Akihide Kamegai

Tenascin: Growth and adhesion modulation—Extracellular matrix degrading function: an in Vitro study

Tenascin (TN), a recently characterised extracellular matrix protein, largely confined to the process with the development of embryo in areas of epithelial-mesenchymal interactions and in areas where there are morphogenetic movements and tissue patterning, has a highly restricted expression in adult tissues. The expression of TN is enhanced in a variety of human neoplastic lesions. However, function(s) and molecular mechanisms of enhanced expression in neoplastic lesions remain unclear. We employed human tongue carcinoma cells (SCCKN), human salivary gland adenocarcinoma cells (SGT-1), normal mouse embryonic fibroblasts (NIH3T3-3) and K-ras-2 transformed fibroblasts (Cle-H3) in an in vitro study to elucidate the biological roles of TN. In in vitro studies, all the cell lines examined had enhanced secretion of TN in the presence of transforming growth factor-beta in a dose-dependent manner and TN itself was found to possess a growth-enhancing activity. Moreover, studies on adhesion of the cell lines on coated substrates of fibronectin (FN), laminin (LN), tenascin (TN), TN/FN and TN/LN showed that all the cells adhere and spread well on FN and LN. However, on TN they attach poorly and remain rounded. The relative concentrations of TN and FN affected the cellular adhesion and morphology. In SCCKN and SGT-1, but not in NIH3T3 and Cle-He3 fibroblasts, a higher concentration of TN inhibited cellular adhesion on fibronectin, suggesting that cells attach poorly on TN, it may interfere with the action of fibronectin, and the relative concentrations of TN, FN or LN may affect cellular adhesion and morphology which may differ in different cell types. When TN was added in the growth medium of exponentially growing cells, the cells lost their cell to cell contact and were seen to be separating. The presence of these extracellular matrix proteins were further tested to determine whether they could modulate the secretion of proteolytic enzymes responsible for extracellular matrix degradation by tumour cells, when the neoplastic cells but not the non-neoplastic cells grown on FN/TN substrate showed positive immunofluorescence for collagenase. FN, LN or TN alone did not induce collagenase in the tumour cells. If the same is true in vivo, although a number of factors and interactions may implicate the ultimate outcome, the enhanced expression of TN in neoplastic lesions may have potential implications for tumour growth, differentiation, cellular adhesion, invasion and metastasis.

Pathologic and enzyme histochemical studies on bone formation induced by bone morphogenetic protein in mouse muscle tissue.

Bone morphogenetic protein (BMP) irreversibly induced the differentiation of mesenchymal-type cells into osteoprogenitor cells for endochondral ossification. During the process of BMP-induced differentiation in mice, 4 cell type (chondroblasts, osteoblasts, chondroclasts, and osteoclasts) were examined for phosphatase and succinate dehydrogenase using a wide range of buffers (4.0 less than or equal to pH less than or equal to 9.2). During the chondroid tissue-forming stage (1 week), chondroblast-like or osteoblast-like cells expressed phosphatase activity at 6.8 less than or equal to pH less than or equal to 9.2; chondroclast-like or osteoclast-like cells expressed phosphatase activity at 4.0 less than or equal to pH less than or equal to 5.8. However, mature chondrocytes found in hyaline cartilage expressed phosphatase activity between 6.6 less than or equal to pH less than or equal to 7.6 (2 weeks). During the process of endochondral ossification, alkaline phosphatase activity decreased in osteoblast-like cells with traces of acid phosphatase activity still detectable. Chondroclastic and osteoclastic giant cells were characterized by intense succinate dehydrogenase activity.

Immunohistochemical evaluation of bone morphogenetic protein (BMP) in mixed tumor of skin

Immunohistochemical reaction of bone morphogenetic protein (BMP) was assessed in 19 cases of skin mixed tumor and 5 cases of skin appendage tumors by using monoclonal antibody raised against BMP. All cases of skin mixed tumor showed positive staining for BMP in modified myoepithelial cells located at the periphery of tubulo-ductal or solid structures, and in plasmacytoid or tumor cells in hyalinous structures. Chondroidally changed cells also showed a strong BMP immunoreactivity. Tumors originating from sweat glands were devoid of BMP immunoreactivity. It is concluded that BMP is synthesized and produced in modified or transformed myoepithelial cells in skin mixed tumor and participates in the process of chondroid changes in the tumor.

Mandibular reconstruction using electrically stimulated periosteum

Electrically stimulated periosteum (ESP) grafting to a 12-year-old female patient who had had a segmental mandibulectomy in the anterior region following a diagnosis of Ewings sarcoma was tried for the mandibular reconstruction. One year after the ESP transplantation, examination of the patient showed that fine, radio-opaque bone formation had occurred. The new technique consists of 1) electrical stimulation of the tibial periosteum with 20 microamperes for five weeks, and then 2) ESP transplantation into a titanium mesh tray placed in the mandibulectomized region. In the present report, these new procedures are detailed, and the process of calcification in the transplanted ESP is discussed.

Immunohistochemical and Enzyme Histochemical Studies in Bone Formation Induced by Bone Morphogenetic Protein (BMP) in Mouse Muscle Tissue

It has been a well-known phenomenon that bone morphogenetic protein (BMP) heterotopically induces bone formation by causing the differentiation of osteoprogenitor cells from mesenchymal cells [1,2]. Two hypotheses of the cell origination and mechanism of bone formation by BMP have been proposed: EMP irreversibly induces the differentiation of perivascular mesenchymal cells into osteoprogenitor cells [3,4]; BMP is capable of stimulating the differentiation of skeletal muscle into cartilage [5–7]. The biological roles of bone formation induced by BMP still remain unclear. In the presnt study, the process of chondro-osteogenesis and cell differentiation for endochondral ossification by BMP implanted into mouse skeletal muscle tissue, were evaluated in terms of the immunohistochemical method for laminin, fibronectin, glycosaminoglycan, and S-100 protein, and the enzyme histochemical method of phosphatase activity with wide range of buffers (pH 4.0 to pH 9.2).

Laminin and fibronectin in bone formation induced by bone morphogenetic protein (BMP) in mouse muscle tissue

The morphological features at the light and electron microscopic level and distribution of fibronectin and laminin using immunohistochemical methods were evaluated in chondro-osteogneic tissue induced by bone morphogenetic protein (BMP) in mouse skeletal muscle. Satellite cells were seen at the muscle fibers adjacent to the site of BMP implantation 3 days after BMP-implantation. At the ultrastructural level, the skeletal muscle had a continuous basal lamina, satellite cells were partially enveloped by basal lamina-like structure, and fibroblast-like cells with abundant mitochondria and endoplasmic reticulum were adjacent to the myofibrils. An enhanced immunoreactivity for laminin and fibronectin was observed at the basement membrane of muscle fibers and extracellular matrix of neighboring connective tissue on day 3. On the 7th day, the enhanced immunoreactivity of laminin was persistent on the muscle fibers adjacent to the BMP and myoblast-like satellite cells, however, fibronectin showed decreased reaction. On the 14th day specimen, the newly formed osteo-chondroid matrix with numerous chondroid cells and osteoblasts showed positive immunoreaction for laminin and fibronectin. During the process of resorption of the induced osteoid tissue, no immunoreactivity for laminin and fibronectin was observed. An enhanced immunoreactivity of these glycoproteins during the early stages of BMP induced chondro-ossification suggests that they play an important role in the differentiation of myo-fibroblasts and organization and integration of ground substances of connective tissue and chondro-osseous matrix.