Eur J Oral Sci 2010; 118: 159167 Printed in Singapore.

All rights reserved

2010 The Authors. Journal compilation 2010 Eur J Oral Sci

European Journal of Oral Sciences

Expression of the T-cell-specic adapter protein in oral epithelium

Kolltveit KM, Schreurs O, strem J, Sland TM, Khuu C, Berge T, Messelt E, Hayashi K, Granum S, Spurkland A, Schenck K. Expression of the T-cell-specic adapter protein in oral epithelium. Eur J Oral Sci 2010; 118: 159167. 2010 The Authors. Journal compilation 2010 Eur J Oral Sci The multifunctional T-cell-specic adapter protein (TSAd) was originally described in T cells but is also expressed in epithelial cells from the respiratory tract and in endothelium. In this study, we found expression of TSAd messenger RNA (mRNA) and protein in both human and murine oral mucosal epithelium as well as in human primary oral keratinocyte cell cultures. In TSAd)/) mice, the mucosa and skin appeared macroscopically normal, but severe disturbances were observed in the ne structures of the basal membrane and intercellular epithelial spaces upon analysis using transmission electron microscopy. Oral epithelial cells from TSAd)/) mice displayed decreased migration compared with cells from wild-type mice, whereas overexpression of TSAd in a human epithelial cell line resulted in impaired proliferation. This study is the rst to show that TSAd is expressed in normal oral mucosa, that it is important for the normal ultrastructural morphology of the epithelium and the basal membrane, and that it is involved in the migration and proliferation of oral keratinocytes.

Kristin M. Kolltveit1,2, Olav Schreurs1, Janet strem2, Tine M. Sland1, Cuong Khuu1, Tone Berge3, Edward Messelt1, Katsuhiko Hayashi4, Stine Granum3, Anne Spurkland3, Karl Schenck1
1

Department of Oral Biology; and2Department of Periodontology, Faculty of Dentistry, University of Oslo, Norway; 3Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, Norway; 4Department of Dentistry, Jikei University School of Medicine, Tokyo, Japan

Kristin M. Kolltveit, Department of Oral Biology, University of Oslo, PO Box 1052 Blindern, N 0316 Oslo, Norway Telefax: +4722840302 E-mail: k.m.kolltveit@odont.uio.no Key words: basal membrane; epithelial cells; migration; proliferation; T-cell-specific adapter protein (TSAd) Accepted for publication January 2010

The mucosal surfaces of the oral cavity and the skin are covered by stratied squamous epithelia of ectodermal origin that form a mechanical and protective barrier for the underlying tissues. When the epithelium is wounded, rapid repair is essential to prevent infection and sustained inammation. In the initial phases of wound healing, neighboring epithelial cells migrate rapidly to, and cover, the wound bed (restitution). Later, proliferation and dierentiation of the keratinocytes dominate (1). Epithelial cell migration and proliferation are regulated by a number of growth factors and cytokines. Epidermal growth factor (EGF), broblast growth factor 2 (FGF-2), platelet-derived growth factor (PDGF), and nerve growth factor-beta (NGF-b) stimulate keratinocyte proliferation, whereas transforming growth factor-beta (TGF-b) is implicated in the suppression of epithelial growth (24). Growth factors also provide signals that stimulate migration by inducing changes in the organization of the actin cytoskeleton. In the oral cavity, several growth factors are secreted in saliva where they act as surveillance peptides, stimulating rapid repair in the event of a breach in the mucosal surface (57). We have previously reported the cloning of the T-cellspecic adapter protein (TSAd) (8), which is also known as p56lck-interacting adapter protein (LAD) or Rlk/Itkbinding protein (RIBP) because it is a binding partner for the Src kinase Lck and for the Tec kinases resting leukocyte kinase (Rlk) and interleukin-2-inducible T-cell kinase (Itk) (9, 10). The TSAd is an intracellular adapter

protein that contains an SH2 domain, a proline-rich SH3-binding motif, 10 potential phosphotyrosine sites, but no catalytic domain (8). The main function of adapter proteins is to serve as intermolecular bridges by bringing signaling proteins together through protein protein interactions (11). Originally, the expression of the TSAd was considered to be restricted to T cells (810), but later studies have shown that the TSAd is also expressed in endothelium and murine lung epithelium (1214). The TSAd protein is encoded by the SH2D2A gene, and a polymorphism in its promoter is associated with genetic susceptibility to the human autoimmune diseases multiple sclerosis and juvenile rheumatoid arthritis (15, 16). T-cell-specic adapter protein-decient mice develop a systemic lupuslike autoimmune disease (17). Recent studies have shown that TSAd deciency is linked to impaired migration of human T cells and murine endothelial cells (13, 18, 19). In the course of studies of ve naturally occurring splice variants of the SH2D2A gene (20,21), we discovered that TSAd was expressed in an epithelial cell line derived from oral mucosa. As TSAd expression has not previously been reported in stratied epithelium, we therefore examined the expression and function of TSAd in oral epithelium. We found that TSAd is important for the normal ultrastructural morphology of the epithelium and the basal membrane (BM). Loss of TSAd expression was associated with decreased migratory capacity and increased proliferation of oral keratinocytes.

