Tumoricidal Activity of Simvastatin in Synergy with RhoA Inactivation in Antimigration of Clear Cell Renal Cell Carcinoma Cells.

Among kidney cancers, clear cell renal cell carcinoma (ccRCC) has the highest incidence rate in adults. The survival rate of patients diagnosed as having metastatic ccRCC drastically declines even with intensive treatment. We examined the efficacy of simvastatin, a lipid-lowering drug with reduced mevalonate synthesis, in ccRCC treatment. Simvastatin was found to reduce cell viability and increase autophagy induction and apoptosis. In addition, it reduced cell metastasis and lipid accumulation, the target proteins of which can be reversed through mevalonate supplementation. Moreover, simvastatin suppressed cholesterol synthesis and protein prenylation that is essential for RhoA activation. Simvastatin might also reduce cancer metastasis by suppressing the RhoA pathway. A gene set enrichment analysis (GSEA) of the human ccRCC GSE53757 data set revealed that the RhoA and lipogenesis pathways are activated. In simvastatin-treated ccRCC cells, although RhoA was upregulated, it was mainly restrained in the cytosolic fraction and concomitantly reduced Rho-associated protein kinase activity. RhoA upregulation might be a negative feedback effect owing to the loss of RhoA activity caused by simvastatin, which can be restored by mevalonate. RhoA inactivation by simvastatin was correlated with decreased cell metastasis in the transwell assay, which was mimicked in dominantly negative RhoA-overexpressing cells. Thus, owing to the increased RhoA activation and cell metastasis in the human ccRCC dataset analysis, simvastatin-mediated Rho inactivation might serve as a therapeutic target for ccRCC patients. Altogether, simvastatin suppressed the cell viability and metastasis of ccRCC cells; thus, it is a potentially effective ccRCC adjunct therapy after clinical validation for ccRCC treatment.

Exerting the Appropriate Application of Methylprednisolone in Acute Spinal Cord Injury Based on Time Course Transcriptomics Analysis.

Methylprednisolone (MP) is an anti-inflammatory drug approved for the treatment of acute spinal cord injuries (SCIs). However, MP administration for SCIs has become a controversial issue while the molecular effects of MP remain unexplored to date. Therefore, delineating the benefits and side effects of MP and determining what MP cannot cure in SCIs at the molecular level are urgent issues. Here, genomic profiles of the spinal cord in rats with and without injury insults, and those with and without MP treatment, were generated at 0, 2, 4, 6, 8, 12, 24, and 48 h post-injury. A comprehensive analysis was applied to obtain three distinct classes: side effect of MP (SEMP), competence of MP (CPMP), and incapability of MP (ICMP). Functional analysis using these genes suggested that MP exerts its greatest effect at 8~12 h, and the CPMP was reflected in the immune response, while SEMP suggested aspects of metabolism, such as glycolysis, and ICMP was on neurological system processes in acute SCIs. For the first time, we are able to precisely reveal responsive functions of MP in SCIs at the molecular level and provide useful solutions to avoid complications of MP in SCIs before better therapeutic drugs are available.

MPT0B169 and MPT0B002, New Tubulin Inhibitors, Induce Growth Inhibition, G2/M Cell Cycle Arrest, and Apoptosis in Human Colorectal Cancer Cells.

We previously synthesized new tubulin inhibitors, MPT0B169 and MPT0B002, which induced growth inhibition and apoptosis in leukemia cells. However, their effects on solid tumor cells have not been determined. In this study, we investigated the effects of MPT0B169 and MPT0B002 on glioblastoma, breast, lung, and colorectal cancer (CRC) cell lines. A cell viability analysis showed that MPT0B169 and MPT0B002 were more effective in inhibiting the proliferation of COLO205 and HT29 CRC cells than U87MG and GBM8401 glioblastoma, MCF-7 and MDA-MB-231 breast cancer, and A549 lung cancer cells. MPT0B169 and MPT0B002 inhibited growth of COLO205 and HT29 cells in dose- and time-dependent manners. A colony-formation assay confirmed the growth inhibitory effects of MPT0B169 and MPT0B002 on COLO205 and HT29 cells. MPT0B169 and MPT0B002 disrupted tubulin polymerization and arrested the cell cycle at the G2/M phase, with a concomitant increase of the cyclin B1 level. MPT0B169 and MPT0B002 induced apoptosis, accompanied by induction of the intrinsic apoptotic pathway, as shown by a reduction in the caspase-9 level and increases in cleaved caspase-3 and cleaved PARP. These results suggest that MPT0B169 and MPT0B002, new tubulin inhibitors, induced growth inhibition, G2/M arrest, and apoptosis in COLO205 and HT29 cells, and they could potentially be anticancer agents for CRC cells.

