Portal de Periódicos da CAPES

Suprabasal Overexpression of the hsRPB7 Gene in Psoriatic Epidermis as Identified by a Reverse Transcriptase-Polymerase Chain Reaction Differential Display Model Comparing Psoriasis Plaque Tissue with Peritonsillar Mucosa

2001; Elsevier BV; Volume: 158; Issue: 2 Linguagem: Inglês

10.1016/s0002-9440(10)63978-8

1525-2191

Raik Böckelmann, P. Neugebauer, Nuschin Djahan Paseban, Martin Hüttemann, Harald Gollnick, Bernd Bonnekoh,

Dermatology and Skin Diseases

In psoriasis an etiopathogenetic vicious circle is nowadays hypothesized that the disease is triggered by skin-specific autoantigen structures, the expression and accessibility of which are positively correlated with the intensity of the hyperproliferation and inflammation in the epidermopapillary compartment driven by autoreactive T cells. Despite the close microanatomical relation between skin and mucosa, clinicians have always been intrigued by the observation that psoriatic affection of the mucosa, if at all existing, is only seen as very rare events in the lips and tongue sparing buccopharyngeal sites. This prompted us to establish an experimental model system comparing psoriatic-involved skin and peritonsillar mucosa from tonsillectomies by a reverse transcriptase-polymerase chain reaction/differential display strategy. Among more than 60 cDNA species to be displayed in psoriasis, but missing in peritonsillar mucosa, one species was identified as coding for the RNA polymerase IIA seventh subunit (hsRPB7 gene) as a most critical factor for DNA to RNA transcription. Immunohistochemistry showed a hitherto unknown, distinctive pattern of hsRPB7 expression that was 1) tissue type-dependent with a surplus in skin keratinocytes and a near absence in peritonsillar mucosa, 2) tightly regulated by the keratinocyte differentiation process with a sharp suprabasal up-regulation in contrast to a basal down-regulation, and 3) substantially augmented in psoriatic-involved skin as compared to normal and psoriatic uninvolved skin. Keratinocytes of actinic keratoses also showed a strong hsRPB7 expression that however did not strictly spare the basal cell layer presumably reflecting the disturbed intraepidermal stratification because of the premalignant status of these precancerous lesions. In psoriasis an etiopathogenetic vicious circle is nowadays hypothesized that the disease is triggered by skin-specific autoantigen structures, the expression and accessibility of which are positively correlated with the intensity of the hyperproliferation and inflammation in the epidermopapillary compartment driven by autoreactive T cells. Despite the close microanatomical relation between skin and mucosa, clinicians have always been intrigued by the observation that psoriatic affection of the mucosa, if at all existing, is only seen as very rare events in the lips and tongue sparing buccopharyngeal sites. This prompted us to establish an experimental model system comparing psoriatic-involved skin and peritonsillar mucosa from tonsillectomies by a reverse transcriptase-polymerase chain reaction/differential display strategy. Among more than 60 cDNA species to be displayed in psoriasis, but missing in peritonsillar mucosa, one species was identified as coding for the RNA polymerase IIA seventh subunit (hsRPB7 gene) as a most critical factor for DNA to RNA transcription. Immunohistochemistry showed a hitherto unknown, distinctive pattern of hsRPB7 expression that was 1) tissue type-dependent with a surplus in skin keratinocytes and a near absence in peritonsillar mucosa, 2) tightly regulated by the keratinocyte differentiation process with a sharp suprabasal up-regulation in contrast to a basal down-regulation, and 3) substantially augmented in