T-cadherin enhances cell-matrix adhesiveness by regulating beta1 integrin trafficking in cutaneous squamous carcinoma cells.

T-cadherin is a glycosyl-phosphatidylinositol (GPI) anchored cadherin molecule. We previously reported that T-cadherin is normally expressed on the basal keratinocytes of the epidermis and is down-regulated in cutaneous squamous cell carcinoma (SCC). We found that expression of T-cadherin in cutaneous squamous carcinoma cells regulated level of surface beta1 integrin, which functioned as extracellular matrix (ECM) receptor. Involvement of T-cadherin in beta1 integrin trafficking was studied using three different stable cell lines with cytomegalovirus (CMV)-driven over-expression, tetracycline (Tet)-inducible expression and RNAi-mediated suppressed expression of T-cadherin. Pulse-chase analysis using a cholesterol-depleting reagent and a tyrosine kinase inhibitor showed that beta1 integrin mainly internalized via caveolae. Over-expression of T-cadherin suppressed the internalization of both beta1 integrin and cholera toxin (CTX), a marker of caveolae-mediated endocytosis. By Western blot analysis of tyrosine-kinase target molecules, we demonstrated a reduced level of EGF receptor (EGFR)-phosphorylation in T-cadherin over-expressing cells. In addition, studies using EGF and EGFR specific inhibitors revealed that EGFR activation stimulated beta1 integrin internalization. Taking these results together, T-cadherin may modulate cell-matrix adhesion in basal keratinocytes as well as invasive potency in SCC by regulating surface level of beta1 integrin.

T-cadherin negatively regulates the proliferation of cutaneous squamous carcinoma cells.

T-cadherin is a unique member of the cadherin superfamily that lacks the transmembrane and cytoplasmic domains, and is instead linked to the cell membrane via a glycosyl-phosphatidylinositol anchor. We previously reported that T-cadherin was specifically expressed on the basal keratinocytes of the epidermis, and the expression of T-cadherin was significantly reduced in invasive cutaneous squamous cell carcinoma (SCC) and in the lesional skin of psoriasis vulgaris. In this study, to obtain an insight into the role of T-cadherin in keratinocytes, we used transfection methods and examined the effect of overexpression or knockdown of T-cadherin in immortalized keratinocyte cell lines derived from SCC. T-cadherin overexpressed cells showed clearly reduced cell proliferation, but the influence of cell-cell adhesiveness and cell mobility was not detected. Using a tetracycline-regulated expression system, we also confirmed that the suppression of cell proliferation was dependent on the expression level of T-cadherin. Cell cycle analysis demonstrated that over expression of T-cadherin induced a delay in the G(2)/M phase. Our findings suggest that T-cadherin acts as an endogenous negative regulator of keratinocyte proliferation and its inactivation is the cause for keratinocyte hyperproliferation in SCC or in psoriasis vulgaris.

Development of both human connective tissue-type and mucosal-type mast cells in mice from hematopoietic stem cells with identical distribution pattern to human body.

The transplantation of primitive human cells into sublethally irradiated immune-deficient mice is the well-established in vivo system for the investigation of human hematopoietic stem cell function. Although mast cells are the progeny of hematopoietic stem cells, human mast cell development in mice that underwent human hematopoietic stem cell transplantation has not been reported. Here we report on human mast cell development after xenotransplantation of human hematopoietic stem cells into nonobese diabetic severe combined immunodeficient (NOD/SCID)/gamma(c)(null) (NOG) mice with severe combined immunodeficiency and interleukin 2 (IL-2) receptor gamma-chain allelic mutation. Supported by the murine environment, human mast cell clusters developed in mouse dermis, but they required more time than other forms of human cell reconstitution. In lung and gastric tract, mucosal-type mast cells containing tryptase but lacking chymase located on gastric mucosa and in alveoli, whereas connective tissue-type mast cells containing both tryptase and chymase located on gastric submucosa and around major airways, as in the human body. Mast cell development was also observed in lymph nodes, spleen, and peritoneal cavity but not in the peripheral blood. Xenotransplantation of human hematopoietic stem cells into NOG mice can be expected to result in a highly effective model for the investigation of human mast cell development and function in vivo.