3-Methylcholanthrene/Aryl-Hydrocarbon Receptor-Mediated Hypertension Through eNOS Inactivation.

Endothelial nitric oxide synthase (eNOS) modulates vascular blood pressure and is predominantly expressed in endothelial cells and activated through the protein kinase B (Akt/PKB)-dependent pathway. We previously reported that 3-methylcholanthrene (3MC) activates the aryl hydrocarbon receptor (AhR) and reduces PI3K/Akt phosphorylation. This study investigated the mechanism underlying the downregulatory effects of 3-MC on nitric oxide (NO) production occurring through the AhR/RhoA/Akt-mediated mechanism. The mechanism underlying the effects of 3-MC on eNOS activity and blood pressure was examined in vitro and in vivo through genetic and pharmacological approaches. Results indicated that 3-MC modified heat shock protein 90 (HSP90), caveolin-1, dynein, and eNOS mRNA and protein expression through the AhR/RhoA-dependent mechanism in mouse cerebral vascular endothelial cells (MCVECs) and that 3-MC reduced eNOS phosphorylation through the AhR/RhoA-mediated inactivation of Akt1. The upregulation of dynein expression was associated with decreased eNOS dimer formation (eNOS dimer; an activated form of the enzyme). Coimmunoprecipitation assay results indicated that 3-MC significantly reduced the interaction between eNOS and its regulatory proteins, including Akt1 and HSP90, but increased the interaction between eNOS and caveolin-1. Immunofluorescence and Western blot analysis revealed that 3-MC reduced the amount of membrane-bound activated eNOS, and a modified Griess assay revealed that 3-MC concomitantly reduced NO production. However, simvastatin reduced 3-MC-mediated murine hypertension. Our study results indicate that AhR, RhoA, and eNOS have major roles in blood pressure regulation. Statin intervention might provide a potential therapeutic approach for reducing hypertension caused by 3-MC. J. Cell. Physiol. 232: 1020-1029, 2017. © 2016 Wiley Periodicals, Inc.

Activating transcription factor 3 protects against pressure-overload heart failure via the autophagy molecule Beclin-1 pathway.

Activating transcription factor 3 (ATF3), a cAMP response element-binding protein/ATF family transcription factors member, has been implicated in the cardiovascular and inflammatory system and is rapidly induced by ischemic-reperfusion injuries. We performed transverse aortic banding (TAB) experiments using ATF3 gene-deleted mice (ATF3(-/-)) and wild-type (WT) mice to determine what effect it might have on heart failure induced by pressure overloading. Compared with the WT mice, ATF3(-/-) mice were found by echocardiography to have decreased left ventricular contractility with loss of normal cardiac hypertrophic remodeling. The ATF3(-/-) mice had greater numbers of terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine nick-end labeling-positive cells and higher levels of activated caspase-3, as well as more apoptosis. Restoration of ATF3 expression in the heart of ATF3(-/-) mice by adenovirus-induced ATF3 treatment significantly improved cardiac contractility after TAB. The results from molecular and biochemical analyses, including chromatin immune-precipitation and in vitro /in vivo promoter assays, showed that ATF3 bound to the ATF/cAMP response element of the Beclin-1 promoter and that ATF3 reduced autophagy via suppression of the Beclin-1-dependent pathway. Furthermore, infusion of tert-butylhydroquinone (tBHQ), a selective ATF3 inducer, increased the expression of ATF3 via the nuclear factor erythroid 2-related transcriptional factor, inhibited TAB-induced cardiac dilatation, and increased left ventricular contractility, thereby rescuing heart failure. Our study identified a new epigenetic regulation mediated by the stress-inducible gene ATF3 on TAB-induced cardiac dysfunction. These findings suggest that the ATF3 activator tBHQ may have therapeutic potential for the treatment of pressure-overload heart failure induced by chronic hypertension or other pressure overload mechanisms.

