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Drebrin, an Actin-Binding, Cell-Type Characteristic Protein: Induction and Localization in Epithelial Skin Tumors and Cultured Keratinocytes

2005; Elsevier BV; Volume: 125; Issue: 4 Linguagem: Inglês

10.1111/j.0022-202x.2005.23793.x

1523-1747

Wiebke K. Peitsch, Ilse Hofmann, Jutta Bulkescher, Michaela Hergt, Herbert Spring, U. Bleyl, Sergij Goerdt, Werner W. Franke,

Cellular Mechanics and Interactions

Isoform E2 of drebrin, an actin-binding protein originally identified in neuronal cells, has recently been identified in diverse non-neuronal cells, mostly in association with cell processes and intercellular junctions. Here, we report on the presence of drebrin in normal human skin, epithelial skin cancers, and cultured keratinocytes. Keratinocytes of normal epidermis contain almost no drebrin but the protein is readily seen in hair follicles. By immunohistochemistry and immunoblot, basal cell carcinomas (BCC) are rich in drebrin, and confocal laser scanning and immunoelectron microscopy show accumulation at adhering junctions, in co-localization with actin and partially with plaque proteins. In squamous cell carcinomas, keratoacanthomas, and in epidermal precancers, drebrin is heterogeneously distributed, appearing as mosaics. Primary keratinocyte cultures contain significant amounts of drebrin enriched at adhering junctions. When epithelium-derived cells devoid of drebrin are transfected with drebrin-enhanced green fluorescent protein, constructs accumulate in the cell periphery, and immunoprecipitation shows complexes with actin. During epidermal growth factor induced formation of cell processes, drebrin retains this junction association, as observed by live cell microscopy. Our results suggest novel functions of drebrin such as an involvement in cell–cell adhesion and tumorigenesis and a potential value in diagnosis of BCC. Isoform E2 of drebrin, an actin-binding protein originally identified in neuronal cells, has recently been identified in diverse non-neuronal cells, mostly in association with cell processes and intercellular junctions. Here, we report on the presence of drebrin in normal human skin, epithelial skin cancers, and cultured keratinocytes. Keratinocytes of normal epidermis contain almost no drebrin but the protein is readily seen in hair follicles. By immunohistochemistry and immunoblot, basal cell carcinomas (BCC) are rich in drebrin, and confocal laser scanning and immunoelectron microscopy show accumulation at adhering junctions, in co-localization with actin and partially with plaque proteins. In squamous cell carcinomas, keratoacanthomas, and in epidermal precancers, drebrin is heterogeneously distributed, appearing as mosaics. Primary keratinocyte cultures contain significant amounts of drebrin enriched at adhering junctions. When epithelium-derived cells devoid of drebrin are transfected with drebrin-enhanced green fluorescent protein, constructs accumulate in the cell periphery, and immunoprecipitation shows complexes with actin. During epidermal growth factor induced formation of cell processes, drebrin retains this junction association, as observed by live cell microscopy. Our results suggest novel functions of drebrin such as an involvement in cell–cell adhesion and tumorigenesis and a potential value in diagnosis of BCC. basal cell carcinoma epidermal growth factor enhanced green fluorescent protein inner root sheath monoclonal antibody outer root sheath phosphate-buffered saline room temperature squamous cell carcinoma Skin carcinomas are the most frequent cancers in the light-skinned population, with an estimated yearly incidence of approximately 100 basal cell carcinomas (BCC) and approximately 20 squamous cell carcinomas (SCC) per 100,000 inhabitants. Important and long-known pathogenetic factors for skin carcinogenesis include ultraviolet (UV) light, radiation, and a diversity of chemical carcinogens. Only recently, molecular pathways leading to development of BCC and SCC have been elucidated. In inherited BCC such as in Gorlin's syndrome and in sporadic BCC, gene mutations of proteins of the Sonic Hedgehog signal transduction pathway have been identified that lead to basaloid hyperproliferations and infiltrative tumors (Hahn et al., 1996Hahn H. Wicking C. Zaphiropoulous P.G. et al.Mutations of the human homolog of Drosophila patched in the nevoid basal cell carcinoma syndrome.Cell. 1996; 85: 841-851Google Scholar; for a recent review see, e.g.,Cohen, 2003Cohen Jr, M.M. The hedgehog signalling network.Am J Med Genet. 