteinase K (Sigma) and washed in 0.1 m triethanolamine buffer containing 0.25% acetic anhydride. Subsequently, sections were hybridized overnight with 2.5 × 106 cpm of labeled antisense or sense probe at 55 °C. Autography was carried out for 14 days. After development of the photographic emulsion, slides were stained with hematoxylin and eosin. The labeled sense strand served as negative control and did not show any labeling of cellular localization.Epidermal Lipid Analysis—Hair was removed from the back of the mice, and skin was isolated by punching. After subcutaneous tissue was removed by scraping on ice, skin pieces were incubated in phosphate-buffered saline at 60 °C for 30 s, and epidermis was isolated by scraping on ice. Lipids were extracted using chloroform:methanol:water (2:4:1,6) overnight, and skin precipitates were stored for further extraction. Four ml chloroform:water (1:1) was added to the supernatant and incubated for 5 min at room temperature followed by centrifugation (2000 rpm). The water phase was discarded, and the organic phase was washed with chloroform:methanol:water (10:10:9). Non-extracted lipids from the original skin precipitates were sequentially extracted using chloroform:methanol (2:1, 1:1, 1:2). Extracts were pooled and evaporated under nitrogen.TLC analysis was performed as described earlier (37Uchida Y. Hara M. Nishio H. Sidransky E. Inoue S. Otsuka F. Suzuki A. Elias P.M. Holleran W.M. Hamanaka S. J. Lipid Res. 2000; 41: 2071-2082Abstract Full Text Full Text PDF PubMed Google Scholar). Total epidermal lipids were separated by HPTLC (Merck) with the following solvent sequence: 1) chloroform to 1.5 cm; 2) chloroform:methanol: acetone 76:16:8 (v/v) to 1 cm; 3) chloroform:methanol:hexyl acetate:acetone 86:4:1:10 (v/v) to 7 cm; 4) chloroform:methanol:acetone 76:20:4 (v/v) to 2 cm; 5) chloroform:methanol:diethyl ether:ethyl acetate:hexyl acetate:acetone 72:4:4:1:4:16 (v/v) to 7,5 cm; 6) n-hexane: diethyl ether:ethyl acetate 80:16:4 (v/v), and 7) n-hexane:diethyl ether:acetic acid 65:35:1 (v/v) to the top of the plate. Lipids were visualized after treatment with cupric acetate-phosphoric acid and heating to 160 °C for 15 min.Hair Lipid Analysis—Hair from adult mice was extracted and filtered twice with 20 ml acetone for 15 min. The two extracts were combined and allowed to evaporate to dryness in glass vials. The amount of dry lipids was calculated by subtracting the weight of empty vials. Equal amounts of lipids (120 μg) were dissolved in acetone and applied to each lane on a Whatman HPTLC silica G plate and separated according to Downing (24Downing D.T. J. Chromatogr. 1968; 38: 91-99Crossref PubMed Scopus (207) Google Scholar). Briefly, the plate was previously run with chloroform, placed in an oven at 105 °C for 30 min, and then immediately cooled down to room temperature. The samples were spotted onto the plate and resolved in hexane. The plates were removed and air dried for 15 min. The plates were then run in toluene until the solvent front reached the end of the plate. After the plates were air dried for 15 min, a third phase containing hexane:ether:acetic acid (70:30:1) was run. The TLC plates were stained with sulfuric acid:ethanol (1:1) and charred at 150 °C. Lipids were analyzed by scanning densitometry of digital pictures taken from the TLC plate by phosphorimaging (Molecular Dynamics) with the ImageQuant program.Mass Spectrometry Analysis—For the fatty acid determinations, 2 ml chloroform:methanol:water (60:30:4.5) extract of hair lipids was evaporated and dissolved in 2 ml 2.5% sulfuric acid in methanol. Acidic methanolysis was performed at 80 °C for 16 h. Formed fatty acid methyl esters were purified by preparative TLC in dichloromethane. Mass spectrometry analysis was performed on Fison MD 800 equipment with on-column injection. A DB 1 (J&W) capillary column 30 m × 0.32 mm was used for the separation.Water Retention Assay and Temperature Measurement—Weight or colonic temperature was measured (RET-3 rectal thermometer; Physitemp Instruments Inc.) once for each adult mouse prior to swimming. Mice were allowed to swim in water at 30 °C for 2 min. Excessive water was eliminated by allowing the mice to walk on paper towels for a few seconds. Weight or colonic temperature was recorded every 5 min at 22 °C. Between each