160

Kolltveit et al. Real-time polymerase chain reaction (PCR) and subsequent data analysis were performed using the Mx4000 Quantitative PCR (qPCR) system (Stratagene) equipped with version 4.0B software. The primers and probes for both TSAd and TATA box-binding protein (TBP) were an assayon-demand (AOD) mix from Applied Biosystems (Applera, Oslo, Norway). Each PCR reaction contained 1 AOD mix, 1 qPCR MasterMix Plus (Eurogentec), and 3 ll of complementary DNA (cDNA) as template. Custom-made primers and probes for the detection of TSAd splice variants have been described previously (20). None of the amplicons exceeded 150 bp. All probes had a dark quencher. Each PCR reaction contained 600 nM primers and 200 nM probe, as well as 1 TaqMan Universal PCR Mastermix (Applied Biosystems). DNA amplication was performed with the following thermal cycling prole: initial denaturation at 95 C for 10 min, followed by 40 cycles of amplication (denaturation at 95 C for 15 s, and annealing and elongation for 1 min at 60 C). Fluorescence data were collected during the annealing stage of amplication. The quantity of TSAd was estimated relative to the quantity of TBP using the MX4000 comparative quantication software. Experiments were performed in duplicate or triplicate for each data point. Immunohistochemistry The following primary antibodies were used for immunohistochemical staining: mouse anti-keratin 14 (NeoMarkers, Thermo Fisher Scientic, Fremont, CA, USA), rabbit anti-collagen IV (Chemicon, Temecula, CA, USA), rat antinidogen (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-b-actin (Abcam, Cambridge, UK), goat anti-integrin a6 (Santa Cruz), and mouse anti-proliferating cell nuclear antigen (PCNA) (Novocastra Laboratories, Newcastle upon Tyne, UK). The anti-human and anti-mouse TSAd serums used for western blotting have been described previously (13, 21). The anti-human TSAd serum used for immunohistochemistry was raised against amino acids 375389 of the C-terminal end of TSAd, with keyhole limpet hemocyanin (KLH) as the hapten carrier and incomplete Freunds adjuvant (IFA) in the immunization procedure. Purication was accomplished using peptidespecic anity chromatography (Eurogentec). Biopsies were xed in 4% buered formaldehyde solution for 24 h, processed through a graded alcohol series oriented, and embedded in paran. Serial, 4-lm-thick sections were cut, backed at 60 C for 2 h, and stored at 4 C until used. Upon use, sections were dewaxed in xylene and rehydrated through a graded alcohol series to distilled water. Phosphate-buered saline (PBS) was used as diluent and for all washing steps between incubations. Antibodies and the Avidin-Biotin Complex (ABC)-reagent were diluted in 1% IgG-free bovine serum albumin (BSA; Jackson Immuno Research Laboratories, West Grove, PA, USA) in PBS (PBS/1% BSA). For the TSAd immunouorescent staining, proteaseinduced epitope retrieval (PIER) was performed by incubation with 20 lg ml)1 of proteinase K at 37 C for 15 min. Potential endogenous binding sites were blocked with 10 lg ml)1 of avidin (Sigma-Aldrich) followed by incubation with 2 mg ml)1 of biotin (Sigma-Aldrich) containing 5% normal goat serum for 20 min at room temperature (22C). Then, the slides were sequentially incubated with 1 lg ml)1 of rabbit anti-TSAd overnight at 4 C, with 7.5 lg ml)1 of biotinylated goat anti-rabbit (BA-1000;

Material and Methods

Mice SH2D2A)/) mice were generated by homologus recombination and backcrossed into C57BL/6 mice background (10). The mice were generously provided by Professor J. A. Bluestone, University of California. SH2D2A+/) C57BL/6 mice were intercrossed (Animal Facilities, University of Oslo) to generate littermate C57BL/6 SH2D2A+/+ mice and SH2D2A)/) mice. The animal studies were approved by the Experimental Animal Board under the ministry of Agriculture of Norway and conducted in conformity with The Norwegian Regulations on animal Experimentation and the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientic Purposes. Mice were killed and biopsies were taken from the buccal mucosa. Biopsies of human normal oral mucosa were collected either during the extraction of third molars or as punch biopsies of the buccal mucosa of healthy volunteers. Permission was given by the regional Ethic Committee to conduct the investigations, and informed consent was obtained from all patients and volunteers. Keratinocyte cultures Epithelial sheets were dissected from punch biopsies of the buccal mucosa. These were immersed either in Cellytic M (Sigma Aldrich, St Louis, MO, USA) for western blotting or in lysis buer of the Absolutely RNA kit (Stratagene, La Jolla, CA, USA). Biopsies isolated from the buccal mucosa that were used for cell culture were immersed in Dulbeccos modied Eagles medium (DMEM) containing 2.7 mg ml)1 of dispase and stored at 4 C overnight. Epithelial sheaths were carefully removed from the connective tissue and cut into small pieces using sterile scalpel blades. The establishment of human keratinocyte cultures has been described previously (7). First-passage or tenth-passage keratinocytes were used for the studies, as indicated in the gure legend (Fig. 1c). Transfections The lung carcinoma cell line A549 [American Type Culture Collection (ATCC), Manassas, VA, USA] was grown in DMEM (Cambrex, Charles City, IA, USA) containing 10% fetal bovine serum (FBS; Cambrex) and antibiotics (Cambrex). Twenty-four hours before transfection at 80% conuency, the cells were trypsinized, diluted with fresh medium (50,000 cells ml)1), and transferred to 24-well plates. Transfections were performed using Lipofectamine 2000 (Invitrogen, Paisley, UK) according to the suppliers instructions. The transfection eciency was about 65%, as assessed by ow cytometry (FACSCalibur; BD, Franklin Lakes, NJ, USA) for the expression of green uorescent protein (GFP). RNA extraction, reverse transcription, and real-time polymerase chain reaction Total RNA was extracted from freshly excised biopsies from the oral mucosa using the Absolutely RNA kit (Stratagene) according to the manufacturers instructions. RNA (250 ng) was transcribed using the Reverse Transcription (RT) Core Kit (Eurogentec, Seraing, Belgium). Random nonamers were used as RT primers.

