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    Decrease PLG Expresion in IV PDF

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    ‘ys Jagous of Bout Camasney 2008 the eran Satyr Bement Meee Boy ne Decreased Profilaggrin Expression in Ichthyosis Vulgaris Is a Result of Selectively Impaired Posttranscriptional Control* (Received for publication, May 31, 1994, and in revised form, November 2, 1994) Wilas Nirunsuksirit, Richard B. Presland{§, Steven G. Brumbaugh!, Beverly A. Dale, and Philip Fleckmant From the ‘Division of Dermatology, Department of Medicine, and Departments of "Biochemistry and Oral Biology and Periodontics, University of Washington, Seattle, Washington 98195 thet val an meet dena der of keratinization characterized by mild hyperkera- tosis and reduced or absent keratohyalin granules in the epidermis. Profilaggrin, a major component of kerato- hyalin granules, is reduced or absent from the skin of individuals with ichthyosis vulgaris. In this report, we have further characterized the molecular basis of low profilaggrin expression, which occurs in this disease. In situ hybridization revealed little profilaggrin mRNA in ichthyosis vulgarie-affected epidermis. In keratinocytes cultured from the epidermis of affected individuals, the abundance of profilaggrin was reduced to less than 10% ‘of normal controls, while the mRNA level was decreased to 30-80% of controls. Expression of K1 and loricrin, other markers of epidermal differentiation, were not affected. Nuclear run-on assays indicated that the de- crease in mRNA levels was not caused by aberrant tran- scription. Nucleotide sequencing of 5'-upstream, 3/-non- coding, and flanking regions of the profilaggrin gene from ichthyosis vulgarisaffected individuals revealed only minor changes, probably due to genetic polymor- phioms. Our results indicate that defective profilaggrin expression in ichthyosis vulgaris is a result of selee- tively impaired posttranscriptional control Xone vulgaris (IV) is an autosomal dominant skin di order reported to cecur in as many as 1 in 250 of the normal population (1). Affected skin appears scaly and is characterized histologically by hyperkeratosis and a decreased or absent granular layer (2). In addition, keratohyalin granules, an ul- trastructural landmark in the granular layer, are either absent or reduced and structurally abnormal (3). Other clinical symp- toms, including hyperlinear palms and soles, a personal or family history of atopy, and keratosis pilaris are often associ- ated with IV (1), Although the disease is well described clini- cally, the etiology is poorly understood. Filaggrin is a cationic protein that aggregates keratin inter- mediate filaments inthe stratum corneum of the epidermis (for review, see Ref, 4). Profilaggrin, the precursor of filaggrin, is first expressed in the granular layer and marks the terminal * Thic work was aupported by National Institutes of Health Grants POI AM 21857 and RST DE 04680, the Endowed Dermatology Research Fund, and the Hammock Trust. The costs of publication ofthis etile ‘were defrayed in part by the payment of page changes. This article mast therefore be hereby marked “advertisement” in accordance with 18 USC, Section 1794 sally to indicate this Ta {To whom correspondence shouldbe addressed Div. of Dermatology, EM-16, University of Washington, Seattle, WA 98196, Tel 208-549 5290; Fax: 206:543-2489, The abbreviations used are: IV, iehthyosia vulgaris; bp, base pai PPCR, polymerase chain teection; HS26, human S26 ribosomal protein; PAGE, polyacrylamide gel electrophoresis; TES, 2(f2-hydroxs,1 bisthydroxymethy)ethyliaminolethanesulfonic acid stages of epidermal differentiation. The phosphorylated pro- filaggrin accumulates in keratobyalin granules and later un- dergoes dephosphorylation and proteolysis to filaggrin. Pro- filaggrin and filaggrin are noticeably decreased or absent from the epidermis of patients with IV (6), We have previously shown that Keratinocytes cultured from affected individuals maintain structural and biochemical phenotypic characteris- tics of the disorder (6). For example, very little profilaggrin is detectable by immunohistochemical staining on Western blots of extracts obtained from IV keratinocytes compared with cot trols, These data are consistent with the absence of keratohs alin in this disorder. This suggests pronounced decrease in profilaggrin synthesis and/or accumulation as is seen in the skin biopsies from affected individuals, Recently, the structure of the human profilaggrin