2003; 123A: 5-28Google Scholar). In addition, there is increasing evidence that defects in cell–cell adhesion might contribute to skin carcinogenesis. For example, downregulation of E-cadherin, the major transmembrane protein of adhering junctions in epithelial cells, has been reported to correlate with development, progression, and metastasis of several kinds of carcinomas (e.g.,Behrens et al., 1989Behrens J. Mareel M.M. Van Roy F.M. Birchmeier W. Dissecting tumor cell invasion: Epithelial cells acquire invasive properties after the loss of uvomorulin-mediated cel–cell adhesion.J Cell Biol. 1989; 108: 2435-2447Google Scholar; Shimoyama and Hirohashi, 1991Shimoyama Y. Hirohashi S. Cadherin intercellular adhesion molecule in hepatocellular carcinomas: Loss of E-cadherin expression in an undifferentiated carcinoma.Cancer Lett. 1991; 57: 131-135Google Scholar; Moll et al., 1993Moll R. Mitze M. Frixen U.H. Birchmeier W. Differential loss of E-cadherin expression in infiltrating ductal and lobular breast carcinomas.Am J Pathol. 1993; 143: 1731-1742Google Scholar; Perl et al., 1998Perl A.K. Wilgenbus P. Dahl U. Semb H. Christofori G. A causal role for E-cadherin in the transition from adenoma to carcinoma.Nature. 1998; 392: 190-193Google Scholar; review:Cavallaro and Christofori, 2004Cavallaro U. Christofori G. Cell adhesion and signalling by cadherins and Ig-CAMs in cancer.Nat Rev Cancer. 2004; 4: 118-132Google Scholar), including SCC of the skin (Llorens et al., 1998Llorens A. Rodrigo I. Lopez-Barcons L. et al.Down-regulation of E-cadherin in mouse skin carcinoma cells enhances a migratory and invasive phenotype linked to matrix metalloproteinase-9 gelatinasen expression.Lab Invest. 1998; 78: 1131-1142Google Scholar; Lozano and Cano, 1998Lozano E. Cano A. Induction of mutual stabilization and retardation of tumor growth by coexpression of plakoglobin and E-cadherin in mouse skin spindle carcinoma cells.Mol Carcinog. 1998; 21: 273-287Google Scholar; Wu et al., 2000Wu H. Lotan R. Menter D. Lippman S.M. Xu X.C. Expression of E-cadherin is associated with squamous differentiation in squamous cell carcinomas.Anticancer Res. 2000; 20: 1385-1390Google Scholar; Papadavid et al., 2002Papadavid E. Pignatelli M. Zakynthinos S. Krausz T. Chu A.C. Abnormal immunoreactivity of the E-cadherin/catenin (alpha-, beta-, and gamma-) complex in premalignant and malignant non-melanocytic skin tumours.J Pathol. 2002; 196: 154-162Google Scholar). Moreover, β-catenin, a constitutive protein of the cytoplasmic plaque of adhering junctions, is part of the Wnt signaling pathway, a cascade of reactions leading to transcription of target genes for proliferation (recently reviewed byNelson and Nusse, 2004Nelson W.J. Nusse R. Convergence of Wnt, beta-catenin, and cadherin pathways.Science. 2004; 303: 1483-1487Google Scholar). Specifically in the skin, activating mutations of β-catenin cause proliferation of cells of the hair follicle sheath and development of pilomatrixomas (Gat et al., 1998Gat U. DasGupta R. Degenstein L. Fuchs E. De novo hair follicle morphogenesis and hair tumors in mice expressing a truncated beta-catenin in skin.Cell. 1998; 95: 605-614Google Scholar; Chan et al., 1999Chan E.F. Gat U. McNiff J.M. Fuchs E. A common human skin tumour is caused by activating mutations in beta-catenin.Nat Genet. 1999; 21: 410-413Google Scholar; Doglioni et al., 2003Doglioni C. Piccinin S. Demontis S. et al.Alterations of beta-catenin pathway in non-melanoma skin tumors: Loss of alpha-ABC nuclear reactivity correlates with the presence of beta-catenin gene mutation.Am J Pathol. 2003; 163: 2277-2287Google Scholar), and this protein might also be involved in the formation of BCC (El-Bahrawy et al., 2003El-Bahrawy M. El-Masry N. Alison M. Poulsom R. Fallowfield M. Expression of beta-catenin in basal cell carcinoma.Br J Dermatol. 