TSAd in oral epithelium Vector Laboratories, Burlingame, CA, USA) for 30 min, and with Cyanine-3 (Cy3)-conjugated streptavidin (PA43001; GE Healthcare, Uppsala, Sweden; 1 lg ml)1 in 10% BSA) for 30 min. Nuclei were stained with 100 ng ml)1 of 4,6-diamidino-2-phenylindole (DAPI) for 10 min and then the slides were coverslipped with Immu-Mount (Shandon, Thermo Fisher Scientic, Waltham, MA, USA). For immunohistochemical staining, endogenous peroxidase was quenched with 0.3% H2O2 in methanol for 30 min. Antigen unmasking of keratin 14, collagen IV, nidogen, integrin a6, and PCNA was performed by heat-induced epitope retrieval (HIER). Sections were immersed in 10 mM citrate buer (pH 6) and heated for 15 min at 100 C in a pressure cooker (Decloaking chamber; Biocare Medical, Concord, CA, USA). After pressure and temperature reduction, the specimens were left to cool for 20 min, washed with distilled water, and equilibrated in PBS. To avoid problems with endogenous mouse immunoglobulins when localizing mouse antibodies to keratin 14 and PCNA on mouse tissues, a mouse-on-mouse kit (BMK-2202; Vector) was used. Blocking steps and incubation with primary and secondary antibodies were performed according to the manufacturers procedure. For staining of non-mouse antibodies, sections were blocked for 20 min with the appropriate normal serum diluted to 5% in PBSA before overnight incubation with the primary antibodies at 4 C. Sections were incubated for 30 min with the following biotinylated secondary antibodies: goat anti-rabbit (BA-1000; Vector), rabbit anti-rat (BA-4001; Vector), or rabbit antigoat (E0466; Dako, Glostrup, Denmark), diluted 1:200 in PBS/1% BSA. The slides were incubated for 30 min with ABCHRP-reagent (Vector) and the signal was visualized using 3,3-diaminobenzidine tetrahydrochloride (SigmaAldrich). Nuclei were counterstained with hematoxylin (Shandon) before the sections were dehydrated and coverslipped using a xylene-based mounting medium (HistoKit, Assistant; Karl Hecht, Sondheim, Germany). Sodium dodecyl sulfatepolyacrylamide gel electrophoresis and western blotting Cells were lysed in Cellytic M (Sigma-Aldrich) containing 1% Halt protease inhibitor cocktail (Thermo Scientic, Rockford, IL, USA) and 0.4% Nonidet P-40 (NP-40). The lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto nitrocellulose membranes (BioRad, Hercules, CA, USA). The nitrocellulose membranes were blocked with Tris-buered saline (TBS, pH 7.4) containing 0.1% Tween 20 (Sigma Aldrich) and 5% skimmed milk, and subsequently incubated with the indicated antibodies in blocking buer containing 1% skimmed milk. Signals were detected using ECF substrate (GE Healthcare, Pittsburgh, PA, USA) and Storm 860 (GE). As a positive control for hTSAd detection in western blotting, lysates from Jurkat Tag cells stably transfected with human (h)TSAd cDNA were included as a control (21). As a positive control for murine (m) TSAd, Jurkat Tag transiently transfected with mTSAd cDNA was used as a control. Fifty micrograms of protein lysate was applied to each well. In situ hybridization Chromogen in situ hybridization (CISH) was performed on formaldehyde xed, paran embedded (FFPE) human and murine tissues using RiboMap and BlueMap kits (Ventana

161

Medical Systems, Illkirch, France) in the Ventana Discovery environment. Oligonucleotide sense and antisense probes were designed and 3 GreenStar hyperlabeled (GeneDetect, Auckland, New Zealand). The biotinylated probe for human TSAd hybridized to nucleotides 834881 was located within the coding sequence of NM-003975. The digoxigenin-labeled probe for murine TSAd hybridized to nucleotides 13271374 located within the coding sequence of NM-021309. Details of the CISH procedure are described in Data S1, Supporting information. Reagents for detection were from Ventana if not mentioned otherwise. Transmission electron microscopy The biopsies were cut into small blocks, xed in 2% glutaraldehyde in 0.2 M cacodylate buer, pH 7.4, postxed in 1% osmium tetroxide, and dehydrated through a graded series of ethanol up to 100%. Tissue blocks were immersed in propylene oxide, twice, each time for 20 min, and were then embedded in Epon (Agar Scientic, Stanstead, Essex, UK). Ultrathin sections were cut on a Leica Ultracut ultramicrotome UCT (Leica, Wetzlar, Germany). The sections were treated with uranyl acetate for 15 min, with lead citrate for 3 min, and were then examined using a Philips CM 120 transmission electron microscope (Philips, Eindhoven, the Netherlands). In vitro migration assay Monolayers of conuent mouse keratinocytes in 6-cm petri dishes (Nalgene, Thermo Fisher Scientic, Fremont, CA, USA) were scratched with a plastic pipette tip to create a cell-free area. The cultures were gently washed three times with keratinocyte serum-free medium (KSFM) (Gibco, Gaithersburg, MD, USA). Three elds of each scratch area were photographed and the surface of the scratched, cellfree area was measured using the public domain program ImageJ 1.34s (National Institutes of Health, Bethesda, MD, USA at http://rsb.info.nih.gov/ij/). The average number of pixels of the scratch closure area in the three elds was calculated. The scratch closure area index was dened as [(scratch area at 0 h scratch area after specic incubation time)/1,000]. Statistical analysis Statistical analysis was performed using the InStat3 software package (InStat3; GraphPad Software, San Diego, CA, USA). The unpaired two-tailed Students t-test was used for comparison of the scratch closure index (n = 3), keratin 14-positive cells (n = 10), and PCNA-positive cells (n = 7), as the values were normally distributed. The unpaired twotailed Students t-test was also used for the comparison of epithelial thickness (n = 14). Average thickness of epithelium was measured as [area (px2)/(length of BM (px)/2,084 (px/lm)] (BM is the basal membrane and px is pixels).

