Immunolocalization of laminin and integrin in regenerating junctional epithelium of mice after gingivectomy

Background and Objective: The expression patterns of adhesive proteins and extracellular matrix proteins in regenerating gingival epithelium after gingivectomy are unknown. The aim of this study was to examine the expression of laminin 1, laminin γ₂ (a specific component of laminin 5), integrin β₄ and integrin α₃ in the regenerating gingival epithelium in order to understand the mechanism of wound healing during reconstitution of the sulcular environment. Material and Methods: The palatal gingivae of the maxillary molars of Institute of Cancer Research mice were excised, and the regenerating tissues were examined 1, 3, 5, 7 and 14 days later. Fresh, non‐fixed and non‐decalcified frozen sections were prepared and stained using immunofluorescence. Results: At 1 day post‐surgery, intense expression of laminin γ₂, integrin β₄ and integrin α₃ was distinct in the frontal margin of the regenerating oral epithelium. Laminin γ₂ was diffusely detected on the root surface and in connective tissues beneath the regenerating oral epithelium at 3 and 5 days. At 7 days, laminin γ₂ was intermittently recognizable in the internal basal lamina (IBL) close to tooth‐facing cells, while laminin γ₂, integrin β₄ and integrin α₃ were observed in the IBL and in the external basal lamina (EBL) of the regenerating junctional epithelium at 14 days. Conclusion: These results suggest that secretion of laminin 5 in the connective tissue may induce epithelial cell migration, and that binding of laminin 5 to integrin α₆β₄ and integrin α₃β₁ in the IBL may provoke cell adhesion and migration of cells facing the tooth on the enamel surface of the regenerating junctional epithelium.     

Changes in the expression of integrins and basement membrane proteins in benign mucous membrane pemphigoid

OBJECTIVE: To determine the location of the subepi-thelial split in benign mucous membrane pemphigoid (BMMP) and its relationship to the anchoring filaments and their receptors. MATERIALS AND METHODS: Frozen sections of lesional and perilesional oral mucosa from 10 cases of BMMP were stained, using an immunofluorescence method, for the β₁, β₄, α₃ and α₆ integrin subunits and for their ligands, laminin I and laminin V (kalinin). In all cases the diagnosis was confirmed by the demonstration of linear staining for IgG at the basement membrane zone. Six specimens of normal mucosa were stained for comparison. RESULTS Staining for integrins, laminin and kalinin in perilesional mucosa was similar to normals, although one case showed loss of α₆ and β₄. In lesional mucosa, laminin and kalinin showed strong linear staining localised to the floor of the bullae. The α₆ and β₄ subunits were expressed only on the roof of the bullae but staining was weak and patchy with areas of loss. In some sections a₆ showed a punctate intracellular distribution similar to IgG. The distribution of α₃ and β₁ was similar to that seen in normals. CONCLUSIONS: In all cases kalinin was found on the connective tissue side of the lesions and α₆β₄ localised to the epithelial side. This shows that the split occurs at a location which separates anchoring filaments from the hemidesmosomes. Loss of the α₆β₄ integrin in the lesions and the similar intracellular staining of α₆ and IgG, suggest that disruption of hemidesmosomes may be a key event in the immunopathogenesis of the lesions and that the α₆ integrin subunit is a potential antigen in oral mucosal BMMP.     

EGF/TGFβ1 co‐stimulation of oral squamous cell carcinoma cells causes an epithelial–mesenchymal transition cell phenotype expressing laminin 332