RhoA-mediated inhibition of vascular endothelial cell mobility: positive feedback through reduced cytosolic p21 and p27.

We previously identified that activation of the aryl hydrocarbon receptor (AhR) by 3-methylcholanthrene (3MC) exerts antiproliferative and antimigratory effects on human umbilical vein endothelial cells (HUVECs) through the upregulation of p21/p27 transcription and RhoA activation. In this study, we investigated the mechanisms of 3MC-mediated downregulation of cytosolic p21/ p27, and the effects of 3MC on RhoA activation and cell migration, in mouse cerebral vascular endothelial cells (MCVECs). Our results indicated that 3MC reduced the phosphorylation of p21/p27 through AhR/RhoA/PTEN-mediated PI3K/Akt inactivation, which reduced cytosolic p21/p27 retention, causing RhoA activation through positive feedback. Downregulation of p21/p27 by siRNA, and cytosolic p21/p27 by the nuclear export blocker leptomycin B, further reduced cell migration in the 3MC-treated cells. Reduced cytosolic p21/p27 expression led to reduced interaction between RhoA and the RhoA inhibitor p190RhoGAP, causing RhoA activation. Treatment with YS-49 activated PI3K/Akt, a downstream target of RhoA, to reduce RhoA/PTEN activation in the 3MC-treated cells, whereas treatment with wortmannin, a PI3K inhibitor, activated RhoA/PTEN. Gain- and loss-of-function analyses revealed that constitutively active (CA) Akt1, but not CA Akt2, inactivated RhoA and stimulated migratory activity. Considering the essential role of RhoA activation in cell migration, we evaluated the potential use of simvastatin, a RhoA inhibitor, as a therapeutic intervention in vivo using matrigel plug formation assays. Our results provide a molecular basis for the therapeutic application of simvastatin to reduce RhoA/PTEN activation, restore cytosolic levels of phosphorylated p21/p27, and induce angiogenic processes.

Mediating effects of aryl-hydrocarbon receptor and RhoA in altering brain vascular integrity: the therapeutic potential of statins.

We have demonstrated previously that focal adhesion kinase (FAK)/RhoA alteration by the aryl-hydrocarbon receptor (AhR) agonist 3-methylcholanthrene (3MC) is involved in the antimigratory effects of 3MC in human umbilical vascular endothelial cells. Here, we identified that signaling properties and molecular mechanisms of RhoA/ß-catenin were both implicated in alterations to blood-brain barrier integrity. The mechanisms of action were the down-regulation of integrin, the extracellular matrix, and adherens junction stability. PTEN phosphorylation by 3MC-mediated AhR/RhoA activation increased the proteasomal degradation of ß-catenin through PKCδ/pGSK3ß-mediated ß-catenin phosphorylation; the crucial roles of AhR/RhoA in this process were verified by using gain- or loss-of-function experiments. The decrease in ß-catenin led to decreased expression of fibronectin and α5ß1 integrin. Additionally, protein interactions among FAK, VE-cadherin, vinculin, and ß-actin were simultaneously decreased, resulting in adherens junction instability. Novel functional TCF/LEF1 binding sites in the promoter regions of fibronectin and α5/ß1 integrin were identified by electrophoretic mobility shift and chromatin immunoprecipitation assays. The results indicate that the binding activities of ß-catenin decreased in mouse cerebrovascular endothelial cells treated with 3MC. In addition, simvastatin and pravastatin treatment reversed 3MC-mediated alterations in mouse cerebrovascular endothelial cells by RhoA inactivation, and the in vitro findings were substantiated by an in vivo blood-brain barrier assay. Thus, endothelial barrier dysfunction due to 3MC occurs through AhR/RhoA-mediated ß-catenin down-regulation, which is reversed by simvastatin treatment in vivo.

