Overexpression of the Oncofetal Fn Variant Containing the EDA Splice-in Segment in the Dermal–Epidermal Junction of Psoriatic Uninvolved Skin
2000; Elsevier BV; Volume: 114; Issue: 4 Linguagem: Inglês
10.1046/j.1523-1747.2000.00871.x
ISSN1523-1747
AutoresKathleen M. Ting, D Rothaupt, Thomas S. McCormick, Craig Hammerberg, Guofen Chen, Anita C. Gilliam, Seth R. Stevens, Lloyd A. Culp, Kevin D. Cooper,
Tópico(s)Skin and Cellular Biology Research
ResumoThe extracellular matrix protein, Fn, has critical functions in cell attachment, migration, differentiation, and proliferation. We have previously shown that fibronectin (Fn) is abnormally expressed and potentiates entry into the cell cycle of basal keratinocytes in uninvolved psoriatic skin, in combination with T cell lymphokines. It is not known what type of Fn is present in psoriatic skin, however, and how this Fn may regulate signaling. Embryonic forms of cellular Fn containing extra domains, designated EDA and EDB, are generated by alternative splicing and are seen in proliferating, developing tissue and in wound healing. Because the EDA segment enhances the integrin binding sequence Arg, Gly, Asp (RGD), which, when present, has been shown to be critical in integrin–extracellular matrix signaling, we were particularly interested in determining whether or not EDA-containing Fn (EDA+Fn) represented the aberrantly expressed Fn in psoriasis. Increased EDA+ Fn protein was demonstrated by immunostaining at the dermal–epidermal junction in clinically uninvolved skin from six of six patients with psoriasis, but not in skin from control subjects. Using reverse transcription polymerase chain reaction an increased ratio of EDA+ Fn versus EDA− Fn mRNA was present in epidermal samples from psoriatic but not control individuals. Interestingly, the EDA+Fn in the psoriatic epidermis had the IIICS region spliced out (EDA+, FDB−, IIICS−, III9+), which was shared with normal epidermis (EDA−, EDB−, IIICS−, III9+). These results suggest a selective predominance of the EDA+ Fn isoform at the dermal–epidermal junction of psoriatic skin. The consistent aberrant localization of EDA+ Fn at the dermal–epidermal junction in uninvolved skin of psoriatics may confer the hyperresponsiveness of psoriatic uninvolved basal keratinocytes for rapid cellular proliferation in response to T cell signals. The extracellular matrix protein, Fn, has critical functions in cell attachment, migration, differentiation, and proliferation. We have previously shown that fibronectin (Fn) is abnormally expressed and potentiates entry into the cell cycle of basal keratinocytes in uninvolved psoriatic skin, in combination with T cell lymphokines. It is not known what type of Fn is present in psoriatic skin, however, and how this Fn may regulate signaling. Embryonic forms of cellular Fn containing extra domains, designated EDA and EDB, are generated by alternative splicing and are seen in proliferating, developing tissue and in wound healing. Because the EDA segment enhances the integrin binding sequence Arg, Gly, Asp (RGD), which, when present, has been shown to be critical in integrin–extracellular matrix signaling, we were particularly interested in determining whether or not EDA-containing Fn (EDA+Fn) represented the aberrantly expressed Fn in psoriasis. Increased EDA+ Fn protein was demonstrated by immunostaining at the dermal–epidermal junction in clinically uninvolved skin from six of six patients with psoriasis, but not in skin from control subjects. Using reverse transcription polymerase chain reaction an increased ratio of EDA+ Fn versus EDA− Fn mRNA was present in epidermal samples from psoriatic but not control individuals. Interestingly, the EDA+Fn in the psoriatic epidermis had the IIICS region spliced out (EDA+, FDB−, IIICS−, III9+), which was shared with normal epidermis (EDA−, EDB−, IIICS−, III9+). These results suggest a selective predominance of the EDA+ Fn isoform at the dermal–epidermal junction of psoriatic skin. The consistent aberrant localization of EDA+ Fn at the dermal–epidermal junction in uninvolved skin of psoriatics may confer the hyperresponsiveness of psoriatic uninvolved basal keratinocytes for rapid cellular proliferation in response to T cell signals. fibronectin Psoriasis is a dynamic process of immune activation and keratinocyte hyperproliferation (Bata-Csorgo et al., 1995Bata-Csorgo Z. Hammerberg C. Voorhees J.J. Cooper K.D. Kinetics and regulation of human keratinocyte stem cell growth in short-term primary ex vivo culture. Cooperative growth factors from psoriatic lesional T lymphocytes stimulate proliferation among psoriatic uninvolved, but not normal, stem keratinocytes.J Clin Invest. 