psoriatic-involved skin as compared to normal and psoriatic uninvolved skin. Keratinocytes of actinic keratoses also showed a strong hsRPB7 expression that however did not strictly spare the basal cell layer presumably reflecting the disturbed intraepidermal stratification because of the premalignant status of these precancerous lesions. The etiology of psoriasis is still unknown.1Griffiths CEM Immunological pathways in psoriasis.in: Roenigk Jr, HH Maibach HI Psoriasis. ed 3. Dekker, New York1998: 341-348Google Scholar, 2Austin LM Coven TR Bhardwaj N Steinmann R Krueger JG Intraepidermal lymphocytes in psoriatic lesions are activated GMP-17(TIA-1)+CD8+CD3+ CTLs as determined by phenotypic analysis.J Cutan Pathol. 1998; 25: 79-88Crossref PubMed Scopus (69) Google Scholar, 3Jenisch S Henseler T Nair RP Guo SW Westphal E Stuart P Kronke M Voorhees JJ Christophers E Elder JT Linkage analysis of human leukocyte antigen (HLA) markers in familial psoriasis: strong disequilibrium effects provide evidence for a major determinant in the HLA-B/-C region.Am J Hum Genet. 1998; 63: 191-199Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 4Nickoloff BJ Skin innate immune system in psoriasis: friend or foe?.J Clin Invest. 1999; 104: 1161-1164Crossref PubMed Scopus (110) Google Scholar, 5Nickoloff BJ The immunologic and genetic basis of psoriasis.Arch Dermatol. 1999; 135: 1104-1110Crossref PubMed Scopus (214) Google Scholar, 6Karasek MA Progress in our understanding of the biology of psoriasis.Cutis. 1999; 64: 319-322PubMed Google Scholar, 7Nickoloff BJ The search for pathogenic T cells and the genetic basis of psoriasis using a severe combined immunodeficient mouse model.Cutis. 2000; 65: 110-114PubMed Google Scholar But there is a lot of at least indirect clinical and experimental evidence that speaks in favor of a predominantly immunological quality of its pathogenesis. Nowadays, autoantigen-directed mechanisms intermingled with microbial (super)-antigen-driven immune-activations are hypothesized to play major roles in psoriasis, with the primary relevance of T-cell actions prevailing over antibody-mediated processes.4Nickoloff BJ Skin innate immune system in psoriasis: friend or foe?.J Clin Invest. 1999; 104: 1161-1164Crossref PubMed Scopus (110) Google Scholar, 5Nickoloff BJ The immunologic and genetic basis of psoriasis.Arch Dermatol. 1999; 135: 1104-1110Crossref PubMed Scopus (214) Google Scholar, 6Karasek MA Progress in our understanding of the biology of psoriasis.Cutis. 1999; 64: 319-322PubMed Google Scholar, 7Nickoloff BJ The search for pathogenic T cells and the genetic basis of psoriasis using a severe combined immunodeficient mouse model.Cutis. 2000; 65: 110-114PubMed Google Scholar, 8Christophers E The immunopathology of psoriasis.Int Arch Allergy Immunol. 1996; 110: 199-206Crossref PubMed Scopus (127) Google Scholar, 9Dudmundsdottir AS Sigmundsdottir H Sigurgeirsson B Good MF Valdimarsson H Jonsdottir I Is an epitope on keratin 17 a major target for autoreactive T lymphocytes in psoriasis?.Clin Exp Immunol. 1999; 117: 580-586Crossref PubMed Scopus (90) Google Scholar, 10Bonnekoh B Huerkamp C Wevers A Geisel J Sebök B Bange F-C Greenhalgh DA Böttger EC Krieg T Mahrle G Up-regulation of keratin 17 expression in human HaCaT keratinocytes by interferon-γ.J Invest Dermatol. 1995; 104: 58-61Crossref PubMed Scopus (47) Google Scholar, 11Nickoloff BJ Wrone-Smith T Superantigens, autoantigens, and pathogenic T cells in psoriasis.J Invest Dermatol. 1998; 110: 459-460Crossref PubMed Scopus (51) Google Scholar In this pathogenetic concept it is a matter of current controversial debates if such putatively expressed HLA-restricted autoantigens are recognized by CD4+ or CD8+lymphocytes.2Austin LM Coven TR Bhardwaj N Steinmann R Krueger JG Intraepidermal lymphocytes in psoriatic lesions are activated GMP-17(TIA-1)+CD8+CD3+ CTLs as determined by phenotypic analysis.J Cutan Pathol. 