Vasudevan Bakthavatchalu

Manganese Superoxide Dismutase: Guardian of the Powerhouse

The mitochondrion is vital for many metabolic pathways in the cell, contributing all or important constituent enzymes for diverse functions such as β-oxidation of fatty acids, the urea cycle, the citric acid cycle, and ATP synthesis. The mitochondrion is also a major site of reactive oxygen species (ROS) production in the cell. Aberrant production of mitochondrial ROS can have dramatic effects on cellular function, in part, due to oxidative modification of key metabolic proteins localized in the mitochondrion. The cell is equipped with myriad antioxidant enzyme systems to combat deleterious ROS production in mitochondria, with the mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD) acting as the chief ROS scavenging enzyme in the cell. Factors that affect the expression and/or the activity of MnSOD, resulting in diminished antioxidant capacity of the cell, can have extraordinary consequences on the overall health of the cell by altering mitochondrial metabolic function, leading to the development and progression of numerous diseases. A better understanding of the mechanisms by which MnSOD protects cells from the harmful effects of overproduction of ROS, in particular, the effects of ROS on mitochondrial metabolic enzymes, may contribute to the development of novel treatments for various diseases in which ROS are an important component.

RelB Enhances Prostate Cancer Growth: Implications for the Role of the Nuclear Factor-κB Alternative Pathway in Tumorigenicity

The nuclear factor-kappaB (NF-kappaB) classic pathway is thought to be critical for tumorigenesis, but little is known about the role of the NF-kappaB alternative pathway in cancer development. Recently, high constitutive nuclear levels of RelB have been observed in human prostate cancer specimens with high Gleason scores. Here, we used four complementary approaches to test whether RelB contributes to tumorigenicity of prostate cancer. Inhibiting RelB in aggressive androgen-independent PC-3 cells by stable or conditional expression of a dominant-negative p100 mutant significantly reduced the incidence and growth rate of tumors. The decrease in tumorigenicity coincided with a reduction in the NF-kappaB target interleukin-8 (IL-8). Consistently, down-regulation of RelB by small interfering RNA targeting also reduced tumor growth and decreased levels of IL-8. Conversely, stable expression of RelB in androgen-responsive LNCaP tumors increased the circulating IL-8 levels. Taken together, these results reveal a tumor-supportive role of RelB, implicate the NF-kappaB alternative pathway as a potential target for preventing prostate cancer, and suggest the use of IL-8 as a marker for prostate cancer prognosis.

Interactions between SIRT1 and AP-1 reveal a mechanistic insight into the growth promoting properties of alumina (Al2O3) nanoparticles in mouse skin epithelial cells

The physicochemical properties of nanomaterials differ from those of the bulk material of the same composition. However, little is known about the underlying effects of these particles in carcinogenesis. The purpose of this study was to determine the mechanisms involved in the carcinogenic properties of nanoparticles using aluminum oxide (Al(2)O(3)/alumina) nanoparticles as the prototype. Well-established mouse epithelial JB6 cells, sensitive to neoplastic transformation, were used as the experimental model. We demonstrate that alumina was internalized and maintained its physicochemical composition inside the cells. Alumina increased cell proliferation (53%), proliferating cell nuclear antigen (PCNA) levels, cell viability and growth in soft agar. The level of manganese superoxide dismutase, a key mitochondrial antioxidant enzyme, was elevated, suggesting a redox signaling event. In addition, the levels of reactive oxygen species and the activities of the redox sensitive transcription factor activator protein-1 (AP-1) and a longevity-related protein, sirtuin 1 (SIRT1), were increased. SIRT1 knockdown reduces DNA synthesis, cell viability, PCNA levels, AP-1 transcriptional activity and protein levels of its targets, JunD, c-Jun and BcL-xl, more than controls do. Immunoprecipitation studies revealed that SIRT1 interacts with the AP-1 components c-Jun and JunD but not with c-Fos. The results identify SIRT1 as an AP-1 modulator and suggest a novel mechanism by which alumina nanoparticles may function as a potential carcinogen.

Helicobacter pylori Infection Induces Anemia, Depletes Serum Iron Storage, and Alters Local Iron-Related and Adult Brain Gene Expression in Male INS-GAS Mice.