1995; 95: 317-327Crossref PubMed Scopus (181) Google Scholar). Inherent functional differences between quiescent (G0) clonogenic (stem cell) keratinocytes of psoriatic versus control individuals is suggested because psoriatic, but not control, ex vivo clonogenic epidermal cells can be induced to hyperproliferate upon addition of the T cell lymphokines interferon-γ, granulocyte-macrophage colony stimulating factor, and interleukin-3 (Bata-Csorgo et al., 1995Bata-Csorgo Z. Hammerberg C. Voorhees J.J. Cooper K.D. Kinetics and regulation of human keratinocyte stem cell growth in short-term primary ex vivo culture. Cooperative growth factors from psoriatic lesional T lymphocytes stimulate proliferation among psoriatic uninvolved, but not normal, stem keratinocytes.J Clin Invest. 1995; 95: 317-327Crossref PubMed Scopus (181) Google Scholar). Differential signaling to psoriatic keratinocytes mediated via integrin–extracellular matrix interactions may be critical to this process, because hyperproliferation is enhanced over 2-fold when uninvolved psoriatic cells, but not control cells, are plated on a Fn matrix prior to the addition of T cell lymphokines (Bata-Csorgo and Hammerberg, 1995Bata-Csorgo Z. Hammerberg C. Intralesional T-lymphocyte activation as a mediator of psoriatic hyperplasia.J Invest Dermatol. 1995; 105: 89S-94SCrossref PubMed Scopus (94) Google Scholar). In addition, we have previously reported that fibronectin (Fn) alone can increase transition from G0 to G1 of the cell cycle in ex vivo keratinocytes from uninvolved skin of psoriatics (Bata-Csorgo et al., 1998Bata-Csorgo Z. Cooper K.D. Ting K.M. Voorhees J.J. Hammerberg C. Fn and alpha5 integrin regulate keratinocyte cell cycling. A mechanism for increased Fn potentiation of T cell lymphokine- driven keratinocyte hyperproliferation in psoriasis.J Clin Invest. 1998; 101: 1509-1518Crossref PubMed Scopus (59) Google Scholar), further supporting a role of Fn and Fn receptor interaction in psoriasis. The major Fn receptor, integrin α5β1, is expressed at low levels on normal basal keratinocytes and diffusely distributed on the cell membrane. In keratinocytes from psoriatic involved and uninvolved patients, however, α5β1 integrins are clearly upregulated and cluster into discrete focal contacts in vitro (De Pellegrini et al., 1992De Pellegrini G. Luca M. Orecchia G. et al.Expression, topography, and function of integrin receptors are severely altered in keratinocytes from involved and uninvolved psoriatic skin.J Clin Invest. 1992; 89: 1783-1795Crossref PubMed Scopus (105) Google Scholar). In vivo, the α5 integrin receptor, but not α2 or α3, is overexpressed in nonlesional psoriatic epidermis (Bata-Csorgo et al., 1998Bata-Csorgo Z. Cooper K.D. Ting K.M. Voorhees J.J. Hammerberg C. Fn and alpha5 integrin regulate keratinocyte cell cycling. A mechanism for increased Fn potentiation of T cell lymphokine- driven keratinocyte hyperproliferation in psoriasis.J Clin Invest. 1998; 101: 1509-1518Crossref PubMed Scopus (59) Google Scholar). During wound healing and in psoriasis, integrins, which are usually located only on basal keratinocytes, display a suprabasal pattern of expression (Hertle et al., 1992Hertle M.D. Kubler M.D. Leigh I.M. Watt F.M. Aberrant integrin expression duirng epidermal wound healing and in psoriatic epidermis.J Clin Invest. 