Angela Rico de Souza

Hydrogen Peroxide Modifies Human Sperm Peroxiredoxins in a Dose-Dependent Manner

Low levels of reactive oxygen species (ROS) modulate signaling pathways required for human sperm activation, but high levels impair sperm function, leading to infertility. Peroxiredoxins (PRDXs) are enzymes with a dual role as ROS scavengers and modulators of ROS-dependent signaling. The present study aimed to characterize PRDXs in human spermatozoa and possible modifications resulting from hydrogen peroxide (H2O2). We found PRDX1, PRDX4, PRDX5, and PRDX6 in both seminal plasma and spermatozoa. Using immunocytochemistry, we demonstrated that these PRDXs are differentially localized in the head, acrosome, mitochondrial sheath, and flagellum. These observations were confirmed by immunoblotting using cytosolic, Triton-soluble and -insoluble, and head and flagella sperm fractions. PRDXs are dose-dependently modified by H2O2, as seen by the formation of disulfide bridges and high-molecular-mass complexes. This first study, to our knowledge, on PRDXs in human spermatozoa indicates that PRDX1, PRDX4, PRDX5, and PRDX6 are modified when spermatozoa are challenged with H2O2. This suggests that PRDXs may protect these cells at high levels of H2O2 but could also control H2O2 levels within different cell compartments so that normal sperm activation can occur.

Genetic Ablation of the Aryl Hydrocarbon Receptor Causes Cigarette Smoke-induced Mitochondrial Dysfunction and Apoptosis

Background: The aryl hydrocarbon receptor (AhR) suppresses lung inflammation and may protect against cigarette smoke-induced apoptosis. Results: Genetic ablation of the AhR increases the sensitivity of lung cells to smoke-induced apoptosis by regulating antioxidant proteins. Conclusion: The AhR regulates pulmonary cell survival. Significance: The AhR control over lung cell survival may be why only some smokers develop chronic lung diseases such as chronic obstructive pulmonary disease. Cigarette smoke is the primary risk factor for chronic obstructive pulmonary disease (COPD). Alterations in the balance between apoptosis and proliferation are involved in the etiology of COPD. Fibroblasts and epithelial cells are sensitive to the oxidative properties of cigarette smoke, and whose loss may precipitate the development of COPD. Fibroblasts express the aryl hydrocarbon receptor (AhR), a transcription factor that attenuates pulmonary inflammation and may also regulate apoptosis. We hypothesized the AhR would prevent apoptosis caused by cigarette smoke. Using genetically deleted in vitro AhR expression models and an established method of cigarette smoke exposure, we report that AhR expression regulates fibroblasts proliferation and prevents morphological features of apoptosis, including membrane blebbing and chromatin condensation caused by cigarette smoke extract (CSE). Absence of AhR expression results in cleavage of PARP, lamin, and caspase-3. Mitochondrial dysfunction, including cytochrome c release, was associated with loss of AhR expression, indicating activation of the intrinsic apoptotic cascade. Heightened sensitivity of AhR-deficient fibroblasts was not the result of alterations in GSH, Nrf2, or HO-1 expression. Instead, AhR−/− cells had significantly less MnSOD and CuZn-SOD expression, enzymes that protects against oxidative stress. The ability of the AhR to suppress apoptosis was not restricted to fibroblasts, as siRNA-mediated knockdown of the AhR in lung epithelial cells also increased sensitivity to smoke-induced apoptosis. Collectively, these results suggest that cigarette smoke induced loss of lung structural support (i.e. fibroblasts, epithelial cells) caused by aberrations in AhR expression may explain why some smokers develop lung diseases such as COPD.

The NF-κB family member RelB regulates microRNA miR-146a to suppress cigarette smoke-induced COX-2 protein expression in lung fibroblasts.