Results
TSAd is expressed in normal human and murine oral epithelium

Messenger RNA (mRNA) for TSAd was detected, using real-time PCR, in all samples examined from human and

162

Kolltveit et al.
A

murine oral mucosal epithelial sheets. The levels were low in epithelial biopsies compared with those in unstimulated CD4+ T cells: hTSAd mRNA in epithelial sheets was about 2% of the level in hCD4+ T cells, whereas mTSAd was about 5% of the level in mCD4+ T cells. In situ hybridization of human and murine oral mucosal biopsy samples revealed that TSAd mRNA was mainly expressed by cells in the basal epithelial cell layers, with some TSAd mRNA-containing single cells occasionally observed in the stratum spinosum (Fig. 1A,B). This staining pattern indicates that approximately 10% of the epithelial biopsy contains TSAd mRNA-positive cells; thus, the low amount of TSAd mRNA observed by real-time PCR is a reection of the low number of positive cells. Five transcript variants of human TSAd, with variable transcript lengths, are known (TSAd 15) (20, 22). In cultured keratinocytes from two donors we found that the dominating transcript was the TSAd-1 variant encoding full-length TSAd. The TSAd-5 variant, encoding a TSAd molecule lacking the major part of the proline-rich region (20), constituted 3% of the total TSAd mRNA. The transcripts representing N-terminal deletions of TSAd (i.e. the TSAd-2 and TSAd-3 variants) constituted 03% of the total amount of TSAd transcripts. T-cell-specic adapter protein expression was examined by western blotting of lysates from epithelial biopsies, both human and murine (Fig. 1C,D). In addition, TSAd expression was examined in keratinocytes grown in culture for 1 and 10 passages. Keratinocytes grown in culture mainly represent the cells of the basal cell layer. In the human samples, a double band of approximately 52 kDa, corresponding to full-length TSAd, was observed, which is similar to what has been observed in Jurkat T cells (21). Interestingly, the strongest TSAd band in the human epithelial specimen was observed at approximately 75 kDa, probably representing a hitherto-unknown modication of the TSAd protein (Fig. 1C). This band was also observed using another TSAd antibody towards a dierent epitope (data not shown). A band of 37 kDa was observed in the biopsy specimen, but not in the cultured keratinocytes. Additionally, a band of 46 kDa was observed in all specimens examined. These bands may correspond to dierent splice variants of TSAd. In the epithelial biopsies isolated from mice, TSAd was observed in the wildtype mice but not in the SH2D2A)/) mice (Fig. 1D). Immunohistochemical staining for TSAd in human mucosa showed intracellular staining of TSAd in the stratum spinosum (Fig. 1E). Staining was also observed in the basal cell layer, but this was unevenly distributed. In some areas an orientation of TSAd expression towards the BM was seen, but this was less marked in other areas. No staining of the supercial epithelial layers was observed (Fig. 1E). The protein-expression pattern thus diered from the mRNA-expression pattern.
SH2D2A)/) mice show abnormal oral epithelium

Fig. 1. Analysis of T-cell-specic adapter protein (TSAd) expression in oral squamous epithelium of humans and mice. In situ hybridization of a human biopsy sample (A) and a murine biopsy sample (B). The blue color indicates positive staining for TSAd messenger RNA (mRNA). Sense controls are shown for comparison. (C) Western blot of human epithelium dissected from a biopsy sample (EB) and keratinocytes isolated from epithelium and grown in culture for 1 (K1) and 10 (K10) passages. Ctr: Jurkat cells stably transfected with hTSAd. (D) Western blot of oral epithelial sheets from SH2D2A+/+ and SH2D2A)/) mice. Ctr: Jurkat cells transiently transfected with mTSAd. (E) Immunohistochemical staining of TSAd in human mucosa. The red color indicates positive staining for TSAd.

In order to examine the role of TSAd in the oral mucosa, we examined mice lacking TSAd. Macroscopically, the

oral mucosa of SH2D2A)/) mice appeared to be normal. There was a tendency towards a thinner epithelium in SH2D2A)/) mice as compared with wild-type mice, but this dierence did not reach statistical signicance (P = 0.077) (Fig. 2). Further microscopic examination of mucosal biopsies revealed several disturbances in the epithelial structure (Fig. 3). In contrast to humans, mice have a keratinized

TSAd in oral epithelium

163

To evaluate, in greater detail, the irregularities in the BM observed in SH2D2A)/) mice, biopsies were examined using transmission electron microscopy (TEM) (Fig. 4). Examination of the oral epithelium of wild-type mice identied a normal, smooth, and continuous BM
Fig. 2. Analysis of epithelial thickness. Hematoxylin staining showing lines for calculation of the area and the length of the basal membrane. A typical example of a SH2D2A)/) mouse.

SH2D2A+/+

SH2D2A/

oral mucosa. SH2D2A)/) mice displayed focal parakeratinization in an otherwise orthokeratinized epithelium, compared with the wild-type mice where the oral epithelium was orthokeratinized (Fig. 3A,B,E,F). In the basal cell layer, SH2D2A)/) mice displayed an irregular cell morphology without the normal cubical shape seen in wild-type mice. At some sites epithelial cells protruded into the underlying connective tissue in the TSAd)/) mice (Fig. 3C,D). Focal areas of vacuolization were observed within the epithelium of the SH2D2A)/) mice (Fig. 3E,F). In addition, a tendency of basal cell hyperplasia (increased number of basal cells) was noted (Fig. 3B,D). However, the counts of basal cells, as marked by staining for keratin 14, indicated no dierence in the numbers of basal cells between wild-type and SH2D2A)/) mice (P = 0.9) (Fig. 3G,H). Because some basal epithelial cells in the TSAd)/) oral mucosa seemed to protrude into the underlying connective tissue, we examined the BM for collagen IV and nidogen, both essential components of the mature basal lamina of oral mucosa. Staining for collagen IV showed a smooth, continuous line along the basal lamina in the wild-type mice, whereas in the SH2D2A)/) mice staining was discontinuous (Fig. 3I,J). Nidogen staining revealed an interrupted line of irregular thickness in the SH2D2A)/) mice as compared with the smooth outline in the wild-type mice (Fig. 3K,L). The basal epithelial cells are connected to the BM by hemidesmosomes, of which the integrin a6b4 receptor is the main component. The integrin a6b4 receptor binds to collagen IV and nidogen. Staining for the integrin a6 chain did not reveal any dierence in the expression between wild-type and SH2D2A)/) mice (Fig. 3M,N).