measurement the mice were kept individually in empty plastic cages. The hair water content was calculated by subtracting the preswim weight from the postswim weight.Trans-epidermal Water Loss (TEWL) Analysis—Mice were anesthetized with 2.5% avertin by intraperitoneal injection at a concentration of 0.014 ml/g body weight. The evaporimeter (EP 1C; Servomed, Göteborg, Sweden) was run for at least 15 min prior to use. The evaporimeter was placed on 1 cm2 of shaved skin on the back of the animal in an open chamber. During the measurement, the evaporimeter was allowed to stabilize for 30 s before the value was recorded. All measurements were performed according to the guidelines from the standardization group of contact dermatitis (25Pinnagoda J. Tupker R.A. Agner T. Serup J. Contact Dermatitis. 1990; 22: 164-178Crossref PubMed Scopus (1049) Google Scholar).RESULTSGeneration of Elovl3-ablated Mice—A SalI fragment, including the transcription start site, exon 1 and 2 of the Elovl3 gene, was deleted by homologous recombination in R1 ES cells due to insertion of a neor gene (Fig. 1A). Targeted ES cells were microinjected into blastocysts of C57BL/6J (B6) mice. Two independent chimeric lines were found to transmit the Elovl3 disruption through the germline, which was confirmed by Southern blot analysis (Fig. 1B). The 5.6-kb ScaI genomic fragment representing the wild-type Elovl3 allele is absent in Elovl3-ablated mice and replaced by a 11.6-kb fragment that hybridizes to the probe (Fig. 1, A and B).To determine the Elovl3 expression, Northern blot analyses were performed with total RNA isolated from brown adipose tissue, skin, and liver, i.e. organs that have been shown to express Elovl3 (7Tvrdik P. Asadi A. Kozak L.P. Nedergaard J. Cannon B. Jacobsson A. J. Biol. Chem. 1997; 272: 31738-31746Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), from wild-type and Elovl3-ablated mice (Fig. 1C). The gene disruption was particularly clear upon cold stimulation, where no Elovl3 mRNA was detected in brown adipose tissue from Elovl3-ablated mice, a site with the highest reported Elovl3 expression (7Tvrdik P. Asadi A. Kozak L.P. Nedergaard J. Cannon B. Jacobsson A. J. Biol. Chem. 1997; 272: 31738-31746Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar).Phenotype Characteristics—Interestingly, the Elovl3-ablated mice did not show any indications of impaired liver or brown adipose tissue function. The striking feature that distinguished Elovl3-ablated mice from wild-type or heterozygous littermates was a tousled and reduced fur content over the whole body (Fig. 1D) that was noticeable within 2 weeks of age when the hair was formed and sustained throughout the life span. In addition, mice older than approximately six months showed irritated skin and distinctive scratch marks on the chin that resembled eczematous skin. The mice grew normally and were fertile. Offspring genotypes obtained from heterozygous F1 intercrosses showed normal Mendelian distribution.Microscopic Distinctions of the Skin in Elovl3-ablated Mice— Grossly, the epidermis of the Elovl3-ablated mice appeared normal, but parts of the epidermis were thicker with observed hypergranulation (Fig. 2, A and B). The nuclei of the hair follicle cells seemed irregularly shaped, especially within the cells in the inner layer of the outer root sheath (Fig. 2, C and D), and as in the epidermis scattered hypergranulation was seen (Fig. 2, A and C). In many of the hair follicles, normal hair was missing in the upper part, though the hair seemed normal in the lower parts of the follicles. However, the most evident finding was a general hyperplasia of the sebaceous glands (Fig. 2E).Fig. 2Histological analysis of the skin from Elovl3-ablated mice and cell-specific expression of Elovl3 mRNA in hair follicles and sebaceous glands in wild-type skin. Microscopic overview of the skin from the back of an Elovl3-ablated mouse (A-E) and wild-type mouse (F-J) at the age of 9 weeks showing epidermis, hair follicles, and dermis. A, in the Elovl3-ablated mouse, the normal hair is missing in the upper part of several hair follicles though the hair seems normal in the lower parts. B, in higher magnification, an increased thickness of the epidermis and hypergranulation is seen both in epidermis and in the hair follicle. C, transverse section of