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2.  Arsenic Inhibits Autophagic Flux, Activating the Nrf2-Keap1 Pathway in a p62-Dependent Manner 
Molecular and Cellular Biology  2013;33(12):2436-2446.
The Nrf2-Keap1 signaling pathway is a protective mechanism promoting cell survival. Activation of the Nrf2 pathway by natural compounds has been proven to be an effective strategy for chemoprevention. Interestingly, a cancer-promoting function of Nrf2 has recently been observed in many types of tumors due to deregulation of the Nrf2-Keap1 axis, which leads to constitutive activation of Nrf2. Here, we report a novel mechanism of Nrf2 activation by arsenic that is distinct from that of chemopreventive compounds. Arsenic deregulates the autophagic pathway through blockage of autophagic flux, resulting in accumulation of autophagosomes and sequestration of p62, Keap1, and LC3. Thus, arsenic activates Nrf2 through a noncanonical mechanism (p62 dependent), leading to a chronic, sustained activation of Nrf2. In contrast, activation of Nrf2 by sulforaphane (SF) and tert-butylhydroquinone (tBHQ) depends upon Keap1-C151 and not p62 (the canonical mechanism). More importantly, SF and tBHQ do not have any effect on autophagy. In fact, SF and tBHQ alleviate arsenic-mediated deregulation of autophagy. Collectively, these findings provide evidence that arsenic causes prolonged activation of Nrf2 through autophagy dysfunction, possibly providing a scenario similar to that of constitutive activation of Nrf2 found in certain human cancers. This may represent a previously unrecognized mechanism underlying arsenic toxicity and carcinogenicity in humans.
doi:10.1128/MCB.01748-12
PMCID: PMC3700105  PMID: 23589329
3.  Arsenic-Mediated Activation of the Nrf2-Keap1 Antioxidant Pathway 
Arsenic is present in the environment and has become a worldwide health concern due to its toxicity and carcinogenicity. However, the specific mechanism(s) by which arsenic elicits its toxic effects has yet to be fully elucidated. The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) has been recognized as the master regulator of a cellular defense mechanism against toxic insults. This review highlights studies demonstrating that arsenic activates the Nrf2-Keap1 antioxidant pathway by a distinct mechanism from that of natural compounds such as sulforaphane (SF) found in broccoli sprouts or tert-butylhyrdoquinone (tBHQ), a natural antioxidant commonly used as a food preservative. Evidence also suggests that arsenic prolongs Nrf2 activation and may mimic constitutive activation of Nrf2, which has been found in several human cancers due to disruption of the Nrf2-Keap1 axis. The current literature strongly suggests that activation of Nrf2 by arsenic potentially contributes to, rather than protects against, arsenic toxicity and carcinogenicity. The mechanism(s) by which known Nrf2 activators, such as the natural chemopreventive compounds SF and lipoic acid, protect against the deleterious effects caused by arsenic will also be discussed. These findings will provide insight to further understand how arsenic promotes a prolonged Nrf2 response, which will lead to the identification of novel molecular markers and development of rational therapies for the prevention or intervention of arsenic-induced diseases. The National Institute of Environmental Health Science (NIEHS) Outstanding New Environmental Scientist (ONES) award has provided the opportunity to review the progress both in the fields of arsenic toxicology and Nrf2 biology. Much of the funding has led to (1) the novel discovery that arsenic activates the Nrf2 pathway by a mechanism different to that of other Nrf2 activators, such as sulforaphane and tert-butylhydroquinone, (2) activation of Nrf2 by chemopreventive compounds protects against arsenic toxicity and carcinogenicity both in vitro and in vivo, (3) constitutive activation of Nrf2 by disrupting Keap1-mediated negative regulation contributes to cancer and chemoresistance, (4) p62-mediated sequestration of Keap1 activates the Nrf2 pathway, and (5) arsenic-mediated Nrf2 activation may be through a p62-dependent mechanism. All of these findings have been published and are discussed in this review. This award has laid the foundation for my laboratory to further investigate the molecular mechanism(s) that regulate the Nrf2 pathway and how it may play an integral role in arsenic toxicity. Moreover, understanding the biology behind arsenic toxicity and carcinogenicity will help in the discovery of potential strategies to prevent or control arsenic-mediated adverse effects.
doi:10.1002/jbt.21463
PMCID: PMC3725327  PMID: 23188707
Nrf2; Arsenic; Keap1; Oxidative stress; p62; Autophagy; Chemoprevention
4.  Nrf2 Is Crucial to Graft Survival in a Rodent Model of Heart Transplantation 
Currently, the sole treatment option for patients with heart failure is transplantation. The battle of prolonging graft survival and modulating innate and adaptive immune responses is still being waged in the clinic and in research labs. The transcription factor Nrf2 controls major cell survival pathways and is central to moderating inflammation and immune responses. In this study the effect of Nrf2 levels in host recipient C57BL/6 mice on Balb/c allogeneic graft survival was examined. Importantly, Nrf2−/− recipient mice could not support the graft for longer than 7.5 days on average, whereas activation of Nrf2 by sulforaphane in Nrf2+/+ hosts prolonged graft survival to 13 days. Several immune cells in the spleen of recipient mice were unchanged; however, CD11b+ macrophages were significantly increased in Nrf2−/− mice. In addition, IL-17 mRNA levels were elevated in grafts transplanted into Nrf2−/− mice. Although Nrf2 appears to play a crucial role in graft survival, the exact mechanism is yet to be fully understood.