2003; 148: 964-970Google Scholar). In addition to their involvement in tumor development and progression, proteins of intercellular junctions can also serve as cell-type "markers" in tumor diagnosis and are thus of particular value in cases of unknown primary tumors and for determination of the degree of differentiation. This has been shown extensively for plaque proteins of desmosomes such as desmoplakin (e.g.,Franke et al., 1983Franke W.W. Moll R. Mueller H. et al.Immunocytochemical identification of epithelium-derived human tumors with antibodies to desmosomal plaque proteins.Proc Natl Acad Sci USA. 1983; 80: 543-547Google Scholar; Moll et al., 1986Moll R. Cowin P. Kapprell H.P. Franke W.W. Desmosomal proteins: New markers for identification and classification of tumors.Lab Invest. 1986; 54: 4-25Google Scholar; Jones and Grelling, 1989Jones J.C. Grelling K.A. Distribution of desmoplakin in normal cultured human keratinocytes and in basal cell carcinoma cells.Cell Motil Cytoskeleton. 1989; 13: 181-194Google Scholar; review:Garrod, 1995Garrod D.R. Desmosomes and cancer.Cancer Surv. 1995; 24: 97-111Google Scholar), the plakophilins (Heid et al., 1994Heid H.W. Schmidt A. Zimbelmann R. et al.Cell type-specific desmosomal plaque proteins of the plakoglobin family: Plakophilin 1 (band 6 protein).Differentiation. 1994; 58: 113-131Google Scholar; Mertens et al., 1999Mertens C. Kuhn C. Moll R. Schwetlick I. Franke W.W. Desmosomal plakophilin 2 as a differentiation marker in normal and malignant tissues.Differentiation. 1999; 64: 277-290Google Scholar), and the various desmosomal cadherins (Parrish et al., 1986Parrish E.P. Garrod D.R. Mattey D.L. Hand L. Steart P.V. Weller R.O. Mouse antisera specific for desmosomal adhesion molecules of suprabasal skin cells, meninges, and meningioma.Proc Natl Acad Sci USA. 1986; 83: 2657-2661Google Scholar; Nuber et al., 1995Nuber U.A. Schäfer S. Schmidt A. Koch P.J. Franke W.W. The widespread human desmocollin Dsc2 and tissue-specific patterns of synthesis of various desmocollin subtypes.Eur J Cell Biol. 1995; 66: 69-74Google Scholar; Schäfer et al., 1996Schäfer S. Stumpp S. Franke W.W. Immunological identification and characterization of the desmosomal cadherin Dsg2 in coupled and uncoupled epithelial cells and in human tissues.Differentiation. 1996; 60: 99-108Google Scholar; for skin tumors seeKrunic et al., 1996Krunic A.L. Garrod D.R. Smith N.P. Orchard G.S. Cvijetic O.B. Differential expression of desmosomal glycoproteins in keratoacanthoma and squamous cell carcinoma of the skin: An immunohistochemical aid to diagnosis.Acta Derm Venereol. 1996; 76: 394-398Google Scholar; Moll et al., 1997Moll I. Kurzen H. Langbein L. Franke W.W. The distribution of the desmosomal protein, plakophilin 1, in human skin and skin tumors.J Invest Dermatol. 1997; 108: 139-146Google Scholar). Similarly, several cell-type-specific components of adhering junctions, notably members of the cadherin and the armadillo families of proteins, have been introduced into pathology (for references, seeMareel et al., 1994Mareel M. Vleminckx K. Vermeulen S. Yan G. Bracke M. van Roy F. Downregulation in vivo of the invasion-suppressor molecule E-cadherin in experimental and clinical cancer.Princess Takamatsu Symp. 1994; 24: 63-80Google Scholar; Tsukita et al., 1994Tsukita S. Tsukita S. Nagafuchi A. Yonemura S. Possible involvement of adherens junction plaque proteins in tumorigenesis and metastasis.Princess Takamatsu Symp. 1994; 24: 38-50Google Scholar; Nakanishi et al., 1997Nakanishi Y. Ochiai A. Akimoto S. et al.Expression of E-cadherin, alpha-catenin, beta-catenin and plakoglobin in esophageal carcinomas and its prognostic significance: Immunohistochemical analysis of 96 lesions.Oncology. 1997; 54: 158-165Google Scholar; Hirohashi and Kanai, 2003Hirohashi S. Kanai Y. Cell adhesion system and human cancer morphogenesis.Cancer Sci. 2003; 94: 575-581Google Scholar). A protein recently identified at adhering junctions as well as at lamellipodia and elongated cell processes of various cell types is drebrin, an actin-binding protein (Peitsch et al., 1999Peitsch W.K. Grund C. Kuhn C. Drebrin is a widespread actin-associating protein enriched at junctional plaques, defining a specific microfilament anchorage system in polar epithelial cells.Eur J Cell Biol. 1999; 78: 767-778Google Scholar,Peitsch et al., 2001Peitsch W.K. Hofmann I. Prätzel S. et al.Drebrin particles: Components in the ensemble of proteins regulating actin dynamics of lamellipodia and filopodia.Eur J Cell Biol. 2001; 80: 567-579Google Scholar; Keon et al., 2000Keon B.H. Jedrzejewski P.T. Paul D.L. Goodenough D.A. Isoform specific expression of the neuronal F-actin binding protein, drebrin, in specialized cells of stomach and kidney epithelia.J Cell Sci. 2000; 113: 325-336Google Scholar). This protein, originally detected in neuronal cells (acronym for developmentally regulated brain protein), can occur in four isoforms, drebrin E1, E2, A, and sA (review:Shirao, 1995Shirao T. The roles of microfilament-associated proteins, drebrins, in brain morphogenesis: A review.J Biochem (Tokyo). 