a hair follicle shows abnormal epithelial cells. D, the inner cell layer of the outer root sheath appears abnormal with irregularly shaped nuclei (arrows). E, hyperplastic and enlarged sebocytes are seen in the sebaceous glands. G, by in situ hybridization very low Elovl3 expression is seen in a cross-section of epidermis of wild-type skin. H, horizontal section showing specific expression of Elovl3 in the epithelial cells of a hair follicle (arrow, black grains). I, a higher magnification of the epithelial cells of the hair follicle showing abundant Elovl3 mRNA signal in the cells of the inner layer of the outer root sheath (ORS) of the hair follicle (arrows). IRS, inner root sheath. J, a strong Elovl3 mRNA signal (black grains) was detected in normal sebocytes. A-E, no Elovl3 mRNA signal was detected in Elovl3-ablated mice. (All sections are counterstained with hematoxylin and eosin).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Cell-specific Expression of Elovl3 in the Skin—Under normal conditions, Elovl3 is sparsely expressed in the skin. To delineate this expression, we performed in situ hybridization analysis. In the wild-type mice, a strong Elovl3 mRNA signal was seen in the cells of the inner layer of the outer root sheath (Fig. 2, H and I) of the hair follicles and in the sebocytes of the sebaceous glands (Fig. 2J). In epidermis the Elovl3 mRNA signal was very low (Fig. 2G), and in fibroblasts no detectable signal was seen. In the Elovl3-ablated mouse, there was no detectable Elovl3 mRNA signal in any cell type (Fig. 2, A-E). No signal was detected in tissues hybridized with the sense probe (data not shown).Disturbed Epidermal Skin Lipid Content of Elovl3-ablated Mice—Regarding the restricted Elovl3 expression in the skin and the abnormal morphology of the hair follicles and pilosebaceous system in the Elovl3-ablated mice, we suspected the lipid composition of the secreted sebum to be affected. Because the sebaceous glands are appendages of the epidermis connected to hair follicles and secrete the sebum onto the skin surface, we analyzed the lipid content of the epidermis, including the outermost part, stratum corneum, by thin layer chromatography. From this it was clear that the major difference between wild-type and Elovl3-ablated mice was observed in the most mobile fractions, which were a mixture of neutral lipids, such as triglycerides and wax and sterol esters, which are normal constituents of the sebum (Fig. 3). In contrast, there was no major difference between the more polar fractions where the different sphingolipid-based ceramide moieties migrate. A similar pattern was also seen when total skin was analyzed (not shown), suggesting a selective unbalance in the neutral lipids of the epidermis and hair.Fig. 3Abnormal content of neutral skin lipids of Elovl3-ablated mice. Equal amounts of epidermal lipids (120 μg) from wild-type (+/+) and Elovl3-ablated (-/-) mice were separated by HPTLC. The arrows and vertical line indicate the transfer area of triglycerides and sterol and wax esters, diacylglycerols and free fatty acids, cholesterol, and ceramides, respectively, according to lipid standards.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Disturbed Lipid Content in the Hair of Elovl3-ablated Mice— Although there was a general tendency toward more acetone-extractable lipids in the hair of Elovl-ablated mice, the total amount of hair lipids did not significantly differ from the wild-type mice (Fig. 4A). When we analyzed the hair lipids by TLC, the fractions containing triglycerides, wax esters, diol esters, and sterol esters formed a different TLC pattern between the two strains of mice (Fig. 4B). The most obvious difference was a shift in the amount of specific lipid components in the area where the triglycerides were localized (Fig. 4B). Densitometric analysis displayed a 10-fold decrease in a relatively more hydrophilic component, which was counteracted by a 10-fold increase in a relatively more hydrophobic component.Fig. 4Abnormal content of hair lipids of Elovl3-ablated mice.A, normal and mutated mice had similar total hair lipid production. Lipid content in the hair was determined in 10 wild-type (+/+) and 8 Elovl3-ablated (-/-) mice, respectively, with values expressed as m
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