J Oral Pathol Med (2011) 40 : 46–54 Epithelial–mesenchymal transition (EMT) is suggested to be crucial for the development of an invasive and metastatic carcinoma cell phenotype. Therefore, the definition of this phenotype is of great clinical interest. We recently evidenced vimentin positive cells in oral squamous cell carcinoma (OSCC) invasive front expressing laminin γ2 chain mRNA implicating an EMT origin of these cells. To further elucidate the nature of these cells, we have investigated the relation between EMT criteria and laminin‐332 expression in a cell culture model of transforming growth factor beta‐1 (TGFβ1)/epithelial growth factor (EGF) long time co‐stimulation. We demonstrate that in contrast to TGFβ1 or EGF alone, co‐stimulation induces phenotype transition in OSCC cells which fulfils the criteria of EMT in terms of vimentin up‐regulation and E‐cadherin down‐regulation on protein level as well as cell scattering. Furthermore, cells displayed a strongly enhanced invasiveness and adhesion to type I‐IV collagens. Phenotype transition is accompanied by an enhanced expression of laminin‐332, especially of its γ2 chain. We further analyse the expression of extracellular matrix related genes by RT‐PCR profiling. With respect to strongly enhanced proteins, data confirm the EMT phenotype of co‐stimulated OSCC cells and expression of laminin‐332. Furthermore, alpha catenin, collagen type 16, the integrin α7 and β1 chains, and MMP11 are suggested as candidates with potential role in EMT in OSCC. In summary we are able to show that EMT in OSCC is mediated by multiple growth factors and is accompanied by laminin γ2 chain up‐regulation evidencing the existence of an intermediate Vim⁺/Ln332⁺ EMT phenotype as seen in situ.     

High-level β1-integrin expression in a subpopulation of highly tumorigenic oral cancer cells

Objectives

The β1 integrin (CD29) is a putative marker for cancerous epithelial stem cells. Cancer stem cells are essential to drive tumor growth, recurrence, and metastasis. We investigated the role of β1-integrin expression in the development of malignant phenotypes of oral squamous cell carcinoma (OSCC).

Materials and methods

Immunostaining was used to analyze the expression levels of β1 integrins in different types of cell colonies and tumor spheres. The results of cell viability and migration assays with and without siRNA knockdown of β1-integrin expression were compared. Cells expressing β1 integrins were evaluated for their tumorigenicity in mice. The expression of β1 integrins in human specimens of oral cancers at different clinical stages was semiquantified based on immunohistochemical staining of the β1-integrin protein.

Results

The expression level of β1 integrins in Meng-1 oral epidermoid carcinoma cells (OECM-1) cells was significantly higher in holoclonal colonies and tumor spheres compared to control cells. The knockdown of β1-integrin expression in OECM-1 cells reduced cell proliferation, migration, and tumor sphere formation. Beta-1 integrin (+) cells were more tumorigenic in the mouse xenograft model than β1 integrin (?) cells. In the human specimens, the expression level of the β1-integrin protein positively correlated with the clinical stage.

Conclusion

The expression of β1 integrin in OECM-1 cells is involved in the development of malignant phenotypes of OSCC.

Clinical relevance

Inhibitors for β1-integrin signaling may be suitable to become target-specific therapies for OSCC.     

The role of α9β1 integrin in modulating epithelial cell behaviour

J Oral Pathol Med (2011) 40 : 755–761 Background: Integrins initiate signalling in response to the extracellular matrix (ECM), which is important in wound healing and cancer. Previous studies have shown that over‐expression of the αvβ6 integrin in oral squamous cell carcinoma (OSCC) cells results in enhanced motility and expression of matrix‐degrading proteases, and the aim of this study was to investigate whether this is also the case for the α9β1 integrin. Methods: H357 OSCCcells were transfected with the α9 integrin subunit and proliferation, adhesion and migration assays were performed on these along with null vector control and wild‐type cells. The effect of ligand engagement on matrix metalloproteinase expression and the plasminogen activator system was measured using ELISA and chromogenic assays. Expression of α9 integrin was examined in oral squamous cell carcinoma tissue by immunohistochemistry. Results: Functionally active α9 integrin mediated specific upregulation of adhesion and migration towards the TNfn3RAA fragment of tenascin‐C but reduced proliferation. Migration towards collagen I was also enhanced in transfected cells. Matrix metalloproteinase‐2 and metalloproteinase‐9 expression was increased upon TNfn3RAA ligand engagement. Cell surface plasmin generation was also enhanced in α9‐expressing cells and was the result of enhanced expression of urokinase receptor. In normal oral mucosa, α9 integrin expression was restricted to the suprabasal and prickle cell layers, and expression was heterogeneous in tumours but present in islands infiltrating connective tissue particularly in moderately and well‐differentiated lesions. Conclusions: The α9β1 integrin may play a key role in modulation of tumour behaviour including enhanced cell migration and expression of matrix‐degrading proteases.     