1998; 25: 79-88Crossref PubMed Scopus (69) Google Scholar, 3Jenisch S Henseler T Nair RP Guo SW Westphal E Stuart P Kronke M Voorhees JJ Christophers E Elder JT Linkage analysis of human leukocyte antigen (HLA) markers in familial psoriasis: strong disequilibrium effects provide evidence for a major determinant in the HLA-B/-C region.Am J Hum Genet. 1998; 63: 191-199Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 12Behrendt C Gollnick H Bonnekoh B Up-regulated perforin expression of CD8+ blood lymphocytes in generalized non-anaphylactic drug eruptions and exacerbated psoriasis.Eur J Dermatol. 2000; 10: 1-5Google Scholar, 13Nickoloff BJ Wrone-Smith T Injection of pre-psoriatic skin with CD4+ T cells induces psoriasis.Am J Pathol. 1999; 155: 145-158Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar Nevertheless, an alternate etiological concept of psoriasis as a disease with an antigen-independent pathogenesis has still to be taken into careful consideration. Only very recently, the possible crucial involvement of components of the innate immune systems including natural killer characteristics of T cells has been brought to the awareness of the scientific community.4Nickoloff BJ Skin innate immune system in psoriasis: friend or foe?.J Clin Invest. 1999; 104: 1161-1164Crossref PubMed Scopus (110) Google Scholar, 5Nickoloff BJ The immunologic and genetic basis of psoriasis.Arch Dermatol. 1999; 135: 1104-1110Crossref PubMed Scopus (214) Google Scholar, 14Nickoloff BJ Wrone-Smith T Bonish B Porcelli SA Response of murine and normal human skin to injection of allogeneic blood-derived psoriatic immunocytes: detection of T cells expressing receptors typically present on natural killer cells, including CD94, CD158, and CD161.Arch Dermatol. 1999; 135: 546-552Crossref PubMed Scopus (124) Google Scholar Moreover, the obvious clinical diversity of psoriasis and its variants lends support to the notion that heterogeneic pathomechanism may co-exist, as well as combinations thereof. Whether the oral mucosa can be specifically affected by psoriasis is an open question.15Morris LF Phillips CM Binnie WH Sander HM Silverman AK Menter MA Oral lesions in patients with psoriasis: a controlled study.Cutis. 1992; 49: 339-344PubMed Google Scholar, 16Sklavounou A Laskaris G Oral psoriasis: report of a case and review of the literature.Dermatologica. 1990; 180: 157-159Crossref PubMed Scopus (29) Google Scholar, 17Van der Wal N Van der Kwast WA Van Dijk E Van der Waal I Geographic stomatitis and psoriasis.Int J Oral Maxillofac Surg. 1988; 17: 106-109Abstract Full Text PDF PubMed Scopus (30) Google Scholar, 18Zhu JF Kaminski MJ Pulitzer DR Hu J Thomas HF Psoriasis: pathophysiology and oral manifestations.Oral Dis. 1996; 2: 135-144Crossref PubMed Scopus (45) Google Scholar This is partly because of the general observation, that signs of a possible psoriatic involvement of the oral mucosa are only seen in rare cases, mostly in conjunction with pustular skin manifestations of psoriasis.19Bork K Hoede N Korting GW Mundschleimhaut- und Lippenkrankheiten. ed 2. Schattauer, New York1993: 55-57Google Scholar, 20Cawson RA Binnie WH Everson JW Color Atlas of Oral Disease. ed 2. Mosby-Wolfe, Hong Kong1993: 1.14-1.15Google Scholar, 21Hornstein OP Erkrankungen des Mundes. Kohlhammer, Berlin1996: 378-382Google Scholar, 22Lotti TM Parish LC Rogers III, RS Oral Diseases. Springer, New York1999: 42-43Google Scholar In such cases, the lips may show an exfoliative psoriatic cheilitis, and usually the tongue presents with an exfoliatio areata linguae (ie, a so-called geographical