Sigesbeckia orientalis Extract Ameliorates the Experimental Diabetic Nephropathy by Downregulating the Inflammatory and Oxidative Stress Signaling Pathways.

Diabetes in children and its complications are on the rise globally, which is accompanied by increasing in diabetes-related complications. Oxidative stress and inflammation induced by elevated blood sugar in diabetic patients are considered risk factors associated with the development of diabetes complications, including chronic kidney disease and its later development to end-stage renal disease. Microvascular changes within the kidneys of DM patients often lead to chronic kidney disease, which aggravates the illness. Sigesbeckia orientalis extract (SOE), reported to have strong antioxidative and excellent anti-inflammatory activities, is used in the modern practice of traditional Chinese medicine. Kidneys from three groups of control mice (CTR), mice with streptozotocin (STZ)-induced diabetes (DM), and mice with STZ-induced DM treated with SOE (DMRx) were excised for morphological analyses and immunohistochemical assessments. Only mice in the DM group exhibited significantly lower body weight, but higher blood sugar was present. The results revealed more obvious renal injury in the DM group than in the other groups, which appeared as greater glomerular damage and tubular injury, sores, and plenty of connective tissues within the mesangium. Not only did the DM group have a higher level of cytokine, tumor necrosis factor, and the oxidative stress marker, 8-hydroxyguanosine expression, but also factors of the nuclear factor pathway and biomarkers of microvascular status had changed. Disturbances to the kidneys in DMRx mice were attenuated compared to the DM group. We concluded that SOE is an effective medicine, with antioxidative and anti-inflammatory abilities, to protect against or attenuate diabetic nephropathy from inflammatory disturbances by oxidative stress and to cure vessel damage in a hyperglycemic situation.

Valacyclovir-associated neurotoxicity among patients on hemodialysis and peritoneal dialysis: A nationwide population-based study.

Whether valacyclovir-associated neurotoxicity (VAN) occurs more frequently in patients with end-stage renal disease (ESRD) on dialysis is unknown. This is the first population-based study to examine the risk of VAN associated with ESRD patients on dialysis. Among 2,284,800 patients diagnosed as having herpes zoster from 2002 to 2016, patients with ESRD on dialysis and individuals with normal renal function were enrolled in this study. Following propensity score matching, we compared the risk of altered mental status between valacyclovir users and non-users in the ESRD and normal renal function cohorts over a 30-day follow-up period. In the ESRD cohort, the incidence of altered mental status was 1.68 and 0.52 per 1,000 person-day in valacyclovir users and non-users, respectively, with an adjusted hazard ratio (HR) of 3.22 (95% confidence interval [CI]: 2.04-4.99, P < 0.001). The incidence of altered mental status of valacyclovir users on hemodialysis (HD) and peritoneal dialysis (PD) was higher than that of non-users. The adjusted HR was 3.20 (95% CI: 1.98-5.15, P < 0.001) for those on HD and 3.44 (95% CI: 1.13-10.49, P = 0.030) for those with PD. However, altered mental status was not observed in patients on HD receiving ≤500 mg of valacyclovir three times per week or in those on PD receiving ≤500 mg of valacyclovir per day. The findings demonstrate that adjusting the valacyclovir dosage and monitoring VAN in patients with HD and PD who have herpes zoster is crucial.

Association Between DPP-4 Inhibitors and Events of Colorectal and Liver Cancers in Patients With Diabetes Receiving Second-Line Agents: A Nested Case-Control Study.