1995; 117: 231-236Google Scholar), only one of which, E2, has so far been identified in non-neuronal cells. In neuronal cells, the drebrins appear to be involved in the formation of cell processes and in synaptic plasticity (e.g.,Takahashi et al., 2003Takahashi H. Sekino Y. Tanaka S. Mizui T. Kishi S. Shirao T. Drebrin-dependent actin clustering in dendritic filopodia governs synaptic targeting of postsynaptic density-95 and dendritic spine morphogenesis.J Neurosci. 2003; 23: 6586-6595Google Scholar). Correspondingly, transfection of the drebrin cDNA can cause formations of long actin-rich protrusions and remodeling of the actin cytoskeleton (e.g.,Shirao et al., 1992Shirao T. Kojima N. Obata K. Cloning of drebrin A and induction of neurite-like processes in drebrin-transfected cells.Neuroreports. 1992; 3: 109-112Google Scholar; Hayashi and Shirao, 1999Hayashi K. Shirao T. Change in the shape of dendritic spines caused by overexpression of drebrin in cultured cortical neurons.J Neurosci. 1999; 19: 3918-3925Google Scholar; Keon et al., 2000Keon B.H. Jedrzejewski P.T. Paul D.L. Goodenough D.A. Isoform specific expression of the neuronal F-actin binding protein, drebrin, in specialized cells of stomach and kidney epithelia.J Cell Sci. 2000; 113: 325-336Google Scholar), whereas antisense cDNA results in suppression of neurite outgrowth (Toda et al., 1999Toda M. Shirao T. Uyemura K. Suppression of an actin-binding protein, drebrin, by antisense transfection attenuates neurite outgrowth in neuroblastoma B104 cells.Brain Res Dev Brain Res. 1999; 114: 193-200Google Scholar). In epithelial and endothelial cells, drebrin has been localized primarily to intercellular junctions (Peitsch et al., 1999Peitsch W.K. Grund C. Kuhn C. Drebrin is a widespread actin-associating protein enriched at junctional plaques, defining a specific microfilament anchorage system in polar epithelial cells.Eur J Cell Biol. 1999; 78: 767-778Google Scholar), whereas in a series of other cell types, it appears to be accumulated in actin filament-rich cell processes (Peitsch et al., 2001Peitsch W.K. Hofmann I. Prätzel S. et al.Drebrin particles: Components in the ensemble of proteins regulating actin dynamics of lamellipodia and filopodia.Eur J Cell Biol. 2001; 80: 567-579Google Scholar,Peitsch et al., 2003Peitsch W.K. Hofmann I. Endlich N. et al.Cell biological and biochemical characterization of drebrin complexes in mesangial cells and podocytes of renal glomeruli.J Am Soc Nephrol. 2003; 14: 1452-1463Google Scholar). Drebrin has so far not been detected in the skin and in skin tumors. Moreover, very little is known about its distribution and function in stratified epithelia. Therefore, we have generated a novel monoclonal drebrin antibody that could be used, in combination with other antibodies, to study the distribution of drebrin in normal skin, epithelial skin tumors, and cultured keratinocytes. In addition, we have transfected cells of a related cell type, the vulvar carcinoma line A431 devoid of endogenous drebrin, with cDNA combining drebrin E2 with enhanced green fluorescent protein (EGFP) to allow the study of this protein by immunocytochemistry and live cell microscopy. In previous studies, we had noted drebrin staining along intercellular junctions of certain epithelial and endothelial cell types (Peitsch et al., 1999Peitsch W.K. Grund C. Kuhn C. Drebrin is a widespread actin-associating protein enriched at junctional plaques, defining a specific microfilament anchorage system in polar epithelial cells.Eur J Cell Biol. 1999; 78: 767-778Google Scholar,Peitsch et al., 2003Peitsch W.K. Hofmann I. Endlich N. et al.Cell biological and biochemical characterization of drebrin complexes in mesangial cells and podocytes of renal glomeruli.J Am Soc Nephrol. 