In primary mammalian cells, oncogenic ras induces premature senescence, depending on an active MEKextracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. It has been unclear how activation of the mitogenic MEK-ERK pathway by ras can confer growth inhibition. In this study, we have found that the stress-activated MAPK, p38, is also activated during the onset of ras-induced senescence in primary human fibroblasts. Constitutive activation of p38 by active MKK3 or MKK6 induces senescence. Oncogenic ras fails to provoke senescence when p38 activity is inhibited, suggesting that p38 activation is essential for ras-induced senescence. Furthermore, we have demonstrated that p38 activity is stimulated by ras as a result of an activated MEK-ERK pathway. Following activation of MEK and ERK, expression of oncogenic ras leads to the accumulation of active MKK3/6 and p38 activation in a MEK-dependent fashion and subsequently induces senescence. Active MEK1 induces the same set of changes and provokes senescence relying on active p38. Therefore, oncogenic ras provokes premature senescence by sequentially activating the MEK-ERK and MKK3/6-p38 pathways in normal, primary cells. These studies have defined the molecular events within the ras signaling cascade that lead to premature senescence and, thus, have provided new insights into how ras confers oncogenic transformation in primary cells.

Nuclear factor E2-related factor 2 (Nrf2) is a cap-n-collar basic leucine zipper (CNC-bZIP) transcription factor that is well established as a master regulator of phase II detoxification and antioxidant gene expression and is strongly expressed in tissues involved in xenobiotic metabolism including liver and kidney. Nrf2 is also abundantly expressed in adipose tissue; however, the exact function of Nrf2 in adipocyte biology is unclear. In the current study we show that targeted knock-out of Nrf2 in mice decreases adipose tissue mass, promotes formation of small adipocytes, and protects against weight gain and obesity otherwise induced by a high fat diet. In mouse embryonic fibroblasts, 3T3-L1 cells, and human subcutaneous preadipocytes, selective deficiency of Nrf2 impairs adipocyte differentiation. Fat cells play an important role in energy storage and metabolism and secrete a variety of factors that influence appetite, insulin sensitivity, inflammation, and many other pathways of biologic and clinical significance (1). Because of the significance of adipocyte biology in the pathogenesis of obesity and related metabolic, cardiovascular, and inflammatory disorders, there has been intense interest in defining the network of transcription factors that controls the expression of genes involved in fat cell development. Peroxisome proliferator-activated receptors (PPARs) 4 are ligand-activated transcription factors that belong to a nuclear hormone receptor family including related molecules that function by forming heterodimers with retinoid X receptors (2). PPAR␥ is expressed abundantly in adipose tissue and is considered to be the dominant transcriptional regulator of adipogenic differentiation (3). Accordingly, much attention has been directed at identifying factors that control PPAR␥ expression during the process of adipogenesis.CCAAT enhancer-binding proteins (C/EBPs) are leucine zipper transcription factors expressed in both white and brown adipose tissue and have been extensively studied for their roles in regulating PPAR␥ activity and adipogenesis (4, 5). C/EBP␤ and C/EBP␦ are transiently expressed at the onset of the adipose differentiation program (6, 7). This phase is then followed by induction of PPAR␥ and C/EBP␣ expression (8 -10). In addition, PPAR␥ and C/EBP␣ form a positive loop by regulating each other's expression (11,12). Although the enforced expression of C/EBP␣ in fibroblasts can trigger adipocytic differentiation, C/EBP␣ is incapable of inducing adipogenesis in the absence of PPAR␥ (13). In contrast, PPAR␥ can induce adipogenic differentiation in C/EBP␣-null cells indicating that PPAR␥ is proximal in effecting adipogenesis (14). Apart from C/EBP␤, -␦, and -␣, relatively few transcription factors have been described that bind to the Ppar␥ promoter and positively regulate Ppar␥ transcription during adipogenesis (15).The nuclear factor erythroid-derived 2-related factor 2 (Nrf2) is a member of the cap-n-collar basic leucine zipper (CNC-bZIP) family of transcription factors and has been shown to pl...