1992; 89: 1892-1901Crossref PubMed Scopus (207) Google Scholar). Transgenic mice overexpressing suprabasal integrins α2, α5, and β1 demonstrate a phenotype similar to psoriasis with cycles of flaking and inflamed skin, further suggesting that disrupted keratinocyte integrin–ligand interactions play a role in inflammatory hyperproliferative states (Carroll et al., 1995Carroll J.M. Rosario R.M. Watt F.M. Suprabasal integrin expression in the epidermis of transgenic mice results in developmental defects and a phenotype resembling psoriasis.Cell. 1995; 83: 957-968Abstract Full Text PDF PubMed Scopus (277) Google Scholar). The mechanism of signaling for increased proliferation by integrin–Fn interaction may occur through α5β1 interaction with the amino acid peptide sequence Arg-Gly-Asp-Ser (RGDS). This sequence stimulates p34/cyclin A activities within 2h of peptide addition and can stimulate growth of nontransformed epithelial cells (Worth et al., 1996Worth R.G. Mayo-Bond L. van de Winkel J.G.J. Todd III., Rf Petty H.R. CR3 (αMβ2; CD11b/CD18) restores IgG-dependent phagocytosis in transfecants expressing a phagocytosis-defective FcgammaRIIA (CD32) tail-minus mutant.J Immunol. 1996; 157: 5660-5665PubMed Google Scholar). Also, Fn signaling via α5β1 can influence clonogenic keratinocyte expansion and cell cycle induction through pathways that are distinct from adhesion, as the addition of an anti-α5 specific antibody 24h after basal keratinocytes are plated on Fn results in significant reduction in the outgrowth of the keratinocyte pool containing stem cells (β1 integrin bright PCNA+ cells) (Bata-Csorgo et al., 1998Bata-Csorgo Z. Cooper K.D. Ting K.M. Voorhees J.J. Hammerberg C. Fn and alpha5 integrin regulate keratinocyte cell cycling. A mechanism for increased Fn potentiation of T cell lymphokine- driven keratinocyte hyperproliferation in psoriasis.J Clin Invest. 1998; 101: 1509-1518Crossref PubMed Scopus (59) Google Scholar). The Fn homodimer has a complex tertiary structure, which only now is being fully appreciated. Previously believed to be a more rigid adhesive structure, Fn is currently thought to exist as a dynamic, flexible composite molecule of various modules that can interact with each other to presumably alter cell function (Main et al., 1992Main A.L. Harvey T.S. Baron M. Boyd J. Campbell I.A. The three-dimensional structure of the tenth type III module of Fn: an insight into RGD-mediated interactions.Cell. 1992; 71: 671-678Abstract Full Text PDF PubMed Scopus (405) Google Scholar;Morla et al., 1994Morla A. Zhang Z. Ruoslahti E. SuperFn is a functionally distinct form of Fn.Nature. 1994; 267: 193-196Crossref Scopus (262) Google Scholar;Ugarova et al., 1995Ugarova T.P. Zamarron C. Veklich Y. Bowditch R.D. Ginsberg M.H. Weisel J.W. Plow E.F. Conformational transitions in the cell binding domain of Fn.Biochem. 1995; 34: 4457-4466Crossref PubMed Scopus (129) Google Scholar;Hocking et al., 1996Hocking D.C. Smith R.K. McKeown-Longo P.J. A novel role for the integrin-binding III-10 module in Fn matrix assembly.J Cell Biol. 1996; 133: 431-444Crossref PubMed Scopus (112) Google Scholar). Fn can exist in 20 different isoforms in humans as a result of alternative splicing at three major splice acceptor sites (Kornblihtt et al., 1996Kornblihtt A.R. Pesce C.G. Alonso C.R. Cramer P. Srebrow A. Werbajh S. Muro A.F. The Fn gene as a model for splicing and transcription studies.FASEB J. 1996; 10: 248-257Crossref PubMed Scopus (168) Google Scholar). Two of the sites are termed ED modules (extra domain), called EDA and EDB. A third site is the IIICS or variable region. It includes an additional three segments subject to variable splicing which also contain the LDV adhesion motif for leukocyte α4β1. Plasma Fn produced by hepatocytes (the normal commercial source of Fn) completely lacks the EDA and EDB segments flanking the cell adhesion motif Arg-Gly-Asp-Ser (RGDS) (Xia and Culp, 1994Xia P. Culp L.A. Adhesion activity in Fn's alternatively spliced domain EDa (EIIA) and its neighboring type III repeats: oncogene-dependent regulation.Exp Cell Res. 1994; 213: 253-265Crossref PubMed Scopus (41) Google Scholar;Chen and Culp, 1996Chen W. Culp L.A. Adhesion mediated by Fn's alternatively spliced Edb (EIIIB) and its neighboring type III repeats.Exp Cell Res. 1996; 223: 9-19Crossref PubMed Scopus (24) Google Scholar;Gumbiner, 1996Gumbiner B.M. Cell adhesion: the molecular basis of tissue architecture and morphogenesis.Cell. 1996; 84: 345-357Abstract Full Text Full Text PDF PubMed