The effect of resveratrol and its methylthio-derivatives on EGFR and Stat3 activation in human HaCaT and A431 cells

Epidermal growth factor receptor (EGFR) interacting with Stat3 is considered to be an attractive therapeutic target. In the current study, we investigated the effect of resveratrol and its two 4′-methylthio-trans-stilbene derivatives (3-M-4′-MTS; S2) (3,5-DM-4″-MTS; S5) on EGFR and Stat3 activation in human immortalized HaCaT keratinocytes and epidermoid carcinoma A431 cells. In the HaCaT cells both derivatives, similarly as resveratrol, decreased the total level of the EGFR receptor. In the A431 cells, resveratrol in the higher dose significantly (p < 0.05) reduced Y1173 and Y1068 EGFR residue phosphorylation, while S2 affected only the phosphorylation of the Y1068 residue. In this cell line, resveratrol in both tested doses and the S2 derivative in the lower concentration significantly diminished Stat3 binding capacity to the DNA consensus site. The effect of the tested compounds on Stat3 activation in HaCaT cells was only slightly affected. These results indicate that methylthiostilbenes are not more potent modulators of the EGFR/Stat3 complex than resveratrol and that introducing an additional methoxy group makes them less effective.     

Oxidative damage and the Nrf2-ARE pathway in neurodegenerative diseases

Oxidative damage contributes to pathogenesis in many neurodegenerative diseases. As the indicator and regulator of oxidative stress, the Nrf2-ARE pathway has been shown dynamic changes and examined for its neuroprotective role in many cases. In this review, we summarize the progress of the Nrf2-ARE pathway in combating toxicity induced from typical misfolded protein aggregates in neurodegenerative diseases, and specifically the effects on the clearance of protein aggregates. This article is part of a Special Issue entitled: Misfolded Proteins, Mitochondrial Dysfunction, and Neurodegenerative Diseases.     

Nrf2-ARE信号通路在神经退行性疾病中的作用研究进展

神经退行性疾病(Neurodegenerative disease)是一类以神经元退行性病变为基础的慢性、进行性、不可逆的神经系统疾病的总称,主要包括阿尔茨海默病(Alzheimer's disease,AD)、帕金森病(Parkinson's disease,PD)、亨廷顿舞蹈病(Huntington disease,HD)、肌萎缩侧索硬化症(amyotrophic lateral sclerosis,ALS)、脊髓肌萎缩症(spinal muscular atrophy,SMA)、不同类型脊髓小脑共济失调(spinal cerebella ataxias,SCA)等。其病因和发病机制十分复杂,其中,氧化应激学说近年来受到了人们的广泛关注和普遍认可。而研究表明,Nrf2-ARE信号通路是体内抗氧化应答机制中最重要的通路之一,其能对氧化应激导致的神经细胞损伤产生保护作用,即阻止神经细胞的病变和凋亡,进而延缓神经退行性疾病的发生发展,因而有望成为神经退行性疾病的有效治疗靶标。本文就Nrf2-ARE信号通路结构特点及Nrf2-ARE信号通路在神经退行性疾病中的作用研究进展作一综述。     

血管内皮Nrf2-ARE通路调控的研究进展与展望

核因子E2相关因子2(nuclear factor E2-related factor 2,Nrf2)是一种对细胞氧化应激十分敏感的基因转录因子,可诱导依赖抗氧化反应元件(ARE)的多种抗氧化蛋白的合成;Nrf2由Kelch样ECH相关蛋白1(Keap1)扣押于胞浆并被转运至26S蛋白酶体降解,从而维持在生理状态下Nrf2的低转录活性;创伤及氧化应激可诱导Nrf2从Keap1解离并转位至胞核,从而启动下游靶基因的转录激活.由蛋白激酶C(PKC)、丝裂原活化蛋白激酶(MAPK)、肌酸激酶2(CK2)等蛋白激酶介导的Nrf2磷酸化、亲电子物质对Keap1巯基的修饰,以及泛素-蛋白酶体系统均与Nrf2-ARE通路的转录调控有关.     

