Tomoko Kakehi

Reactive Oxygen Species Derived from NOX1/NADPH Oxidase Enhance Inflammatory Pain

The involvement of reactive oxygen species (ROS) in an augmented sensitivity to painful stimuli (hyperalgesia) during inflammation has been suggested, yet how and where ROS affect the pain signaling remain unknown. Here we report a novel role for the superoxide-generating NADPH oxidase in the development of hyperalgesia. In mice lacking Nox1 (Nox1−/Y), a catalytic subunit of NADPH oxidase, thermal and mechanical hyperalgesia was significantly attenuated, whereas no change in nociceptive responses to heat or mechanical stimuli was observed. In dorsal root ganglia (DRG) neurons of Nox1+/Y, pretreatment with chemical mediators bradykinin, serotonin, or phorbol 12-myristate 13-acetate (PMA) augmented the capsaicin-induced calcium increase, whereas this increase was significantly attenuated in DRG neurons of Nox1−/Y. Concomitantly, PMA-induced translocation of PKCε was markedly perturbed in Nox1−/Y or Nox1+/Y DRG neurons treated with ROS-scavenging agents. In cells transfected with tagged PKCε, hydrogen peroxide induced translocation and a reduction in free sulfhydryls of full-length PKCε but not of the deletion mutant lacking the C1A domain. These findings indicate that NOX1/NADPH oxidase accelerates the translocation of PKCε in DRG neurons, thereby enhancing the TRPV1 activity and the sensitivity to painful stimuli.

NOX enzymes and diabetic complications

Several molecular mechanisms have been identified that mediate the tissue-damaging effects of hyperglycemia. These are increased flux through the polyol and hexosamine pathways, increased formation of advanced glycation end products, activation of protein kinase C, and augmented generation of reactive oxygen species (ROS). Increased production of ROS not only causes cellular damage but also activates the signal transduction cascade that activates specific target genes. Based on recent experimental data, it is now accepted that increased NADPH oxidase activity in tissues vulnerable to hyperglycemia takes place downstream of the advanced glycation end products and protein kinase C pathways, two of the primary mechanisms involved in the pathogenesis of diabetic complications. Thus, compounds that suppress NADPH oxidase activity may offer therapeutic benefits to ameliorate diabetic complications, highlighting the significance of NADPH oxidase as a new therapeutic target.

Effects of different anti-tau antibodies on tau fibrillogenesis: RTA-1 and RTA-2 counteract tau aggregation.

Tau is the major antigenic component of neurofibrillary pathology in tauopathy, including Alzheimers disease. Although conversion of soluble tau to an insoluble polymerized fibrillar form is a key factor in the pathogenesis of tauopathy, the mechanism of the change is unclear and no inhibitors of fibril formation are available. Monoclonal antibodies against the 1st or 2nd repeat of the microtubule binding domain, but not the C‐terminal 16 residues, completely inhibited tau aggregation into PHF. Furthermore, they did not inhibit tau‐induced tubulin assembly. Thus, they are useful to investigate tau protein conversion and will be useful therapeutic lead materials.

NADPH oxidase NOX1 is involved in activation of protein kinase C and premature senescence in early stage diabetic kidney.

Increased oxidative stress and activation of protein kinase C (PKC) under hyperglycemia have been implicated in the development of diabetic nephropathy. Because reactive oxygen species derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, NOX1 accelerate the translocation of PKC isoforms, NOX1 is postulated to play a causative role in the development of diabetic nephropathy. Hyperglycemia was induced in wild-type and Nox1-deficient mice (KO) by two doses of streptozotocin injection. At 3 weeks after the induction of hyperglycemia, glomeruli and cortical tubules were isolated from kidneys. The mRNA level of Nox1 was significantly upregulated in the renal cortex at 3 weeks of hyperglycemia. Urinary albumin and expression of inflammatory or fibrotic mediators were similarly elevated in diabetic wild-type and KO; however, increases in glomerular volume and mesangial matrix area were attenuated in diabetic KO. Nox1 deficiency significantly reduced the levels of renal thiobarbituric acid-reacting substances and 8-hydroxydeoxyguanosine, membranous translocation of PKCα/β, activity of PKC, and phosphorylation of p38 mitogen-activated protein kinase in the diabetic kidney. Furthermore, increased staining of senescence-associated β-galactosidase in glomeruli and cortical tubules of diabetic mice was significantly suppressed in KO. Whereas the levels of cyclin-dependent kinase inhibitors, p16(INK4A) and p21(Cip1), were equivalent between the genotypes, increased levels of p27(Kip1) and γ-H2AX, a biomarker for DNA double-strand breaks, were significantly attenuated in isolated glomeruli and cortical tubules of diabetic KO. Taken together, NOX1 modulates the p38/p27(Kip1) signaling pathway by activating PKC and promotes premature senescence in early stage diabetic nephropathy.

Myocyte enhancer factor 2B is involved in the inducible expression of NOX1/NADPH oxidase, a vascular superoxide-producing enzyme

NADPH oxidase is a major source of the superoxide produced in cardiovascular tissues. Expression of NOX1, a catalytic subunit of NADPH oxidase, is induced by various vasoactive factors, including angiotensin II, prostaglandin (PG) F2α and platelet‐derived growth factor (PDGF). To clarify the molecular basis of this transcriptional activation, we delineated the promoter region of the NOX1 gene. RT‐PCR and 5′‐rapid amplification of cDNA ends‐based analyses revealed a novel 5′‐terminal exon of the rat NOX1 gene located approximately 28 kb upstream of the exon containing the start codon. Both PGF2α and PDGF enhanced the transcriptional activity of the − 3.6 kb 5′‐flanking region of the NOX1 gene in A7r5 cells, a rat vascular smooth muscle cell line. A PGF2α‐response element was located between −146 and −125 in the 5′‐flanking region containing a consensus binding site for myocyte enhancer factor 2 (MEF2), to which binding of MEF2 was augmented by PGF2α. Gene silencing of MEF2B by RNA interference significantly suppressed the expression of NOX1, while silencing of activating transcription factor (ATF)‐1, previously implicated in up‐regulation of NOX1, abolished the PGF2α‐ or PDGF‐induced expression of MEF2B. These results indicate that superoxide production in vascular smooth muscle cells is regulated by the ATF‐1–MEF2B cascade by induction of the expression of the NOX1 gene.