Candesartan and epigallocatechin‐3‐gallate ameliorate gentamicin‐induced renal damage in rats through p38‐MAPK and NF‐κB pathways

This study pointed to estimate the possible protective impacts of candesartan and/or epigallocatechin‐3‐gallate (EGCG) against gentamicin‐induced nephrotoxicity. The current work revealed that gentamicin significantly elevated relative kidney weight and the serum level of creatinine and urea. Also, renal level of malondialdehyde was significantly increased with a concurrent decrease in renal glutathione‐S‐transferase and superoxide dismutase activities. Moreover, renal levels of nuclear factor‐kappa B (NF‐κB) and p38 mitogen‐activated protein kinase (p38‐MAPK) were increased together with the elevation of tumor necrosis factor‐alpha and interleukin‐1 beta levels after gentamicin treatment. In addition, caspase‐3 expression was elevated, and histological examination revealed extreme alterations enlightening inflammation, degeneration, and necrosis. Pretreatments with candesartan and/or EGCG attenuated gentamicin‐induced nephrotoxicity. Importantly, the altered expression of p38‐MAPK and NF‐κB may play a significant role in the protective mechanisms exerted by candesartan and EGCG. Coadministration of candesartan and EGCG exhibited more profound response compared with the monotherapy.     

Induction of Heme Oxygenase-1 by Na+-H+ Exchanger 1 Protein Plays a Crucial Role in Imatinib-resistant Chronic Myeloid Leukemia Cells

Resistance toward imatinib (IM) and other BCR/ABL tyrosine kinase inhibitors remains troublesome in the treatment of advanced stage chronic myeloid leukemia (CML). The aim of this study was to estimate the reversal effects of down-regulation of Na⁺/H⁺ exchanger 1 (NHE1) on the chemoresistance of BCR-ABL-positive leukemia patients'' cells and cell lines. After treatment with the specific NHE1 inhibitor cariporide to decrease intracellular pH (pH_i), the heme oxygenase-1 (HO-1) levels of the K562R cell line and cells from IM-insensitive CML patients decreased. HO-1, as a Bcr/Abl-dependent survival molecule in CML cells, is important for the resistance to tyrosine kinase inhibitors in patients with newly diagnosed CML or IM-resistant CML. Silencing PKC-β and Nrf-2 or treatment with inhibitors of p38 pathways obviously blocked NHE1-induced HO-1 expression. Furthermore, treatment with HO-1 or p38 inhibitor plus IM increased the apoptosis of the K562R cell line and IM-insensitive CML patients'' cells. Inhibiting HO-1 enhanced the activation of caspase-3 and poly(ADP-ribose) polymerase-1. Hence, the results support the anti-apoptotic role of HO-1 induced by NHE1 in the K562R cell line and IM-insensitive CML patients and provide a mechanism by which inducing HO-1 expression via the PKC-β/p38-MAPK pathway may promote tumor resistance to oxidative stress.     

The Expression of Fn14 via Mechanical Stress-activated JNK Contributes to Apoptosis Induction in Osteoblasts

Bone mass is maintained by the balance between the activities of bone-forming osteoblasts and bone-resorbing osteoclasts. It is well known that adequate mechanical stress is essential for the maintenance of bone mass, whereas excess mechanical stress induces bone resorption. However, it has not been clarified how osteoblasts respond to different magnitudes of mechanical stress. Here we report that large-magnitude (12%) cyclic stretch induced Ca²⁺ influx, which activated reactive oxygen species generation in MC3T3-E1 osteoblasts. Reactive oxygen species then activated the ASK1-JNK/p38 pathways. The activated JNK led to transiently enhanced expression of FGF-inducible 14 (Fn14, a member of the TNF receptor superfamily) gene. Cells with enhanced expression of Fn14 subsequently acquired sensitivity to the ligand of Fn14, TNF-related weak inducer of apoptosis, and underwent apoptosis. On the other hand, the ASK1-p38 pathway induced expression of the monocyte chemoattractant protein 3 (MCP-3) gene, which promoted chemotaxis of preosteoclasts. In contrast, the ERK pathway was activated by small-magnitude stretching (1%) and induced expression of two osteogenic genes, collagen Ia (Col1a) and osteopontin (OPN). Moreover, activated JNK suppressed Col1a and OPN induction in large-magnitude mechanical stretch-loaded cells. The enhanced expression of Fn14 and MCP-3 by 12% stretch and the enhanced expression of Col1a and OPN by 1% stretch were also observed in mouse primary osteoblasts. These results suggest that differences in the response of osteoblasts to varying magnitudes of mechanical stress play a key role in switching the mode of bone metabolism between formation and resorption.     