Integrin Expression and Migration of Adenoid Cystic Carcinoma Cells in Response to Basement Membrane Components

Adenoid cystic carcinoma iAdCC jis more aggressive than oral squamous cell carcinoma iSCC j and has a proclivity to invade nerve and endothelial sheaths containing basement membrane iBM j . In the present study, we found that AdCC cell lines showed higher motility responses to BM proteins, especially type IV collagen, compared to oral SCC cell lines. Flow cytometric analysis demonstrated that AdCC cell lines showed generally similar pattern of integrin subunits to oral SCC lines. Although AdCC cell lines expressed slightly higher levels of α₂β₁ and α₃β₁ integrins than SCC cell lines, these differences were not as impressive as those seen in haptotaxis assays to ECM proteins, and in particular to type IV collagen. Antibody blocking experiments clearly showed that the AdCC motility response to type IV collagen is mainly mediated through α₂β₁ integrin. In conclusion, such high motility response of AdCC cell lines could not be explained by only the surface levels of integrins, and study on integrin functionality will be more important to elucidate high invasiveness of AdCC cells.     

Stromal laminin chain distribution in normal,hyperplastic and malignant oral mucosa: relation to myofibroblast occurrence and vessel formation

J Oral Pathol Med (2010) 39 : 290–298 Background: The contribution of stromal laminin chain expression to malignant potential, tumour stroma reorganization and vessel formation in oral squamous cell carcinoma (OSCC) is not fully understood. Therefore, the expression of the laminin chains α2, α3, α4, α5 and γ2 in the stromal compartment/vascular structures in OSCC was analysed. Methods: Frozen tissue of OSCC (9× G1, 24× G2, 8× G3) and normal (2×)/hyperplastic (11×) oral mucosa was subjected to laminin chain and α‐smooth muscle actin (ASMA) immunohistochemistry. Results were correlated to tumour grade. The relation of laminin chain positive vessels to total vessel number was assessed by immunofluorescence double labelling with CD31. Results: Stromal laminin α2 chain significantly decreases and α3, α4, α5 and γ2 chains and also ASMA significantly increase with rising grade. The amount of stromal α3, α4 and γ2 chains significantly increased with rising ASMA positivity. There is a significant decrease in α3 chain positive vessels with neoplastic transformation. Conclusions: Mediated by myofibroblasts, OSCC development is associated with a stromal up‐regulation of laminin isoforms possibly contributing to a migration promoting microenvironment. A vascular basement membrane reorganization concerning α3 and γ2 chain laminins during tumour angioneogenesis is suggested.     

Immunolocalization of fibronectin and vitronectin receptors in human gingival fibroblasts spreading on titanium surfaces