Diseases due to cigarette smoke exposure, including chronic obstructive pulmonary disease (COPD) and lung cancer, are associated with chronic inflammation typified by the increased expression of cyclooxygenase-2 (COX-2) protein. RelB is an NF-κB family member that suppresses cigarette smoke induction of COX-2 through an unknown mechanism. The ability of RelB to regulate COX-2 expression may be via miR-146a, a miRNA that attenuates COX-2 in lung fibroblasts. In this study we tested whether RelB attenuation of cigarette smoke-induced COX-2 protein is due to miR-146a. Utilizing pulmonary fibroblasts deficient in RelB expression, together with siRNA knock-down of RelB, we show the essential role of RelB in diminishing smoke-induced COX-2 protein expression despite robust activation of the canonical NF-κB pathway and subsequent induction of Cox-2 mRNA. RelB did not regulate COX-2 protein expression at the level of mRNA stability. Basal levels of miR-146a were significantly lower in Relb-deficient cells and cigarette smoke increased miR-146a expression only in Relb-expressing cells. Inhibition of miR-146a had no effects on Relb expression or induction of Cox-2 mRNA by cigarette smoke but significantly increased COX-2 protein. These data highlight the potential of a RelB-miR-146a axis as a novel regulatory pathway that attenuates inflammation in response to respiratory toxicants.

Aryl Hydrocarbon Receptor (AhR) Attenuation of Subchronic Cigarette Smoke-induced Pulmonary Neutrophilia Is Associated with Retention of Nuclear RelB and Suppression of Intercellular Adhesion Molecule-1 (ICAM-1)

Cigarette smoke is associated with chronic and enhanced pulmonary inflammation characterized by increased cytokine production and leukocyte recruitment to the lung. Although the aryl hydrocarbon receptor (AhR) is well-known to mediate toxic effects of manmade environmental contaminants, the AhR has emerged as a suppressor of acute cigarette smoke-induced neutrophilia by a mechanism involving the NF-κB protein RelB. Yet individuals who smoke often smoke for many years and vary in their cigarette consumption. As there is currently no information on the AhR prevention of lung inflammation, including neutrophilia, due to varied and prolonged exposure regimes, we exposed control and AhR(-/-) mice to cigarette smoke for 2 weeks (subchronic exposure) utilizing low and high exposure protocols and evaluated pulmonary inflammation. Subchronic cigarette smoke exposure significantly increased pulmonary neutrophilia dose-dependently in AhR(-/-) mice. Surprisingly, there was no difference between smoke-exposed AhR(+/-) and AhR(-/-) mice in the expression of cytokines associated with neutrophil recruitment. Expression of pulmonary intercellular adhesion molecule-1 (ICAM-1), an adhesion molecule involved in neutrophil migration and retention, was higher in pulmonary endothelial cells from AhR(-/-) mice. Although total lung RelB expression was increased by cigarette smoke, nuclear RelB was significantly lower in subchronically exposed AhR(-/-) mice. Inhibition of AhR activity by CH-223191 in endothelial cells potentiated ICAM-1 expression and prevented RelB nuclear translocation but had no effect on neutrophil adhesion. These data support that genetic absence of the AhR contributes to heightened pulmonary neutrophilia in response to ongoing cigarette smoke exposure. Interindividual variations in AhR expression may enhance the susceptibility to cigarette smoke-induced diseases.

T cells demonstrate a Th1-biased response to native β2-glycoprotein I in a murine model of anti-phospholipid antibody induction

Anti-phospholipid syndrome (APS) is an autoimmune disorder characterized by the presence of autoantibody (AAb) to phospholipid (PL)-binding proteins, such as β2-glycoprotein I (β2GPI), and clinical manifestations including thrombosis and/or recurrent pregnancy loss. β2GPI-reactive T cells are clearly implicated in the generation of these AAb, but the mechanism responsible for their activation remains unclear. We hypothesized that immunization of mice with human β2GPI, in the context of a potent innate immune activator lipopolysaccharide (LPS), would generate not only high titers of anti-PL AAb, but also a strong β2GPI-specific T cell response. Healthy, nonautoimmune C57BL/6 mice were immunized repeatedly with human β2GPI in the presence of LPS. High titers of anti-PL to β2GPI appeared after the second immunization, with T cell reactivity to β2GPI detectable only after the fourth immunization. Splenic T cells from these mice proliferated in response to native β2GPI, alone or bound to anionic PL. These T cells produced IL-2 and IFN-γ, but not IL-4 or IL-10, indicating a Th1 bias of the β2GPI-specific response. These findings suggest that T cells responsive to β2GPI may become activated in APS patients by exposure to their cognate Ag in the context of innate immune activation and a pro-inflammatory environment.