Objective: Plasma dipeptidyl peptidase-4 (DPP4) levels were significantly lower in patients with colorectal and liver cancers, and animal studies also showed DPP4 inhibitors (DPP4is) have procarcinogenic effects in colorectal cancer. Until now, whether DPP4is therapy affects the progression of liver cancer and colorectal cancer in patients with T2DM has not been well investigated. We investigated the association between cumulative defined daily dose (cDDD) of DPP4is exposure and risks of liver and colorectal cancers in patients with type 2 diabetes. Materials and Methods: We identified 268,520 patients with diabetes receiving DPP4is as second-line agents between March 1, 2009, and December 31, 2013, from Taiwan's National Health Insurance Research Database, Taiwan Cancer Registry, and National Death Registry of Taiwan. The amount of DPP4is were divided into three groups (low, medium, and high) based on the interquartile range of the cDDD of the DPP4is. Results: The data showed that the low cDDD of DPP-4is was associated with a reducing risk of colorectal cancer [adjusted odds ratio (OR), 0.49; 95% CI, 0.32-0.75; P=0.001]. However, the high cDDD of DPP-4is was associated with an increasing risk of colorectal cancer (adjusted OR, 1.86; 95% CI, 1.32-2.61; P<0.001). No association between DPP4is use and liver cancer risk was observed. Conclusions: This nested case study revealed a J-shaped association between the cDDD of DPP-4is and colorectal cancer risk, but not liver cancer risk. Therefore, the effects of long-term DPP4is use on colorectal cancer risk warrant further study.

L-Carnitine reduces reactive oxygen species/endoplasmic reticulum stress and maintains mitochondrial function during autophagy-mediated cell apoptosis in perfluorooctanesulfonate-treated renal tubular cells.

We previously reported that perfluorooctanesulfonate (PFOS) causes autophagy-induced apoptosis in renal tubular cells (RTCs) through a mechanism dependent on reactive oxygen species (ROS)/extracellular signal-regulated kinase. This study extended our findings and determined the therapeutic potency of L-Carnitine in PFOS-treated RTCs. L-Carnitine (10 mM) reversed the effects of PFOS (100 µM) on autophagy induction and impaired autophagy flux. Furthermore, it downregulated the protein level of p47Phox, which is partly related to PFOS-induced increased cytosolic ROS in RTCs. Moreover, L-Carnitine reduced ROS production in mitochondria and restored PFOS-impeded mitochondrial function, leading to sustained normal adenosine triphosphate synthesis and oxygen consumption and reduced proton leakage in a Seahorse XF stress test. The increased inositol-requiring enzyme 1α expression by PFOS, which indicated endoplasmic reticulum (ER) stress activation, was associated with PFOS-mediated autophagy activation that could be attenuated through 4-phenylbutyrate (5 mM, an ER stress inhibitor) and L-Carnitine pretreatment. Therefore, by reducing the level of IRE1α, L-Carnitine reduced the levels of Beclin and LC3BII, consequently reducing the level of apoptotic biomarkers including Bax and cleaving PARP and caspase 3. Collectively, these results indicate that through the elimination of oxidative stress, extracellular signal-regulated kinase activation, and ER stress, L-Carnitine reduced cell autophagy/apoptosis and concomitantly increased cell viability in RTCs. This study clarified the potential mechanism of PFOS-mediated RTC apoptosis and provided a new strategy for using L-Carnitine to prevent and treat PFOS-induced RTC apoptosis.

Glucocorticoid protection of oligodendrocytes against excitotoxin involving hypoxia-inducible factor-1alpha in a cell-type-specific manner.