2003; 14: 1452-1463Google Scholar). But not much was known about drebrin in stratified squamous epithelia, including the epidermis, and tumors derived therefrom. Therefore, examination of the presence, localization, and dynamics of this protein in human skin and related tissues as well as SCC was overdue. As, in our former studies, the only available monoclonal antibody (mab) to drebrin (clone M2F6) had worked well on cultured cells but sometimes showed reactions that were too faint or of dubious significance on tissue sections, we decided to generate a novel mab against drebrin that reliably reacted with all isoforms (mab Mx823; see Materials and Methods). This antibody was used in parallel with the mab M2F6, a guinea-pig antiserum directed against the same peptide (dreb4.1 gp) and two further guinea-pig drebrin antisera (dreb254.2 gp and drebE2/A.2 gp). Immunostaining on cryostat and paraffin-embedded sections of normal human skin (Figure 1) showed that the epidermis was essentially negative for drebrin (Figure 1a). Drebrin was, however, enriched in the ducts of eccrine sweat glands, especially along their cell–cell boundaries (Figure 1b), whereas sebaceous glands did not display detectable amounts. In addition, intercellular junctions of hair follicles were stained, both in the bulbs (Figure 1c) and, in upper parts, in a layer apparently corresponding to the outer root sheath (ORS; Figure 1d). As previously reported (Peitsch et al., 1999Peitsch W.K. Grund C. Kuhn C. Drebrin is a widespread actin-associating protein enriched at junctional plaques, defining a specific microfilament anchorage system in polar epithelial cells.Eur J Cell Biol. 1999; 78: 767-778Google Scholar), small blood vessels in the dermis were also drebrin-positive (asterisks in Figures 1a and b). This specific distribution pattern was observed in normal human skin from different parts of the body such as the face, scalp, trunk, and foot sole, and with all the various drebrin antibodies used. To further specify the localization of drebrin within hair follicles, double-label confocal microscopy was performed on cryostat sections of scalp skin, using drebrin antibodies in combination with marker proteins for the different hair follicle compartments. Immunostaining of such sections for drebrin and hair keratin 6hf, an intermediate filament protein specific for the companion layer (Winter et al., 1998Winter H. Langbein L. Praetzel S. et al.A novel human type II cytokeratin, K6hf, specifically expressed in the companion layer of the hair follicle.J Invest Dermatol. 1998; 111: 955-962Google Scholar), showed strong drebrin reactions in the companion layer. In the ORS, drebrin labeling was somewhat weaker (Figures 2a –a″, transverse section; Figure 2b, longitudinal section) and depended on the specific type of fixation (see Materials and Methods). By contrast, when hair follicles were double labeled with antibodies against drebrin and keratin 6irs1, a marker protein for the inner root sheath (IRS;Langbein et al., 2002Langbein L. Rogers M.A. Praetzel S. Aoki N. Winter H. Schweizer J. A novel epithelial keratin, hK6irs1, is expressed differentially in all layers of the inner root sheath, including specialized huxley cells (Flugelzellen) of the human hair follicle.J Invest Dermatol. 2002; 118: 789-799Google Scholar), a differential distribution of both proteins and absence of drebrin from the IRS was noted (data not shown). The same observation was made for drebrin antibodies in combination with a marker specific for the Huxley layer of the IRS, hair keratin 6irs4 (Figure 2c;Langbein et al., 2003bLangbein L. Rogers M.A. Praetzel S. Winter H. Schweizer J. K6irs1, K6irs2, K6irs3, and K6irs4 represent the inner-root-sheath-specific type II epithelial keratins of the human hair follicle.J Invest Dermatol. 