Aryl hydrocarbon receptor-dependent regulation of miR-196a expression controls lung fibroblast apoptosis but not proliferation.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor implicated in the regulation of apoptosis and proliferation. Although activation of the AhR by xenobiotics such as dioxin inhibits the cell cycle and control apoptosis, paradoxically, AhR expression also promotes cell proliferation and survival independent of exogenous ligands. The microRNA (miRNA) miR-196a has also emerged as a regulator of proliferation and apoptosis but a relationship between the AhR and miR-196a is not known. Therefore, we hypothesized that AhR-dependent regulation of endogenous miR-196a expression would promote cell survival and proliferation. Utilizing lung fibroblasts from AhR deficient (AhR(-/-)) and wild-type (AhR(+/+)) mice, we show that there is ligand-independent regulation of miRNA, including low miR-196a in AhR(-/-) cells. Validation by qRT-PCR revealed a significant decrease in basal expression of miR-196a in AhR(-/-) compared to AhR(+/+) cells. Exposure to AhR agonists benzo[a]pyrene (B[a]P) and FICZ as well as AhR antagonist CH-223191 decreased miR-196a expression in AhR(+/+) fibroblasts concomitant with decreased AhR protein levels. There was increased proliferation only in AhR(+/+) lung fibroblasts in response to serum, corresponding to a decrease in p27(KIP1) protein, a cyclin-dependent kinase inhibitor. Increasing the cellular levels of miR-196a had no effect on proliferation or expression of p27(KIP1) in AhR(-/-) fibroblasts but attenuated cigarette smoke-induced apoptosis. This study provides the first evidence that AhR expression is essential for the physiological regulation of cellular miRNA levels- including miR-196a. Future experiments designed to elucidate the functional relationship between the AhR and miR-196a may delineate additional novel ligand-independent roles for the AhR.

Aryl hydrocarbon receptor-dependent retention of nuclear HuR suppresses cigarette smoke-induced cyclooxygenase-2 expression independent of DNA-binding.

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that responds to man-made environmental toxicants, has emerged as an endogenous regulator of cyclooxygenase-2 (Cox-2) by a mechanism that is poorly understood. In this study, we first used AhR-deficient (AhR−/−) primary pulmonary cells, together with pharmacological tools to inhibit new RNA synthesis, to show that the AhR is a prominent factor in the destabilization of Cox-2 mRNA. The destabilization of Cox-2 mRNA and subsequent suppression of cigarette smoke-induced COX-2 protein expression by the AhR was independent of its ability to bind the dioxin response element (DRE), thereby differentiating the DRE-driven toxicological AhR pathway from its anti-inflammatory abilities. We further describe that the AhR destabilizes Cox-2 mRNA by sequestering HuR within the nucleus. The role of HuR in AhR stabilization of Cox-2 mRNA was confirmed by knockdown of HuR, which resulted in rapid Cox-2 mRNA degradation. Finally, in the lungs of AhR−/− mice exposed to cigarette smoke, there was little Cox-2 mRNA despite robust COX-2 protein expression, a finding that correlates with almost exclusive cytoplasmic HuR within the lungs of AhR−/− mice. Therefore, we propose that the AhR plays an important role in suppressing the expression of inflammatory proteins, a function that extends beyond the ability of the AhR to respond to man-made toxicants. These findings open the possibility that a DRE-independent AhR pathway may be exploited therapeutically as an anti-inflammatory target.

RelB attenuates cigarette smoke extract-induced apoptosis in association with transcriptional regulation of the aryl hydrocarbon receptor