40

40

60

60

60

60

40

40

Fig. 3. Analysis of epithelial morphology in T-cell-specic adapter protein wild type mice and knock out mice by immunohistochemistry. Results from the SH2D2A+/+ mice are shown in panels A, C, E, G, I, K, M, O, and Q; and results from the SH2D2A)/) mice are shown in panels B, D, F, H, J, L, N, P, and R. (A and B) Hematoxylin staining; the square indicates the area magnied and shown in panels C and D. (E and F) Hematoxylin and eosin staining, the large arrows indicate areas with vacuolization; the small arrow indicates the area with focal parakeratinization. (G and H) Keratin-14 staining. (I and J) Collagen IV staining; the square indicates the area magnied and shown in panels K and L. (M and N) Nidogen staining; the square indicates the area magnied and shown in panels O and P. (Q and R) Integrin a6. In panels G to R the brown color indicates positive staining. One representative sample of each staining procedure is shown out of three SH2D2A+/+ mice and three SH2D2A)/) mice.

40

40

60

164
A

Kolltveit et al.
B

from SH2D2A)/) mice, and none were seen in the areas with the membrane defects described above (Fig. 4B). The above-described changes in the BM area of the SH2D2A)/) mice were not uniformly present. There were also normal areas where the BM area had the same appearance as that in the wild-type mice.
TSAd is involved in proliferation and migration of oral keratinocytes

13 500

13 500

24 500

24 500

Fig. 4. Ultrastructural analysis of the basal membrane using transmission electron microscopy (TEM). (A and B) Sections of the oral mucosa of T-cell-specic adapter protein (SH2D2A)+/+ and SH2D2A)/) mice (magnication 13,500). Squares indicate the areas shown in panels C and D (magnication 24,500). (C) Arrows indicate hemidesmosomes. (D) The arrow indicates normal hemidesmosomes, the arrowhead indicates thickening of basal membrane, double arrows indicate micro blisters, and the thin arrow indicates loss of continuity of basal membrane.

Altered attachment of cells to the BM, as observed in the oral epithelium from SH2D2A)/) mice, is often associated with alteration of proliferative activity. We therefore examined biopsies from the oral mucosa of the SH2D2A)/) mice and the wild-type mice for the expression of PCNA (Fig. 5AC). More epithelial basal cells from the oral mucosa of SH2D2A)/) mice stained with anti-PCNA serum compared with epithelium from normal oral murine mucosa (P = 0.01) (Fig. 5C), indicating that in the absence of TSAd, keratinocytes are hyperproliferative. As TSAd deciency was associated with increased proliferation of keratinocytes, the eect on cellular proliferation of keratinocytes overexpressing TSAd was examined. For this purpose, the human epithelial cell line A549 was transfected with cDNA encoding GFP-tagged full-length TSAd, and the average transfection eciency was 60% (data not shown). Cells expressing GFP alone were used as negative controls. The transfected cells were observed and counted every day for 5 d. On day 1 the total cell numbers were equal, but from day 2 onwards the proliferation of TSAd-transfected cells was lower compared with that of the control cells (Fig. 5F). T-cell-specic adapter protein has been found to regulate chemokine-induced T-cell migration (18) as well as growth-dependent endothelial cell migration (13). The migratory capacity of keratinocytes isolated from the buccal mucosa of TSAd)/) mice and wild-type mice was therefore compared in an in vitro scratch assay. TSAd)/) keratinocytes showed a signicantly lower scratch closure index than cells isolated from wild-type mice (P = 0.05) (Fig. S1). This eect was not caused by decreased proliferation, as the proliferation in SH2D2A)/) keratinocytes was increased (Fig. 5C).

with hemidesmosomes distributed evenly along the membrane, as well as a regular pattern of intercellular desmosomes (Fig. 4A,C). Lamina densa and lamina lucida were well dened (Fig. 4A,C). By contrast, in the oral epithelium from SH2D2A)/) mice, the epithelial cells were irregularly shaped in the basal area, with parts of the cells protruding into the underlying connective tissue (Fig. 4B,D), consistent with what was observed by light microscopy (Fig. 3B). Furthermore, the BM in the SH2D2A)/) mice was irregularly shaped, with partial thickening of the lamina densa in some areas (Fig. 4B,D). At other sites, the lamina densa was interrupted (Fig. 4B,D). Separation of the basal cell membrane from the basal lamina in the lamina lucida was seen in the form of micro-blisters (Fig. 4D). Hemidesmosomes, which connect the basal epithelial cells to the BM, were unevenly distributed in the oral epithelium

Discussion
T-cell-specic adapter protein is a multifunctional adapter protein originally described in T cells. This study is the rst observation that TSAd also is expressed in the oral multilayered squamous epithelium, both in humans and in mice. We found that TSAd was required for maintaining the integrity of the BM. In normal stratied epithelia, keratinocytes are attached to the underlying BM by hemidesmosomes, multiprotein adhesion complexes that provide stable adhesion of the epithelia to the underlying extracellular matrix. Stable attachment of basal keratinocytes to the BM is fundamental for maintaining epithelial integrity because diseases in which hemidesmosome components