2003; 120: 512-522Google Scholar). Drebrin and cytokeratin 14, one of the intermediate filament proteins synthesized in the ORS, were found in the same layer but, as expected, in a different localization: Drebrin was accumulated along cell boundaries, whereas cytokeratin 14 was positive throughout the cytoplasm (Figure 2d). As the gene expression patterns of several junction-associated proteins are modified in skin tumors, our next aim was to examine the distribution of drebrin in such tumors and in precancerous lesions. Sections of frozen and paraffin-embedded BCC, SCC, keratoacanthomas, and precursor lesions such as actinic keratoses were systematically examined, using drebrin-specific antibodies (Table I). In BCC, intense drebrin reactivity was observed in all specimens examined (Figure 3a). This conspicuously positive reaction was seen throughout the entire tumor on formaldehyde-fixed cryostat sections and paraffin-embedded tissues. On cryostat sections fixed with acetone, the carcinoma cells located at the periphery of the BCC sometimes appeared enhanced, whereas reactions within the tumors were slightly weaker (not shown).Table IDrebrin distribution in epithelial skin tumors and precancerous lesions, compared with psoriasis and seborrheic keratosesSpecimenNumber of samplesDrebrin distributionBCC13 (from 12 patients)Twelve of 13 (both nodular and sclerodermiforme BCC): Intense and homogenous staining at cell borders throughout the tumor Nodular11One nodular BCC (recurrence): intense and homogeneous staining in one area, whereas other regions show only weak cell border staining Sclerodermiforme2SCC11 (from 10 patients)Eleven of 11: significant cell border staining in certain cell groups, other cell groups drebrin-negative Well differentiated (G1)6G1 tumors: staining occasionally increased staining near horn pearls Moderately differentiated (G2)4G2 and G3 tumors: in some tumors, staining at invasive margins; in average, less drebrin-positive cell groups than in SCC of G1 Poorly differentiated (G3)1 (G2–3)Keratoacanthomas2Two of two: cell borders intensely drebrin-positive in certain regions; other areas completely negativeActinic keratoses5Five of five: some dysplastic keratinocytes in the basal epidermis drebrin-positive; number of reactive cells and intensity variablePsoriasis9Nine of 9: no drebrin reactions within the epidermis; small dermal blood vessel show cell border staining Psoriasis vulgaris8 Psoriasis pustulosa palmoplantaris1Seborrheic keratoses4Four of four: drebrin-negativeBCC, basal cell carcinoma; SCC, squamous cell carcinoma. Open table in a new tab BCC, basal cell carcinoma; SCC, squamous cell carcinoma. In contrast to the strong and near-homogeneous drebrin immunostaining of BCC, the drebrin distribution appeared to be mostly inhomogeneous in SCC: usually, rather intense drebrin reactions were noted along cell borders in certain parts of the tumor, whereas other parts of the same tumor appeared to be negative for drebrin (Figure 3b). The drebrin-positive tumor regions were mostly found at the invasive tumor front, but sometimes also occurred near horn pearls of differentiated squamous carcinomas, in a distribution that did not display a correlation to the specific degree of differentiation. To examine whether the drebrin-rich cells within an SCC represented the highly proliferative fraction, double immunolabeling was performed with drebrin antibodies in combination with others reactive with the proliferation marker Ki67. Reactions for Ki67 were found in numerous cells at the invasive tumor margins, but the Ki67-positive cells were not identical to the drebrin-positive ones (data not shown). The same phenomenon as in SCC, i.e. mosaicism of intensely drebrin-positive and drebrin-negative tumor regions, was observed in keratoacanthomas (Figures 3c,d). Precancerous lesions such as actinic keratoses showed similar heterogeneity. Here, in some regions, the basal keratinocytes revealed some cell border drebrin staining, whereas other basal regions and the upper epidermal layers remained drebrin-negative (Table I). To clarify whether drebrin synthesis might be correlated with epidermal hyperproliferation in general, biopsy tissues from patients with psoriasis were immunostained. In all of the psoriasis samples examined, the epidermis was practically devoid of drebrin, indicating that drebrin most likely is not related to hyperproliferation per se. Moreover, none of the seborrheic keratoses examined revealed significant amounts of drebrin (Table I). We wondered whether the staining pattern observed here was also applicable for other actin-binding proteins. Therefore, we immunostained sections of human skin and skin tumors with antibodies to ezrin, a protein sharing certain features with drebrin such as a prolin-rich, profilin-binding domain and the