Podoplanin is indispensable for cell motility and platelet-induced epithelial-to-mesenchymal transition-related gene expression in esophagus squamous carcinoma TE11A cells.

BACKGROUND: The transmembrane glycoprotein podoplanin (PDPN) is upregulated in some tumors and has gained attention as a malignant tumor biomarker. PDPN molecules have platelet aggregation-stimulating domains and, are therefore, suggested to play a role in tumor-induced platelet activation, which in turn triggers epithelial-to-mesenchymal transition (EMT) and enhances the invasive and metastatic activities of tumor cells. In addition, as forced PDPN expression itself can alter the propensity of certain tumor cells in favor of EMT and enhance their invasive ability, it is also considered to be involved in the cell signaling system. Nevertheless, underlying mechanisms of PDPN in tumor cell invasive ability as well as EMT induction, especially by platelets, are still not fully understood. METHODS: Subclonal TE11A cells were isolated from the human esophageal squamous carcinoma cell line TE11 and the effects of anti-PDPN neutralizing antibody as well as PDPN gene knockout on platelet-induced EMT-related gene expression were measured. Also, the effects of PDPN deficiency on cellular invasive ability and motility were assessed. RESULTS: PDPN-null cells were able to provoke platelet aggregation, suggesting that PDPN contribution to platelet activation in these cells is marginal. Nevertheless, expression of platelet-induced EMT-related genes, including vimentin, was impaired by PDPN-neutralizing antibody as well as PDPN deficiency, while their effects on TGF-ß-induced gene expression were marginal. Unexpectedly, PDPN gene ablation, at least in either allele, engendered spontaneous N-cadherin upregulation and claudin-1 downregulation. Despite these seemingly EMT-like alterations, PDPN deficiency impaired cellular motility and invasive ability even after TGF-ß-induced EMT induction. CONCLUSIONS: These results suggested that, while PDPN seems to function in favor of maintaining the epithelial state of this cell line, it is indispensable for platelet-mediated induction of particular mesenchymal marker genes as well as the potentiation of motility and invasion capacity.

A pull-down and slot blot-based screening system for inhibitor compounds of the podoplanin-CLEC-2 interaction.

Podoplanin, a transmembrane glycoprotein, is overexpressed in certain types of tumors and induces platelet aggregation by binding to C-type lectin-like receptor 2 (CLEC-2) on the platelet membrane. Activated platelets release granule components, which in turn, trigger epithelial-mesenchymal transition and confer invasive capacity to the tumor cells. Therefore, blocking the podoplanin-CLEC-2 interaction by a small-molecule compound is a potential therapeutic strategy to prevent cancer metastasis and invasion. To effectively identify such inhibitory compounds, we have developed a pull-down-based inhibitory compound screening system. An immunoglobulin Fc domain-CLEC-2 fusion protein was used as a bait to capture podoplanin derived from podoplanin-overexpressing HeLa cells in the presence and absence of the test compound. The protein complex was then pulled down using protein A beads. To shorten the turnaround time, increase throughput, and decrease the workload for the operators, centrifugal filter units were employed to separate free and bound podoplanin, instead of using customary aspiration-centrifugation washing cycles. Slot blotting was also utilized in lieu of gel electrophoresis and electrical transfer. Thus, the use of our pull down screening system could facilitate the effective selection of potential inhibitor compounds of the podoplanin-CLEC-2 interaction for cancer therapy. Importantly, our methodology is also applicable to targeting other protein-protein interactions.

Introduction of human ß-defensin-3 into cultured human keratinocytes and fibroblasts by infection of a recombinant adenovirus vector.

Cultured epidermal autografts and cultured skin substitute are vulnerable to infection. Human beta defensin (HBD)-3 is an antimicrobial peptide that exhibits a wide-spectrum antimicrobial activity against gram-positive/negative bacteria and fungi. This study determined whether normal human keratinocytes (NHKs) and human dermal fibroblasts (HDFs) transfected with the HBD-3 gene secrete HBD-3 peptide with an antimicrobial activity. An adenovirus vector with an HBD-3 cDNA inserted downstream of the CMV promoter (ADhBD3) was created. The HBD-3 gene was introduced into NHKs and HDFs via ADhBD3 infection. HBD-3 gene expression in each type of transfected cells was evaluated by RT-PCR. The presence of HBD-3 peptide in the culture supernatants of each type of transfected cells was evaluated by Western blotting. The antimicrobial activities of the culture supernatants of each type of transfected cells against several bacterial strains were also measured. Both NHKs and HDFs infected with ADhBD3 expressed the HBD-3 gene and secreted HBD-3 peptide into culture supernatants. These supernatants exhibited a strong bacteriocidal activity against a Staphylococcus aureus reference strain and methicillin-resistant S. aureus (MRSA). NHKs and HDFs transfected with the HBD-3 gene secrete HBD-3 peptide with an antimicrobial activity against S. aureus and MRSA.

