Jason M. Hansen

Commensal bacteria modulate cullin-dependent signaling via generation of reactive oxygen species

The resident prokaryotic microflora of the mammalian intestine influences diverse homeostatic functions of the gut, including regulation of cellular growth and immune responses; however, it is unknown how commensal prokaryotic organisms mechanistically influence eukaryotic signaling networks. We have shown that bacterial coculture with intestinal epithelial cells modulates ubiquitin‐mediated degradation of important signaling intermediates, including β‐catenin and the NF‐κB inhibitor IκB‐α. Ubiquitination of these proteins as well as others is catalyzed by the SCFβTrCP ubiquitin ligase, which itself requires regulated modification of the cullin‐1 subunit by the ubiquitin‐like protein NEDD8. Here we show that epithelia contacted by enteric commensal bacteria in vitro and in vivo rapidly generate reactive oxygen species (ROS). Bacterially induced ROS causes oxidative inactivation of the catalytic cysteine residue of Ubc12, the NEDD8‐conjugating enzyme, resulting in complete but transient loss of cullin‐1 neddylation and consequent effects on NF‐κB and β‐catenin signaling. Our results demonstrate that commensal bacteria directly modulate a critical control point of the ubiquitin–proteasome system, and suggest how enteric commensal bacterial flora influences the regulatory pathways of the mammalian intestinal epithelia.

Compartmental oxidation of thiol-disulphide redox couples during epidermal growth factor signalling

Exogenously added ROS (reactive oxygen species) cause generalized oxidation of cellular components, whereas endogenously generated ROS induced by physiological stimuli activate discrete signal transduction pathways. Compartmentation is an important aspect of such pathways, but little is known about its role in redox signalling. We measured the redox states of cytosolic and nuclear Trx1 (thioredoxin-1) and mitochondrial Trx2 (thioredoxin-2) using redox Western blot methodologies during endogenous ROS production induced by EGF (epidermal growth factor) signalling. The glutathione redox state was measured by HPLC. Results showed that only cytosolic Trx1 undergoes significant oxidation. Thus EGF signalling involves subcellular compartmental oxidation of Trx1 in the absence of a generalized cellular oxidation.

Lactobacillus rhamnosus blocks inflammatory signaling in vivo via reactive oxygen species generation.

Uncontrolled inflammatory responses in the immature gut may play a role in the pathogenesis of many intestinal inflammatory syndromes that present in newborns or children, such as necrotizing enterocolitis (NEC), idiopathic inflammatory bowel diseases (IBD), or infectious enteritis. Consistent with previous reports that murine intestinal function matures over the first 3 weeks of life, we show that inflammatory signaling in the neonatal mouse gut increases during postnatal maturation, with peak responses occurring at 2-3 weeks. Probiotic bacteria can block inflammatory responses in cultured epithelia by inducing the generation of reactive oxygen species (ROS), which inhibit NF-kappaB activation through oxidative inactivation of the key regulatory enzyme Ubc12. We now report for the first time that the probiotic Lactobacillus rhamnosus GG (LGG) can induce ROS generation in intestinal epithelia in vitro and in vivo. Intestines from immature mice gavage fed LGG exhibited increased GSH oxidation and cullin-1 deneddylation, reflecting local ROS generation and its resultant Ubc12 inactivation, respectively. Furthermore, prefeeding LGG prevented TNF-alpha-induced intestinal NF-kappaB activation. These studies indicate that LGG can reduce inflammatory signaling in immature intestines by inducing local ROS generation and may be a mechanism by which probiotic bacteria can prevent NEC in premature infants or reduce the severity of IBD in children.

Attenuation of Angiotensin II–Induced Vascular Dysfunction and Hypertension by Overexpression of Thioredoxin 2

Reactive oxygen species increase in the cardiovascular system during hypertension and in response to angiotensin II. Because mitochondria contribute to reactive oxygen species generation, we sought to investigate the role of thioredoxin 2, a mitochondria-specific antioxidant enzyme. Mice were created with overexpression of human thioredoxin 2 (TghTrx2 mice) and backcrossed to C57BL/6J mice for ≥6 generations. Twelve-week-old male TghTrx2 or littermate wild-type mice were made hypertensive by infusion of angiotensin II (400 ng/kg per minute) for 14 days using osmotic minipumps. Systolic arterial blood pressure was not different between TghTrx2 and wild-type animals under baseline conditions (101±1 respective 102±1 mm Hg). The angiotensin II–induced hypertension in wild-type mice (145±2 mm Hg) was significantly attenuated in TghTrx2 mice (124±1 mm Hg; P<0.001). Aortic endothelium-dependent relaxation was significantly reduced in wild-type mice after angiotensin II infusion but nearly unchanged in transgenic mice. Elevated vascular superoxide and hydrogen peroxide levels, as well as expression of NADPH oxidase subunits in response to angiotensin II infusion, were significantly attenuated in TghTrx2 mice. Mitochondrial superoxide anion levels were augmented after angiotensin II infusion in wild-type mice, and this was blunted in TghTrx2 mice. Angiotensin II infusion significantly increased myocardial superoxide formation, heart weight, and cardiomyocyte size in wild-type but not in TghTrx2 mice. These data indicate a major role for mitochondrial thioredoxin 2 in the development of cardiovascular alterations and hypertension during chronic angiotensin II infusion. Thioredoxin 2 may represent an important therapeutic target for the prevention and treatment of hypertension and oxidative stress.

