IntroductionReceptor activator of NF-B (nuclear factor-B) ligand (RANKL; also called TRANCE [tumor necrosis factor (TNF) activationinduced cytokine], ODF [osteoclast differentiation factor], and OPGL [osteoprotegerin ligand]) 1-4 is a key factor stimulating the differentiation and activation of osteoclasts and, therefore, is essential for bone remodeling. 5 The binding of RANKL to its receptor RANK leads to recruitment of TNF receptor-associated factor 6 (TRAF6) to the cytoplasmic domain of RANK, thereby resulting in the activation of distinct signaling cascades mediated by mitogen-activated protein (MAP) kinases, including c-Jun N-terminal kinase (JNK), p38 MAP kinase (p38), and extracellular signal-regulated kinase (ERK). 6 It has been shown that JNK1-activated c-Jun signaling in cooperation with nuclear factor of activated T cells (NFAT) is key to RANKL-regulated osteoclast differentiation. 7 In addition, stimulation of p38 results in the downstream activation of the mi/Mitf (microphthalmia/microphthalmia transcription factor), which controls the expression of the genes encoding tartrate-resistant acid phosphatase (TRAP) and cathepsin K, indicating the importance of p38 signaling cascades. 6 Although our understanding of signaling pathways associated with osteoclast differentiation has advanced considerably recently, the mechanism of RANKL-mediated osteoclastogenesis, specifically the molecular linkage between TRAF6 and MAP kinases, is still unknown.At high concentrations, reactive oxygen species (ROSs) cause oxidative stress that has been viewed as deleterious phenomena, including inflammatory response, apoptosis, or ischemia. 8 Recent studies, however, indicate that small nontoxic amounts of ROS may play a role as a second messenger in the various receptor signaling pathways. [9][10][11][12][13] Osteoclasts have shown to be activated by ROSs to enhance bone resorption, 14 but little attention has been given to the role of ROSs in differentiation of macrophages and monocytes into osteoclasts. Signaling molecules such as JNK and p38, which are known to be essential for osteoclast differentiation, 6,7 are sensitive to activation by ROSs. 11,12 Thus, we hypothesized that signaling cascade(s) can be modulated by ROSs in bone marrow monocyte-macrophage lineage (BMM) cells.Here, we show that RANKL generates ROSs in BMM cells. Examination of the mechanism by which RANKL generates ROSs revealed the involvement of TRAF6, Rac1, and NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 1 (Nox1). These data suggest that RANKL-mediated ROS production serves to regulate RANKL signaling pathways, including JNK and p38 activation required for osteoclast differentiation. Materials and methods Reagents and plasmids2Ј,7Ј-dichlorofluorescein diacetate (DCFH-DA) was purchased from Molecular Probes (Leiden, The Netherlands); all other chemicals and FLAG (five NH2-terminally deleted epitope-tagged) epitope (M2) were from Expression constructs encoding FLAG-tagged wild-type RANK, HAtagged TRAF6 (amino acids [aa] 289-530), and...

Z (Glu342 --> Lys) and S(iiyama) (Ser53 --> Phe) genetic variations of human alpha1-antitrypsin (alpha1-AT) cause a secretion blockage in the hepatocytes, leading to alpha1-AT deficiency in the plasma. Using in vitro folding analysis, we have shown previously that these mutations interfere with the proper folding of polypeptides. To understand the fundamental cause for the secretion defect of the Z and S(iiyama) variants of alpha1-AT, we investigated in vivo folding and stability of these variant alpha1-AT using the secretion system of yeast Saccharomyces cerevisiae. Various thermostable mutations suppressing the folding block of the Z variant in vitro corrected the secretion defect as well as the intracellular degradation in the yeast secretion system. Significantly, the extent of suppression in the secretion defect of Z protein was proportional to the extent of suppression in the folding defect, assuring that the in vivo defect associated with the Z variant is primarily derived from the folding block. In contrast, the folding and secretion efficiency of S(iiyama) was not much improved by the same mutations. In addition, none of the rarely secreted S(iiyama) alpha1-AT carrying the stabilizing mutations for the wild type and Z variant were active. It appears that the major defect in S(iiyama) variant is the loss of stability in contrast to the kinetic block of folding in the Z variant.

Because the mucosal epithelia are in constant contact with large numbers of microorganisms, these surfaces must be armed with efficient microbial control systems. Here, we show that the Drosophila nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme, dual oxidase (dDuox), is indispensable for gut antimicrobial activities. Adult flies in which dDuox expression is silenced showed a marked increase in mortality rate even after a minor infection through ingestion of microbe-contaminated food. This could be restored by the specific reintroduction of dDuox, demonstrating that this oxidase generates a unique epithelial oxidative burst that limits microbial proliferation in the gut. Thus, oxidant-mediated antimicrobial responses are not restricted to the phagocytes, but rather are used more broadly, including in mucosal barrier epithelia.