The effectiveness of basic fibroblast growth factor in fibrin-based cultured skin substitute in vivo.

Cultured skin substitute (CSS), comprised keratinocytes and fibroblasts in a biopolymer matrix, is useful for adjunctive burn therapy. However, the vascularization of CSS is much slower than split-thickness autografts, because it lacks a vascular plexus. This study evaluated the influence of basic fibroblast growth factor (bFGF) on fibrin-based CSS grafting in vivo. Fibrin-based CSS treated with 0, 0.26, 1.3, 6.5, 13, or 130 microg/cm bFGF was transplanted into athymic mice, and macroscopic and histologic examinations of the graft were performed on day 21 posttransplantation. Engrafted CSS of the 0.26 to 6.5 microg/cm bFGF treatment groups were similar to the untreated control. However, the engrafted area was significantly suppressed in the 13 microg/cm bFGF treatment group, and the 130 microg/cm bFGF treatment group was not engrafted. Neovascularization of CSS was significantly increased in the 1.3 microg/cm bFGF treatment group compared with the control (P < .05). The number of human fibroblastic cells in CSS that were positive for vimentin increased significantly in the 0.26 and 1.3 microg/cm bFGF treatment groups (P < .01). CSS treated with 0.26 to 6.5 microg/cm bFGF showed normal epidermis with keratinizing stratified squamous epithelium, whereas the thickness of the epidermis and proliferation of keratinocytes in the basal layer was decreased. These results demonstrated that bFGF treatment (1.3 microg/cm) in fibrin-based CSS may enhance angiogenesis and fibroblast proliferation after transplantation.

Effect of dermal fibroblasts on long-term maintenance of regenerating xenotransplanted human esophageal epithelial cells in immunodeficient mice.

OBJECTIVE: Artificial esophagus of various materials has been tried without satisfactory results. Effects of fibroblasts derived from human dermis on cultured human esophageal epithelial cells were investigated regarding their preservation over time. METHODS: Human esophageal epithelial cells (EE) were subcutaneously injected either alone or with human esophageal fibroblasts (EF) or human dermal fibroblasts (DF) into the flank of the BALB/cA-nu, scid mice of 8 to 12 weeks of age. At 10 days, 3, 6 and 48 weeks after transplantation, randomly chosen mice were sacrificed and the rates of nodule formation at the injected sites were compared. Nodules were examined histologically by hematoxylin and eosin staining and immunohistochemically with anti- Ki-67 and anti-p63 antibodies. RESULTS: The rate of subcutaneous epidermoid cyst formation at 10 days was 33% for EE, and 67% for EE with EF, and 100% for EE with DF transplantation. At 6 weeks after transplantation, subcutaneous cysts were not found for EE alone or EE with EF, but were detected in 100% of the mice transplanted EE with DF and still preserved at 12 months. CONCLUSIONS: Long-term preservation of regenerated esophageal epithelium in vivo after transplantation of cultured esophageal epithelial cells is possible by co-transplantation of human dermal fibroblasts.

Induction of vascular endothelial growth factor by fibrin as a dermal substrate for cultured skin substitute.

In the initial phase of wound healing, endogenous fibrin clots are known to form a provisional matrix and to promote angiogenesis. Growth factors such as vascular endothelial growth factor (VEGF) increase in wounds to stimulate angiogenesis. However, it remains unknown whether VEGF is induced when fibrin is used as a dermal substrate for cultured skin substitutes. The authors investigated the effect of fibrin gel as a dermal substrate for a cultured skin substitute, using human keratinocytes and dermal fibroblasts. A collagen-cultured skin substitute was also examined for comparison. VEGF in the culture supernatant in both types was measured by enzyme-linked immunosorbent assay, and VEGF mRNA was determined semiquantitatively by reverse-transcriptase polymerase chain reaction after 2 days of incubation. Experiments were performed using 12 cultured skin substitutes: four for histologic examination before transplantation, four for VEGF assay in vitro, and four for the transplantation to athymic mice. Three independent experiments were performed for each step. VEGF concentration in the fibrin-cultured supernatant was 84.3 +/- 11.8 pg/ml, whereas it was 27.8 +/- 4.68 pg/ml in the case of the collagen substrate. The relative levels of VEGF mRNA were 1.088 +/- 0.100 and 0.698 +/- 0.226, respectively. In in vivo transplantation, the fibrin-type cultured skin substitute showed an excellent take on the wound bed, and a normally proliferating keratinocyte layer with emergence of vascular endothelial cells in the transplanted floor was seen 3 days after transplantation. Vascular endothelial cells, which were identified using alkaline phosphatase stain, were significantly increased in the fibrin-type cultured skin substitute. The use of fibrin as a dermal substrate for cultured skin substitute increases the secretion of VEGF, improves regeneration of mature epidermal structure after in vivo transplantation, and promotes the migration of vascular endothelial cells.