A monoclonal antibody established from the immunization of basal cell carcinoma (BCC) tissues reacts to the intercellular space of BCC and hair follicles.

To detail the histogenetic relationship between basal cell carcinoma (BCC) and hair follicles, a monoclonal antibody, coded as MMKB-1 and established from immunization of mice by human basal cell carcinoma (BCC) tissues, was immunohistochemically studied in tissues of BCC and squamous cell carcinoma (SCC) of the skin, as well as in normal human skin tissues. On 1M-NaCl-split normal human skin tissues, it reacted against both the epidermal and the dermal sides of the basement membrane zone (BMZ). This monoclonal antibody reacted to the BMZ and intercellular space (ICS) of the solid, superficial, and fibrosing types of BCC cell nests, but, in SCC tumor cell nests, it reacted exclusively to the BMZ. Immunoelectron microscopic studies revealed that the corresponding antigen of the monoclonal antibody was distributed in the hemidesmosomes and the anchoring fibrils along the BMZ of the normal human skin and the desmosomes of the BCC cell nest. The monoclonal antibody also reacted to the ICS of the lower outer root sheath and hair matrix. We discussed the histogenesis of BCC and hair follicles, referring to the results of the staining patterns of MMKB-1 monoclonal antibody and to other studies suggesting a histogenetic relationship between BCC and hair follicles.

Loss of T-cadherin (CDH13, H-cadherin) expression in cutaneous squamous cell carcinoma.

We previously reported that T-cadherin (CDH13, H-cadherin), a unique cadherin molecule, was expressed on the basal cell layer in normal murine and human epidermis. In the present study, T-cadherin expression in archival human skin specimens comprising a spectrum of human squamous cell neoplasia was investigated. T-cadherin expression was observed in both normal epidermal basal cells and adnexal epithelial cells of formalin-fixed and paraffin-embedded tissue sections. Western immunoblotting also revealed that mature T-cadherin protein was expressed in cultured human skin tissue equivalent. Atypical keratinocytes in 27 of 53 specimens of actinic keratosis and 23 of 30 specimens of Bowen's disease expressed T-cadherin. In contrast, T-cadherin was focally expressed in 6 of 56 invasive cutaneous squamous cell carcinomas. To explore the molecular mechanism of down-regulation of T-cadherin expression in invasive squamous cell carcinoma, loss of heterozygosity, genetic alternations, and methylation status in the 5' region of the T-cadherin gene were investigated. Loss of heterozygosity at intron 1 of the T-cadherin gene was observed in 8 of 28 informative cases of invasive squamous cell carcinoma. Although no structural genomic alternations were found by sequence analysis, aberrant promoter methylation of the T-cadherin gene was found in 12 of 28 invasive squamous cell carcinomas. T-cadherin expression was restored in cultured A431 cells, in which aberrant methylation was found by treatment with the demethylating agent 5'-aza-2-deoxycytidine. These findings suggest that a combination of deletion and aberrant methylation of the T-cadherin gene may play a role in loss of gene expression in a considerable number of invasive cutaneous squamous cell carcinomas.

Expression of desmoglein 1 compensates for genetic loss of desmoglein 3 in keratinocyte adhesion.

The desmoglein compensation hypothesis, namely that one desmoglein can compensate for loss of function of another, has been proposed to explain the tissue specificity of the autoantibody-induced loss of cell adhesion in pemphigus. To validate this hypothesis genetically, we used desmoglein-3 knockout mice (DSG3-/-) that lose their telogen hair prematurely due to loss of adhesion between keratinocytes of the telogen hair club and the outer root sheath, where the only desmoglein expressed in normal mice is desmoglein-3. To determine if desmoglein-1 could substitute for the function of desmoglein-3 in telogen hair, we produced transgenic mice that express desmoglein-1 driven off the keratin 14 promoter, and then bred the transgene (TG) into DSG3-/- mice. Immunoblotting showed transgene expression in skin, and immunofluorescence showed desmoglein-1 in the telogen club of DSG3-/-TG+ but not DSG3-/-TG- mice. DSG3-/-TG- mice lost telogen hair with each wave of telogen, whereas DSG3-/-TG+ mice had markedly delayed and decreased hair loss. DSG3-/- mice also show low weights due to blisters in the oral mucosa. Surprisingly, DSG3-/-TG+ mice showed similar low weights, because the transgene, although expressed in skin, was not well expressed in oral mucous membranes. These studies show that desmoglein-1 can compensate for loss of desmoglein-3-mediated adhesion, and provide genetic evidence confirming the desmoglein compensation hypothesis.