Glucocorticoids are commonly used in treating diseases with white matter lesions, including demyelinating diseases and spinal cord injury (SCI). However, glucocorticoids are ineffective in gray matter injuries, such as head injury and stroke. The differential glucocorticoid effects in white and gray matter injuries are unclear. We report here a novel mechanism of methylprednisolone (MP), a synthetic glucocorticoid widely used for treating multiple sclerosis and SCI, in protecting oligodendrocytes (OLGs) against AMPA-induced excitotoxicity, which has been implicated in the white matter injuries and diseases. The cytoprotective action of MP in OLGs is causally related to its upregulation of a neuroprotective cytokine erythropoietin (Epo). MP transactivation of Epo expression involves dual transcription factors: glucocorticoid receptor (GR) and hypoxia-inducible factor-1alpha (HIF-1alpha). Coimmunoprecipitation, chromatin immunoprecipitation analysis, yeast two-hybrid analysis, and structure modeling of three-dimensional protein-protein interactions confirm that MP induces interaction between GR DNA binding domain and HIF-1alpha PAS domain, with subsequent recruitment of HIF-1beta to transactivate Epo expression in OLGs. In contrast, MP activates GR but does not induce GR-HIF-1alpha interaction, HIF-1alpha binding to Epo enhancer/promoter, or Epo expression in cultured cortical neurons. The OLG-specific GR-HIF-1alpha transactivation of Epo provides novel insights into the development of more effective therapies for diseases affecting the white matter.

Adiponectin-mediated heme oxygenase-1 induction protects against iron-induced liver injury via a PPARα dependent mechanism.

Protective effects of adiponectin (APN; an adipocytokine) were shown against various oxidative challenges; however, its therapeutic implications and the mechanisms underlying hepatic iron overload remain unclear. Herein, we show that the deleterious effects of iron dextran on liver function and iron deposition were significantly reversed by adiponectin gene therapy, which was accompanied by AMP-activated protein kinase (AMPK) phosphorylation and heme oxygenase (HO)-1 induction. Furthermore, AMPK-mediated peroxisome proliferator-activated receptor-α (PPARα) activation by APN was ascribable to HO-1 induction. Additionally, we revealed direct transcriptional regulation of HO-1 by the binding of PPARα to a PPAR-responsive element (PPRE) by various experimental assessments. Interestingly, overexpression of HO-1 in hepatocytes mimicked the protective effect of APN in attenuating iron-mediated injury, whereas it was abolished by SnPP and small interfering HO-1. Furthermore, bilirubin, the end-product of the HO-1 reaction, but not CO, protected hepatocytes from iron dextran-mediated caspase activation. Herein, we demonstrate a novel functional PPRE in the promoter regions of HO-1, and APN-mediated HO-1 induction elicited an antiapoptotic effect and a decrease in iron deposition in hepatocytes subjected to iron challenge.

Perfluorooctane sulfonate induces autophagy-associated apoptosis through oxidative stress and the activation of extracellular signal-regulated kinases in renal tubular cells.

Perfluorooctane sulfonate (PFOS) is among the most abundant organic pollutants and is widely distributed in the environment, wildlife, and humans. Its toxic effects and biological hazards are associated with its long elimination half-life in humans. However, how it affects renal tubular cells (RTCs) remains unclear. In this study, PFOS was observed to mediate the increase in reactive oxygen species (ROS) generation, followed by the activation of the extracellular-signal-regulated kinase 1/2 (ERK1/2) pathway, which induced autophagy in RTCs. Although PFOS treatment induced autophagy after 6 h, prolonged treatment (24 h) reduced the autophagic flux by increasing lysosomal membrane permeability (LMP), leading to increased p62 protein accumulation and subsequent apoptosis. The increase in LMP was visualized through increased green fluorescence with acridine orange staining, and this was attenuated by 3-methyladenine, an autophagy inhibitor. N-acetyl cysteine and an inhibitor of the mitogen-activated protein kinase kinases (U0126) attenuated autophagy and apoptosis. Taken together, these results indicate that ROS activation and ROS-mediated phosphorylated ERK1/2 activation are essential to activate autophagy, resulting in the apoptosis of PFOS-treated RTCs. Our findings provide insight into the mechanism of PFOS-mediated renal toxicity.

Mechanisms of adiponectin-mediated COX-2 induction and protection against iron injury in mouse hepatocytes.