Scopus (2819) Google Scholar). EDA expression has been proposed to alter the conformational state of the Fn molecule, affecting adjacent RGDS binding to other molecules including integrins. Due to the inclusion of EDA sequences in the Fn molecule, the peptides in the RGD sequence are placed in a conformation that is more accessible, thereby increasing cell sensitivity to the adhesion sequence motif (Mugnai et al., 1988Mugnai G. Lewandowska K. Carnemolla B. Zardi L. Culp L.A. Modulation of matrix adhesive responses of human neuroblastoma cells by neighboring sequences in the Fns.J Cell Biol. 1988; 106: 931-943Crossref PubMed Scopus (39) Google Scholar;Manabe et al., 1997Manabe R-I. Oh -e N. Maeda T. Fukada T. Sekiguchi K. Modulation of cell-adhesive activity of Fn by the alternatively spliced EDA segment.J Cell Biol. 1997; 139: 295-307Crossref PubMed Scopus (156) Google Scholar). In vivo, EDA+ Fn expression is abundant in developing tissues, especially at basement membranes, but in adults it is usually found only in endothelia, tumors, and granulation tissue of wounds (Kornblihtt et al., 1984Kornblihtt A.R. Vibe-Pedersen K. Baralle F.E. Human Fn: cell specific alternative mRNA splicing generates polypeptide chains differing in the number of internal repeats.Nucleic Acids Res. 1984; 12: 5853Crossref PubMed Scopus (159) Google Scholar;Vartio et al., 1987Vartio T. Laitenen L. Narvanen O. Cutolo M. Thornell L-E. Zardi L. Virtanen I. Differential expression of the ED sequence-containing form of cellular Fn in embryonic and adult human tissues.J Cell Sci. 1987; 88: 419-430PubMed Google Scholar;ffrench-Constant et al., 1989ffrench-Constant C. Van De Water L. Dvorak H.F. Hynes O. Reappearance of an embryonic pattern of Fn splicing during wound healing in the adult rat.J Cell Biol. 1989; 109: 903-914Crossref PubMed Scopus (379) Google Scholar;Brown et al., 1993Brown L.F. Dubin D. Lavigne L. Logan B. Dvorak H.F. Van De Water L. Macrophages and fibroblasts express embryonic Fns during cutaneous wound healing.Am J Pathol. 1993; 142: 793-801PubMed Google Scholar). Recently, we have described the upregulation and deposition of EDA+ Fn following ultraviolet B treatment of normal skin (Yoshida et al., 1999Yoshida Y. Kang K. Chen G. Gilliam A.C. Cooper K.D. Cellular Fn is induced in ultraviolet-exposed human skin and induces IL-10 production by monocytes/macrophages.J Invest Dermatol. 1999; 113: 49-55Crossref PubMed Scopus (12) Google Scholar). The finding that EDA+ Fn is consistently produced in association with remodeling or growth of tissue or organs suggests an important role for this specific splice variant of Fn to affect tissue growth by alteration of RGDS site conformation. Taken together, the above data prompted the hypothesis that the EDA splice variant containing Fn (EDA+ Fn) is involved in the hyperproliferative disease psoriasis. Our results show distinct deposition of EDA+ Fn at the dermal–epidermal junction in uninvolved skin from patients with psoriasis and increased EDA+ Fn mRNA expression in the epidermal fraction of skin from psoriasis patients. This may reflect an underlying abnormality in the extracellular matrix of uninvolved psoriatic skin that may predispose keratinocytes to proliferate after appropriate stimuli. Keratome and 4mm punch biopsies were obtained after local anesthesia with buffered 1% lidocaine with epinephrine (1:100,000) from buttock skin of patients with psoriasis and from the same areas of normal healthy volunteers, according to approved internal review board protocols for tissue procurement and informed consent. Topical and systemic therapies for psoriasis were discontinued 2wk and 4wk respectively before obtaining skin specimens. Tissue specimens included stable inflammatory plaques (involved psoriatic skin) and normal appearing skin within 20cm of an active plaque but not closer than 5cm (uninvolved psoriatic skin) (n=6). Total RNA was extracted from keratome biopsies obtained from uninvolved and involved buttock skin of psoriasis patients and normal volunteers as follows: the keratome was incubated for 20min in dispase (Collaborative Biomedical Products, Bedford, MA) at 37°C; epidermis was then separated