ω‐hydroxyundec‐9‐enoic acid induces apoptosis by ROS mediated JNK and p38 phosphorylation in breast cancer cell lines

ω‐Hydroxyundec‐9‐enoic acid (ω‐HUA), a plant secondary metabolite, exhibits anti‐fungal activity. However, its effect on breast cancer cells is unknown. Here, we investigated the anti‐ breast cancer activity of ω‐HUA and its underlying mechanism. Treatment of human breast cancer cell lines, MDA‐MB‐231 and MDA‐MB‐435, with ω‐HUA induced apoptotic cell death with increased cleaved caspase‐3 and poly (ADP‐ribose) polymerase (PARP) levels, and p38 and JNK phosphorylation. Inhibition of these mitogen‐activated protein kinase (MAPK) pathways using specific inhibitors or siRNA, for p38 and JNK, respectively, blocked the ω‐HUA‐induced apoptosis in a dose‐dependent manner. Moreover, pretreatment of the cells with antioxidant N‐acetyl cysteine (NAC) inhibited ω‐HUA‐induced increased reactive oxygen species (ROS) levels, cleaved caspase‐3 and cleaved PARP, and phosphorylated JNK, phosphorylated p38, and increased cell viability and colony‐forming ability. MDA‐MB‐231 xenograft model showed that the ω‐HUA‐treated group exhibited greater tumor regression and significantly reduced tumor weight compared to that exhibited by the vehicle‐administered group. Collectively, ω‐HUA‐induced intracellular ROS generation induced breast cancer cell apoptosis through JNK and p38 signaling pathway activation, resulting in tumor regression. The results suggested that ω‐HUA is an effective supplement for inhibiting human breast cancer growth.     

Isoform‐specific cleavage of 14‐3‐3 proteins in apoptotic JURL‐MK1 cells

The proteins of 14‐3‐3 family are substantially involved in the regulation of many biological processes including the apoptosis. We studied the changes in the expression of five 14‐3‐3 isoforms (β, γ, ε, τ, and ζ) during the apoptosis of JURL‐MK1 and K562 cells. The expression level of all these proteins markedly decreased in relation with the apoptosis progression and all isoforms underwent truncation, which probably corresponds to the removal of several C‐terminal amino acids. The observed 14‐3‐3 modifications were partially blocked by caspase‐3 inhibition. In addition to caspases, a non‐caspase protease is likely to contribute to 14‐3‐3's cleavage in an isoform‐specific manner. While 14‐3‐3 γ seems to be cleaved mainly by caspase‐3, the alternative mechanism is essentially involved in the case of 14‐3‐3 τ, and a combined effect was observed for the isoforms ε, β, and ζ. We suggest that the processing of 14‐3‐3 proteins could form an integral part of the programmed cell death or at least of some apoptotic pathways. J. Cell. Biochem. 106: 673–681, 2009. © 2009 Wiley‐Liss, Inc.     

The 14‐3‐3 protein GF14f negatively affects grain filling of inferior spikelets of rice (Oryza sativa L.)

In rice (Oryza sativa L.), later flowering inferior spikelets (IS), which are located on proximal secondary branches, fill slowly and produce smaller and lighter grains than earlier flowering superior spikelets (SS). Many genes have been reported to be involved in poor grain filling of IS, however the underlying molecular mechanisms remain unclear. The present study determined that GF14f, a member of the 14‐3‐3 protein family, showed temporal and spatial differences in expression patterns between SS and IS. Using GF14f–RNAi plants, we observed that a reduction in GF14f expression in the endosperm resulted in a significant increase in both grain length and weight, which in turn improved grain yield. Furthermore, pull‐down assays indicated that GF14f interacts with enzymes that are involved in sucrose breakdown, starch synthesis, tricarboxylic acid (TCA) cycle and glycolysis. At the same time, an increase in the activity of sucrose synthase (SuSase), adenosine diphosphate‐glucose pyrophosphorylase (AGPase), and starch synthase (StSase) was observed in the GF14f–RNAi grains. Comprehensive analysis of the proteome and metabolite profiling revealed that the abundance of proteins related to the TCA cycle, and glycolysis increased in the GF14f–RNAi grains together with several carbohydrate intermediates. These results suggested that GF14f negatively affected grain development and filling, and the observed higher abundance of the GF14f protein in IS compared with SS may be responsible for poor IS grain filling. The study provides insights into the molecular mechanisms underlying poor grain filling of IS and suggests that GF14f could serve as a potential tool for improving rice grain filling.     