Adiponectin (APN)-mediated cyclooxygenase (COX)-2 induction is known to have various protective effects on cardiovascular diseases. However, the molecular mechanisms of APN-mediated COX-2 induction and its protection against iron-mediated injury in hepatocytes are still unclear. Herein, we show that AMP-mediated peroxisome proliferator-activated receptor (PPAR)alpha activation was attributable to COX-2 induction by APN, which was further confirmed by identifying novel functional PPAR responsive elements (PPREs) in the mouse COX-2 promoter region. Prostaglandin (PG)I2 and PGE2, metabolites of COX-2, time-dependently increased in hepatocytes treated with APN. Interestingly, beraprost and misoprostol, respective agonists for PGI2 and PGE2, mimicked the protective effects of APN in iron-mediated inflammation in hepatocytes. The iron dextran-activated nuclear factor (NF)-kappaB pathway was correlated with the increased production of inflammatory cytokines including tumor necrosis factor-alpha, intercellular adhesion molecule-1, and monocyte chemotactic protein-1. This was eliminated by administration of APN, whereas blockage of PPARalpha activation, an upstream regulator of COX-2 induction by APN, and COX-2 activation reversed the anti-inflammatory effect of APN, suggesting the crucial role of COX-2 in this event. Herein, we demonstrate that APN-mediated COX-2 induction through a PPARalpha-dependent mechanism, and COX-2 exerted an anti-inflammatory effect of APN in hepatocytes subjected to iron challenge.

Aryl-hydrocarbon receptor-dependent alteration of FAK/RhoA in the inhibition of HUVEC motility by 3-methylcholanthrene.

We previously demonstrated the antiproliferative and antiangiogenic effects of 3-methylcholanthrene (3MC), an aryl-hydrocarbon receptor (AhR) agonist, in human umbilical vascular endothelial cells (HUVECs). Herein, we unraveled its molecular mechanisms in inhibiting HUVEC motility. 3MC down-regulated FAK, but up-regulated RhoA, which was rescued by AhR knockdown. It led us to identify novel AhR binding sites in the FAK/RhoA promoters. Additionally, 3MC increased RhoA activity via suppression of a negative feedback pathway of FAK/p190RhoGAP. With an increase in membrane-bound RhoA, subsequent stress fiber and focal adhesion complex formation was observed in 3MC-treated cells, and this was reversed by a RhoA inhibitor and AhR antagonists. Notably, these compounds significantly reversed 3MC-mediated anti-migration in a transwell assay. The in vitro findings were further confirmed using an animal model of Matrigel formation in Balb/c mice. Collectively, AhR's genomic regulation of FAK/RhoA, together with RhoA activation, is ascribable to the anti-migration effect of 3MC in HUVECs.

Long-Term Effects of Ketoanalogues on Mortality and Renal Outcomes in Advanced Chronic Kidney Disease Patients Receiving a Low-Protein Diet.

The effects of ketoanalogues (KA) supplementation on mortality and progression to dialysis in patients with pre-dialysis stage 5 chronic kidney disease (CKD) receiving a low-protein diet (LPD) remain ambiguous. From Taiwan's National Health Insurance Research Database during 1996-2011, 165 patients with pre-dialysis CKD on an LPD (0.6 g/kg/day) with KA supplementation were matched with 165 patients with pre-dialysis CKD on an LPD without KA supplementation. Of the 165 patients with advanced CKD receiving KA supplementation, 34 (20.6%) died, and 124 (75.2%) underwent long-term dialysis during the study period. There was no significant difference in mortality between the KA-user group and the KA-nonuser group (adjusted hazard ratio [HR], 1.41; 95% confidence interval [CI], 0.68-2.93; p = 0.355). KA supplementation significantly increased long-term dialysis risk (adjusted HR, 1.41; 95% CI, 1.04-1.90; p = 0.025) and combined outcome risk (defined as long-term dialysis and death; adjusted HR, 1.37; 95% CI, 1.02-1.83; p = 0.034). KA supplementation also increased long-term dialysis risk (adjusted HR, 1.49; 95% CI, 1.00-2.20; p = 0.048) in the subgroup of pre-dialysis patients with diabetes mellitus (DM), but not in those patients without DM. In conclusion, KA supplementation might increase long-term dialysis risk in patients with advanced CKD receiving an LPD, but it did not increase mortality.

