Reactive oxygen species (ROS) generated in some non-phagocytic cells are implicated in mitogenic signalling and cancer. Many cancer cells show increased production of ROS, and normal cells exposed to hydrogen peroxide or superoxide show increased proliferation and express growth-related genes. ROS are generated in response to growth factors, and may affect cell growth, for example in vascular smooth-muscle cells. Increased ROS in Ras-transformed fibroblasts correlates with increased mitogenic rate. Here we describe the cloning of mox1, which encodes a homologue of the catalytic subunit of the superoxide-generating NADPH oxidase of phagocytes, gp91phox. mox1 messenger RNA is expressed in colon, prostate, uterus and vascular smooth muscle, but not in peripheral blood leukocytes. In smooth-muscle cells, platelet-derived growth factor induces mox1 mRNA production, while antisense mox1 mRNA decreases superoxide generation and serum-stimulated growth. Overexpression of mox1 in NIH3T3 cells increases superoxide generation and cell growth. Cells expressing mox1 have a transformed appearance, show anchorage-independent growth and produce tumours in athymic mice. These data link ROS production by Mox1 to growth control in non-phagocytic cells.

Abstract-Monocyte chemoattractant protein-1 (MCP-1) is an important component of the inflammatory response of the vessel wall and has been shown to be regulated by cytokines, such as tumor necrosis factor-␣ (TNF-␣). However, the precise signaling pathways leading to MCP-1 induction have not been fully elucidated in vascular smooth muscle cells (VSMCs). Cytokine signal transduction involves protein kinases as well as reactive oxygen species (ROS). The relation between these 2 factors is not clear. In this study, we show that TNF-␣ induces a parallel phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38MAPK) and increases MCP-1 mRNA expression in cultured VSMCs. Inhibition of ERK1/2 but not p38MAPK caused a partial attenuation of MCP-1 induction (43Ϯ10% inhibition). Incubation of VSMCs with multiple antioxidants (diphenylene iodonium, liposomal superoxide dismutase, catalase, N-acetylcysteine, dimethylthiourea, and pyrrolidine dithiocarbamate) had no effect on TNF-␣-mediated MCP-1 upregulation. However, simultaneous blockade of the ERK1/2 and ROS pathways by using PD098059 combined with diphenylene iodonium or N-acetylcysteine potently enhanced the ability of MAPK kinase inhibitors to abrogate MCP-1 mRNA expression (100Ϯ2% inhibition). Thus, parallel ROS-dependent and ERK1/2-dependent pathways converge to regulate TNF-␣-induced MCP-1 gene expression in VSMCs. These data unmask a complex but organized integration of ROS and protein kinases that mediates cytokine-induced vascular inflammatory gene expression.

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We aim to establish a 3D model of cartilage wound healing, and explore the involvement of chondrocytes in its repair. To characterize chondrocyte involvement in wound healing, an in vitro 3D model composed of chondrocyte mixing with either type II/I collagen or type I collagen matrix was established. The "defects" measuring 5 mm in diameter were made on each collagen matrix-chondrocyte construct to mimic in vivo cartilage defects. The effects of basic fibroblast growth factor (bFGF) on chondrocytes migration and differentiation were studied. The migration and Glucosaminoglycan (GAG) synthesis of chondrocytes in the defect areas were observed by microscopy after Alcian-blue staining. In the presence of bFGF, GAG expression increased significantly when chondrocytes were cultured in type II/I collagen matrix compared to type I collagen matrix. However, mild GAG accumulation was also found when cells were cultured in either type I or type II/I collagens without bFGF. In a 3D model of cartilage wound healing, bFGF promote chondrocyte proliferation, migration and differentiation in the presence of type II/I collagen matrix, and showed potential to regulate wound healing. These wound healing models may provide feasible methods to explore various drugs prior to human trials.

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