doi:10.1155/2013/919313
PMCID: PMC3603380  PMID: 23533698
5.  Skeletal muscle molecular alterations precede whole-muscle dysfunction in NYHA Class II heart failure patients 
Background
Heart failure (HF), a debilitating disease in a growing number of adults, exerts structural and neurohormonal changes in both cardiac and skeletal muscles. However, these alterations and their affected molecular pathways remain uncharacterized. Disease progression is known to transform skeletal muscle fiber composition by unknown mechanisms. In addition, perturbation of specific hormonal pathways, including those involving skeletal muscle insulin-like growth factor-1 (IGF-1) and insulin-like growth factor-binding protein-5 (IGFB-5) appears to occur, likely affecting muscle metabolism and regeneration. We hypothesized that changes in IGF-1 and IGFB-5 mRNA levels correlate with the transformation of single–skeletal muscle fiber myosin heavy chain isoforms early in disease progression, making these molecules valuable markers of skeletal muscle changes in heart failure.
Materials and methods
To investigate these molecules during “early” events in HF patients, we obtained skeletal muscle biopsies from New York Heart Association (NYHA) Class II HF patients and controls for molecular analyses of single fibers, and we also quantified isometric strength and muscle size.
Results
There were more (P < 0.05) single muscle fibers coexpressing two or more myosin heavy chains in the HF patients (30% ± 7%) compared to the control subjects (13% ± 2%). IGF-1 and IGFBP-5 expression was fivefold and 15-fold lower in patients with in HF compared to control subjects (P < 0.05), respectively. Strikingly, there was a correlation in IGF-1 expression and muscle cross-sectional area (P < 0.05) resulting in a decrease in whole-muscle quality (P < 0.05) in the HF patients, despite no significant decrease in isometric strength or whole-muscle size.
Conclusion
These data indicate that molecular alterations in myosin heavy chain isoforms, IGF-1, and IGFB-5 levels precede the gross morphological and functional deficits that have previously been associated with HF, and may be used as a predictor of functional outcome in patients.
doi:10.2147/CIA.S37879
PMCID: PMC3508558  PMID: 23204842
muscle quality; hybrid fibers; IGF1 and IGFBP-5
6.  Therapeutic Potential of Nrf2 Activators in Streptozotocin-Induced Diabetic Nephropathy 
Diabetes  2011;60(11):3055-3066.
OBJECTIVE
To determine whether dietary compounds targeting NFE2-related factor 2 (Nrf2) activation can be used to attenuate renal damage and preserve renal function during the course of streptozotocin (STZ)-induced diabetic nephropathy.
RESEARCH DESIGN AND METHODS
Diabetes was induced in Nrf2+/+ and Nrf2−/− mice by STZ injection. Sulforaphane (SF) or cinnamic aldehyde (CA) was administered 2 weeks after STZ injection and metabolic indices and renal structure and function were assessed (18 weeks). Markers of diabetes including blood glucose, insulin, polydipsia, polyuria, and weight loss were measured. Pathological alterations and oxidative damage in glomeruli were also determined. Changes in protein expression of the Nrf2 pathway, as well as transforming growth factor-β1 (TGF-β1), fibronectin (FN), collagen IV, and p21/WAF1Cip1 (p21) were analyzed. The molecular mechanisms of Nrf2-mediated protection were investigated in an in vitro model using human renal mesangial cells (HRMCs).
RESULTS
SF or CA significantly attenuated common metabolic disorder symptoms associated with diabetes in Nrf2+/+ but not in Nrf2−/− mice, indicating SF and CA function through specific activation of the Nrf2 pathway. Furthermore, SF or CA improved renal performance and minimized pathological alterations in the glomerulus of STZ-Nrf2+/+ mice. Nrf2 activation reduced oxidative damage and suppressed the expression of TGF-β1, extracellular matrix proteins and p21 both in vivo and in HRMCs. In addition, Nrf2 activation reverted p21-mediated growth inhibition and hypertrophy of HRMCs under hyperglycemic conditions.
CONCLUSIONS
We provide experimental evidence indicating that dietary compounds targeting Nrf2 activation can be used therapeutically to improve metabolic disorder and relieve renal damage induced by diabetes.
doi:10.2337/db11-0807
PMCID: PMC3198067  PMID: 22025779
7.  Desmoplakin and talin2 are novel mRNA targets of Fragile X Related Protein-1 in cardiac muscle 
Circulation research  2011;109(3):262-271.