tendency to accumulate in actin-rich cell protrusions and in the cell cortex (for a review, seeBretscher et al., 2002Bretscher A. Edwards K. Fehon R.G. ERM proteins and merlin: Integrators at the cell cortex.Nat Rev Mol Cell Biol. 2002; 3: 586-599Google Scholar). In normal skin, ezrin was enriched at the junctions of keratinocytes in all epidermal layers, in sebaceous glands, eccrine sweat glands, and hair follicles, and also at cell borders in endothelia of small blood vessels (data not shown). Immunostaining of different epithelial skin tumors and precancers showed strong ezrin reactions at cell boundaries and, although weaker, in the cytoplasm of BCC, SCC, keratoacanthomas, and actinic keratoses, homogeneously throughout the lesions (not shown). When cryostat sections of BCC were double stained for drebrin and actin, almost complete co-localization of both proteins along intercellular borders was found (Figures 4a –a″). Double immunostaining with different marker proteins of adhering junction plaques such as α-catenin, β-catenin, and plakoglobin showed a far-reaching overlap with drebrin (data not shown). In contrast, the desmosomal plaque protein, desmoplakin, was completely differently distributed, showing the typical punctuate desmosomal arrays whereas drebrin immunostaining displayed a more linear and, in some regions, distinctive interdesmosomal pattern (Figures 4b –b″). When BCC were labeled for proteins of tight junctions such as ZO-1, ZO-2, and occludin, no significant reaction was noted. Claudin-1 was enriched along cell boundaries, but mostly differently distributed from drebrin (not shown; for localization of claudin-1 in SCC, seeLangbein et al., 2003aLangbein L. Pape U.F. Grund C. et al.Tight junction-related structures in the absence of a lumen: Occludin, claudins and tight junction plaque proteins in densely packed cell formations of stratified epithelia and squamous cell carcinomas.Eur J Cell Biol. 2003; 82: 385-400Google Scholar). With antibodies against the gap junction protein connexin 43 faint, if any, staining and no overlap with drebrin was seen in BCC (see alsoTada and Hashimoto, 1997Tada J. Hashimoto K. Ultrastructural localization of gap junction protein connexin 43 in normal human skin, basal cell carcinoma, and squamous cell carcinoma.J Cutan Pathol. 1997; 24: 628-635Google Scholar), whereas keratinocytes in the overlying epidermis displayed intense typical gap junction-like reactions, serving as an internal control (not shown). As other authors have recently observed co-localization and biochemical interaction of drebrin and connexin 43 at gap junctions of astrocytes and Vero cells (Butkevich et al., 2004Butkevich E. Hulsmann S. Wenzel D. Shirao T. Duden R. Majoul I. Drebrin is a novel connexin-43 binding partner that links gap junctions to the submembrane cytoskeleton.Curr Biol. 2004; 14: 650-658Google Scholar), we double immunostained various cultured cells, including primary human keratinocytes, mammary carcinoma cells of line MCF-7, and endothelial human umbilical vein endothelial cells for drebrin and connexin 43 but, again, mutually exclusive localization was noted. As our double label laser scanning results indicated accumulation of drebrin near adhering junctions, we also performed immunoelectron microscopy (Figures 5b –e). On sections of BCC, immunogold labeling for drebrin was detected in the microfilament network underlying the plaques of adhering junctions, usually close to the plaques, whereas desmosomes were not significantly labeled. For comparison, immunoelectron microscopy with antibodies to β-catenin was performed, which showed a typical junctional plaque reaction, typically closer to the plasma membrane than drebrin (data not shown). To compare the amounts of drebrin in the skin and in skin tumors, we performed immunoblot analysis of total protein lysates from normal skin (Figures 6a –a ″,lane 1), scalp (lane 2), BCC (lane 3), squamous carcinomas (lane 4), melanoma metastases (lane 5), and leiomyosarcoma (lane 6). An immunoreactive 120 kDa band corresponding to drebrin was hardly detecTable In normal skin and scalp but was drastically increased in all tumors examined. Comparably high amounts of drebrin were found in lysates of BCC, corresponding to our immunostaining results, and in SCC, despite the microscopically obse