Iron deficiency anemia (IDA) affects > 500 million people worldwide, and is linked to impaired cognitive development and function in children. Helicobacter pylori, a class 1 carcinogen, infects about half of the world’s population, thus creating a high likelihood of overlapping risk. This study determined the effect of H. pylori infection on iron homeostasis in INS-GAS mice. Two replicates of INS-GAS/FVB male mice (n = 9-12/group) were dosed with H. pylori (Hp) strain SS1 or sham dosed at 6–9 weeks of age, and were necropsied at 27–29 weeks of age. Hematologic and serum iron parameters were evaluated, as was gene expression in gastric and brain tissues. Serum ferritin was lower in Hp SS1-infected mice than uninfected mice (p < 0.0001). Infected mice had a lower red blood cell count (p<0.0001), hematocrit (p < 0.001), and hemoglobin concentration (p <0.0001) than uninfected mice. Relative expression of gastric hepcidin antimicrobial peptide (Hamp) was downregulated in mice infected with Hp SS1 compared to sham-dosed controls (p<0.001). Expression of bone morphogenic protein 4 (Bmp4), a growth factor upstream of hepcidin, was downregulated in gastric tissue of Hp SS1-infected mice (p<0.001). Hp SS1-infected mice had downregulated brain expression of tyrosine hydroxylase (Th) (p = 0.02). Expression of iron-responsive genes involved in myelination (myelin basic protein (Mbp) and proteolipid protein 2 (Plp2)) was downregulated in infected mice (p = 0.001 and p = 0.02). Expression of synaptic plasticity markers (brain derived neurotrophic factor 3 (Bdnf3), Psd95 (a membrane associated guanylate kinase), and insulin-like growth factor 1 (Igf1)) was also downregulated in Hp SS1-infected mice (p = 0.09, p = 0.04, p = 0.02 respectively). Infection of male INS-GAS mice with Hp SS1, without concurrent dietary iron deficiency, depleted serum ferritin, deregulated gastric and hepatic expression of iron regulatory genes, and altered iron-dependent neural processes. The use of Hp SS1-infected INS-GAS mice will be an appropriate animal model for further study of the effects of concurrent H. pylori infection and anemia on iron homeostasis and adult iron-dependent brain gene expression.

Manganese superoxide dismutase deficiency triggers mitochondrial uncoupling and the Warburg effect

Manganese superoxide dismutase (MnSOD) is a mitochondrially localized primary antioxidant enzyme, known to be essential for the survival of aerobic life and to have important roles in tumorigenesis. Here, we show that MnSOD deficiency in skin tissues of MnSOD-heterozygous knockout (Sod2+/−) mice leads to increased expresson of uncoupling proteins (UCPs). When MnSOD is deficient, superoxide radical and its resulting reactive oxygen species (ROS) activate ligand binding to peroxisome proliferator-activated receptor alpha (PPARα), suggesting that the activation of PPARα signaling is a major mechanism underlying MnSOD-dependent UCPs expression that consequently triggers the PI3K/Akt/mTOR pathway, leading to increased aerobic glycolysis. Knockdown of UCPs and mTOR suppresses lactate production and increases ATP levels, suggesting that UCPs contribute to increased glycolysis. These results highlight the existence of a free radical-mediated mechanism that activates mitochondria uncoupling to reduce ROS production, which precedes the glycolytic adaptation described as the Warburg Effect.

Down-regulation of programmed cell death 4 leads to epithelial to mesenchymal transition and promotes metastasis in mice.

In this study, we demonstrated that knockdown of programmed cell death 4 (Pdcd4), a novel tumour suppressor, decreased the expressions of epithelial-specific proteins and increased the expressions of mesenchymal-specific proteins in vitro and in vivo, suggesting that knockdown of Pdcd4 results in epithelial to mesenchymal transition (EMT). Knockdown of Pdcd4 increased the rate of wound closure and migration capacity in wound-healing assays and Boyden chamber migration assays, respectively, indicating that Pdcd4 knockdown promotes cell migration. Pdcd4 knockdown also altered the adhesion capacity of GEO cells to extracellular matrix including laminin, collagen IV and fibronectin. To test whether knockdown of Pdcd4 promotes metastasis in vivo, parental, control and Pdcd4 knockdown cells were injected into the caecal wall (orthotopic implantation) of nude mice. Tumours are formed on caecum in all injected mice. However, only mice injected with Pdcd4 knockdown cells developed hepatic and local lymph node metastases. Immunohistochemical staining analyses showed that c-Myc and Snail/Slug expressions were up-regulated in the tumours derived from injection of Pdcd4 knockdown cells. These results implicated that promotion of metastasis by Pdcd4 knockdown was contributed by up-regulation of c-Myc and Snail/Slug in nude mice. Taken together, our data demonstrated that knockdown of Pdcd4 leads to EMT, alternation of adhesion and promotion of migration and metastasis.