tongue or benign migratory glossitis). The latter manifestation is histologically characterized by intraepithelial microabscesses of neutrophilic leukocytes also known as a quite pathognomonic feature of psoriatic skin affection.19Bork K Hoede N Korting GW Mundschleimhaut- und Lippenkrankheiten. ed 2. Schattauer, New York1993: 55-57Google Scholar, 20Cawson RA Binnie WH Everson JW Color Atlas of Oral Disease. ed 2. Mosby-Wolfe, Hong Kong1993: 1.14-1.15Google Scholar, 23Terui T Ozawa M Tagami H Role of neutrophils in induction of acute inflammation in T-cell-mediated immune dermatosis, psoriasis: a neutrophil-associated inflammation-boosting loop.Exp Dermatol. 2000; 9: 1-10Crossref PubMed Scopus (122) Google Scholar Attempts have been made to explain the rarity of thus still questionable psoriatic involvement of oral mucosa by pointing to the fact that in this tissue compartment the epithelial proliferation rate reaches under physiological conditions already such a high level that hypothetically may not be further increased in a psoriasis-typical manner.21Hornstein OP Erkrankungen des Mundes. Kohlhammer, Berlin1996: 378-382Google Scholar But this explanation seems to be insufficient to a certain extent, as it relates only to epithelial hyperproliferation without addressing the phenomenon that an inflammatory infiltrate as another histological hallmark of psoriatic skin manifestation is usually missing in the oral mucosa of psoriasis patients. Therefore an alternate hypothesis might be raised postulating: 1) the missing expression or accessibility of putative psoriasis-relevant autoantigens or 2) the lack of psoriasis-determining antigen-independent alterations of gene expression, respectively, in oral mucosa as possible decisive reasons for its common noninvolvement in the psoriatic disease process. Most interestingly, the manifestation of psoriasis in a split-skin graft transplanted into the oral cavity has recently been reported emphasizing the crucial pathogenetic role of the epidermodermal compartment in psoriasis.24Dimitrakopoulos I Lazaridis N Scordalaki A Dermal psoriasis involving an oral split-skin graft. Case report.Aust Dent J. 1998; 43: 321-323Crossref PubMed Scopus (11) Google Scholar Given these considerations, we have established an experimental model comparing directly the gene expression between psoriatic plaque tissue and oral peritonsillar mucosa by a differential display/reverse transcriptase polymerase chain reaction (DD/RT-PCR) approach. As reported herein, this strategy led to the identification of more than 60, until now unknown, cDNA species up-regulated in the psoriatic plaque as compared to the mucosa background. Additionally, this comparison showed an overexpression of the transcription-related hsRPB7 gene in psoriasis, which was analyzed by immunohistochemistry in detail. Our recent nonradioactive modification25Böckelmann R Bonnekoh B Gollnick H Optimized visualization and PCR reamplification of differentially displayed cDNA bands detected by silver staining in polyacrylamide gels as established in the model of dithranol-treated keratinocytes.Skin Pharmacol Appl Skin Physiol. 1999; 12: 54-63Crossref PubMed Scopus (7) Google Scholar of the original DD/RT-PCR protocol26Liang P Pardee AB Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction.Science. 1992; 257: 967-971Crossref PubMed Scopus (4745) Google Scholar was used as a technique for an optimized visualization and PCR reamplification of differentially displayed cDNA bands detected by silver staining. In brief, tissue specimens from skin lesions of plaque psoriasis, normal skin, and tonsillectomy-derived peritonsillar mucosa27Neugebauer P Bonnekoh B Wevers A Michel O Mahrle G Krieg T Stennert E Human keratinocyte culture from the peritonsillar mucosa.Eur Arch Otorhinolaryngol. 