from dermis, rinsed in phosphate-buffered saline and placed in triZol reagent (Gibco BRL, Gaithersburg, MD) for RNA extraction. Tissue was homogenized with a hand-held electronic tissue homogenizer and total RNA was obtained according to the manufacturer's protocol. cDNA was generated by reverse transcription using random hexamers and MMLV reverse transcriptase. Specific primers for Fn splice variants and their respective RT-PCR product sizes are as follows (see Figure 3): EDA sense, 5′-GGA GAG AGT CAG CCT CTG GTT CAG-3′ EDA antisense, 5′-TGT CCA CTG GGC GCT CAG GCT TGT G-3′ (374 bp EDA+, 104 bp EDA-); EBD sense, 5′-CGG CCT GGA GTA CAA TGT CAG TGT-3′ EDB antisense, 5′AGG TGA CAC GCA TGG TGT CTG GA-3′ (413 bp EDB+, 143 bp EDB-); III-9 sense, 5′-GCC TGG TAC AGA ATA TGT AGT G-3′ III-9 antisense, 5′-ATC CCA GCT GAT CAG TAG GCT GGT G-3′ (419 bp total Fn) (Tavian et al., 1994Tavian D. DePetro G. Colombi M. Portolani N. Giulini S.M. Gardella R. Barlati S. RT-PCR detection of Fn EDA+ and EDB+ mRNA isoforms: molecular markers for hepatocellular carcinoma.Int J Cancer. 1994; 56: 820-825Crossref PubMed Scopus (48) Google Scholar); IIICS sense, 5′-ACC GTG TGG GTA CAG GTG-3′ IIICS antisense, 5′-GTC ACA GAG GCT ACT AT-3′ (348 bp III-CS region) (Oyama et al., 1993Oyama F. Hirohashi S. Sakamoto M. Titani K. Sekiguchi K. Coordinate oncodevelopmental modulation of alternative splicing of Fn pre-messenger RNA at ED-A, ED-B, and CS1 regions in human liver tumors.Cancer Res. 1993; 53: 2005-2011PubMed Google Scholar). Amplification of β-actin with specific primer sets (sense, 5′-CAC CCT GAA GTA CCC CAT CGA-3′, antisense, 5′-CTC CTT AAT GTC ACG CAC GAT TTC-3′) served as a positive PCR control. PCR was carried out using 100 pmol of the specific primer sets, 2 units of Taq DNA polymerase and 5μl of the RT reaction product. The protocol for the amplification was as follows: denaturation at 94°C for 5min, annealing at 56°C for 30s, and extension at 72°C for 60s for one cycle followed by 28 cycles at 94°C for 30s, 56°C for 30s, and 72°C for 60s; followed by 94°C for 30s, annealing at 56°C for 30s, and terminal extension at 72°C for 5min. Four millimeter punch biopsies were immediately frozen in liquid nitrogen and stored at -80°C until cryosectioning. Immunostaining was performed on acetone-fixed frozen sections of these biopsies embedded in O.C.T. compound (Tissue-Tek, Sakura Finetek Torrance, CA). The following 1° antibodies were used: MoAb to the extra domain of cellular Fn (EDA sequence) (ICN Biomedicals, Costa Mesa, CA); MoAb to the IIICS-C or IIISC-B regions (Serotec, Raleigh, NC, clones 10D8 and 18D8 respectively); MoAb to PCNA (DAKO, Carpinteria, CA, clone PC10). Fluorescein isothiocyanate conjugated goat anti-mouse IgGl1 (Boehringer Mannheim, Indianapolis, IN) served as secondary antibody, and the appropriate isotype antibodies (Boehringer Mannheim) were used as controls. Detection was by diaminobenzidine reaction (ABC Vectastain, Vector Laboratories, Burlingame, CA) and counterstained with hematoxylin. When immunofluorescence was used to label protein, slides were counterstained with Evan's blue (Sigma, St Louis, MO). Double immunofluorescence was performed using primary antibodies with differing isotypic variants and isotype-specific tagged secondary antibodies. Images from immunostaining experiments were photographed on Kodak (Rochester, NJ) 160T film (bright field studies) or P1600 film (immunofluorescence studies) through a Zeiss (Thornwood, NY) Axiophot microscope at the indicated magnifications. The images were scanned (SprintScan, Polaroid, Cambridge, MA) and formatted in Adobe Photoshop and Illustrator. Band intensities of RT-PCR reactions were quantitated from scanned photographs of the original gels, digitized, and analyzed using the OPTIMAS 6.1 (Bothell, WA) image analysis software. In some cases, the band intensity was directly quantitated from PCR gels via the BioRad (Hercules, CA) Gel Doc system. The derived densities of bands were compared using Student's t test. Means are indicated with the standard deviation. To determine whether EDA+ Fn protein is abnormally expressed in psoriasis, we performed immunohistochemistry using