Endocrinopathy and Aging in Ferrets

Ferrets have become more popular as household pets and as animal models in biomedical research in the past 2 decades. The average life span of ferrets is about 5-11 years with onset of geriatric diseases between 3-4 years including endocrinopathies, neoplasia, gastrointestinal diseases, cardiomyopathy, splenomegaly, renal diseases, dental diseases, and cataract. Endocrinopathies are the most common noninfectious disease affecting middle-aged and older ferrets. Spontaneous neoplasms affecting the endocrine system of ferrets appear to be increasing in prevalence with a preponderance toward proliferative lesions in the adrenal cortex and pancreatic islet cells. Diet, gonadectomy, and genetics may predispose ferrets to an increased incidence of these endocrinopathies. These functional proliferative lesions cause hypersecretion of hormones that alter the physiology and metabolism of the affected ferrets resulting in a wide range of clinical manifestations. However, there is an apparent dearth of information available in the literature about the causal relationship between aging and neoplasia in ferrets. This review provides a comprehensive overview of the anatomy and physiology of endocrine organs, disease incidence, age at diagnosis, clinical signs, pathology, and molecular markers available for diagnosis of various endocrine disorders in ferrets.

GPR4 deficiency alleviates intestinal inflammation in a mouse model of acute experimental colitis

GPR4 is a proton-sensing G protein-coupled receptor that can be activated by extracellular acidosis. It has recently been demonstrated that activation of GPR4 by acidosis increases the expression of numerous inflammatory and stress response genes in vascular endothelial cells (ECs) and also augments EC-leukocyte adhesion. Inhibition of GPR4 by siRNA or small molecule inhibitors reduces endothelial cell inflammation. As acidotic tissue microenvironments exist in many types of inflammatory disorders, including inflammatory bowel disease (IBD), we examined the role of GPR4 in intestinal inflammation using a dextran sulfate sodium (DSS)-induced acute colitis mouse model. We observed that GPR4 mRNA expression was increased in mouse and human IBD tissues when compared to control intestinal tissues. To determine the function of GPR4 in intestinal inflammation, wild-type and GPR4-deficient mice were treated with 3% DSS for 7days to induce acute colitis. Our results showed that the severity of colitis was decreased in GPR4-deficient DSS-treated mice in comparison to wild-type DSS-treated mice. Clinical parameters, macroscopic disease indicators, and histopathological features were less severe in the DSS-treated GPR4-deficient mice than the DSS-treated wild-type mice. Endothelial adhesion molecule expression, leukocyte infiltration, and isolated lymphoid follicle (ILF) formation were reduced in intestinal tissues of DSS-treated GPR4-null mice. Collectively, our results suggest GPR4 provides a pro-inflammatory role in the inflamed gut as the absence of GPR4 ameliorates intestinal inflammation in the acute experimental colitis mouse model.

Helicobacter pylori infection and low dietary iron alter behavior, induce iron deficiency anemia, and modulate hippocampal gene expression in female C57BL/6 mice

Helicobacter pylori (H.pylori), a bacterial pathogen, is a causative agent of gastritis and peptic ulcer disease and is a strong risk factor for development of gastric cancer. Environmental conditions, such as poor dietary iron resulting in iron deficiency anemia (IDA), enhance H.pylori virulence and increases risk for gastric cancer. IDA affects billions of people worldwide, and there is considerable overlap between regions of high IDA and high H.pylori prevalence. The primary aims of our study were to evaluate the effect of H.pylori infection on behavior, iron metabolism, red blood cell indices, and behavioral outcomes following comorbid H. pylori infection and dietary iron deficiency in a mouse model. C57BL/6 female mice (n = 40) were used; half were placed on a moderately iron deficient (ID) diet immediately post-weaning, and the other half were maintained on an iron replete (IR) diet. Half were dosed with H.pylori SS1 at 5 weeks of age, and the remaining mice were sham-dosed. There were 4 study groups: a control group (-Hp, IR diet) as well as 3 experimental groups (-Hp, ID diet; +Hp, IR diet; +Hp,ID diet). All mice were tested in an open field apparatus at 8 weeks postinfection. Independent of dietary iron status, H.pylori -infected mice performed fewer exploratory behaviors in the open field chamber than uninfected mice (p<0.001). Hippocampal gene expression of myelination markers and dopamine receptor 1 was significantly downregulated in mice on an ID diet (both p<0.05), independent of infection status. At 12 months postinfection, hematocrit (Hct) and hemoglobin (Hgb) concentration were significantly lower in +Hp, ID diet mice compared to all other study groups. H.pylori infection caused IDA in mice maintained on a marginal iron diet. The mouse model developed in this study is a useful model to study the neurologic, behavioral, and hematologic impact of the common human co-morbidity of H. pylori infection and IDA.