The adhesion and spreading of human gingival fibroblasls on glass and differently processed titanium surfaces was studied by immunolocalization of vinculin and the alpha and beta subunits of the fibronectin (α₅β₁) and vitronectin (α_vβ3) receptors. Vinculin-containing focal contacts were present both at 4 and 24 h of spreading in cells grown on glass or electropolished or etched titanium surfaces but not in cells spreading on sandblasted titanium surfaces. Immunostaining for the α5 and β1 subunits of the fibronectin receptor showed only a diffuse membrane fluorescence after 4 h of cell spreading irrespective of the growth surface. The α_v and β₃ subunits of the vitronectin receptor were at this stage detected in focal contacts in cells spreading on glass or electropolished or etched titanium surfaces. In cells spreading on sandblasted titanium surfaces, however, the vitronectin receptor had only a diffuse distribution. In cells that had been allowed to spread for 24 h on glass or electropolished or etched titanium surfaces the α₅ and β₁ integrin subunits were either diffusely distributed or showed a localization typical of extracellular matrix contacts. The α_v and β₃ integrin subunits were, as earlier, localized to typical focal contacts in cells grown on glass or electropolished or etched titanium surfaces. Cells attached to sandblasted titanium surfaces still expressed all the integrin subunits only diffusely. The results show that the surface texture of the substratum can affect the expression of integrin subunits in human gingival fibroblasts. As evidenced by the recruitment of integrin subunits to focal and extracellular matrix contacts, smooth or finely grooved titanium surfaces appear to be optimal in supporting the attachment of human gingival fibroblasts.     

Integrin expression in human dental pulp cells and their role in cell attachment on extracellular matrix proteins

Integrins are a family of heterodimeric glycoproteins consisting of alpha and beta subunits that noncovalently interact to form cell surface adhesion receptors. The objective of this study was to identify integrins in human dental pulp cells and determine their role in human dental pulp cell attachment to the biological active molecules, laminin and fibronectin. Integrin expression was studied by immunoblot and immunoprecipitation using monoclonal integrin antibodies. The role of integrin in human dental pulp cell adhesion on laminin and fibronectin was determined by inhibition of cell adhesion with those antibodies. This study found human dental pulp cells expressed alpha 1, alpha 3, alpha 5, alpha 6, alpha v, and beta 1 integrin subunits. The adhesion of human dental pulp cells to laminin and fibronectin was not inhibited by monoclonal antibody to any subunit, except that anti-beta 1 antibody inhibited pulp cells adhesion on laminin. These data provide information for further studying the role of integrins in dental pulp cell biological function.     

Simvastatin alters fibroblastic cell responses involved in tissue repair

Cáceres M, Romero A, Copaja M, Díaz‐Araya G, Martínez J, Smith PC. Simvastatin alters fibroblastic cell responses involved in tissue repair. J Periodont Res 2011; 46: 456–463. © 2011 John Wiley & Sons A/S Background and Objective: Statins have been used to control hypercholesterolemia. However, these drugs also exert pleiotropic effects that include the modulation of inflammation and cell signaling. The present study has analyzed the effects of simvastatin on several cell responses involved in tissue repair, including cell adhesion, cell migration and invasion, actin cytoskeleton remodeling and cell viability. Material and Methods: Primary cultures of gingival fibroblasts were stimulated with simvastatin. Cell adhesion was evaluated using a colorimetric assay. Cell spreading was evaluated microscopically. Cell migration and invasion were assessed using a scratch wound‐healing assay and a bicameral cell culture system, respectively. Changes in actin cytoskeleton and focal adhesion assembly were evaluated through immunofluorescence for actin, vinculin and active β1 integrin. Rac activation was evaluated by means of a pull‐down assay. Cell viability was assessed using a colorimetric assay that determines mitochondrial functionality. Data analysis was performed using the Mann–Whitney U‐test. Results: Simvastatin diminished cell adhesion and spreading over a fibronectin matrix. It also altered the closure of scratch wounds induced on cell monolayers and cell invasion through a Transwell system. Simvastatin‐treated cells displayed an altered lamellipodia with poorly developed focal adhesion contacts and reduced levels of β1 integrin activation. During cell spreading, simvastatin diminished Rac activation. Conclusion: The present study shows that simvastatin may alter cell migration by disrupting the cell signaling networks that regulate the actin cytoskeleton dynamics. This mechanism may affect the response of gingival mesenchymal cells during wound healing.     