Expression of T-cadherin in Basal keratinocytes of skin.

T-cadherin is a unique member of the cadherin superfamily that shares the ectodomain organization with classical cadherins, but lacks both transmembrane and cytoplasmic regions and is instead anchored to the plasma membrane through a glycosyl-phosphatidylinositol (GPI) moiety. The function of T-cadherin has not been revealed yet. The special structure of T-cadherin might endow this molecule with specific intracellular targeting properties and functions that are distinct from classical cadherins. T-cadherin was originally cloned from chicken embryo brain and then was also found in mouse and human nervous and cardiovascular systems; however, T-cadherin in the keratinocytes and skin tissue is still an unknown area that remains to be explored. To test whether the unusual truncated T-cadherin is expressed in keratinocytes, we performed the reverse transcriptase-polymerase chain reaction of T-cadherin, as well as several classical cadherins (E-, P-, and N-cadherin), on the mouse keratinocyte cell line Pam212, fibroblast NIH3T3, and melanoma cell B16. The result indicated that mouse keratinocytes expressed the mRNA of truncated T-cadherin apart from classical cadherins, E-, and P-cadherin. To confirm the expression of T-cadherin in mouse keratinocytes, immunocytochemistry staining was carried out on Pam212 cells by using rabbit anti-T-cadherin antibody and rat antimouse E- and P-cadherin antibody. The result of immunofluorescence staining proved that T-cadherin was expressed in mouse keratinocytes. In order to analyze the distribution patterns of T-cadherin and classical cadherins on the keratinocytes, 3D scanning was performed by using a confocal microscope. From the Z-sections and XZ-sections, it was clearly demonstrated that T-cadherin was distributed diffusely on the whole cell surface, while E- and P-cadherin were concentrated on the cell-cell contacts. To examine the expression and the localization of T-cadherin on skin tissue, the frozen sections of the mouse back skin were immunohistochemically labeled by using anti-T-cadherin antibody. It was found that T-cadherin was intensively expressed only on the basal cell layer of the mouse skin. Apart from mouse keratinocytes and mouse skin, we further examined the expression of T-cadherin in human keratinocytes and human skin by western blot, immunocytochemistry, and immunohistochemistry staining. The same results were achieved with human samples. In this study, we found and verified that T-cadherin was expressed on the mouse and human keratinocytes and specifically localized on the basal cell layer of skin. The nature of T-cadherin function and its mechanism of localization at the basal cell layer of skin are important issues to be addressed concerning this unique member of the cadherin family and its physiologic and pathologic roles in the skin.

Neutrophils from MMP-9- or neutrophil elastase-deficient mice show no defect in transendothelial migration under flow in vitro.

Recent evidence has suggested a role for neutrophil proteases during certain inflammatory responses. We demonstrated previously that neutrophil proteases can degrade components of the adherens junctions during neutrophil-endothelial adhesion. We tested the hypothesis that degradation of VE-cadherin at lateral junctions by elastase or MMP-9 facilitates neutrophil transendothelial migration. Neutrophils from MMP-9 or elastase null mice and strain-matched control mice expressed high levels of LFA-1, Mac-1, and L-selectin on their cell surface. Under flow conditions, wild-type and deficient neutrophils rolled, arrested, and transmigrated activated murine endothelium. There was no difference in the total numbers of interacting neutrophils or in the percentage of transmigrated cells. In addition, deficient neutrophils remained capable of degrading murine endothelial VE-cadherin. These results indicate that although neutrophil proteases may play a role in the acute inflammatory response, neutrophil elastase or MMP-9 is not essential for neutrophil transendothelial migration in this murine system.