Effect of c‐Ski on atrial remodelling in a rapid atrial pacing canine model

Atrial fibrosis is an important factor in the initiation and maintenance of atrial fibrillation (AF); therefore, understanding the pathogenesis of atrial fibrosis may reveal promising therapeutic targets for AF. In this study, we successfully established a rapid atrial pacing canine model and found that the inducibility and duration of AF were significantly reduced by the overexpression of c‐Ski, suggesting that this approach may have therapeutic effects. c‐Ski was found to be down‐regulated in the atrial tissues of the rapid atrial pacing canine model. We artificially up‐regulated c‐Ski expression with a c‐Ski–overexpressing adenovirus. Haematoxylin and eosin, Masson's trichrome and picrosirius red staining showed that c‐Ski overexpression alleviated atrial fibrosis. Furthermore, we found that the expression levels of collagen III and α‐SMA were higher in the groups of dogs subjected to right‐atrial pacing, and this increase was attenuated by c‐Ski overexpression. In addition, c‐Ski overexpression decreased the phosphorylation of smad2, smad3 and p38 MAPK (p38α and p38β) as well as the expression of TGF‐β1 in atrial tissues, as shown by a comparison of the right‐atrial pacing + c‐Ski‐overexpression group to the control group with right‐atrial pacing only. These results suggest that c‐Ski overexpression improves atrial remodelling in a rapid atrial pacing canine model by suppressing TGF‐β1–Smad signalling and p38 MAPK activation.     

Ablation of the 14‐3‐3gamma Protein Results in Neuronal Migration Delay and Morphological Defects in the Developing Cerebral Cortex

14‐3‐3 proteins are ubiquitously‐expressed and multifunctional proteins. There are seven isoforms in mammals with a high level of homology, suggesting potential functional redundancy. We previously found that two of seven isoforms, 14‐3‐3epsilon and 14‐3‐3zeta, are important for brain development, in particular, radial migration of pyramidal neurons in the developing cerebral cortex. In this work, we analyzed the function of another isoform, the protein 14‐3‐3gamma, with respect to neuronal migration in the developing cortex. We found that in utero 14‐3‐3gamma‐deficiency resulted in delays in neuronal migration as well as morphological defects. Migrating neurons deficient in 14‐3‐3gamma displayed a thicker leading process stem, and the basal ends of neurons were not able to reach the boundary between the cortical plate and the marginal zone. Consistent with the results obtained from in utero electroporation, time‐lapse live imaging of brain slices revealed that the ablation of the 14‐3‐3gamma proteins in pyramidal neurons slowed down their migration. In addition, the 14‐3‐3gamma deficient neurons showed morphological abnormalities, including increased multipolar neurons with a thicker leading processes stem during migration. These results indicate that the 14‐3‐3gamma proteins play an important role in radial migration by regulating the morphology of migrating neurons in the cerebral cortex. The findings underscore the pathological phenotypes of brain development associated with the disruption of different 14‐3‐3 proteins and will advance the preclinical data regarding disorders caused by neuronal migration defects. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 600–614, 2016     

2, 3, 7, 8‐Tetrachlorodibenzo‐p‐dioxin promotes endothelial cell apoptosis through activation of EP3/p38MAPK/Bcl‐2 pathway

Endothelial injury or dysfunction is an early event in the pathogenesis of atherosclerosis. Epidemiological and animal studies have shown that 2, 3, 7, 8‐tetrachlorodibenzo‐p‐dioxin (TCDD) exposure increases morbidity and mortality from chronic cardiovascular diseases, including atherosclerosis. However, whether or how TCDD exposure causes endothelial injury or dysfunction remains largely unknown. Cultured human umbilical vein endothelial cells (HUVECs) were exposed to different doses of TCDD, and cell apoptosis was examined. We found that TCDD treatment increased caspase 3 activity and apoptosis in HUVECs in a dose‐dependent manner,at doses from 10 to 40 nM. TCDD increased cyclooxygenase enzymes (COX)‐2 expression and its downstream prostaglandin (PG) production (mainly PGE₂ and 6‐keto‐PGF_1α) in HUVECs. Interestingly, inhibition of COX‐2, but not COX‐1, markedly attenuated TCDD‐triggered apoptosis in HUVECs. Pharmacological inhibition or gene silencing of the PGE₂ receptor subtype 3 (EP3) suppressed the augmented apoptosis in TCDD‐treated HUVECs. Activation of the EP3 receptor enhanced p38 MAPK phosphorylation and decreased Bcl‐2 expression following TCDD treatment. Both p38 MAPK suppression and Bcl‐2 overexpression attenuated the apoptosis in TCDD‐treated HUVECs. TCDD increased EP3‐dependent Rho activity and subsequently promoted p38MAPK/Bcl‐2 pathway‐mediated apoptosis in HUVECs. In addition, TCDD promoted apoptosis in vascular endothelium and delayed re‐endothelialization after femoral artery injury in wild‐type (WT) mice, but not in EP3^?/? mice. In summary, TCDD promotes endothelial apoptosis through the COX‐2/PGE₂/EP3/p38MAPK/Bcl‐2 pathway. Given the cardiovascular hazard of a COX‐2 inhibitor, our findings indicate that the EP3 receptor and its downstream pathways may be potential targets for prevention of TCDD‐associated cardiovascular diseases.