Abstract Chronic obstructive pulmonary disease (COPD) is a chronic and prevalent respiratory disease caused primarily by long term inhalation of cigarette smoke. A major hallmark of COPD is elevated apoptosis of structural lung cells including fibroblasts. The NF‐&kgr;B member RelB may suppress apoptosis in response to cigarette smoke, but its role in lung cell survival is not known. RelB may act as a pro‐survival factor by controlling the expression of superoxide dismutase 2 (SOD2). SOD2 is also regulated by the aryl hydrocarbon receptor (AhR), a ligand‐activated transcription factor that suppresses cigarette smoke‐induced apoptosis. As the AhR is also a binding partner for RelB, we speculate that RelB suppresses cigarette smoke‐induced apoptosis by regulating the AhR. Using an in vitro model of cigarette smoke exposure (cigarette smoke extract [CSE]), we found that CSE down‐regulated RelB expression in mouse lung fibroblasts, which was associated with elevated levels of cleaved PARP. Genetic ablation of RelB elevated CSE‐induced apoptosis, including chromatin condensation, and reduced mitochondrial function. There was also more reactive oxygen species production in RelB‐/‐ cells exposed to CSE. While there was no alteration in Nrf2 expression or localization between RelB‐/‐ and wild type cells in response to CSE, RelB‐/‐ cells displayed significantly decreased AhR mRNA and protein expression, concomitant with loss of AhR target gene expression (Cyp1a1, Cyp1b1, Nqo1). Finally, we found that RelB binds to the Ahr gene at 3 sites to potentially increase its expression via transcriptional induction. These data support that RelB suppresses cigarette smoke‐induced apoptosis, potentially by increasing the AhR. Together, these two proteins may comprise an important cell survival signaling pathway that reduces apoptosis upon cigarette smoke exposure. Graphical abstract Figure. No Caption available. HighlightsRelB is a member of the alternative NF‐&kgr;B pathway with diverse biological function.RelB suppresses basal and cigarette smoke extract‐induced lung cell apoptosis.RelB promotes the expression of anti‐oxidant genes that are also regulated by the AhR.AhR expression and activation are controlled by RelB via RelB binding to the AhR gene.Together, RelB and AhR represent a pathway essential in promoting lung health against respiratory toxicants.

Aryl hydrocarbon receptor (AhR)-dependent regulation of pulmonary miRNA by chronic cigarette smoke exposure

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor historically known for its toxic responses to man-made pollutants such as dioxin. More recently, the AhR has emerged as a suppressor of inflammation, oxidative stress and apoptosis from cigarette smoke by mechanisms that may involve the regulation of microRNA. However, little is known about the AhR regulation of miRNA expression in the lung in response to inhaled toxicants. Therefore, we exposed Ahr−/− and Ahr+/− mice to cigarette smoke for 4 weeks and evaluated lung miRNA expression by PCR array. There was a dramatic regulation of lung miRNA by the AhR in the absence of exogenous ligand. In response to cigarette smoke, there were more up-regulated miRNA in Ahr−/− mice compared to Ahr+/− mice, including the cancer-associated miRNA miR-96. There was no significant change in the expression of the AhR regulated proteins HuR and cyclooxygenase-2 (COX-2). There were significant increases in the anti-oxidant gene sulfiredoxin 1 (Srxn1) and FOXO3a- predicted targets of miR-96. Collectively, these data support a prominent role for the AhR in regulating lung miRNA expression. Further studies to elucidate a role for these miRNA may further uncover novel biological function for the AhR in respiratory health and disease.

Pulmonary and diaphragmatic pathology in collagen type I α1 mutant mice with osteogenesis imperfecta

BackgroundOsteogenesis imperfecta (OI) is most often caused by mutations in type I collagen genes. Respiratory complications have been largely attributed to spine and ribcage deformities. We hypothesized that direct involvement of the pulmonary parenchyma and/or diaphragm by the disease may occur.MethodsIn Col1a1Jrt/+ mice, a model of severe dominant OI, mean linear intercept length (Lm) was used to assess the distal airspace size. Cross-sectional area (CSA) and myosin heavy chain (MyHC) phenotype of the diaphragm muscle fibers, as well as contractile properties, were determined. OI mice were also treated with neutralizing antibodies against transforming growth factor-β (TGF-β).ResultsDistal airspace enlargement occurred in OI mice (Lm +27%). Diaphragmatic thickness and fiber number were reduced, with increases in fast-twitch type IIx/IIb MyHC fibers. Ex vivo force generation (normalized for CSA) of the diaphragm was also significantly reduced. The increased Lm values found in OI mice were not prevented by anti-TGF-β antibody treatment.ConclusionsThe Col1a1Jrt/+ mouse model of OI demonstrates: (1) pulmonary airspace enlargement not driven by TGF-β; and (2) reduced muscle mass and intrinsic contractile weakness of the diaphragm. These results suggest a complex and multifaceted basis for respiratory complications in OI that cannot be solely attributed to bone manifestations.