Apoptosis occurs via the ceramide recycling pathway in human HaCaT keratinocytes

Keratinocytes contain abundant ceramides compared to other cells. However, studies on these cells have mainly focused on the barrier function of ceramide, while their other roles, such as those in apoptosis or cell cycle arrest, have not been well addressed. In this study, we investigated the apoptosis-inducing effect of exogenously added cell-permeable ceramides in HaCaT keratinocytes. We found that N-hexanoyl sphingosine (C6-ceramide) induced apoptosis efficiently through the accumulation of long chain ceramides. On the other hand, N-acetyl sphingosine (C2-ceramide) induced neither apoptosis nor accumulation of long chain ceramides. We also found that exogenously added C6-ceramide was hydrolyzed to sphingosine and then reacylated in long chain ceramides (ceramide recycling pathway), but that C2-ceramide was not hydrolyzed and thus not recycled. We propose that this is the basis for the chain length-specific heterogeneity observed in ceramide-induced apoptosis in these cells. These results also imply that keratinocytes utilize exogenous sphingolipids or ceramides to coordinate their own ceramide compositions.     

Retinoic acid induces transforming growth factor-beta 2 in cultured keratinocytes and mouse epidermis.

We have studied the functional interaction between retinoic acid and transforming growth factor-beta (TGF-beta), using the mouse epidermis as a model system. Treatment with retinoic acid increases expression of TGF-beta 2 in cultured keratinocytes in vitro, as well as in the epidermis in vivo. This TGF-beta 2 is secreted in a biologically active form that can bind to surface receptors, in contrast to most other conditions in which TGF-beta is secreted in a latent form. Specific antibodies to TGF-beta 2 partially reverse the ability of retinoic acid to inhibit DNA synthesis in cultured keratinocytes. The regulation of TGF-beta 2 expression by retinoic acid may have important physiological and pharmacological roles in the maintenance of epidermal homeostasis.     

Effects of different exercise durations on Keap1-Nrf2-ARE pathway activation in mouse skeletal muscle

Abstract

The purpose of this study was to investigate the effects of acute exercise stress on the nuclear factor-erythroid2 p45-related factor 2 (Nrf2)/antioxidant response element (ARE) transactivation, Kelch-like ECH-associated protein 1 (Keap1) cytosolic protein and Nrf2 nucleoprotein expressions, Nrf2 target genes mRNA expressions, and glutathione redox (GSH/GSSG) ratio level; with a particular focus on the changes in Keap1-Nrf2-ARE pathway activation following different durations of exercise. Wild-type mice (C57BL/6J, two months old) were separated into one-hour and six-hour treadmill running groups, as well as a non-exercise control group (n = 10 in each group). Measurements of Nrf2/ARE transactivation, Nrf2 nucleoprotein expressions, Keap1 cytosolic protein expression, Nrf2 target genes’ mRNA expressions (superoxide dismutase-1 [SOD1], superoxide dismutase-2 [SOD2], γ-glutamyl cysteine ligase-modulatory [GCLm], γ-glutamyl cysteine ligase-catalytic [GCLc], glutathione reductase [GR], glutathione peroxidase-1 [Gpx1], catalase [CAT], and hemoxygenase-1 [Ho-1]), and GSH/GSSG ratio were carried out immediately after exercise. The results showed significant increases in Keap1-Nrf2-ARE pathway activation and the mRNA expressions of six measured enzymes in skeletal muscle after six hours of exercise; while in the one-hour exercise group, there was no change in Keap1-Nrf2-ARE pathway activation and only two enzymes’ mRNA expressions were increased. It is suggested that the changes in Keap1-Nrf2-ARE pathway activation and its target genes’ mRNA expressions were dependent on the exercise duration, with longer duration associated with higher responses.     