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LPS, the primary constituent of the outer membrane of Gram-negative bacteria, is recognized by TLR4. Binding of TLR4 to LPS triggers various cell signaling pathways including NF-κB activation and reactive oxygen species (ROS) production. In this study, we present the data that LPS-induced ROS generation and NF-κB activation are mediated by a direct interaction of TLR4 with (NAD(P)H oxidase 4 (Nox) 4), a protein related to gp91phox (Nox2) of phagocytic cells, in HEK293T cells. Yeast two hybrid and GST pull-down assays indicated that the COOH-terminal region of Nox4 interacted with the cytoplasmic tail of TLR4. Knockdown of Nox4 by transfection of small interference RNA specific to the Nox4 isozyme in HEK293T cells expressing TLR4 along with MD2 and CD14 resulted in inhibition of LPS-induced ROS generation and NF-κB activation. Taken together, these results indicate that direct interaction of TLR4 with Nox4 is involved in LPS-mediated ROS generation and NF-κB activation.

Ligand-receptor interactions can generate the production of hydrogen peroxide (H(2)O(2)) in cells, the implications of which are becoming appreciated. Fluctuations in H(2)O(2) levels can affect the intracellular activity of key signaling components including protein kinases and protein phosphatases. Rhee et al. discuss recent findings on the role of H(2)O(2) in signal transduction. Specifically, H(2)O(2) appears to oxidize active site cysteines in phosphatases, thereby inactivating them. H(2)O(2) also can activate protein kinases; however, although the mechanism of activation for some kinases appears to be similar to that of phosphatase inactivation (cysteine oxidation), it is unclear how H(2)O(2) promotes increased activation of other kinases. Thus, the higher levels of intracellular phosphoproteins observed in cells most likely occur because of the concomitant inhibition of protein phosphatases and activation of protein kinases.

Reactive oxygen species (ROS) including superoxide anion and hydrogen peroxide (H(2)O(2)) are thought to be byproducts of aerobic respiration with damaging effects on DNA, protein, and lipid. A growing body of evidence indicates, however, that ROS are involved in the maintenance of redox homeostasis and various cellular signaling pathways. ROS are generated from diverse sources including mitochondrial respiratory chain, enzymatic activation of cytochrome p450, and NADPH oxidases further suggesting involvement in a complex array of cellular processes. This review summarizes the production and function of ROS. In particular, how cytosolic and membrane proteins regulate ROS generation for intracellular redox signaling will be detailed.

We report our studies on root gravitropism indicating that reactive oxygen species (ROS) may function as a downstream component in auxin-mediated signal transduction. A transient increase in the intracellular concentration of ROS in the convex endodermis resulted from either gravistimulation or unilateral application of auxin to vertical roots. Root bending was also brought about by unilateral application of ROS to vertical roots pretreated with the auxin transport inhibitor N-1-naphthylphthalamic acid. Furthermore, the scavenging of ROS by antioxidants (N-acetylcysteine, ascorbic acid, and Trolox) inhibited root gravitropism. These results indicate that the generation of ROS plays a role in root gravitropism.Since Cholodny (1926) and Went (1926) discovered that directional auxin transport occurs upon gravistimulation, the mechanism of auxin transport is well established. According to the mechanism, the gravitropic stimulation induces asymmetric auxin movement, and the localized auxin in turn causes gravitropic curvature (Young et al., 1990; Dolan, 1998; Rosen et al., 1999). These results indicate that auxin is indeed essential for gravitropism. Several lines of evidence suggest that the second messengers, Ca 2ϩ and inositol 1,4,5-triphosphate (IP 3 ), are involved in root gravitropism (Lee et al., 1983; Perera et al., 1999). However, the relationship between auxin and second messengers is still unknown.Although reactive oxygen species (ROS) such as superoxide anions and H 2 O 2 are generally considered to be toxic byproducts of respiration, recent evidence suggests that the production of ROS might be an integral component of intracellular signaling (Krieger-Brauer and Kather, 1992; Finkel, 1998; Rhee et al., 2000). In mammalian cells, a variety of extracellular stimuli have been shown to induce a transient increase in the intracellular concentration of ROS, and specific inhibition of the ROS generation results in a complete blockage of stimulus-dependent signaling (Sundaresan et al., 1995; Bae et al., 1997). Also, several lines of evidence suggest that ROS serve as signaling molecules in plants. It has been shown that ROS mediate systemic signal networks for plant defense (Chen et al