Rationale
The proper function of cardiac muscle requires the precise assembly and interactions of numerous cytoskeletal and regulatory proteins into specialized structures that orchestrate contraction and force transmission. Evidence suggests that post-transcriptional regulation is critical for muscle function, but the mechanisms involved remain understudied.
Objective
To investigate the molecular mechanisms and targets of the muscle-specific Fragile X mental retardation, autosomal homolog 1 (FXR1), an RNA binding protein whose loss leads to perinatal lethality in mice and cardiomyopathy in zebrafish.
Methods and Results
Using RNA immunoprecipitation approaches we found that desmoplakin and talin2 mRNAs associate with FXR1 in a complex. In vitro assays indicate that FXR1 binds these mRNA targets directly and represses their translation. Fxr1 KO hearts exhibit an upregulation of desmoplakin and talin2 proteins, which is accompanied by severe disruption of desmosome as well as costamere architecture and composition in the heart, as determined by electron microscopy and deconvolution immunofluorescence analysis.
Conclusions
Our findings reveal the first direct mRNA targets of FXR1 in striated muscle and support translational repression as a novel mechanism for regulating heart muscle development and function, in particular the assembly of specialized cytoskeletal structures.
doi:10.1161/CIRCRESAHA.111.244244
PMCID: PMC3163600  PMID: 21659647
Cytoskeletal dynamics; mRNA binding proteins; Desmosome; Heart development
8.  Age-Related Changes in Relative Expression of Real-Time PCR Housekeeping Genes in Human Skeletal Muscle 
The purpose of this investigation was to examine the expression of three commonly used housekeeping genes—glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β2-microglobulin (β2M), and RNA polymerase 2a (polR2a)—in elderly (E) compared to young (Y) subjects. Nine young subjects (22.7 ± 3.4 yrs) and 11 elderly subjects (73.0 ± 9.5 yrs) underwent a percutaneous skeletal muscle biopsy of the vastus lateralis. Equal concentrations of isolated mRNA from these samples were used to perform real-time polymerase chain reaction with primer/probe combinations specific to each gene of interest. The expression of GAPDH, β2M, and polR2a was obtained as the value of cycle threshold (CT). An independent t-test with a level of significance at p ≤ 0.05 was used to determine differences between groups. There was no difference in average CT of GAPDH between groups (p=0.869) (Y = 16.92 ± 2.25 vs. E = 17.08 ± 2.09) and polR2a (p = 0.089) (Y = 28.00 ± 0.89 vs. E = 26.73 ± 1.91). However, there was a significant difference (p ≤ 0.05) in the average CT of β2M (Y =21.79 ± 0.44 vs. E = 21.05 ± 0.51). The results indicate that special consideration needs to be made when selecting housekeeping genes for comparisons in real-time reverse-transcriptase polymerase chain reaction, depending upon the age of the populations of interest.
PMCID: PMC2291775  PMID: 16741243
Housekeeping genes; real-time reverse-transcriptase polymerase chain reaction (RT-PCR); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); β2-microglobulin (β2M); RNA polymerase 2a (polR2a)
9.  A Comparison of Thermoregulation With Creatine Supplementation Between the Sexes in a Thermoneutral Environment 
Journal of Athletic Training  2004;39(1):50-55.
Objective:
To compare the effect of creatine supplementation on thermoregulation in males and females during exercise in a thermoneutral environment.
Design and Setting:
Male and female subjects participated in 30 minutes of cycle ergometry in nonsupplemented (NS) and creatine-supplemented (Cr) conditions at 70% to 75% of predetermined peak oxygen consumption.
Subjects:
Ten male and ten female subjects were evaluated with and without creatine supplementation.
Measurements:
Analyses were performed during exercise for core temperature and mean skin temperature using two 2 × 2 × 7 mixed-factorial analyses of variance (ANOVAs). We compared mean differences between NS and Cr conditions and sex for heart rate, systolic blood pressure, and diastolic blood pressure using 3 2 × 2 × 4 mixed-factorial ANOVAs. Three 2 × 2 mixed-factorial ANOVAs were computed to examine differences between sex and conditions for the following variables: nude body weight and blood urea nitrogen before and after exercise and urine specific gravity.
Results:
Significant time effects were found for core temperature, skin temperature, heart rate, and diastolic blood pressure. Time effect and difference between the sexes for systolic blood pressure were both significant. Differences in nude body weight and blood urea nitrogen before and after exercise were greater for males, but there was no difference between conditions. No significant difference between sex and condition for urine specific gravity was noted.
Conclusions:
Short-term creatine supplementation did not affect thermoregulation between the sexes when exercising in a thermoneutral environment. Differences in changes in nude body weight before and after exercise may be due to a higher sweating rate in males versus females. Differences in blood urea nitrogen before and after exercise between the sexes may be due to a reduced glomerular filtration rate coupled with greater muscle creatine breakdown in males.
PMCID: PMC385262  PMID: 15085212
males; females; hypohydration; exercise

Results 1-9 (9)