1996; 253: 245-251Crossref PubMed Scopus (15) Google Scholar were frozen in liquid nitrogen and then homogenized on ice (Polytron PT3000, Kinematica AG). Total RNA was isolated by the standard guanidinium isothiocyanate method (RNAzol B), and mRNA was purified by a single run through an oligo(dT)-cellulose spun column (Pharmacia). The cDNA resulting from reverse transcription with anchored primers T12AN was used as a template in a 20-μl PCR reaction containing 1 μmol/L of the T12AN primer and an arbitrary 10-mer primer28Bauer D Müller H Reich J Riedel H Ahrenkiel V Warthoe P Strauss M Identification of differentially expressed mRNA species by an improved display technique (DDRT-PCR).Nucleic Acids Res. 1993; 21: 4272-4280Crossref PubMed Scopus (507) Google Scholar each, 2.5 U AmpliTaq DNA polymerase (Perkin Elmer), 200 μmol/L dNTP, and 2 mmol/L MgCl2. Kinetics of the PCR reactions were set to 10 minutes at 94°C, 41 cycles of 30 seconds at 94°C, 30 seconds at 42°C, and 60 seconds at 72°C with a 10 minutes termination step at 72°C. The PCR-amplified cDNA species were separated electrophoretically on polyester film-backed 10% polyacrylamide gels (CleanGel, ETC) under nondenaturing flatbed conditions at 15°C. The cDNA bands were detected by an optimized silver staining as described in detail earlier.25Böckelmann R Bonnekoh B Gollnick H Optimized visualization and PCR reamplification of differentially displayed cDNA bands detected by silver staining in polyacrylamide gels as established in the model of dithranol-treated keratinocytes.Skin Pharmacol Appl Skin Physiol. 1999; 12: 54-63Crossref PubMed Scopus (7) Google Scholar From the differential display gels those cDNA bands were cut out that were identified for psoriatic tissue but found to be missing for normal oral mucosa when comparing corresponding lanes running side-by-side. The cDNA polyacrylamide material was intensely chopped by scalpel cuttings and squashed by a micropestle. The extraction was performed in a 0.5 mol/L ammonium acetate/1 mmol/L ethylenediaminetetraacetic acid solution (pH 8.0) at 37°C overnight under vigorous shaking. After centrifugation, the supernatant was glass microfiber-filtered (2-μm pore size, Whatman) to remove polyacrylamide remnants. The cDNA was ethanol-precipitated, redissolved, ethanol-reprecipitated, and washed, as well as vacuum-dried for the subsequent PCR reamplification under the conditions given above.25Böckelmann R Bonnekoh B Gollnick H Optimized visualization and PCR reamplification of differentially displayed cDNA bands detected by silver staining in polyacrylamide gels as established in the model of dithranol-treated keratinocytes.Skin Pharmacol Appl Skin Physiol. 1999; 12: 54-63Crossref PubMed Scopus (7) Google Scholar The PCR reamplification product was run in a 0.8% low-melting agarose gel, subcloned (pCR2.1-TOPO; Invitrogen) and submitted to a nonradioactive cycle-sequencing reaction (Thermo Sequenase, Amersham). Sequencing was performed by using UV-polymerized gels (Repro Gel Long Read) in conjunction with an automated sequencer (ALFexpress, Pharmacia). For this purpose, biopsies of psoriatic plaques, peritonsillar mucosa, uninvolved psoriatic, and normal skin as well as seborrheic and actinic keratoses (n = 3 to 6 donors each) were placed in frozen specimen-embedding medium (catalog no. 6769006; Shandon, Pittsburgh, PA), quickly frozen on a freezing block at −55 to −60°C, cut into 5-μm slices using a microtome, and fixed with acetone (4°C, 10 minutes). The slides were incubated with a murine monoclonal anti-RPB7-antibody (dilution 1:500; BAb Co., Richmond, CA)29Thompson NE Steinberg TH Aronson DB Burgess RR Inhibition of in vivo and in vitro transcription