TSAd in oral epithelium

A
SH2D2A+/+

165

SH2D2A/

GFP-TSAd

GFP

Fig. 5. Analysis of cell growth rates in T-cell-specic adapter protein (SH2D2A))/) mice. (A and B) Representative immunohistochemical staining for proliferating cell nuclear antigen (PCNA); brown indicates positive staining. (C) Number of PCNA-positive cells per millimetre of basal membrane. (D and E) Morphology of A549green uorescent protein (GFP)TSAd and A549GFP cultures 3 d post transfection in bright eld microscope. (F) Cell numbers following transfection with TSAdGFP and GFP, data from three independent transfection experiments.

are aected lead to a variety of skin blistering disorders (23). Modulation of hemidesmosomal function is crucial for the dierentiation and migration of keratinocytes during wound healing (23, 24). In stratied epithelia, the components of hemidesmosomes are the integrin a6b4 heterodimer, CD151, BP180, BP230, and plectin (23). Integrin a6b4 is the principal adhesion receptor and binds to laminin-5, a component of the BM. Intracellularly, integrin a6b4 is attached to keratin laments and, in migrating cells, indirectly to F-actin in lopodia and lamellipodia (25). Laminin-5 assembles with other proteins, including collagen IV, nidogen, perlecan, and agrin, thereby forming the central BM scaolding (25). In order to bind to laminin, integrin a6b4 in epithelial cells must form a complex with laminin-binding protein (LBP) (26). In the TSAd-decient mice, we found that the orientation of the cells in the basal cell layer of the oral mucosa was disturbed and that there were regions showing a disorganized BM. In those regions, no hemidesmosomes were seen. No dierences in the expression of integrin a6 could be seen between wild-type mice and SH2D2A)/) mice and therefore failure in the binding between keratinocytes and the BM is probably not caused by loss of expression of the hemidesmosomal receptor. The disturbances observed might rather be related to a newly described role of TSAd. In T cells it has recently been shown that binding of TSAd to LBP is required for the binding of LBP to integrin a6b1 and subsequent binding to laminin-5 (19). If TSAd in epithelial cells plays a similar role in the formation of the integrin a6b4LBP complex and in subsequent binding to laminin-5, TSAd deciency in the SH2D2A)/) mice may disturb the formation of hemidesmosomes and

thereby explain the defects in their BM. This notion is further supported by the irregular staining pattern of collagen IV and nidogen observed in the present study. Both nidogen and collagen IV localize to the lamina densa (27), which we found to be interrupted at the ultrastructural level. The disorganized BM and irregular staining pattern of the BM proteins collagen IV and nidogen in SH2D2A)/) mice were observed in some areas, whereas in other areas the BM had the same appearance as in wild-type mice. Although the SH2D2A)/) mice had ultrastructural defects in their BM, their skin and oral mucosa appeared normal. This indicates that there is redundancy in the signaling pathways involved. In this study we chose to focus on the disturbances observed in the basal cell layer and the interaction between the basal cell layer and the BM. However, different disturbances were also observed throughout the stratum spinosum and these will be investigated further. Another interesting nding was that TSAd mRNA is expressed only in the basal cell layers, whereas the protein is also observed in the stratum spinosum. Oral mucosa has an epithelial turnover of approximately 25 d, which indicates that TSAd protein in this tissue has a prolonged half-life. The half-time of TSAd protein is currently not known, but preliminary data from small interfering (si)RNA experiments indicate that the halflife of TSAd protein could be around 24 h in T cells. Altered migration of TSAd was observed in western blotting in the human biopsy specimens but not in the cultured keratinocytes. This suggests that TSAd may be modied, making it more stable in the stratum spinosum. These observations need further investigation.

166

Kolltveit et al.

The TSAd may be involved in regulating the proliferation of oral keratinocytes: the epithelium of SH2D2A)/) mice exhibited a higher number of proliferating basal cells and overexpression of TSAd in epithelial cell cultures repressed proliferation. The TSAd has been shown to bind to Smad2/3 and to modulate TGF-b-dependent Smad signaling (28). Activity through the Smad2/3IKKa (I kappa-b-kinase-alfa) complex represses proliferation and controls epidermal dierentiation (29). It is therefore possible that TSAd may regulate epithelial proliferation through TGF-b signaling. Disruption of TSAd expression resulted in impaired migration of oral keratinocytes. Cellular migration requires re-organization of the actin cytoskeleton. In endothelial cells, TSAd binds to vascular endothelial growth factor receptor 2 (VEGFR-2) and this is critical for VEGF-mediated actin re-organization (13). In epithelial cells, re-organization of the actin cytoskeleton, and subsequent migration, is promoted by TGF-b- and EGF-signaling (30, 31). The TSAd has been found to interact in signaling pathways downstream of both TGFb receptor (TGF-bR) and EGF receptor (EGFR) (28, 32, 33). In T cells, TSAd mediates chemokine-dependent migration through coupling of the G protein b subunit and the focal adhesion kinases Pyk2 and paxillin (18), as well as laminin-mediated migration through binding to LBP (19). Thus, TSAd may participate at more than one level in the signaling pathways inducing migration in epithelial cells. In wound healing, early migration of epithelial cells (restitution) is crucial. In this phase, proliferation is repressed because the initial primary task of the cells is to migrate, not to proliferate (34). As TSAd appears to constrain proliferation and to facilitate migration, it may have a role in the early phases of wound healing. The skin and oral mucosa of the SH2D2A)/) mice had a normal appearance, but the ultrastructural abnormalities in the BM and their epithelial hyperproliferative state are reminiscent of changes seen in some common human skin diseases. The blistering and the breach in continuity in the BM observed in the present study, closely resemble ultrastructural changes that can be observed in the skin of patients with lupus erythematosus and in the skin of a mouse model of systemic lupus erythematosus (SLE) (MRL/l strain) (3538). Furthermore, in psoriatic skin lesions, the lamina densa can be interrupted, as observed in the SH2D2A)/) mice in the present study (39). In oral lichen planus, disturbances of the basal lamina, such as increased width of the lamina densa and loss of hemidesmosomes, can be seen (40, 41). It remains to be examined whether such changes can be linked to deciencies in TSAd expression. A polymorphism in the TSAd promoter results in reduced expression of TSAd and is associated with susceptibility for multiple sclerosis and juvenile rheumatoid arthritis (15, 16). This promoter genotype could probably aect TSAd expression also in epithelial cells. It is therefore possible that this polymorphism may contribute to the development of skin and mucosal diseases. In summary, in this study we have shown that TSAd is expressed in normal oral epithelium in humans and in