gene has Deen reported by two different laboratories (7,8). The gene (see Fig. 84) contains three exons interrupted by two introns of 9,719 and 570 bp, respectively, The 5'-noncoding region (75 bp) is divided into two exons separated by the large intron. The coding rogion begins in the second exon and continues in the third exon, where 10-12 highly repetitive flaggrin sequences of exactly 972 bp reside. The number of flaggrin repeats varies ‘between individuals and is inherited in a Mendelian fashion (9), The amino terminus of profilaggrin contains a calcium binding domain consisting of two EF-hands resembling those present in the S-100 family of proteins (7). It has recently been ‘shown that the S-100 domain of profilaggrin binds ealeium (8, 10). Hence, profilaggrin may not only function as a keratin ‘aggrogating protein, but it may also play a critical role in the regulation of ealeium-dependent events during epidermal differentiation (7) To investigate further the association between decreased profilaggrin expression and IV, we studied profilaggrin mRNA levels in vivo by in situ hybridization and utilized a human epidermal keratinocyte culture system as an in vitro model. We analyzed profilaggrin expression at the protein, steady-state mRNA, and transcriptional levels in keratinocytes cultured from individuals with IV as well as from appropriate, unaf- fected family members and age- and sex-matched normal con- trols. Our data indicate that selectively impaired posttran- scriptional regulation results in reduced profilaggrin mRNA and protein in IV. IALS AND METHODS Diagnosis of Ichthyosis Vulgoris—Probanda were identified from ps tients sen in the University of Washington Medical Centar Dermstol- ogy Clinics and from individuals referred by clinical dermatologists {hom the community. Subjects who met published clinical ertera for IV (0, 1) were biopsied from the extensor sarface ofthe arm after obtaa- ‘ng informed consent. Biopsies were Fixed in methyl Carnoy’s for light microscopy and immunoeytachemistey and fixed in helf-strength Kar nnovaky' for electron microscopy ax deseribed previously (12), Individ- ‘uals withthe clinical eriteria for TV who had one or fewer layers of| 871 72 tranular cells in hematoxylin and eosinstained sections (D, who had fbeont or attenuated. ctaining with the anti-proflaggrin antibody AKHI with normal staining with the ent-keratahyalin antibody AKH2 (13) and who had no keratohyalin granules when examined by electron ‘ieroscopy (9,8) were considered to be affected In this report, results from affected, unrelated individuals from three different families are presented. Related, unaffected family members served as controls for fo cases, and an unrelated, age- and sex-matched subjects for the other. ‘in Situ Hybridization—Biopsies wore obtained from the extensor surface of the arm and snap frozen in Tieaue-Tek® OCT (Mile, Ine) fembedding medium, Ten-micron frozen eactions were fixed briefly in paraformaldehyde and processed by standard techniques (14) with the following modifications; proteinase K digestion was omitted, an initial {Eh wash at room temperature with 4 % SSC 1» SSC: 0.15 ac sodium chloride, 0156 sodium strata, pH 70) was added after hybridization, fand 10 mor dithiothretol was added to all rinses, Hybridization was with a "S-labeled riboprobe generated from a human flaggrin repeat Cloned into pGEM-1 (Promega Corp). The plasmid was linearized with ‘Heol, and antisense riboprobe was generated with the use of TT RNA polymerase in the presence of [a™*S]UTP (1,000 Ciimmal) (DuPont NEN, Cell Culture—Adult human keratinocytes were obtained from bistor biopsies as described previously (6). Keratinocytes were cultured on mitomycin C-treated 973 cells and maintained ina humidified 6% CO, itmosphere at 389°C in Dulbecco's modified Eagle's medium (Life Technologies) containing 20% fetal all serum, hydrocortisone, cholera toxin, and epidermal growth factor. Colls wore fed 3 times a wook and 24 h before harvesting, To ensure proflaggrin expression, cella Were harvested 3 days after cells reached confluence. Protein Extraction, SDS:PAGE, and Western Blotting—Total urea: ‘Tes-soluble protein extracts (15) were obtained from keratinocytes cultured in parallel with thore used for the tolation of RNA and nuclei Equal protein loadings were separated on discontinuous 7.5-16% SDS- PAGE gels, and proteins were blotted to nitrocellulose (Schleicher and Schuell). Proflaggrin was detected with polyclonal anti-human pro- Slaggrin/Glaggrin antiserum (16), K1 with monoclonal antibody ABS (a fenerous gift of T-T. Sun, New York University Medical Cente, Ref 17, and loririn with antibody raised against a synthetic peptide cor- responding to the 14 most carboxy-terminal reidves of mouse lorierin Known to erose-reset with human Joirin (18) Plasmids, Probes, PCR Primers, and Genamie DNA—1) The profla arin ending probe was a 972-p Mlaggrin repeat of exon 3 (7 2) The profilaggrin 5° upstream region (885 bp) was generated from hhoman genomic DNA using the following oligonveleatides* (Ref. 5, GenBank 0196943). 