by monoclonal antibodies prepared against wheat germ RNA polymerase II that react with the heptapeptide repeat of eukaryotic RNA polymerase II.J Biol Chem. 1989; 264: 11511-11520Abstract Full Text PDF PubMed Google Scholar, 30Baskaran R Dahmus ME Wang JYJ Tyrosine phosphorylation of mammalian RNA polymerase II carboxyl-terminal domain.Proc Natl Acad Sci USA. 1993; 90: 11167-11171Crossref PubMed Scopus (199) Google Scholar with a subsequent detection by an alkaline phosphatase Vectastain ABC kit (catalog no. AK-5002; Vector Laboratories Inc., Burlingame, CA) in conjunction with a Vector red substrate kit (catalog no. SK-5100, Vector Laboratories, Inc.). Endogenous alkaline phosphatase activity was blocked by adding its inhibitor levamisole to the substrate solution. Nuclei were counterstained with Mayer’s hemalaun solution (catalog no. 1.09249.0500; Merck, Darmstadt, Germany). Mounting of the slides was performed by the use of Kaiser’s glycerin gelatin (catalog no. 1.09242.0100, Merck). Technical negative controls were performed by omitting the first antibody. The study identified more than 60 cDNA species that were expressed in the psoriatic plaque but not in the normal buccopharyngeal mucosa. When analyzing these cDNA data by comparison with GenBank data from the National Center for Biotechnology Information using the BLAST 2.0 search routine (www.ncbi.nlm.nih.hov/cgi-bin/BLAST), the main body of the cDNA species was found to contain until now unknown sequence information. The novel data were registered at GenBank receiving appropriate accession numbers and are communicated in Table 1.Table 1Panel of 65 Novel cDNA Species Found to Be Up-Regulated in Psoriatic-Involved Skin as Compared to Normal Peritonsillar Mucosa when Performing a Differential Display/RT-PCR AnalysisAF126046: 151 bpAF136806: 235 bpAF136805: 267 bpAF143341: 328 bpAF126047: 164 bpAF126041: 236 bpAF143338: 267 bpAF143357: 331 bpAF143354: 164 bpAF143340: 237 bpAF143334: 271 bpAF143350: 332 bpAF126948: 183 bpAF126039: 244 bpAF143361: 271 bpAF143355: 341 bpAF125380: 184 bpAF143347: 244 bpAF113845: 275 bpAF116185: 346 bpAF143344: 185 bpAF126040: 247 bpAF143335: 276 bpAF143339: 349 bpAF143351: 187 bpAF143343: 248 bpAF143337: 281 bpAF126950: 374 bpAF127574: 190 bpAF119787: 251 bpAF143333: 282 bpAF118927: 408 bpAF143362: 194 bpAF126946: 251 bpAF116186: 284 bpAF119788: 425 bpAF118928: 199 bpAF143367: 252 bpAF118929: 291 bpAF143353: 465 bpAF125379: 209 bpAF126044: 253 bpAF143363: 305 bpAF143352: 505 bpAF126045: 210 bpAF143349: 260 bpAF126949: 311 bpAF126044: 521 bpAF143366: 220 bpAF143360: 260 bpAF143365: 323 bpAF126947: 554 bpAF125378: 225 bpAF143356: 261 bpAF127575: 324 bpAF126042: 555 bpAF143364: 226 bpAF143358: 262 bpAF136807: 327 bpAF143342: 660 bpAF143347: 230 bpAF143359: 262 bpAF143336: 327 bpAF143346: 777 bpAF143345: 232 bpSequence data were registered at GenBank under the appropriate accession numbers as indicated beside the bp length of the fragments. Open table in a new tab Sequence data were registered at GenBank under the appropriate accession numbers as indicated beside the bp length of the fragments. A certain cDNA species with a 338-bp length, which appeared to be overexpressed in the psoriatic plaque as compared to oral mucosa as well as normal skin (Figure 1), was found to be identical with the hsRPB7 gene (GenBank identification U52427; corresponding positions 5553 to 5890) as a human homologue of yeast RPB7 coding the seventh subunit of RNA polymerase II.31Schoen TJ Chandrasekharappa SC Guru SC Mazuruk K Chader GJ Rodriguez IR Human gene for the RNA polymerase II seventh subunit (hsRPB7): structure, expression and chromosomal localization.Biochim Biophys Acta. 1997; 1353: 39-49Crossref PubMed Scopus (10) Google Scholar, 