mice. Normal expression of TSAd is necessary for maintaining the integrity of the BM and the epithelium. Impaired TSAd expression increases the proliferation, and impairs migration, of oral keratinocytes.
Acknowledgements We thank Hanne Weidemann and Amandeep Singh Dhesi (Department of Oral Biology, Faculty of Dentistry, University of Oslo) for excellent technical assistance.

References
1. Hakkinen L, Uitto VJ, Larjava H. Cell biology of gingival wound healing. Periodontol 2000 2000; 24: 127152. 2. Barrientos S, Stojadinovic O, Golinko MS, Brem H, TomicCanic M. Growth factors and cytokines in wound healing. Wound Repair Regen 2008; 16: 585601. 3. Bachman KE, Park BH. Dual nature of TGF-beta signaling: tumor suppressor vs. tumor promoter. Curr Opin Oncol 2005; 17: 4954. 4. Hayashi K, Karatsaidis A, Schreurs O, Bjornland T, Sugisaki M, Schenck K. NGF and its receptors TrkA and p75NTR in the epithelium of oral lichen. J Oral Pathol Med 2008; 37: 241248. 5. Zelles T, Purushotham KR, Macauley SP, Oxford GE, Humphreys-Beher MG. Saliva and growth factors: the fountain of youth resides in us all. J Dent Res 1995; 74: 18261832. 6. Hayashi K, Storesund T, Schreurs O, Khuu C, Husvik C, Karatsaidis A, Helgeland K, Martin-Zanca D, Schenck K. Nerve growth factor beta/pro-nerve growth factor and their receptors in normal human oral mucosa. Eur J Oral Sci 2007; 115: 344354. 7. Storesund T, Hayashi K, Kolltveit KM, Bryne M, Schenck K. Salivary trefoil factor 3 enhances migration of oral keratinocytes. Eur J Oral Sci 2008; 116: 135140. 8. Spurkland A, Brinchmann JE, Markussen G, Pedeutour F, Munthe E, Lea T, Vartdal F, Aasheim HC. Molecular cloning of a T cell-specific adapter protein (TSAd) containing an Src homology (SH) 2 domain and putative SH3 and phosphotyrosine binding sites. J Biol Chem 1998; 273: 4539 4546. 9. Choi YB, Kim CK, Yun Y. Lad, an adapter protein interacting with the SH2 domain of p56lck, is required for T cell activation. J Immunol 1999; 163: 52425249. 10. Rajagopal K, Sommers CL, Decker DC, Mitchell EO, Korthauer U, Sperling AI, Kozak CA, Love PE, Bluestone JA. RIBP, a novel Rlk/Txk- and itk-binding adaptor protein that regulates T cell activation. J Exp Med 1999; 190: 1657 1668. 11. Flynn DC. Adaptor proteins. Oncogene 2001; 20: 62706272. 12. Wu LW, Mayo LD, Dunbar JD, Kessler KM, Ozes ON, Warren RS, Donner DB. VRAP is an adaptor protein that binds KDR, a receptor for vascular endothelial cell growth factor. J Biol Chem 2000; 275: 60596062. 13. Matsumoto T, Bohman S, Dixelius J, Berge T, Dimberg A, Magnusson P, Wang L, Wikner C, Qi JH, Wernstedt C, Wu J, Bruheim S, Mugishima H, Mukhopadyay D, Spurkland A, Claesson-Welsh L. VEGF receptor-2 Y951 signaling and a role for the adapter molecule TSAd in tumor angiogenesis. EMBO J 2005; 24: 23422353. 14. Park D, Choi YB, Han MK, Kim UH, Shin J, Yun Y. Adaptor protein Lad relays PDGF signal to Grb2 in lung cells: a tissue-specific PDGF signal transduction. Biochem Biophys Res Commun 2001; 284: 275281. 15. Dai KZ, Harbo HF, Celius EG, Oturai A, Sorensen PS, Ryder LP, Datta P, Svejgaard A, Hillert J, Fredrikson S, Sandberg-Wollheim M, Laaksonen M, Myhr KM, Nyland H, Vartdal F, Spurkland A. The T cell regulator gene SH2D2A contributes to the genetic susceptibility of multiple sclerosis. Genes Immun 2001; 2: 263268. 16. Smerdel A, Dai KZ, Lorentzen AR, Flato B, Maslinski S, Thorsby E, Frre , Spurkland A. Genetic association