a monoclonal antibody specific for EDA+ Fn. Staining of sections from skin of normal individuals did not show significant localization of EDA+ Fn to the dermal–epidermal junction area (Figure 1a), (n=6). The papillary dermis contained cells with modest pericellular staining, as did the horizontal vascular plexus, whereas the epidermis was negative for EDA+ Fn. In contrast, uninvolved psoriatic skin consistently (in six of six samples) demonstrated localization of EDA+ Fn to the dermal–epidermal junction in a linear pattern (Figure 1b) in addition to the normal pattern of papillary dermal pericellular and horizontal plexus staining (not shown). Interestingly, skin from patients with psoriasiform atopic dermatitis also demonstrated deposition of increasing EDA+ Fn near the capillary loops (Figure 1c). Involved psoriatic skin demonstrated a more irregular deposition of EDA+ Fn at the dermal–epidermal junction but showed prominent staining of the elongated vertically oriented capillary loops of the papillary dermis (Figure 2a). Staining with fluorescein or rhodamine-conjugated secondary antibodies further delineated EDA+ Fn to be finely distributed along the dermal–epidermal junction, including projections from dermal papillary structures traversing the dermal–epidermal junction and penetrating the basal epidermis into the basolateral keratinocyte surface in specimens taken from psoriatic plaques (Figure 2b), and to a lesser extent uninvolved tissue (Figure 2d).Figure 2Cellular Fn penetrates the basal epidermis and surrounds basal keratinocytes in psoriasis involved tissue. Psoriasis involved (A, B) and uninvolved (C, D) tissue was stained with anti-cellular EDA+ specific antibody (clone DH1). The primary antibody was detected using secondary specific antibodies conjugated to either rhodamine (A, C) or fluorescein isothiocyanate (B, D). Localized staining at the dermal–epidermal junction is visible in the sections presented. Positive staining of endothelial cells (vessel) is also evident (A), as shown by the striped arrows. Transient amplifying cells (PCNA positive staining, green) are shown in contact with the basal layer of the dermal–epidermal junction/EDA Fn positive staining (A), indicated with white arrows. Fn staining also appears to penetrate the basal epidermis from the dermal papillary area and surround basal keratinocytes, as shown with white arrows in (B). Sections (B) and (D) were counterstained with Evan's blue, as described for single staining under Materials and methods.View Large Image Figure ViewerDownload (PPT) We also performed double immunostaining experiments showing co-localization of EDA+ Fn and PCNA expressing keratinocytes, rare in normal skin but common in psoriatic skin due to the increased proportions of progenitor and transient amplifying cells in cycle. Basal PCNA+ cells are dim relative to superbasal cells that have accumulated protein over several rapid cell cycles (Figure 2a). PCNA+ basal cells were observed in areas which also expressed dense EDA+ Fn (Figure 2). PCNA staining was not evident in uninvolved tissue (Figure 2c). Monoclonal antibodies for the IIICS regions B and C did not show any morphologic variation in staining patterns between the three types of samples (control, uninvolved, and involved psoriatic skin), but did demonstrate more intense overall staining in psoriatic dermis around endothelia (data not shown). IIICS Fn was not expressed in the epidermis of any tissue samples as assessed by RT-PCR. To more precisely define the Fn splice forms in psoriasis, we conducted RT-PCR analysis for various Fn splice variant isoforms (Figure 3). EDA+Fn message was amplified from skin obtained from control and psoriatic patients (involved and uninvolved areas). To localize the particular message to either the dermal or epidermal fractions, RT-PCR was performed on skin that had been separated via enzyme digestion with dispase into either dermal or epidermal fractions prior to total RNA isolation. The separated skin revealed clear separation of dermal and epidermal tissue when examined microscopically with hematoxylin and eosin staining. EDA+Fn (374 bp fragment) was detected to a significant