Biogenic nanoselenium particles activate Nrf2-ARE pathway by phosphorylating p38, ERK1/2, and AKT on IPEC-J2 cells

As the intestinal epithelium is vulnerable to oxidative stress because of frequent enterocyte renewal and continuous exposure to exogenous agents, it is meaningful to figure out how the epithelial cells exert antioxidant function. We previously synthesized a novel biogenic nanoselenium (BNS) particles and proved that BNS could effectively improve intestinal antioxidative function through activating Nrf2-ARE pathway. The objective of the present study was to investigate the mechanism by which BNS activate Nrf2-ARE pathway on the physiological function of intestinal epithelial cells. In the present study, we demonstrated that treatment of IPEC-J2 cells with BNS particles not only elevated the levels of downstream proteins of nuclear factor (erythroid-derived-2)-like 2 (Nrf2) such as heme oxygenase-1 and NQO-1 in a time-dependent manner which started to weaken at 12 hr after treatment but also significantly activated Nrf2, mitogen-activated protein kinase (MAPK), and protein kinase B (AKT) pathway in a time-dependent manner within 24 hr. BNS particles significantly increased the content of phosphorylated-Nrf2, without evident influence on the level of Kelch-like ECH-associated protein 1 (Keap1). Moreover, BNS also induced the activation of p38, extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase, and AKT while phosphorylating Nrf2. Using specific protein kinase inhibitors, we found that the Nrf2-phosphorylating and antioxidative effects of BNS particles were abolished when p38, ERK1/2, and AKT were significantly inhibited. Overall, our data demonstrated that BNS particles activated Nrf2-ARE pathway through p38, ERK1/2, and AKT mediated-phosphorylation of Nrf2 to improve the antioxidant function of intestinal epithelial cells     

Enhancement of dopaminergic activity and region-specific activation of Nrf2-ARE pathway by intranasal supplements of testosterone propionate in aged male rats

The potential influence of intranasal testosterone propionate (InTP) supplements on mesodopaminergic system in aged male rats was investigated by analyzing the exploratory and motor behaviors as well as dopamine neurobiochemical indices. Meanwhile, oxidative stress parameters and pathway of nuclear factor erythroid 2-related factor 2 (Nrf2)-binding antioxidant response elements (Nrf2-ARE) were examined to check whether the Nrf2-ARE pathway was involved in the InTP-induced alteration of mesodopaminergic system in aged male rats. The exploratory and motor behavioral deficits, as well as the reduced expression of dopamine, tyrosine hydroxylase, and dopamine transporter, which indicated the declined activity of mesodopaminergic system, were ameliorated in rats administered with 12-week InTP. The results indicated that chronic InTP supplements could effectively influence the brain function activity in a way opposite to the effect of aging on the mesodopaminergic system of rats. The increased levels of Nrf2, heme oxygenase-1, and NAD(P)H:quinone oxidoreductase-1 in the substantia nigra and ventral tegmental area, but not in the hippocampus of InTP-administered aged male rats, indicated that the ameliorative effect of InTP supplements on mesodopaminergic system might be related to the region-specific activation of the Nrf2-ARE pathway.     

Protective effect of oxidative stress in HaCaT keratinocytes expressing E7 oncogene

Summary. In a previous study, we established a stable cell line which constitutively expresses E7 in HaCaT human keratinocyte cell line and identified various relevant factors including oxygen modulators affected by the E7 oncogene. E7-expressing HaCaT cells (HaCaT/E7) appeared to be more resistant to H₂O₂-induced cell death. Here, we demonstrate how E7 oncogene would modulate oxidative stress-induced cell death. In addition, we verified the increased expression of catalase in the HaCaT/E7 by Western blot analysis. The results suggest that the E7 oncogene would induce higher resistance to ROS-induced cell injury in the E7-infected cells via the upregulation of catalase. To investigate these paradoxical effects of high concentrations of H₂O₂ (500 μM–1 mM), we examined their effects on receptor mediated apoptosis, cell death via the mitochondrial pathway and modulation of apoptosis related factors. Our results revealed that HaCaT keratinocytes infected with HPV 16 E7 oncogene modulated expressions of catalase, Bcl-xL, IL-18, Fas, Bad, and cytochrome c as well as NF-κB, resulting in the resistance to oxidative stress-induced cell death. Authors’ address: Dr. Do-Young Yoon, Laboratory of Cell and Immunobiochemistry, Department of Bioscience and Biotechnology, Konkuk University, Hwayang-dong 1, Gwangjin-gu, Seoul 143-701, Korea