Enteric commensal bacteria potentiate epithelial restitution via reactive oxygen species-mediated inactivation of focal adhesion kinase phosphatases

The mechanisms by which enteric commensal microbiota influence maturation and repair of the epithelial barrier are relatively unknown. Epithelial restitution requires active cell migration, a process dependent on dynamic turnover of focal cell-matrix adhesions (FAs). Here, we demonstrate that natural, commensal bacteria stimulate generation of reactive oxygen species (ROS) in intestinal epithelia. Bacteria-mediated ROS generation induces oxidation of target cysteines in the redox-sensitive tyrosine phosphatases, LMW-PTP and SHP-2, which in turn results in increased phosphorylation of focal adhesion kinase (FAK), a key protein regulating the turnover of FAs. Accordingly, phosphorylation of FAK substrate proteins, focal adhesion formation, and cell migration are all significantly enhanced by bacterial contact in both in vitro and in vivo models of wound closure. These results suggest that commensal bacteria regulate cell migration via induced generation of ROS in epithelial cells.

A Novel Hypothesis for Thalidomide-Induced Limb Teratogenesis: Redox Misregulation of the NF-κB Pathway

Several hypotheses have been proposed to explain the mechanisms of thalidomide teratogenesis, although none adequately accounts for the observed malformations and explains the basis for species specificity. Recent observations that thalidomide increases the production of free radicals and elicits oxidative stress, coupled with new insights into the redox regulation of nuclear transcription factors, lead to the suggestion that thalidomide may act through redox misregulation of the limb outgrowth pathways. Oxidative stress, as marked by glutathione depletion/oxidation and a shift in intracellular redox potential toward the positive, occurs preferentially in limbs of thalidomide-sensitive rabbits, but not in resistant rats. DNA binding of nuclear factor kappa-B (NF-kappaB), a redox-sensitive transcription factor and key regulator of limb outgrowth, was shown to be significantly attenuated in rabbit limb cells and could be restored following the addition of a free radical spin-trapping agent, phenyl N-tert-butyl nitrone. The inability of NF-kappaB to bind to its DNA promoter results in the failure of limb cells to express fibroblast growth factor (FGF)-10 and twist in the limb progress zone (PZ) mesenchyme, which in turn attenuates expression of FGF-8 in the apical ectodermal ridge (AER). Failure to establish an FGF-10/FGF-8 feedback loop between the PZ and AER results in the truncation of limb outgrowth. We hypothesize that species-selective alterations in redox microenvironment caused by free radical production from thalidomide results in attenuation of the NF-kappaB-mediated gene expression that is responsible for limb outgrowth.

Prohibitin Is a Novel Regulator of Antioxidant Response That Attenuates Colonic Inflammation in Mice

BACKGROUND & AIMS Increased free radicals and/or impaired antioxidant defenses have been shown to play a pathogenetic role in human and animal models of inflammatory bowel disease. Our previous studies showed that prohibitin (PHB) levels are decreased during colitis and that cultured intestinal epithelial cells overexpressing PHB are protected from oxidative stress. This study investigated the effect of intestinal epithelial cell-specific PHB overexpression on oxidative stress associated with experimental colitis and the potential mechanism by which PHB functions as an antioxidant using PHB transgenic mice. METHODS Colitis was induced using 2 established mouse models (Salmonella typhimurium and dextran sodium sulfate) in PHB transgenic mice and wild-type littermates. Oxidative stress was determined by measuring glutathione and protein carbonyl levels in the cecum or colon. Nuclear factor erythroid 2-related factor 2 (Nrf2), a transcriptional regulator of oxidant responses, expression, and activation, was assessed in colon mucosa and cultured intestinal epithelial cells overexpressing PHB. RESULTS Cells overexpressing PHB showed sustained Nrf2 nuclear accumulation and DNA binding during oxidant stress. PHB transgenic mice exhibited decreased oxidative stress and colitis and increased Nrf2 messenger RNA expression, nuclear protein translocation, and DNA binding compared with wild-type littermates during colitis. CONCLUSIONS These results show that PHB is a regulator of Nrf2 expression in intestinal epithelial cells during oxidative conditions and prevents inflammation-associated oxidative stress and injury through sustained activation of Nrf2. Our data show that PHB is a novel regulator of antioxidants and suggest that restoration of PHB levels represents a potential therapeutic approach in inflammatory bowel disease.

