Peroxiredoxin 6 suppresses Muc5ac overproduction in LPS-induced airway inflammation through H2O2-EGFR-MAPK signaling pathway.

Mucus hypersecretion is a prominent mechanism in airway inflammation. Muc5ac is a major component of mucus and can be activated by reactive oxygen species (ROS). Peroxiredoxin 6 (Prdx6) highly expresses in airway epithelium and protects the airway from oxidative stress. In this study, we investigated the roles of Prdx6 in lipopolysaccharide (LPS)-induced mucin production in mice. We found that the levels of H2O2 and the Muc5ac mRNA were significantly increased in Prdx6 (-/-) mice compared to those in C57BL/6J mice after LPS instillation, which were markedly inhibited by epithelial growth factor receptor (EGFR) inhibitor Elrotinib. In vitro studies showed that mRNA levels of Prdx6 were decreased while H2O2 and Muc5ac were increased in a dose-dependent manner after LPS exposure, with significant increase in Prdx6 knockdown bronchial epithelial cells compared with those in normal epithelial cells. LPS-induced Muc5ac release was significantly inhibited by EGFR inhibitor, p38 inhibitor and JNK inhibitor, but not ERK1/2 inhibitor, indicating that the H2O2-EGFR-MAPK pathway is likely involved in the responses. This study indicated that Prdx6 decreased LPS-induced Muc5ac increase and played important roles in mucin hypersecretion after LPS exposure.

Dual role of the antioxidant enzyme peroxiredoxin 6 in skin carcinogenesis.

The antioxidant enzyme peroxiredoxin 6 (Prdx6) is a key regulator of the cellular redox balance, particularly under stress conditions. We identified Prdx6 as an important player in different phases of skin carcinogenesis. Loss of Prdx6 in mice enhanced the susceptibility to skin tumorigenesis, whereas overexpression of Prdx6 in keratinocytes of transgenic mice had the opposite effect. The tumor-preventive effect of Prdx6, which was observed in a human papilloma virus 8-induced and a chemically induced tumor model, was not due to alterations in keratinocyte proliferation, apoptosis, or in the inflammatory response. Rather, endogenous and overexpressed Prdx6 reduced oxidative stress as reflected by the lower levels of oxidized phospholipids in the protumorigenic skin of Prdx6 transgenic mice and the higher levels in Prdx6-knockout mice than in control animals. In contrast to its beneficial effect in tumor prevention, overexpression of Prdx6 led to an acceleration of malignant progression of existing tumors, revealing a dual function of this enzyme in the pathogenesis of skin cancer. Finally, we found strong expression of PRDX6 in keratinocytes of normal human skin and in the tumor cells of squamous cell carcinomas, indicating a role of Prdx6 in human skin carcinogenesis. Taken together, our data point to the potential usefulness of Prdx6 activators or inhibitors for controlling different stages of skin carcinogenesis.

Deletion of peroxiredoxin 6 potentiates lipopolysaccharide-induced acute lung injury in mice.

OBJECTIVE: To investigate the role and signaling pathway of peroxiredoxin 6, a newly identified peroxidase, in lipopolysaccharide-induced acute lung injury. DESIGN: Prospective, randomized, controlled study. SETTING: Research laboratory. SUBJECTS: Peroxiredoxin 6 (-/-) and wild-type C57BL/6 mice. INTERVENTIONS: Wild-type or peroxiredoxin 6 (-/-) mice were challenged by intratracheal instillation of lipopolysaccharide (5 mg/kg) for 4 hrs or 24 hrs for lung injury measurement. In other studies, peritoneal macrophages, isolated from wild-type and peroxiredoxin 6 (-/-) mice, were preincubated in presence or absence of mitogen-activated protein kinases inhibitors for 30 mins before being stimulated with lipopolysaccharide (1 µg/mL) for 4 hrs. MEASUREMENTS AND MAIN RESULTS: Bronchoalveolar lavage myeloperoxidase activity and the lung injury score were significantly increased in peroxiredoxin 6 (-/-) mice compared with wild-type mice after lipopolysaccharide instillation at both 4 hrs and 24 hrs. Hydrogen peroxide and malondialdehyde levels, as well as nuclear factor-κB activities, tumor necrosis factor-α, interleukin-1ß, and matrix metalloproteinase-9 messenger RNA, protein concentration, and activities were significantly increased whereas total antioxidative capability was markedly decreased in lungs of peroxiredoxin 6 (-/-) mice compared with wild-type mice. In vitro studies showed intracellular reactive oxygen species levels and release of tumor necrosis factor-α, interleukin-1, and matrix metalloproteinase-9 were significantly increased in macrophages from peroxiredoxin 6 (-/-) mice compared with that from wild-type mice after lipopolysaccharide stimulation. Cytokines release was partially suppressed by extracellular signal-regulated kinase and c-Jun N-terminal kinase inhibitors, but not by the p38 mitogen-activated protein kinase inhibitor. CONCLUSIONS: Deletion of peroxiredoxin 6 exaggerates lipopolysaccharide-induced acute lung injury and inflammation with increased oxidative stress, inflammatory responses, and matrix degradation, all of which were partially dependent on nuclear factor-κB, extracellular signal-regulated kinase, and c-Jun N-terminal kinase pathways.

Peroxiredoxin 6 differentially regulates acute and chronic cigarette smoke–mediated lung inflammatory response and injury.