5'-TGGTAGGAGGCACAATGT.3" and 5'-GAGC- CTGCTGGGTACTGA-S', Amplification using PCR was performed us- ‘ng Tag polymerase (Promega Corp). Conditions for PCR were 84°C for Tin, 50°C for I min, and 72°C for 1 min, for 30 3) The profilaggrin--noncoding region (517 bp) was generated from Jnuman genomic DNA using an upstream oligonucleotide that included the atop codon (underlined) of human profilaggrin gene (9): 5 -GATAC- 'TATTACTATGAGTAAGA'S' and 5-GACATCTAATTCTGGCCATGG- 2 Amplification using PCR war performed using Tag polymerase (Pro- ‘mea Corp.) Conditions for PCR were 94°C for I min, 42°C for I min, land 72°C fer 1 min, for 30 cycles, '#) The Ki eDNA clone (@ gift of Dr. D. Roop, Baylor College of ‘Medicine, Houston, TX) i a subclone of PK456 (110 bp) and contains the BamHIL-Petlfragmont of pk, which encodes the earboxyi-terminal ‘end domain and 3"-nonceding region of the human KI gene (19) '5) The loririn-specifie probe (120 bp) was prepared based on the _¥-noncoding sequence of human loricrin (20) ung the following olgo- nucleotides: §-GTACCACGGAGGCGAAGGAGT-3" and 5-GGTTGG. GAGGTAGTTGTACAG-S". Conditions used for PCR were 94°C for 1 ‘min, §9°C for 1 min, and 72°C for 1 min, for 30 cycles. 16) The glyceraldehyde-?-phosphate dehydrogenase, pHCGAP plasmid was obtained from ATCC, "The eDNA for human $26 ribosomel protein (HS26) was a gift of Dr. P, Fort, Montpelier Cedex 2, France '8) Genomic DNA was prepared from cultured Gbreblasta of normal and IV-alfected individuals as described previously (20) "Northern Analysis—Total cellular RNA was prepared from eultured keratinocytes using the guanidine thiocyanate-acid phen! method (22) Equal amounts of total RNA (10 ng) were separated on 1¢ glyoral gels, blotted onto GeneScreen Plas membranes (DuPont NEN) and hybrid: * RB. Presland, unpublished data Profilaggrin Expression in Ichthyosis Vulgaris ized toa nek-translated or random primed prabe overnight at 60°C in the buffer recommended by the manufacturer except that 200 pg/ml sonicated denatured salmon sperm DNA was included, Fitere were washed 3 times in 2 % SSC, 01% SDS, followed by 2 times in O.1% SDSO.1 x SSC at 65°C, All Northern blots were reprobed with give: ceraldchyde-®-phosphate dehydrogenase and HS26 cDNA to ascertain ‘equal ENA loadings. The autoradiographs were scanned using the Phosphorimager (Molecular Dynamics, Sunnyvale, CA) and quantified using image analysia softwere (ImageQuant Version 3.22, Molecular Dynamics) All comparison between hybridization signals was based on ‘equal glyceraldehyde 3-phoaphate dehydrogenase or HS26 signal Tolation of Nuclei and Nuclear Runcon Aseaye—First or second pateage human Keratinocytes were cultured to 3 days post-confuence ‘Nuclei wore isolated by a modification of tho mothod of Greenberg and Bender (23). Keratinocytes were harvested from five to six 100-mm plates, resuapended in 1% Nonidet P-40 lyse buffer, vortexed briefly, ‘and severed with sterile seissors. Cells were broken using 40 strokes of ‘2 Dounce homogenizer, and nucei were isolated, snap frozen in liquid nitrogen, and stored at 70°C. Nuelet were incubated with run-on Feaction buffer in the presence of 250 Ci of a-"PIGTP (8,000 Cima) {or 30 min, Labeled RNA wes iolated and purified through a SuperS- lect column (§' ~ 3', Boulder, CO), Equivalent "P-labeled RNA (as {determined by trichloroacetic cid preepitation) was hybridized to plas mid DNA that had bien linearized, denatured, and immobilized onto nitrocellulose using a sat blotting apparatus (Bio-Rad). Hybridization was carried out in 1m of TES buffer with 100 mg/ml denatured sonicated salmon sperm DNA for 48-72 h at 65°C, fllowed by two ‘washes in 2 ~ SSC, 0.1% SDS and two washes in 0.1 x SSC, 0.1% SDS ‘2065°C for $0 min, Exposure to x-ray fm was for 3-7 days. Nuclear rrun-on was also caried out in the presence of 0.5% sarkosy (24) in ‘order to distinguish between polymerase initiation and elongation, ‘cAmanitin (2 ug/ml) was used to show thet in utro transcription was due