A reducing redox environment promotes C2C12 myogenesis—implications for regeneration in aged muscle

Intracellular redox potential of skeletal muscle becomes progressively more oxidized with aging, negatively impacting regenerative ability. We examined the effects of oxidizing redox potential on terminal differentiation of cultured C2C12 myoblasts. Redox potentials were manipulated by changing the culture O2 environment, by free radical scavenging, or addition of H2O2. Intracellular reactive oxygen species (ROS) production was higher in 20% environmental O2 and in this condition, redox potential became progressively oxidized compared to cultures in 6% O2. Treatment with a ROS trapping agent (phenyl‐N‐tert‐butylnitrone, PBN) caused reducing redox potentials and enhanced C2C12 differentiation, while addition of 25 micromolar H2O2 to cells in 20% O2 dramatically slowed differentiation. Under these most oxidative conditions, quantitative PCR showed a significant decrease in myogenic basic helix—loop—helix transcription factor expression compared to cultures treated with PBN or grown in 6% O2. Thus, oxidative intracellular environments impair myoblast differentiation, while reducing environments favor myogenesis.

Disruption of endothelial peroxisome proliferator-activated receptor-γ reduces vascular nitric oxide production

Vascular endothelial cells express the ligand-activated transcription factor, peroxisome proliferator-activated receptor-gamma (PPARgamma), which participates in the regulation of metabolism, cell proliferation, and inflammation. PPARgamma ligands attenuate, whereas the loss of function mutations in PPARgamma stimulate, endothelial dysfunction, suggesting that PPARgamma may regulate vascular endothelial nitric oxide production. To explore the role of endothelial PPARgamma in the regulation of vascular nitric oxide production in vivo, mice expressing Cre recombinase driven by an endothelial-specific promoter were crossed with mice carrying a floxed PPARgamma gene to produce endothelial PPARgamma null mice (ePPARgamma(-/-)). When compared with littermate controls, ePPARgamma(-/-) animals were hypertensive at baseline and demonstrated comparable increases in systolic blood pressure in response to angiotensin II infusion. When compared with those of control animals, aortic ring relaxation responses to acetylcholine were impaired, whereas relaxation responses to sodium nitroprusside were unaffected in ePPARgamma(-/-) mice. Similarly, intact aortic segments from ePPARgamma(-/-) mice released less nitric oxide than those from controls, whereas endothelial nitric oxide synthase expression was similar in control and ePPARgamma(-/-) aortas. Reduced nitric oxide production in ePPARgamma(-/-) aortas was associated with an increase in the parameters of oxidative stress in the blood and the activation of nuclear factor-kappaB in aortic homogenates. These findings demonstrate that endothelial PPARgamma regulates vascular nitric oxide production and that the disruption of endothelial PPARgamma contributes to endothelial dysfunction in vivo.

Misregulation of gene expression in the redox‐sensitive NF‐κb‐dependent limb outgrowth pathway by thalidomide

Thalidomide is known to induce oxidative stress, but mechanisms have not been described through which oxidative stress could contribute to thalidomide‐induced terata. Oxidative stress modulates intracellular glutathione (GSH) and redox status and can perturb redox‐sensitive processes, such as transcription factor activation and/or binding. Nuclear factor‐kappa B (NF‐κB), a redox‐sensitive transcription factor involved in limb outgrowth, may be modulated by thalidomide‐induced redox shifts. Thalidomide‐resistant Sprague‐Dawley rat embryos (gestation day [GD] 13) treated with thalidomide in utero showed no changes in GSH distribution in the limb but thalidomide‐sensitive New Zealand White rabbit embryos (GD 12) showed selective GSH depletion in the limb bud progress zone (PZ). NF‐κB and regulatory genes that initiate and maintain limb outgrowth and development, such as Twist and Fgf‐10, are selectively expressed in the PZ. Green fluorescent protein (GFP) reporter vectors containing NF‐κB binding promoter sites were transfected into both rat and rabbit limb bud cells (LBCs). Treatment with thalidomide caused a preferential decrease in GFP expression in rabbit LBCs but not in rat LBCs. N‐acetylcysteine and α‐N‐t‐phenylbutyl nitrone (PBN), a free radical trapping agent, rescued GFP expression in thalidomide‐treated cultures compared with cultures that received thalidomide only. In situ hybridization showed a preferential decrease in Twist, Fgf‐8, and Fgf‐10 expression after thalidomide treatment (400 mg/kg per day) in rabbit embryos. Expression in rat embryos was not affected. Intravenous cotreatment with PBN and thalidomide (gavage) in rabbits restored normal patterns and localization of Twist, Fgf‐8, and Fgf‐10 expression. These findings show that NF‐κB binding is diminished due to selective thalidomide‐induced redox changes in the rabbit, resulting in the significant attenuation of expression of genes necessary for limb outgrowth.