Hisataka Kitano

Discoidin domain of Del1 protein contributes to its deposition in the extracellular matrix

The extracellular matrix (ECM) acts as a critical factor during morphogenesis. Because the organization of the ECM directly influences the structure of tissues and organs, a determination of the way that ECM organization is regulated should help to clarify morphogenesis. We have analyzed the assembly of Del1, an ECM protein produced by endothelial cells in embryos, in the ECM. Del1 consists of three epidermal growth factor repeats (E1–E3) at its N-terminus and two discoidin domains (C1, C2) at its C-terminus. Experiments with various deletion mutants of Del1 have revealed that fragments containing the C-terminus of C1, which has a lectin-like structure, direct deposition in the ECM. The efficiency of deposition varies according to the presence of other domains in Del1. A fragment containing E3 and C1 has the strongest deposition activity, whereas fragments containing C2, which is highly homologous to C1, have low deposition activity. Digestion of ECM with hyaluronidase from bovine testis releases Del1 from the ECM, suggesting that glycosaminoglycans are involved in the deposition of Del1. In vivo gene transfer experiments have shown that fusion with the deposition domain of Del1 dramatically alters the distribution of exogenous proteins in mice. Thus, the extent of Del1 deposition may modify the organization of the ECM.

The extracellular matrix protein Del1 induces apoptosis via its epidermal growth factor motif

Mouse Del1 is an extracellular matrix protein mainly expressed in the developing embryo. Del1 has three EGF motifs and two discoidin domains. The second EGF motif reportedly contains an RGD sequence that binds to integrin receptors. Here, we provide evidence that Del1 protein induces cell death in vitro. Chromatin condensation and DNA laddering were observed, suggestive of apoptosis. The results of analysis using the TUNEL method and annexin V staining were also consistent with apoptosis. The apoptosis-inducing activity of Del1 could be mapped to the third EGF motif, which fitted the consensus sequence CX(D/N)XXXX(F/Y)XCXC, wherein the aspartic acid residue (D) could be beta-hydroxylated. As little as twenty-five picomolar of recombinant E3 could induce apoptosis.

Overexpression of integrin αv facilitates proliferation and invasion of oral squamous cell carcinoma cells via mek/erk signaling pathway that is activated by interaction of integrin αvβ8 with type I collagen

To examine the role of integrin αv subunit in the progression of squamous cell carcinoma (SCC), oral SCC cells were stably transfected with integrin αv cDNA. Integrin αv transfectants exhibited the enhancement of proliferation on type Ⅰ collagen, and seemed to have a high ability to invade type Ⅰ collagen gel. Overexpression of integrin αv led to rapid phosphorylation of focal adhesion kinase (FAK), mitogen‑activated protein kinase kinase 1/2 (MEK1/2) and extracellular signal‑regulated kinase 1/2 (ERK1/2) in SCC cells on type Ⅰ collagen. The downregulation of integrin β8 in integrin αv transfectants by its specific antisense oligonucleotide led to a decrease in type Ⅰ collagen‑stimulated activation of FAK and the MEK/ERK signaling pathway, and also suppressed the proliferation on type Ⅰ collagen and the invasiveness into type Ⅰ collagen gel. Moreover, the expression of integrin β8 was induced following transfection with integrin αv cDNA. These results indicated that the overexpression of integrin αv induces integrin αvβ8 heterodimer formation and the binding of integrin αvβ8 to type Ⅰ collagen might enhance the proliferation and invasion of SCC cells via the activation of the MEK/ERK signaling pathway.

Coagulation factor IX regulates cell migration and adhesion in vitro.

Coagulation factor IX is thought to circulate in the blood as an inactive zymogen before being activated in the coagulation process. The effect of coagulation factor IX on cells is poorly understood. This study aimed to evaluate the effects of intact coagulation factor IX and its cleavage fragments on cell behavior. A431 cells (derived from human squamous cell carcinoma), Pro5 cells (derived from mouse embryonic endothelial cells), Cos7 cells, and human umbilical vein endothelial cells were utilized in this study. The effects of coagulation factor IX and its cleavage fragments on cell behavior were investigated in several types of experiments, including wound‐healing assays and modified Boyden chamber assays. The effect of coagulation factor IX depended on its processing; full‐length coagulation factor IX suppressed cell migration, increased adhesion to matrix, and enhanced intercellular adhesion. In contrast, activated coagulation factor IX enhanced cell migration, suppressed adhesion to matrix, and inhibited intercellular adhesion. An activation peptide that is removed during the coagulation process was found to be responsible for the activity of full‐length coagulation factor IX, and the activity of activated coagulation factor IX was localized to an EGF domain of the coagulation factor IX light chain. Full‐length coagulation factor IX has a sedative effect on cells, which is counteracted by activated coagulation factor IX in vitro. Thus, coagulation factor IX may play roles before, during, and after the coagulation process.