TNF-alpha stimulation increases dental pulp stem cell migration in vitro through integrin alpha-6 subunit upregulation

ObjectiveThe dissemination of stem cells into tissues requiring inflammatory and reparative response is fundamentally dependent upon their chemotactic migration. Expression of TNF-α is up regulated in inflamed pulps. Dental pulp cells are also known to express integrin α6 subunit. Expression of integrin subunit α6 has been linked to the acquisition of migratory potential in a wide variety of cell types in both pathological and physiological capacities. Therefore, in this study we examined the effects of a pleiotropic cytokine TNF-α on the migration of hDPSCs and investigated its relationship with expression of integrin α6 in hDPSCs during chemotactic migration.DesignhDPSC cultures were established. Protein expression profile of α6 integrin subunit was determined. Effect of exogenous TNF-α (50 ng/ mL) on hDPSCs’ migration potential was evaluated by transwell inserts and in vitro scratch assay. Upregulation/downregulation of TNF-α mediated migration was assayed in presence/absence of integrin α6 respectively. To suppress integrin α6 expression, cells were transfected with integrin α6 siRNA and then cell migration and cytoskeletal changes were evaluated.ResultsOur results showed significant increase of hDPSCs’ migration after stimulation with TNF-α. By knockdown of integrin α6, which is upregulated by TNF-α, we observed a decrease in the TNF-α directed chemotaxis of hDPSCs.ConclusionIn this study, we show that activation of integrin α6 brought about by TNF-α led to an increase in migratory activity in DPSCs in vitro thus describing a novel association between a cytokine TNF-α and α6 chain of an adhesion receptor integrin in regulating migration of hDPSCs.     

Streptococcus mutans antigen I/II binds to α5β1 integrins via its A-domain and increases β1 integrins expression on periodontal ligament fibroblast cells

The cell wall anchored protein I/II of Streptococcus mutans plays a significant role in colonizing the dental structures and induces the synthesis of proinflammatory cytokines after binding to α5β1 integrins of host cells. Whilst the signalling pathways triggered by bound protein I/II and leading to the release of proinflammatory cytokines has been extensively studied, the molecular mechanisms of binding of protein I/II to this host cell factor remain more elusive. Using a panel of purified recombinant polypeptides corresponding to defined domains of the streptococcal protein I/II, we aimed to better characterize protein I/II-α5β1 integrins interaction. Our results show that the A region plays a major role in binding of protein I/II to α5β1 integrin. Using specific inhibitors, we demonstrated that this interaction requires β subunits of the integrin and that the glycosylated residues of the integrin interact with the V-region of the protein I/IIf. Periodontal fibroblasts (PDL-Fb) express β1-integrins on their cell membrane. Stimulation with protein I/II increases the expression of β1-integrins. These data suggest a modulatory effect of the streptococcal protein I/II on expression of integrins by PDL-Fb. This might have important implication for understanding the role of PDL-Fb cells in periradicular inflammation.     

The Role of Integrin αv in Proliferation and Differentiation of Human Dental Pulp Cell Response to Calcium Silicate Cement

Introduction

It has been proved that integrin αv activity is related to cell proliferation, differentiation, migration, and organ development. However, the biological functions of integrin αv in human dental pulp cells (hDPCs) cultured on silicate-based materials have not been explored. The aim of this study was to investigate the role of integrin αv in the proliferation and odontogenic differentiation of hDPCs cultured with the effect of calcium silicate (CS) cement and β-tricalcium phosphate (TCP) cement.

Methods

In this study, hDPCs were cultured on CS and TCP materials, and we evaluated fibronectin (FN) secretion and integrin αv expression during the cell attachment stage. After small interfering RNA transfection targeting integrin αv, the proliferation and odontogenesis differentiation behavior of hDPCs were analyzed.