TSAd in oral epithelium

between juvenile rheumatoid arthritis and polymorphism in the SH2D2A gene. Genes Immun 2004; 5: 310312. Drappa J, Kamen LA, Chan E, Georgiev M, Ashany D, Marti F, King PD. Impaired T Cell Death and Lupus-like Autoimmunity in T Cell-specific Adapter Protein-deficient Mice. J Exp Med 2003; 198: 809821. Park D, Park I, Lee D, Choi YB, Lee H, Yun Y. The adaptor protein Lad associates with the G protein beta subunit and mediates chemokine-dependent T-cell migration. Blood 2007; 109: 51225128. Park E, Choi Y, Ahn E, Park I, Yun Y. The adaptor protein, LAD/TSAd mediates Laminin-dependent T cell migration via association with the 67 kDa Laminin Binding Protein (LBP). Exp Mol Med 2009; 41: 728736. Granum S, Sundvold-Gjerstad V, Dai KZ, Kolltveit KM, Hildebrand K, Huitfeldt HS, Lea T, Spurkland A. Structure function analysis of SH2D2A isoforms expressed in T cells reveals a crucial role for the proline rich region encoded by SH2D2A exon 7. BMC Immunol 2006; 7: 15. Sundvold V, Torgersen KM, Post NH, Marti F, King PD, Rottingen JA, Spurkland A, Lea T. T cell-specific adapter protein inhibits T cell activation by modulating Lck activity. J Immunol 2000; 165: 29272931. Dai KZ, Vergnaud G, Ando A, Inoko H, Spurkland A. The SH2D2A gene encoding the T-cell-specific adapter protein (TSAd) is localized centromeric to the CD1 gene cluster on human Chromosome 1. Immunogenetics 2000; 51: 179185. Litjens SH, de Pereda JM, Sonnenberg A. Current insights into the formation and breakdown of hemidesmosomes. Trends Cell Biol 2006; 16: 376383. Mercurio AM, Rabinovitz I, Shaw LM. The alpha 6 beta 4 integrin and epithelial cell migration. Curr Opin Cell Biol 2001; 13: 541545. Yurchenco PD, Amenta PS, Patton BL. Basement membrane assembly, stability and activities observed through a developmental lens. Matrix Biol 2004; 22: 521538. Ardini E, Tagliabue E, Magnifico A, Buto S, Castronovo V, Colnaghi MI, Menard S. Co-regulation and physical association of the 67-kDa monomeric laminin receptor and the alpha6beta4 integrin. J Biol Chem 1997; 272: 23422345. DiPersio CM, Hodivala-Dilke KM, Jaenisch R, Kreidberg JA, Hynes RO. alpha3beta1 Integrin is required for normal development of the epidermal basement membrane. J Cell Biol 1997; 137: 729742. Richard KC, Bertolesi GE, Dunfield LD, McMaster CR, Nachtigal MW. TSAd interacts with Smad2 and Smad3. Biochem Biophys Res Commun 2006; 347: 266272. Descargues P, Sil AK, Sano Y, Korchynskyi O, Han G, Owens P, Wang XJ, Karin M. IKKalpha is a critical coregulator of a Smad4-independent TGFbeta-Smad2/3 signaling pathway that controls keratinocyte differentiation. Proc Natl Acad Sci U S A 2008; 105: 24872492. Boland S, Boisvieux-Ulrich E, Houcine O, Baeza-Squiban A, Pouchelet M, Schoevaert D, Marano F. TGF beta 1 promotes actin cytoskeleton reorganization and migratory phenotype in epithelial tracheal cells in primary culture. J Cell Sci 1996; 109: 22072219. Kurten RC, Chowdhury P, Sanders RC Jr, Pittman LM, Sessions LW, Chambers TC, Lyle CS, Schnackenberg BJ,

167

17.

32.

18.

33.

19.

34. 35.

20.

21.

36.

22.

37. 38.

23. 24. 25. 26.

39.

40. 41.

27.

Jones SM. Coordinating epidermal growth factor-induced motility promotes efficient wound closure. Am J Physiol Cell Physiol 2005; 288: C109C121. Sun W, Wei X, Kesavan K, Garrington TP, Fan R, Mei J, Anderson SM, Gelfand GW, Johnson GL. MEK kinase 2 and the adaptor protein Lad regulate extracellular signalregulated kinase 5 activation by epidermal growth factor via Src. Mol Cell Biol 2003; 23: 22982308. Sun W, Kesavan K, Schaefer BC, Garrington TP, Ware M, Johnson NL, Gelfand GW, Johnson GL. MEKK2 associates with the adapter protein Lad/RIBP and regulates the MEK5BMK1/ERK5 pathway. J Biol Chem 2001; 276: 50935100. Raja, Sivamani K, Garcia MS, Isseroff RR. Wound reepithelialization: modulating keratinocyte migration in wound healing. Front Biosci 2007; 12: 28492868. Murphy JK, Stephens C, Hartley T, Das AK, Hughes GR, McKee PH. Subacute cutaneous lupus erythematosusthe annular variant. A histological and ultrastructural study of five cases. Histopathology 1991; 19: 329336. Lourenco SV, de Carvalho FR, Boggio P, Sotto MN, Vilela MA, Rivitti EA, Nico MM. Lupus erythematosus: clinical and histopathological study of oral manifestations and immunohistochemical profile of the inflammatory infiltrate. J Cutan Pathol 2007; 34: 558564. Kobayasi T, Asboe-Hansen G. Ultrastructure of systemic lupus erythematosus. Dermal connective tissue. Acta Derm Venereol 1974; 54: 2328. Horiguchi Y, Furukawa F, Hamashima Y, Imamura S. Ultrastructural observations of skin lesions in MRL mice dermoepidermal junction. Arch Dermatol Res 1984; 276: 229 234. Esrefoglu M, Gul M, Seyhan M. Ultrastructural findings and tumor necrosis factor-alpha and intercellular adhesion molecule-1 expression in psoriasis patients before and after oral cyclosporin A therapy. Ultrastruct Pathol 2006; 30: 95102. Shklar G, Flynn E, Szabo G. Basement membrane alterations in oral lichen planus. J Invest Dermatol 1978; 70: 4550. Bouloc A, Vignon-Pennamen MD, Caux F, Teillac D, Wechsler J, Heller M, Lebbe C, Flageul B, Morel P, Dubertret L, Prost C. Lichen planus pemphigoides is a heterogeneous disease: a report of five cases studied by immunoelectron microscopy. Br J Dermatol 1998; 138: 972980.

28. 29.

Supporting Information
Additional Supporting Information may be found in the online version of this article: Figure S1. In vitro scratch assay, cultures analyzed using a brighteld microscope. Data S1. Details of the CISH procedure. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

30.

31.

This document is a scanned copy of a printed document. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material.