extent in epidermal fractions of psoriatic uninvolved skin in four of four subjects tested, and not in control epidermis (n=5). EDA+ Fn in psoriatic involved lesional skin was also observed (Figure 4). The EDA− splice variant of Fn was the dominant isoform in the superficial dermal RNA extracts from control, uninvolved, and lesional skin (Figure 4). Quantification of band density revealed that the mean ratio of EDA+ Fn to EDA− Fn in the psoriatic uninvolved epidermal fraction was 1.575±0.65, n=4. This compared with a mean ratio of 0.57±0.19 for control skin, n=4, p=0.05.Figure 4EDA+mRNA expression appears restricted to the epidermal fraction in both involved and uninvolved psoriatic skin. RT-PCR amplification of the splice variant EDA+ Fn from separated epidermal (E)/dermal(D) skin fractions from control (N), psoriasis uninvolved (PU) or involved (PI) skin. RNA that had been homogenized in Trizol was reverse transcribed and the cDNA was then amplified with primer sets specific for the EDA+ Fn splice in the region (as detailed in Materials and methods). Prominent EDA+ Fn message is demonstrated in the epidermal fraction of both psoriasis involved and uninvolved skin.View Large Image Figure ViewerDownload (PPT) The same order of samples was used to detect splice variants containing EDB (Figure 5a), III-9 (Figure 5b), IIICS (Figure 5c), and β-actin control (Figure 5d). Figure 5a shows no amplification of EDB+ Fn in any keratome samples tested. III-9 is a marker for total Fn as it flanks the RGDS containing III-10 module and is included in all Fn. It is interesting to note that there is amplification of total Fn mainly in the dermis, as expected, but a consistent signal in the epidermis, including control epidermis (Figure 5b), confirming the production of EDA-Fn in normal epidermis. IIICS+ Fn is not expressed in the epidermis, but is clearly spliced-in in dermal Fn, in control and psoriatic skin (Figure 5c). Taken together, control epidermis contains EDA−, EDB−, IIICS−, III-9+ Fn, whereas psoriatic epidermis contains EDA+, EDB−, IIICS−, III-9+ Fn, producing cells. By contrast, the superficial dermis of both normal and psoriatic skin contains cells making EDA−, EDB−, IIICS+, III-9+ Fn. Psoriasis is a polygenic disease comprising inherent defects at both the genetic and immunological level with a sporadic mode of inheritance that is not completely understood. The role of extracellular matrix components in psoriasis may be critical, as integrin expression patterns on keratinocytes isolated from psoriasis patients are clearly altered compared with those of normal individuals (Hertle et al., 1992Hertle M.D. Kubler M.D. Leigh I.M. Watt F.M. Aberrant integrin expression duirng epidermal wound healing and in psoriatic epidermis.J Clin Invest. 1992; 89: 1892-1901Crossref PubMed Scopus (207) Google Scholar;De Pellegrini et al., 1992De Pellegrini G. Luca M. Orecchia G. et al.Expression, topography, and function of integrin receptors are severely altered in keratinocytes from involved and uninvolved psoriatic skin.J Clin Invest. 1992; 89: 1783-1795Crossref PubMed Scopus (105) Google Scholar;Petzelbauer and Wolff, 1992Petzelbauer P. Wolff K. 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Cooperative growth factors from psoriatic lesional T lymphocytes stimulate proliferation among psoriatic uninvolved, but not normal, stem keratinocytes.J Clin Invest. 1995; 95: 317-327Crossref PubMed Scopus (181) Google Scholar;Gottlieb et al., 1995Gottlieb S.L. Gilleaudeau P. Johnson R. Estes L. Woodworth T.G. Gottlieb A.B. Krueger J.G. Response of psoriasis to a lymphocyte-selective toxin (DAB389IL-2) suggests a primary immune, but not keratinocyte, pathogenic basis.Nature Med. 1995; 1: 442-447Crossref PubMed Scopus (566) Google Scholar;Bata-Csorgo et al., 1998Bata-Csorgo Z. Cooper K.D. Ting K.M. Voorhees J.J. Hammerberg C. Fn and alpha5 integrin regulate keratinocyte cell cycling. A mechanism for increased Fn potentiation of T cell lymphokine- driven keratinocyte hyperproliferation in psoriasis.J Clin Invest. 1998; 101: 1509-1518Crossref PubMed Scopus (59) Google Scholar). Alterations in integrin e
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