The Del1 deposition domain can immobilize 3α-hydroxysteroid dehydrogenase in the extracellular matrix without interfering with enzymatic activity

Developing methods that result in targeting of therapeutic molecules in gene therapies to target tissues has importance, as targeting can increase efficacy and decrease off target-side-effects. Work from my laboratory previously showed that the extracellular matrix protein Del1 is organized in the extracellular matrix (ECM) via the Del1 deposition domain (DDD). In this work, a fusion protein with DDD was made to assay the ability to immobilize an enzyme without disrupting enzymatic function. A prostatic cancer-derived cell line LNCap that grows in an androgen-dependent manner was used with 3α-hydroxysteroid dehydrogenase (3 αHD), which catalyzes dihydrotestosterone (DHT). Plasmids encoding a 3αHD:DDD fusion were generated and transfected into cultured cells. The effects of 3αHD immobilized in the ECM by the DDD were evaluated by monitoring growth of LNCap cells and DHT concentrations. It was demonstrated that the DDD could immobilize an enzyme in the ECM without interfering with function.

Efficient nonviral gene therapy with FasL and Del1 fragments in mice

The expression of FasL in cancer cells is currently being explored as a potential cancer therapy. Because high levels of FasL are necessary for effective treatment, current methods typically rely on the use of highly efficient viral vectors. However, because viral vector‐based gene therapy is associated with certain risks, the development of effective nonviral routes for gene delivery would be useful. The present study aimed to improve FasL gene therapy with a nonviral vector by taking advantage of the E3 and C1 domains of Del1 protein, which induces apoptosis and localizes to the extracellular matrix.

The first EGF domain of coagulation factor IX attenuates cell adhesion and induces apoptosis

Activated coagulation factor IX (FIX) attenuated cell adhesion to the extracellular matrix (ECM) and induced apoptosis. This activity was localized to the first epidermal growth factor (EGF) domain, EGF-F9. Experiments with caspase-3 inhibitors revealed that attenuation of adhesion and apoptosis by EGF-F9 were dependent on caspase-3.

Improvement of FasL Gene Therapy In Vitro by Fusing the FasL to Del1 Protein Domains

Gene delivery, transfection, cytotoxicity, and many other factors influence the ability of gene therapy to treat cancer. In addition, as with pharmacologic agents, longer exposure to higher concentrations of gene products should intensify their effects (Wada et al., 2007). Cytotoxic gene products, such as FasL and TRAIL, may remain in tissues after the death of the transfected cells, and they are known to induce apoptosis in both transfected cells and neighbouring cells (Hyer et al., 2003; Kagawa et al., 2001). They have been examined for use in cancer gene therapy and its effects have been examined in vitro and in vivo (Elojeimy et al., 2006; Griffith et al., 2009). FasL delivered via a viral vector can reduce tumour size and improve prognosis in an explanted tumour model.

Pedunculated Haemangioma of the Hard Palate

Abstract Haemangioma is a common benign tumour of the oral cavity, with the tongue, lips, and buccal mucous membranes being the most common sites. However, pedunculated haemangioma of the oral cavity is extremely rare. This report is of a 75-year-old woman with pedunculated haemangioma of the hard palate. A 15 × 15 × 5 mm-tumour was found to the right of the hard palate on examination, which increased in size after 2 months to 25 × 20 × 10 mm. The lesion was excised under local anaesthesia. Histopathological examination revealed vascular differentiation, with small and large vascular channels separated by thin-walled vessels, in the tumour. The diagnosis was capillary haemangioma. Interestingly, the tumour displayed a peduncle leading to the hard palate.

Mandibular Ameloblastic Fibro-odontoma

Abstract We present the case of a 6-year-old boy diagnosed with ameloblastic fibro-odontoma in the mandibular premolar region. The patient underwent excision of the tumour under general anaesthesia. On histopathological study, the tumour was found to be comprised of odontogenic epithelium, mesenchymal tissue, dentin, adamantine and dental cementum, surrounded by ameloblastic fibroma. Interestingly, most of the tumour was comprised of hard tissues, including adamantine, dentin and dental cementum, unlike most ameloblastic fibro-odontomas in which soft tissues dominate.