32Khazak V Sadhale PP Woychik NA Brent R Golemis EA Human RNA polymerase II subunit hsRPB7 functions in yeast and influences stress survival and cell morphology.Mol Biol Cell. 1995; 6: 759-775Crossref PubMed Scopus (67) Google Scholar, 33Khazak V Estojak J Cho H Majors J Sonoda G Testa JR Golemis EA Analysis of the interaction of the novel RNA polymerase II (pol II) subunit hsRPB4 with its partner hsRPB7 and with pol II.Mol Cell Biol. 1998; 18: 1935-1945Crossref PubMed Scopus (58) Google Scholar Because of the until now unknown expression pattern of the hsRPB7 protein in psoriasis, we undertook a thorough immunohistological analysis giving the following results. In peritonsillar mucosa there was no substantial expression of hsRPB7 detectable (Figure 2E). For psoriatic plaques we found a strong hsRPB7 expression mostly in the cytoplasm of the keratinocytes in the upper two thirds of the subcorneal epidermis, ie, in the stratum spinosum, whereas the basal and, in some instances, also the first suprabasal cell layers were spared (Figure 2, A–C). Normal epidermis and uninvolved psoriatic skin showed only a weak to moderate expression of hsRPB7 also restricted to the suprabasal layers (Figure 2D). Overall, we did not observe any substantial hsRPB7 expression in the capillaries, inflammatory cells, or fibrocytic elements of the subepidermal or subepithelial compartments of involved and uninvolved psoriatic as well as normal skin nor peritonsillar mucosa, respectively. To investigate the specificity of these findings for the psoriatic disease process, the immunohistological study was extended to some other forms of hyperkeratotic diseases. The hsRPB7 expression proved to be weak to moderate in seborrheic keratoses (n = 3) as well as in one case of ichthyosis vulgaris (n = 1), but to be very strong in actinic keratoses (n = 3) representing precancerous lesions. Notably, in actinic keratoses the basal cell layers were not so strictly spared from the hsRPB7 expression. Overall, a distinct pattern of RPB7 expression was observed, nearly completely missing in the peritonsillar mucosa epithelium, and being restricted to the suprabasal epidermis under nonpremalignant conditions with a much higher intensity in involved psoriatic skin as compared to normal and uninvolved psoriatic skin. With regard to the contribution to the further elucidation of the etiopathogenesis of psoriasis, DD-RT/PCR techniques have already been used to compare involved and uninvolved psoriatic skin.34Rivas MV Jarvis ED Morisaki S Carbonaro H Gottlieb AB Krueger JG Identification of aberrantly regulated genes in diseased skin using the cDNA differential display technique.J Invest Dermatol. 1997; 108: 188-194Crossref PubMed Scopus (62) Google Scholar We now propose an alternate model comparing psoriatic plaque tissue and peritonsillar mucosa based on the clinical notion that despite the close microanatomical relation between both tissues, the latter is never involved by the psoriatic disease process. The DD/RT-PCR comparison of psoriatic-involved skin with normal peritonsillar mucosa is an attractive research objective aiming at the identification of gene products that might be critically involved in the etiopathogenesis of psoriasis with regard to 1) the epidermal hyperproliferation, 2) the vascular inflammatory compartment, as well as 3) putatively involved autoantigens. The project led to the discovery of so far unknown DNA sequence information that has to be further characterized and identified at the nucleotide and protein level. The main outcome of our study is the hitherto unknown differential expression of the hsRPB7 gene in psoriatic and normal skin as well as peritonsillar mucosa. The corresponding product, which we picked up by DD-RT/PCR