Expression of antioxidant and phase 2 xenobiotic metabolizing enzyme genes is regulated through cis-acting sequences known as antioxidant response elements. Transcriptional activation through the antioxidant response elements involves members of the CNC (Cap 'n' Collar) family of basic leucine zipper proteins including Nrf1 and Nrf2. Nrf2 activity is regulated by Keap1-mediated compartmentalization in the cell. Given the structural similarities between Nrf1 and Nrf2, we sought to investigate whether Nrf1 activity is regulated similarly to Nrf2. Nrf1 also resides normally in the cytoplasm of cells. Cytoplasmic localization however, is independent of Keap1. Colocalization analysis using green fluorescent protein-tagged Nrf1 and subcellular fractionation of endogenous Nrf1 and fusion proteins indicate that Nrf1 is primarily a membranebound protein localized in the endoplasmic reticulum. Membrane targeting is mediated by the N terminus of the Nrf1 protein that contains a predicted transmembrane domain, and deletion of this domain resulted in a predominantly nuclear localization of Nrf1 that significantly increased the activation of reporter gene expression. Treatment with tunicamycin, an endoplasmic reticulum stress inducer, caused an accumulation of a smaller form of Nrf1 that correlated with detection of Nrf1 in the nucleus by biochemical fractionation and immunofluorescent analysis. These results suggest that Nrf1 is normally targeted to the endoplasmic reticulum membrane and that endoplasmic reticulum stress may play a role in modulating Nrf1 function as a transcriptional activator.Oxidative stress occurs as a result of imbalance between antioxidant defense and the production of reactive oxygen species (1). Sustained oxidative stress causes cellular damage and dysfunction, and it has been linked to various diseases. Antioxidant and phase 2 metabolizing enzymes protect cells from oxidants and electrophiles (2). Transcriptional regulation of these protective genes is regulated in part through DNA sequences known as antioxidant response elements (ARE) 2 (3). Members of small Maf and "Cap 'n' Collar" type of basic leucine zipper (CNC-bZIP) proteins mediate transcriptional regulation through the ARE (4). The CNC-bZIP family comprises a number of structurally related proteins that bind DNA as heterodimers. This family includes p45NFE2, Nrf1, Nrf2, Nrf3, Bach1, and Bach2 (5-9). Loss of function analysis in Nrf2 knock-out mice has demonstrated its importance in protection against oxidative stress-induced pathology in various organs (10 -15). These and other studies have established that Nrf2 is an important regulator of ARE function. In mouse, Nrf1 is indispensable for viability during embryonic development (16). Analysis of Nrf1-deficient fibroblasts suggests that it is also involved in the oxidative stress response (17). Nrf1 has been shown to activate expression of both the catalytic and regulatory subunits of glutamyl-cysteine ligase genes (Gclc and Gclm) through AREs in the promoters of these genes (17, 18). The si...

Rice grain with excessive cadmium (Cd) is a major source of dietary Cd intake and a serious threat to health for people who consume rice as a staple food. The development of elite rice cultivars with consistently low Cd content is challenging for conventional breeding approaches, and new strategies urgently need to be developed. Here, we report the development of new indica rice lines with low Cd accumulation and no transgenes by knocking out the metal transporter gene OsNramp5 using CRISPR/Cas9 system. Hydroponic culture showed that Cd concentrations in shoots and roots of osnramp5 mutants were dramatically decreased, resulting in rescue of impaired growth in high Cd condition. Cd-contaminated paddy field trials demonstrated that Cd concentration in osnramp5 grains was consistently less than 0.05 mg/kg, in contrast to high Cd concentrations from 0.33 mg/kg to 2.90 mg/kg in grains of Huazhan (the wild-type indica rice). In particular, the plant yield was not significantly affected in osnramp5 mutants. Furthermore, we developed promising hybrid rice lines with extremely low Cd content in grains. Our work supplies a practical approach to developing Cd pollution-safe indica rice cultivars that minimizes Cd contamination risk in grains.

Therapeutic nanoreactors have been proposed to treat cancers through in situ transformation of low-toxicity prodrugs into toxic therapeutics in the body. However, the in vivo applications are limited by low tissue-specificity and different tissue distributions between sequentially injected nanoreactors and prodrugs. Herein, we construct a block copolymer prodrug-based polymersome nanoreactor that can achieve novel orchestrated oxidation/chemotherapy of cancer via specific activation at tumor sites. The block copolymers composed of poly(ethylene glycol) (PEG) and copolymerized monomers of camptothecin (CPT) and piperidine-modified methacrylate [P(CPTMA-co-PEMA)] were optimized to self-assemble into polymersomes in aqueous solution for encapsulation of glucose oxidase (GOD) to obtain GOD-loaded polymersome nanoreactors (GOD@PCPT-NR). GOD@PCPT-NR maintained inactive in normal tissues upon systemic administration. After deposition in tumor tissues, tumor acidity-triggered protonation of PPEMA segments resulted in high permeability of the polymersome membranes and oxidation reaction of diffused glucose and O under the catalysis of GOD. The activation of the reaction generated HO, improving the oxidative stress in tumors. Simultaneously, a high level of HO further activated PCPTMA prodrugs, releasing active CPT drugs. High tumor oxidative stress and released CPT drugs synergistically killed cancer cells and suppressed tumor growth via oxidation/chemotherapy. Our study provides a new strategy for engineering therapeutic nanoreactors in an orchestrated fashion for cancer therapy.