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Background: Enamel matrix derivative (EMD) protein has been clinically used for periodontal regeneration, but the molecular mechanisms are not clear. Previous studies suggested that the activation of phosphoinositide 3-kinase (PI 3-kinase) plays a key role in facilitating cell migration. Given that the migration of osteoblasts is one of the key steps in the wound-healing processes, we hypothesized that EMD protein would stimulate osteoblast migration by activating Pl 3-kinase. In this study, we tested this hypothesis using MG-63 cells as model systems to evaluate mechanisms of migration by stimulation with EMD protein. Methods: Confluent MG-63 cells were mechanically scratched using a sterilized 1-mm pipette tip that removed the cells within a circular area. The wells were incubated for 24 hours in various stimulation conditions (25, 50, or 100 µg/ml EMD protein) with or without the PI 3-kinase inhibitor wortmannin (1, 10, and 100 nM) or LY294002 (1, 10, and 100 µM). Migrated cells in the wound section were counted by randomly selecting three areas from one well. The activation of PI 3-kinase by EMD protein was evaluated by the phosphorylation of Akt using Western blot analysis. Results: Although EMD protein did not affect proliferation, it enhanced migration into wounds on MG-63 cells. We showed that EMD protein enhanced the phosphorylation of Akt in a dose-dependent manner. We demonstrated that the PI 3-kinase inhibitors wortmannin and LY294002 blocked migration into wounds and the phosphorylation of Akt enhanced by EMD protein in MG-63 cells. Conclusion: These results demonstrated that the activation of PI 3-kinase plays a key role in the EMD protein-stimulated migration of osteoblasts. J Periodontal 2009;80:1631-1637. [ABSTRACT FROM AUTHOR]

Background: The tetraspan protein CD63, originally described as a stage-specific melanoma antigen but also present in a number of normal cells, regulates melanoma cell growth in nude mice, motility in serum containing media, and adhesion to several extracellular matrix proteins. CD63 has been reported to associate with β1 and β2 integrins, but the mechanism of signal transduction by CD63 is not clear. This study examined whether CD63 is associated with protein kinase and can transmit signals in melanoma cells. Methods: Immunoprecipitation and radiolabeling were used to test for association of protein kinase activity with CD63. Adhesion of cells to monoclonal antibodies immobilized to microtiter plates was used to examine the ability of CD63 to transmit signals. Results: CD63 was capable of transmitting a signal in melanoma cells that required extracellular calcium. In the absence of extracellular calcium at the time of binding to the CD63 mAb, the cell was no longer responsive to stimulation by CD63. Immunoprecipitation studies demonstrated protein kinase activity associated with CD63, and phosphoamino acid analysis revealed that most of this protein kinase activity was due to serine kinase activity. Conclusion: The current study suggests that serine protein kinase activity associated with CD63 may play a role in signaling by CD63 in melanoma cells. [ABSTRACT FROM AUTHOR]

Hyaluronan (HA) is a cell-surface glycosaminoglycan that has been implicated in cancer progression. Cells isolated from metastatic colon carcinoma (SW620) produce greater amounts of pericellular HA than cells isolated from a primary tumor (SW480). Inhibition of hyaluronan synthases (HAS) by transfection with antisense cDNA decreases HA production. Because adhesion to the extracellular matrix (ECM) is required for invasion and metastasis, we hypothesized that pericellular HA mediates adhesion to ECM proteins such as laminin, collagen, and fibronectin and that inhibition of HA production or removal of HA by digestion with hyaluronidase would impair adhesion.SW480, SW620, and antisense transfectants (SW620 cells transfected with vector alone, antisense HAS2, antisense HAS3, and both antisense HAS2 and HAS3) were assessed for adhesion to laminin, Type 1 collagen, or fibronectin-coated plates. To confirm that adhesion was mediated by HA, cells were treated with or without hyaluronidase prior to the assays.Metastatic SW620 cells adhered well to laminin; SW480 cells demonstrated 46% less adhesion (P < 0.05; Student’s t test). SW620 cell adhesion to Type 1 collagen and fibronectin was >50% less than adhesion to laminin. Inhibition of HAS2 and/or HAS3 or pretreatment with hyaluronidase significantly decreased adhesion of SW620 cells to laminin (P < 0.05), suggesting that adhesion was dependent upon pericellular HA.Metastatic SW620 cells that produce large amounts of pericellular HA adhered well to laminin. Inhibition of HAS2 and/or HAS3 expression, or hyaluronidase digestion of pericellular HA significantly inhibited adhesion. These data suggest that HA promotes adhesion to laminin and may thereby facilitate invasion of the basement membrane and metastasis in colon carcinoma. [Copyright &y& Elsevier]