Author Correction: Simvastatin reduces the carcinogenic effect of 3-methylcholanthrene in renal epithelial cells through histone deacetylase 1 inhibition and RhoA reactivation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

PPARdelta-mediated p21/p27 induction via increased CREB-binding protein nuclear translocation in beraprost-induced antiproliferation of murine aortic smooth muscle cells.

We previously showed that an increase in the peroxisome proliferator-activated receptor-delta (PPARdelta), together with subsequent induction of inducible nitric oxide synthase (iNOS) by beraprost (BPS), inhibits aortic smooth muscle cell proliferation. Herein, we delineated the mechanisms of the antiproliferative effects of BPS through the induction of p21/p27. BPS concentration dependently induced the p21/p27 promoter- and consensus cAMP-responsive element (CRE)-driven luciferase activities, which were significantly suppressed by blocking PPARdelta activation. Surprisingly, other than altering the CRE-binding protein (CREB), BPS-mediated PPARdelta activation increased nuclear localization of the CREB-binding protein (CBP), a coactivator, which was further confirmed by chromatin immunoprecipitation. Furthermore, novel functional PPAR-responsive elements (PPREs) next to CREs in the rat p21/p27 promoter regions were identified, where PPARdelta interacted with CREB through CBP recruitment. BPS-mediated suppression of restenosis in mice with angioplasty was associated with p21/p27 induction. Herein, we demonstrate for the first time that BPS-mediated PPARdelta activation enhances transcriptional activation of p21/p27 by increasing CBP nuclear translocation, which contributes to the vasoprotective action of BPS.

Antioxidation and anti-inflammation by haem oxygenase-1 contribute to protection by tetramethylpyrazine against gentamicin-induced apoptosis in murine renal tubular cells.

BACKGROUND: Gentamicin, a widely used antibiotic for the treatment of bacterial infection, can cause nephrotoxicity. Tetramethylpyrazine (TMP) is a compound purified from the rhizome of Ligusticum wallichi (called chuanxiong in Chinese). Besides its protection against ischaemia-reperfusion injury and nephritis in mice, we previously reported that TMP reverses gentamicin-induced apoptosis in rat kidneys. Haem oxygenase-1 (HO-1) induction by TMP has also been shown to attenuate myocardial ischaemia/reperfusion injury in rats. METHODS: We used rat renal tubular (NRK-52E) cells, transformed cells with HO-1 overexpression or knockdown, and an adenovirus carrying the HO-1 gene (Adv-HO-1) as gene therapy targeting murine kidneys to explore the role of HO-1 in protection by TMP against gentamicin-induced toxicity both in vitro and in vivo. We evaluated the protective effects of HO-1 on several apoptotic parameters induced by gentamicin: cleaved caspases-3 and -9, cycloxygenase-2 (Cox-2) and subcellular localization of nuclear factor kappa B-p65 (NF-kappaB-p65), Bcl-xl and HS-1-associated protein (Hax-1) in NRK-52E cells. RESULTS: NRK-52E cells treated with TMP exhibited transcriptional upregulation of the HO-1 protein by approximately twofold. Overexpression of HO-1 in NRK-52E cells significantly increased mitochondrial protein levels of the antiapoptotic molecules, Bcl-xL and Hax-1, and markedly decreased the NADPH oxidase activity and proinflammatory molecules, NF-kappaB-p65 and Cox-2, which might decrease gentamicin-induced activation of caspases-9 and -3. Conversely, NRK-52E cells with HO-1 knockdown significantly exacerbated gentamicin-induced tubular cell apoptosis. Additionally, the concomitant HO-1 induction by TMP was also evident in vivo, and HO-1 therapy markedly attenuated gentamicin-induced renal apoptosis to a similar extent as TMP pretreatment. CONCLUSIONS: Collectively, we suggest that HO-1 induced by TMP might, at least in part, protect against gentamicin-induced nephrotoxicity through antiapoptotic and anti-inflammatory mechanisms, and that it may have therapeutic potential for patients with renal disease. This is also the first demonstration that HO-1 increases Hax-1 mitochondrial localization.