O-005 YI Microbiota Drives Inflammation by Altering Intestinal Lamina Propria Macrophage Phenotype in a Novel IL10R-Deficient Model of Very Early Onset IBD:

Background:Rare mutations in IL-10 or its receptors (IL10R) lead to severe very early onset (VEO)/infantile IBD in humans suggesting that the IL10/IL10R pathway is indispensable for mucosal immune homeostasis in the developing gut. Mice deficient in IL10/IL10R also develop spontaneous colitis. We recently reported that IL10R signaling in macrophages is critical for mucosal homeostasis in adult mice and humans (Shouval et al, Immunity 2014). However, the identification of IL10-dependent regulatory mechanisms in infants remains elusive. The goal of this study is to dissect key IL10R-dependent mechanisms that initiate mucosal homeostasis in the maturing infant colon. Methods:We performed age-dependent analysis of pro- and anti-inflammatory genes expression in the colon of developing littermate Il10rb−/− and Il10rb+/− (control) 129SvEv mice. We analyzed fecal microbiome from 2 to 14 week old control and Il10rb−/− mice using 16S rRNA sequencing. Flow cytometry was performed to assess various lamina propria (LP) immune cell populations. Antibiotics were given in drinking water starting in utero to investigate the influence of microbial communities on gene expression, LP immune populations, and inflammation. The role of pro-inflammatory macrophages (Ly6C+, CCR2+) in the development of pro-inflammatory signatures was assessed employing a CCR2-depleting antibody. Finally, we analyzed the development of colitis in crosses between C57BL/6 mice lacking IL10R&agr; and those carrying a genetic locus driving colitis susceptibility, Cdcs1, neither of which independently develop colitis in infancy. Results:Colitis was observed as early as 4 weeks in Il10rb−/− 129SvEv mice. An increase in colonic IL-10 expression was observed in both Il10rb−/− and control mice during the third week of life, and interestingly this was accompanied by a dramatic increase in proinflammatory cytokine expression including IL12p40, IL17A, and IFN&ggr; within Il10rb−/− but not control mice. Increases in pro-inflammatory cytokines were paralleled by the expansion of Ly6C+ monocytes and decreases in anti-inflammatory Ly6C− macrophages within the LP. Fecal microbiome analysis demonstrated a significant increase in microbial diversity, and acquisition of Helicobacter sp. at 3 weeks of age. Interestingly, while antibiotic treatment of Il10rb−/− mice attenuated expression of proinflammatory gene expression and expansion of Ly6C+ monocytes, depletion of Ly6C+ monocytes with CCR2 neutralizing antibodies did not diminish pro-inflammatory gene expression. Finally, while IL10R&agr;-deficient mice on C57BL/6 background do not develop colitis, severe spontaneous colitis developed in Cdcs1+/+Il10ra−/− mice on the C57BL/6 background by 3 weeks of age. Conclusions:Microbial colonization induces effector proinflammatory gene expression in the developing colon that is controlled by a concomitant induction of IL10. While antibiotics inhibited both inflammatory gene expression and Ly6C+ monocyte accumulation, depletion of these Ly6C+ monocytes did not inhibit inflammatory gene expression suggesting an alternative source for inflammatory cytokines in this model. We hypothesize that microbial-dependent skewing of intestinal Ly6C− macrophages rather than accumulation of Ly6C+ monocytes in the absence of IL10R may play a central role in driving colonic inflammation in neonates that lack IL10R. We suggest that further mechanistic studies using the described novel IL10R-deficient neonatal models may lead to novel insights into the pathogenesis of both neonatal and adult onset IBD.