Results

The results indicate that CS releases Si ion–increased FN secretion and adsorption, which promote cell attachment more effectively than TCP. The CS cement facilitates FN and αv subintegrin expression. However, the FN adsorption and integrin expression of TCP are similar to that observed in the control dish. Integrin αv small interfering RNA inhibited odontogenic differentiation of hDPCs with the decreased formation of mineralized nodules on CS. It also down-regulated the protein expression of multiple markers of odontogenesis and the expression of dentin sialophosphoprotein protein.

Conclusions

These results establish composition-dependent differences in integrin binding and its effectiveness as a mechanism regulating cellular responses to biomaterial surface.     

Alteration of the actin cytoskeleton and localisation of the α6β1 and α3 integrins during regeneration of the rat submandibular gland

ObjectivesActin filaments, which are regulated by signal transduction via integrins, play important roles in the regulation of cell differentiation and polarity. The aim of this study was to assess alterations in the cytoskeleton and the localisation of integrins during regeneration of the rat submandibular gland.DesignAfter obstruction for 7 days, the regenerating glands were collected at days 0, 1, 3, 7, 14 after duct release for analysis of regeneration. Alterations in the actin filaments were examined using phalloidin, which specifically binds to filamentous actin (F-actin), and the distributions of the α6β1 and α3 integrins were examined immunohistochemically.ResultsF-actin was strongly localised at the apical region in the intercalated ducts of normal and day-14 glands and in duct-like structures during the regenerative process. Thereafter, actin accumulated at the basement membrane in mature acinar cells. A temporo-spatial correlation was found between the apical distribution of F-actin and α3 integrin staining. Diffuse α6β1 integrin staining, which occurred at a distal site in α3 integrin-positive cells, was observed in immature cells at day 3. At day 14, α6β1 integrin was detected at the basement membrane in terminal differentiated acinar cells.ConclusionThese findings suggest that duct-like structures have the same properties as intercalated ducts, that alterations in α3 to α6β1 integrins regulate the generation of acinar cells from duct-like structures, and that the α6β1 integrin is involved in the differentiation of acinar cells during regeneration of the rat submandibular gland.     

Re‐epithelialization of wounds

Shortly after wounding, epithelial keratinocytes become activated through the combined effects of the exposure to pro‐migratory matrix molecules within the wound site and to growth factors that are released by other wound cells and from the blood clot as well as by wound‐generated electrical fields. Within 24 hours, they start migrating from the wound edges into the fibrin–fibronectin‐rich blood clot. They also deposit their own matrix molecules that facilitate their motility, including EDA fibronectin, laminin‐332, and tenascin‐C as well as express their receptors (mainly integrins). Basal keratinocytes adjacent to the wound site start proliferating 48–72 hours after the injury, contributing to the migrating cell pool. Re‐epithelialization is stimulated by a number of cytokines and growth factors such as members of the epidermal growth factor, transforming growth factor‐β, and keratinocyte growth factor families that promote keratinocyte migration and proliferation. Furthermore, re‐epithelialization is dependent on regulated expression of proteases, including plasmin and matrix metalloproteinases, which break down extracellular matrix to allow keratinocyte invasion into wound provisional matrix as well as release and activate matrix‐bound growth factors. Thus, wound re‐epithelialization is a complex process that requires coordinated expression of several new extracellular matrix molecules, their receptors, and proteinases, and when dysregulated, can result in failures to re‐epithelialize and formation of chronic wounds.     

Initial attachment of human oral keratinocytes cultured on zirconia or titanium

Initial attachment of human oral keratinocytes (HOKs) cultured on mirror-surfaced commercially pure titanium (Ti) or yttria-stabilized tetragonal zirconia polycrystals (TZP) was investigated. Numbers of viable attached HOKs, their mRNAs and proteins expression of laminin γ2 and integrin β4 were evaluated using the WST-1 assay, quantitative real-time PCR and enzyme-linked immunosorbent assay, respectively. Localization of laminin γ2 and integrin β4 was observed using immunofluorescent staining. Cell spreading was evaluated by measuring the perimeter of actin on fluorescent stained images, and cell morphology was examined using scanning electron microscopy. At 1 h TZP elicited less of initial attachment than Ti in terms of mRNAs expression and proteins expression of laminin γ2 and integrin β4 (p<0.05). However, at 48 h TZP was showed similar initial attachment in comparison to Ti. Therefore, it was suggested that TZP has a potential to form epithelial attachment like Ti.     

