Nerve growth factor (NGF) stimulation of pheochromocytoma PC12 cells transiently increased the intracellular concentration of reactive oxygen species (ROS).This increase was blocked by the chemical antioxidant N-acetylcysteine and a flavoprotein inhibitor, diphenylene iodonium. NGF responses of PC12 cells, including neurite outgrowth, tyrosine phosphorylation, and AP-1 activation, was inhibited when ROS production was prevented by N-acetylcysteine and diphenylene iodonium. The expression of dominant negative Rac1N17 blocked induction of both ROS generation and morphological differentiation by NGF. The ROS produced appears to be H 2 O 2 , because the introduction of catalase into the cells abolished NGF-induced neurite outgrowth, ROS production, and tyrosine phosphorylation. These results suggest that the ROS, perhaps H 2 O 2 , acts as an intracellular signal mediator for NGF-induced neuronal differentiation and that NGF-stimulated ROS production is regulated by Rac1 and a flavoprotein-binding protein similar to the phagocytic NADPH oxidase.Reactive oxygen species (ROS) 1 that cause oxidative stress have generally been viewed as cytotoxic depending on the dose (1, 2). ROS are responsible for the host defense mechanism in neutrophils (3) and possess carcinogenic potential associated with tumor promotion (4, 5). Recent studies, however, indicate that small nontoxic amounts of ROS may play a normal role as a second messenger in the various signaling pathways (1). The production of ROS such as superoxide (O 2 . ) and hydrogen peroxide (H 2 O 2 ) was observed in a number of cells stimulated with cytokines such as transforming growth factors-␤1 (6, 7), interleukin-1 (8), and tumor necrosis factor ␣ (9) or peptide growth factors such as platelet-derived growth factor (PDGF) (10) and epidermal growth factor (EGF) (11). H 2 O 2 has been shown to mediate PDGF-induced cellular DNA synthesis of rat vascular smooth muscle cells (10). Ras-dependent cell growth requires generation of the O 2 . free radical through a pathway involving Rac1 (12). Although the role of ROS has been extensively studied in mitogenesis, inflammation, and apoptosis (1), little is known about its functional role in the differentiation process. The differentiation process in the nervous system is regulated by the action of differentiation and growth factors including NGF. NGF induces the growth arrest of PC12 cells and promotes their differentiation into sympathetic neuron-like cells (13). NGF binding to its receptor tyrosine kinase, TrkA, initiates various molecular interactions including tyrosine phosphorylation of proteins and the action of the Ras/Raf/MEK/MAPK pathway (14,15). NGF induces the production of reactive nitric oxide (NO), and NO is required for NGF-induced cytostasis and differentiation (16), suggesting that free radical molecules such as NO and ROS may exert a regulatory role in certain types of cellular differentiation. In the current study, we focused on the role of ROS and a small GTP-binding protein, Rac1, in the NGF-induced neuron...

Mos is an upstream activator of mitogenactivated protein kinase (MAPK) and, in mouse oocytes, is responsible for metaphase II arrest. This activity has been likened to its function in Xenopus oocytes as a component of cytostatic factor. Thus, Mos-deficient female mice are less fertile and oocytes derived from these animals fail to arrest at metaphase II Here we show that maturing MOS-'-oocytes fail to activate MAPK throughout meiosis, while p34edc2 kinase activity is normal until late in metaphase II when it decreases prematurely. Phenotypically, the first meiotic division of MOS-/-oocytes frequently resembles mitotic cleavage or produces an abnormally large polar body. In these oocytes, the spindle shape is altered and the spindle fails to translocate to the cortex, leading to the establishment of an altered cleavage plane. Moreover, the first polar body persists instead of degrading and sometimes undergoes an additional cleavage, thereby providing conditions for parthenogenesis. These studies identify meiotic spindle formation and programmed degradation of the first polar body as new and important roles for the Mos/MAPK pathway.The mos protooncogene encodes a protein serine/threonine kinase (1) and Mos is expressed at high levels in oocytes undergoing meiotic maturation (2,3). InXenopus oocytes, Mos has been shown to function as a meiotic initiator (4, 5) and an active component of cytostatic factor (CSF) (6), an activity that is responsible for the arrest of an unfertilized egg at metaphase II of meiosis (7). More recently, mitogen-activated protein kinase (MAPK), which is highly activated throughout oocyte maturation (8-12), has been identified as one of the major downstream targets of Mos (13)(14)(15)(16)(17). Mos has been implicated in the activation and stabilization of p34cdc2 kinase as maturation promoting factor (MPF) (4,(18)(19)(20), and evidence for the mutual dependency between MPF and the Mos/MAPK pathway in Xenopus oocytes has been reported (8,21,22).More recently, it has been shown that oocytes from mice homozygously deficient in mos (MOS-/-) fail to arrest at metaphase II and undergo parthenogenetic activation (23,24). These observations clearly demonstrated that Mos is an active component of CSF, but left unclear whether Mos functions prior to metaphase II arrest as it does in Xenopus.We generated MOS-'-mice and confirmed previous reports (23,24) that oocytes from these animals fail to arrest at metaphase II and instead undergo parthenogenetic activation.Also we show that maturing oocytes from MOS-1-mice fail to activate MAPK, while p34cdc2 kinase activation is normal until metaphase, when it decreases prematurely. Moreover, in MOS-'-oocytes, we observe that the first polar bodies can be abnormally large and sometimes undergo an additional cleavage instead of undergoing rapid degeneration. Thus, in addition to CSF activity, Mos/MAPK are required for regulating the size and degradation of the first polar body. MATERIALS AND METHODSGeneration of MOS-1-Knockout Mice. A replacement-type ...

The met proto-oncogene product (Met) and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), have been implicated in cell mitogenic response, cell motility, and the promotion of the ordered spatial arrangement of tissue. By means of confocal laser-scanning microscopy, it was shown that Met is expressed in cells bordering lumen-like structures that resemble ducts in the human mammary cell line T47D. In human breast tissue biopsies, Met staining was intense in normal cells bordering mammary ducts but was reduced in adjacent tumor tissue. Met staining in lumen-forming organs colocalizes with staining of antibody to phosphotyrosine, which suggests that the Met receptor and its substrates may be activated in lumen structures or ducts. HGF/SF treatment of human epithelial carcinoma cell lines resulted in the formation of lumen-like structures in vitro. Reduced expression of Met could be related to the extent of tumor cell differentiation.