to RNA polymerase I activity (29). o RESULTS In Situ Analysis of Profilaggrin mRNA in IV and Normal Epidermis—It has been shown previously by immunohisto- ‘chemical techniques using an anti-human profilaggrin anti- body that immunoreactive profilaggrin and flagerin are mark- ‘edly decreased in the epidermis of IV-affected individuals ‘compared with normal controls (5). To determine if the de- crease is due to a reduction in profilaggrin mRNA, in situ hybridization using a “S-labeled riboprobe transcribed from a ‘human filaggrin repeat was performed. A dramatic decrease in silver grains in the granular layer of affected IV epidermis compared with normal skin was seen (Fig. 1) showing reduced levels of proflaggrin mRNA in the epidermis of IV-affected individuals and confirming studies at the protein level (5)..No significant labeling of the epidermis was seen with the sense strand riboprobe (data not shown), Decreased Profilaggrin Expression in IV Keratinocytes— Western analysis of profilaggrin, K1, and lorierin in protein, extracts from keratinocytes obtained from 1V-affected individ- uals compared with normal controls is demonstrated in Fig. 2. It should be noted that cultured human keratinocytes express profilaggrin at confluence but do not process it to flaggrin as normally occurs during terminal differentiation in vivo (6). It typically appears as a broad smear on SDS gels. The anti ‘human profilaggrin antibody detected very little proflaggrin in ‘extracts from affected individuals, in agreement with previous findings (6). We visually estimated the amount of immunore- active protein in keratinocyte extracts from affected individu- als to be less than 10% of normal controls. This result paral- Jeled the loss or reduction of keratohyalin granules observed in ‘he corresponding skin biopsies (data not shown). In contrast, the expression of neither lorierin, another marker of the late stages of epidermal differentiation also localized in keratohya- Jin granules, nor K1, a marker of euprabasal differentiation, were reduced (Fig. 2). The results strongly suggest that the defect in keratinocytes obtained from IV-affected individuals is specific to profilaggrin. ‘Steady State Level of the Profilaggrin mRNA in IV Keratino- Profilaggrin Expression in Ichthyosis Vulgaris, Fic. 1. In situ hybridization of skin from IV-affected individ: uals and controls, Biopsies of unaffected (C) and affected (IV) ind ‘duals were hybridized with "S-labeled antisense lagugein riboprobe Reduced silver grains were viewed by light mieroscopy. , basal layer ‘Sy spinous layer G, granular layer, C, cornified layer; Bar = 30 jm, ‘ytes—To gain insight into the etiology of the disease, proflag sin mRNA levels in keratinocytes from affected individuals and controls was determined, Northern analysis of total RNA har- vested from confluent keratinocytes of three unrelated IV sub- jects and controls was conducted with a probe from a filaggrin repeat within exon 3 ofthe proflaggrin gene (Fig. 34), The level of profilaggrin mRNA in affected cells was markedly reduced ‘compared with the normal counterparts (Fig. 3), Similar results| ‘were obtained when exon 2, which contains part of the EF-hand domain, was used as a probe (data not shown). The intensity of each hybridization signal was quantified using the Phosphorlm- ‘ager and the level of mRNA normalized to the internal glyceral- dehyde-3-phosphate dehydrogenase control. The levels of pro- filaggrin mRNA were 30, 61, and 43% of their corresponding, controls (from let to right panel). When loricrin- and K1-specific probes were tsed on the same blots, no differences in RNA levels ‘were seen between normal and affected (Pig. 33). These results ‘were consistent with those of the Western analysis, although compared with the normal counterparts, the levels of immuno- reactive profilaggrin protein were much lower in IV keratino- cytes than the levels of profilaggrin mRNAs, ‘The basis of comparison relied on the use of glyceraldehyde- S-phosphate dehydrogenase to normalize RNA on the blots However, recent evidence suggests that glyceraldehyde-3-phos- phate dehydrogenase expression may vary as a function of proliferative state and physiological conditions within the cell, (25-27), Keratinocytes used in this study were at posteonflu- fence, when proliferation is reduced (28). Our assumption was that the rate of proliferation of normal and IV-affected kerati- 873 FCO NCWCWN a 200 — prorG 96 67 43 FG 67 Bk KS 7K6 43 - °° 3

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