and identified by sequencing, contains part of exon 7, all of intron 7, and part of exon 8 from the hsRPB7 gene. Therefore this cDNA species may represent a pre-mRNA as well as an alternatively spliced form of hsRPB7 mRNA, in which intron 7 is not spliced out, leading to a modification in the C-terminus of the protein. As a pivotal part of the DNA transcription machinery the hsRPB7 protein was found to be nearly absent in the peritonsillar mucosa epithelium and to be expressed suprabasally in the epidermis with a much higher abundance in involved psoriatic skin as compared to uninvolved psoriatic and normal skin. Overexpression of hsRPB7 did not seem to be strictly psoriasis-specific because we could observe this phenomenon also in actinic keratoses. However, there was a difference in the pattern of hsRPB7 expression between psoriatic plaques and actinic keratoses. Most interestingly, the latter expressed hsRPB7 also in major parts of the basal cell layer as a presumptive special feature of the disturbances related to the premalignant transformation occurring in these precancerous lesions. Originally the RPB7 gene product has been characterized as a 19-kd subunit of the RNA polymerase II oligopolypeptide complex in the yeast Saccharomyces cerevisae functioning as an essential factor for cell growth and viability.35McKune K Richards KL Edwards AM Young RA Woychik NA RPB7, one of two dissociable subunits of yeast RNA polymerase II, is essential for cell viability.Yeast. 1993; 9: 295-299Crossref PubMed Scopus (77) Google Scholar Surface plasmon resonance studies demonstrated that the yeast RPB7 protein together with RPB4 stabilizes a pre-initiation complex consisting of promoter DNA, TATA box-binding protein, transcription factor TFIIB, as well as the RNA polymerase II being responsible for mRNA generation as a prerequisite for protein translation.36Jensen GJ Meredith G Bushnell DA Kornberg RD Structure of wild-type yeast RNA polymerase II and location of Rpb4 and Rpb7.EMBO J. 1998; 17: 2353-2358Crossref PubMed Scopus (58) Google Scholar Only recently the existence of an evolutionarily conserved human homologue to RPB7 was shown, ie, hsRPB7.31Schoen TJ Chandrasekharappa SC Guru SC Mazuruk K Chader GJ Rodriguez IR Human gene for the RNA polymerase II seventh subunit (hsRPB7): structure, expression and chromosomal localization.Biochim Biophys Acta. 1997; 1353: 39-49Crossref PubMed Scopus (10) Google Scholar, 32Khazak V Sadhale PP Woychik NA Brent R Golemis EA Human RNA polymerase II subunit hsRPB7 functions in yeast and influences stress survival and cell morphology.Mol Biol Cell. 1995; 6: 759-775Crossref PubMed Scopus (67) Google Scholar, 33Khazak V Estojak J Cho H Majors J Sonoda G Testa JR Golemis EA Analysis of the interaction of the novel RNA polymerase II (pol II) subunit hsRPB4 with its partner hsRPB7 and with pol II.Mol Cell Biol. 1998; 18: 1935-1945Crossref PubMed Scopus (58) Google Scholar Until now the knowledge about the regulation of mRNA and protein expression of hsRPB7 in the skin under physiological and pathophysiological conditions is rather limited. Our data point to an up-regulation of hsRPB7 protein expression and thus DNA-mRNA transcription during the psoriasis dependently disturbed keratinocyte differentiation process in the upper epidermis. This finding seems to be another, novel facet reflecting the disturbed intraepidermal cell differentiation process in psoriasis. A very striking feature under psoriatic and normal tissue conditions was the spared hsRPB7 expression in the basal epidermal cells, an obvious cell differentiation-related phenomenon getting lost under premalignant conditions that awaits further characterization. We thank Mrs. J. Leipold for excellent technical assistance.