Peroxiredoxin 6 (Prdx6) exerts its protective role through peroxidase activity against H2O2 and phospholipid hydroperoxides. We hypothesized that targeted disruption of Prdx6 would lead to enhanced susceptibility to cigarette smoke (CS)-mediated lung inflammation and/or emphysema in mouse lung. Prdx6 null (Prdx6⁻/⁻mice exposed to acute CS showed no significant increase of inflammatory cell influx or any alterations in lung levels of proinflammatory cytokines compared to wild-type (WT) mice. Lung levels of antioxidant enzymes were significantly increased in acute CS-exposed Prdx6⁻/⁻ compared to WT mice. Overexpressing (Prdx6⁻/⁻) mice exposed to acute CS showed significant decrease in lung antioxidant enzymes associated with increased inflammatory response compared to CS-exposed WT mice or air-exposed Prdx6⁻/⁻ mice. However, chronic 6 months of CS exposure resulted in increased lung inflammatory response, mean linear intercept (Lm), and alteration in lung mechanical properties in Prdx6⁻/⁻ when compared to WT mice exposed to CS. These data show that targeted disruption of Prdx6 does not lead to increased lung inflammatory response but is associated with increased antioxidants, suggesting a critical role of lung Prdx6 and several compensatory mechanisms during acute CS-induced adaptive response, whereas this protection is lost in chronic CS exposure leading to emphysema.

Protein expression profiling of lens epithelial cells from Prdx6-depleted mice and their vulnerability to UV radiation exposure.

Oxidative stress is one of the causative factors in progression and etiology of age-related cataract. Peroxiredoxin 6 (Prdx6), a savior for cells from internal or external environmental stresses, plays a role in cellular signaling by detoxifying reactive oxygen species (ROS) and thereby controlling gene regulation. Using targeted inactivation of the Prdx6 gene, we show that Prdx6-deficient lens epithelial cells (LECs) are more vulnerable to UV-triggered cell death, a major cause of skin disorders including cataractogenesis, and these cells display abnormal protein profiles. PRDX6-depleted LECs showed phenotypic changes and formed lentoid body, a characteristic of terminal cell differentiation and epithelial-mesenchymal transition. Prdx6(-/-) LECs exposed to UV-B showed higher ROS expression and were prone to apoptosis compared with wild-type LECs, underscoring a protective role for Prdx6. Comparative proteomic analysis using fluorescence-based difference gel electrophoresis along with mass spectrometry and database searching revealed a total of 13 proteins that were differentially expressed in Prdx6(-/-) cells. Six proteins were upregulated, whereas expression of seven proteins was decreased compared with Prdx6(+/+) LECs. Among the cytoskeleton-associated proteins that were highly expressed in Prdx6-deficient LECs was tropomyosin (Tm)2beta. Protein blot and real-time PCR validated dramatic increase of Tm2beta and Tm1alpha expression in these cells. Importantly, Prdx6(+/+) LECs showed a similar pattern of Tm2beta protein expression after transforming growth factor (TGF)-beta or H(2)O(2) treatment. An extrinsic supply of PRDX6 could restore Tm2beta expression, demonstrating that PRDX6 may attenuate adverse signaling in cells and thereby maintain cellular homeostasis. Exploring redox-proteomics (Prdx6(-/-)) and characterization and identification of abnormally expressed proteins and their attenuation by PRDX6 delivery should provide a basis for development of novel therapeutic interventions to postpone ROS-mediated abnormal signaling deleterious to cells or tissues.

Peroxiredoxin-6 protects against mitochondrial dysfunction and liver injury during ischemia-reperfusion in mice.

Hepatic ischemia-reperfusion (I/R) injury is an important complication of liver surgery and transplantation. Mitochondrial function is central to this injury. To examine alterations in mitochondrial function during I/R, we assessed the mitochondrial proteome in C57Bl/6 mice. Proteomic analysis of liver mitochondria revealed 234 proteins with significantly altered expression after I/R. From these, 13 proteins with the greatest expression differences were identified. One of these proteins, peroxiredoxin-6 (Prdx6), has never before been described in mitochondria. In hepatocytes from sham-operated mice, Prdx6 expression was found exclusively in the cytoplasm. After ischemia or I/R, Prdx6 expression disappeared from the cytoplasm and appeared in the mitochondria, suggesting mitochondrial trafficking. To explore the functional role of Prdx6 in hepatic I/R injury, wild-type and Prdx6-knockout mice were subjected to I/R injury. Prdx6-knockout mice had significantly more hepatocellular injury compared with wild-type mice. Interestingly, the increased injury in Prdx6-knockout mice occurred despite reduced inflammation and was associated with increased mitochondrial generation of H(2)O(2) and dysfunction. The mitochondrial dysfunction appeared to be related to complex I of the electron transport chain. These data suggest that hepatocyte Prdx6 traffics to the mitochondria during I/R to limit mitochondrial dysfunction as a protective mechanism against hepatocellular injury.

Peroxiredoxin 6 is required for blood vessel integrity in wounded skin.

Peroxiredoxin 6 (Prdx6) is a cytoprotective enzyme with largely unknown in vivo functions. Here, we use Prdx6 knockout mice to determine its role in UV protection and wound healing. UV-mediated keratinocyte apoptosis is enhanced in Prdx6-deficient mice. Upon skin injury, we observe a severe hemorrhage in the granulation tissue of knockout animals, which correlates with the extent of oxidative stress. At the ultrastructural level endothelial cells appear highly damaged, and their rate of apoptosis is enhanced. Knock-down of Prdx6 in cultured endothelial cells also increases their susceptibility to oxidative stress, thus confirming the sensitivity of this cell type to loss of Prdx6. Wound healing studies in bone marrow chimeric mice demonstrate that Prdx6-deficient inflammatory and endothelial cells contribute to the hemorrhage phenotype. These results provide insight into the cross-talk between hematopoietic and resident cells at the wound site and the role of reactive oxygen species in this interplay.