Shuichi Furuta

NADPH oxidase 4 contributes to transformation phenotype of melanoma cells by regulating G2-M cell cycle progression.

Generation of reactive oxygen species (ROS) has been implicated in carcinogenic development of melanoma, but the underlying molecular mechanism has not been fully elucidated. We studied the expression and function of the superoxide-generating NADPH oxidase (Nox)4 in human melanoma cells. Nox4 was up-regulated in 13 of 20 melanoma cell lines tested. Silencing of Nox4 expression in melanoma MM-BP cells by small interfering RNAs decreased ROS production and thereby inhibited anchorage-independent cell growth and tumorigenecity in nude mice. Consistently, a general Nox inhibitor, diphenylene iodonium, and antioxidants vitamine E and pyrrolidine dithiocarbamate blocked cell proliferation of MM-BP cells. Flow cytometric analysis indicated that Nox4 small interfering RNAs and diphenylene iodonium induced G(2)-M cell cycle arrest, which was also observed with another melanoma cell line, 928mel. This was accompanied by induction of the Tyr-15 phosphorylated, inactive form of cyclin-dependent kinase 1 (a hallmark of G(2)-M checkpoint) and hyperphosphorylation of cdc25c leading to its increased binding to 14-3-3 proteins. Ectopic expression of catalase, a scavenger of ROS, also caused accumulation of cells in G(2)-M phase. Immunohistochemistry revealed that expression of Nox4 was detected in 31.0% of 13 melanoma patients samples, suggesting the association of Nox4 expression with some steps of melanoma development. The findings suggest that Nox4-generated ROS are required for transformation phenotype of melanoma cells and contribute to melanoma growth through regulation of G(2)-M cell cycle progression.

Ras is involved in the negative control of autophagy through the class I PI3-kinase.

Ras proteins exert a pivotal regulatory function in signal transduction involved in cell proliferation and their activation mutation leads to malignant cell transformation. However, the role of Ras proteins in autophagy, an intracellular protein degradation process in cell growth control is unknown. In the present study, we demonstrate that the degradation of long-lived proteins in NIH3T3 cells in response to nutrient starvation was significantly suppressed by oncogenic RasVal12 transformation in a rapamycin (mTOR inhibitor)-sensitive manner. Morphologic observations also show the decrease in the formation of autophagic vacuoles upon the Ras transformation. Furthermore, epidermal growth factor or serum downregulated the protein degradation induced by serum starvation and the dominant-negative RasAsn17 mutant counteracted this suppressive effect, indicating that Ras mediates the growth factor downregulation of autophagy. The suppression of protein degradation by the activated RasVal12 was mediated by the class I phosphatidyl inositol 3-kinase (PI3-kinase), but not either or Raf Ral GDS. Consistent with this, RasVal12 and class I PI3-kinase inhibited the rate of autophagic sequestration of LDH. These data suggest that Ras plays a critical role as a negative regulator for nutrient deprivation-induced autophagy through the class I PI3-kinase signaling pathway.

Reactive Oxygen Generated by NADPH Oxidase 1 (Nox1) Contributes to Cell Invasion by Regulating Matrix Metalloprotease-9 Production and Cell Migration

A mediating role of the reactive oxygen species-generating enzyme Nox1 has been suggested for Ras oncogene transformation phenotypes including anchorage-independent cell growth, augmented angiogenesis, and tumorigenesis. However, little is known about whether Nox1 signaling regulates cell invasiveness. Here, we report that the cell invasion activity was augmented in K-Ras-transformed normal rat kidney cells and attenuated by transfection of Nox1 small interference RNAs (siRNAs) into the cells. Diphenyleneiodonium (DPI) or Nox1 siRNAs blocked up-regulation of matrix metalloprotease-9 at both protein and mRNA levels in K-Ras-transformed normal rat kidney cells. Furthermore, DPI and Nox1 siRNAs inhibited the activation of IKKα kinase and the degradation of IκBα, suppressing the NFκB-dependent matrix metalloprotease-9 promoter activity. Additionally, epidermal growth factor-stimulated migration of CaCO-2 cells was abolished by DPI and Nox1 siRNAs, indicating the requirement of Nox1 activity for the motogenic effect of epidermal growth factor. This Nox1 action was mediated by down-regulation of the Rho activity through the low molecular weight protein-tyrosine phosphatase-p190RhoGAP-dependent mechanism. Taken together, our findings define a mediating role of Nox1-generated reactive oxygen species in cell invasion processes, most notably metalloprotease production and cell motile activity.