Smad2 overexpression enhances adhesion of gingival epithelial cells

ObjectiveGingival epithelial cells play an important role in preventing the initiation of periodontitis, by their hemidesmosomal adhesion to the tooth root surface. Adhesion requires integrin-extracellular matrix (ECM) interactions that are intricately regulated by transforming growth factor-β (TGF-β) signaling. However, the mechanisms underlying the interplay between adhesion molecules and TGF-β, especially the respective roles of Smad2 and Smad3, remain elusive. In this study, we examined the effects of Smad overexpression on gingival epithelial cell adhesion and expression profiles of integrin and ECM-related genes.MethodsHuman gingival epithelial cells immortalized by the SV40 T-antigen were transfected with Smad2- and Smad3-overexpression vectors. A cell adhesion assay involving fluorescence detection of attached cells was performed using the ArrayScan imaging system. Real-time PCR was performed to examine the kinetics of integrin and ECM gene expression. In vitro and in vivo localization of adhesion molecules was examined by immunofluorescence analysis.ResultsBy using SB431542, a specific inhibitor of the TGF-β type I receptor, Smad2/3 signaling was confirmed to be dominant in TGF-β1-induced cell adhesion. The Smad2-transfectant demonstrated higher potency for cell adhesion and integrin expression (α2, α5, β4, and β6) than the Smad3-transfectant, whereas little or no change in ECM expression was observed in either transfectant. Moreover, the gingival epithelium of transgenic mice that overexpressed Smad2 driven by the keratin 14 promoter showed increased integrin α2 expression.ConclusionThese findings indicate the crucial role of Smad2 in increased adhesion of gingival epithelial cells via upregulation of integrin α2.     

Differential expression of perlecan receptors, α‐dystroglycan and integrin β1, before and after invasion of oral squamous cell carcinoma

J Oral Pathol Med (2011) 40 : 552–559 Objectives: The deposition of perlecan, a heparan sulfate proteoglycan, is enhanced within oral carcinoma in situ (CIS) foci, while it dynamically switches from CIS foci to the stromal space in squamous cell carcinoma (SCC). Because α‐dystroglycan and integrin β1 have been identified as two of the perlecan receptors, we wanted to determine their differential distributions before and after invasion of oral SCC. Methods: Eighty‐two surgical tissue specimens of oral SCC containing different precancerous stages were examined by immunohistochemistry for perlecan, α‐dystroglycan, integrin β1, and Ki‐67. In addition, α‐dystroglycan mRNA signals were localized by in situ hybridization. Results: In normal epithelia, α‐dystroglycan and integrin β1 were localized on the cell membrane of basal cells, while perlecan was faintly present in the intercellular spaces of parabasal cells. In epithelial dysplasia and CIS, α‐dystroglycan and perlecan were well co‐localized in the epithelial layer, especially in its lower half, and this co‐localization was mostly overlapped with Ki‐67‐positive (+) cell zones. However, in SCC, α‐dystroglycan was localized neither within carcinoma cell nests nor in the stroma, while perlecan disappeared from SCC foci but emerged in the stromal space, leaving integrin β1+ and Ki‐67+ cells only to the periphery of SCC foci. α‐Dystroglycan mRNA signals were basically identical to the α‐dystroglycan protein localizations. Conclusion: The findings suggest that α‐dystroglycan and integrin β1 act as perlecan receptors in oral precancerous lesions prior to invasion, and that the perlecan signals via the two different receptors function in cellular differentiation and proliferation of CIS cells, respectively.