ROS‐generating oxidases Nox1 and Nox4 contribute to oncogenic Ras‐induced premature senescence

Activated oncogenes induce premature cellular senescence, a permanent state of proliferative arrest in primary rodent and human fibroblasts. Recent studies suggest that generation of reactive oxygen species (ROS) is involved in oncogenic Ras‐induced premature senescence. However, the signaling mechanism controlling this oxidant‐mediated irreversible growth arrest is not fully understood. Here, we show that through the Ras/MEK pathway, Ras oncogene up‐regulated the expression of superoxide‐generating oxidases, Nox1 in rat REF52 cells and Nox4 in primary human lung TIG‐3 cells, leading to an increase in intracellular level of ROS. Ablation of Nox1 and Nox4 by small interfering RNAs (siRNAs) blocked the RasV12 senescent phenotype including β‐galactosidase activity, growth arrest and accumulation of tumor suppressors such as p53 and p16Ink4a. This suggests that Nox‐generated ROS transduce senescence signals by activating the p53 and p16Ink4a pathway. Furthermore, Nox1 and Nox4 siRNAs inhibited both Ras‐induced DNA damage response and p38MAPK activation, whereas overexpression of Nox1 and Nox4 alone was able to induce senescence. The involvement of Nox1 in Ras‐induced senescence was also confirmed with embryonic fibroblasts derived from Nox1 knockout mice. Together, these findings suggest that Nox1‐ and Nox4‐generated ROS play an important role in Ras‐induced premature senescence, which may involve DNA damage response and p38MAPK signaling pathways.

Cloning and expression of cDNA for a newly identified isozyme of bovine liver 3-hydroxyacyl-CoA dehydrogenase and its import into mitochondria.

cDNA for a heretofore undescribed mitochondrial 3-hydroxyacyl-CoA dehydrogenase, designated as the type II enzyme with different molecular and catalytic properties, compared to those of the classical mitochondrial beta-oxidation enzyme (type I enzyme), was cloned from a bovine liver cDNA library. Nucleotide sequence of the cDNA encoded 261 amino acids with a subunit molecular weight of 27,140. The deduced primary structure of the type II enzyme showed no significant homology to the reported amino acid sequence of the classical 3-hydroxyacyl-CoA dehydrogenases. On SDS-PAGE, no differences in subunit molecular weights were observed among the in vitro translation products, the recombinant type II enzyme produced in Escherichia coli and the purified enzyme. NH2-terminal and COOH-terminal amino acid sequence analysis of the purified type II enzyme revealed that the mature enzyme had not been proteolytically processed. The in vitro translation products of the type II enzyme were efficiently incorporated into isolated rat liver mitochondria, without changes in size, thereby suggesting that unlike other mitochondrial enzymes of fatty acid beta-oxidation, the type II enzyme had no cleavable signal peptide upon import into mitochondria.

Cell-free synthesis of the enzymes of peroxisomal β-oxidation

Three enzymes of peroxisomal β-oxidation of rat liver were synthesized in a cell-free protein-synthesizing system derived from rabbit reticulocyte lysate. The invitro products of acyl-CoA oxidase and enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase multifunctional protein were similar in size to or slightly larger than the subunit of the respective mature enzymes. The invitro product of peroxisomal 3-ketoacyl-CoA thiolase was about 3,000 daltons larger than the mature subunit. The hepatic levels of translatable mRNAs coding for these three enzymes were about 10 times higher in rats fed a di(2-ethylhexyl)phthalate-containing diet than in control animals.

Turnover of enzymes of peroxisomal β-oxidation in rat liver

Male Wistar rats were given a diet containing 2% (w/w) di-ethylhexyl)-phthalate (DEHP), a peroxisomal proliferator, for 4 weeks. The activities of enzymes of peroxisomal beta-oxidation and of catalase were markedly increased by the DEHP administration. The time required to reach halfway to the maximal induction for enzymes of peroxisomal beta-oxidation was 5--7 days, whereas that for catalase was 3 days. A separate DEHP group was placed on the control diet after 14 days of feeding with the DEHP diet. On the withdrawal of DEHP, activities of enzymes of the beta-oxidation system and of catalase decreased to the control levels with a half-life of 2--3 days. Responses of some mitochondrial enzymes involved in fatty acid oxidation are also described.

Riboflavin deficiency and β-oxidation systems in rat liver

Weanling rats were fed a riboflavin-deficient diet. The mitochondrial fatty acid oxidation in liver was depressed in riboflavin deficiency but restored after supplementation of riboflavin. Among the enzymes involved in this system, only the acyl-CoA dehydrogenase (EC 1.3.99.2 and 1.3.99.3) activities varied with the change in fatty acid oxidation. An accumulation of the apoforms of acyl-CoA dehydrogenases was found in riboflavin deficiency. The levels of electron transfer flavoprotein and other enzymes involved in the β-oxidation system remained unchanged. The peroxisomal fatty acid oxidation and levels of individual enzymes of this system remained constant. No accumulation of the apoform of acyl-CoA oxidase was observed under simple, riboflavin-deficient conditions. However, accumulation of a large amount of apo-acyl-CoA oxidase was observed when the peroxisomal system was induced by administration of a peroxisome proliferator, di(2-ethylhexyl)phthalate, under riboflavin-deficient conditions.

Induction of peroxisomal β-oxidation by the administration of acetylsalicylic acid

Abstract The effects of acetylsalicylic acid (ASA) on the fatty acid oxidation system of liver peroxisomes were examined. Male Wistar rats were fed powdered diet containing 1% ( w w ) ASA for 10 days. Peroxisomal palmitoyl-CoA oxidation of rat liver, which is cyanide insensitive, increased by sevenfold after the administration of ASA. Activities of component enzymes of this system were also increased. The effects of salicylic acid (SA) on the induction of peroxisomal β-oxidation were nearly the same as those of ASA. p -Aminosalicylic acid (PAS) and gentisic acid (GA), however, did not cause the alteration of this system. The activity-time curve of the peroxisomal β-oxidation system was studied. The time to reach half-way to maximal induction by the administration of ASA was 3.5 days and the half-life of the system, estimated by drug withdrawal, was 2.0 days. A dose-dependent increase in activity of the system was observed. The results demonstrate that ASA results in proliferation of a peroxisomal fatty acyl-CoA oxidation system in the rat liver, and that the effects of ASA on this system are nearly the same as those of other peroxisomal proliferators which are unrelated in the chemical structures.

Light Chain 3 associates with a Sos1 guanine nucleotide exchange factor: its significance in the Sos1-mediated Rac1 signaling leading to membrane ruffling.

A 19 kDa protein was identified to associate with the Dbl oncogene homology domain of Sos1 (Sos–DH) and was purified from rat brains by GST–Sos–DH affinity chromatography. Peptide sequencing revealed that the protein is identical to light chain 3 (LC3), a microtubule-associated protein. LC3 coimmunoprecipitated with Sos1, and GST–LC3 was capable of forming complexes with Sos1 in in vitro GST-pull down assay. Furthermore, LC3 was colocalized with Sos1 in cells, as determined by immunohistochemistry. While Sos1 stimulated the guanine nucleotide exchange reaction on Rac1, LC3 suppressed the ability of Sos1 to activate Rac1 in in vitro experiments using COS cell lysates. Consistent with this, overexpression of LC3 decreased the level of active GTP-bound Rac1 in COS cells. Sos1 expression induced membrane ruffling, a downstream target for Rac1, but LC3 expression inhibited this biological effect of Sos1. These findings suggest that LC3 interacts with Sos1 and thereby negatively regulates the Sos1-dependent Rac1 activation leading to membrane ruffling