Article

The role of caveolin-1 in PCB77-induced eNOS phosphorylation in human-derived endothelial cells

Department of Pediatrics, University of Kentucky, Lexington, Kentucky, United States
AJP Heart and Circulatory Physiology (Impact Factor: 3.84). 01/2008; 293(6):H3340-7. DOI: 10.1152/ajpheart.00921.2007
Source: PubMed

ABSTRACT

Polychlorinated biphenyls (PCBs) may contribute to the pathology of atherosclerosis by activating inflammatory responses in vascular endothelial cells. Endothelial nitric oxide synthase (eNOS) is colocalized with caveolae and is a critical regulator of vascular homeostasis. PCBs may be proatherogenic by causing dysfunctional eNOS signaling. The objective of this study was to investigate the role of caveolin-1 in PCB-induced endothelial dysfunction with a focus on mechanisms associated with eNOS signaling. Cells derived from an immortalized human vascular endothelial cell line were treated with PCB77 to study nitrotyrosine formation through eNOS signaling. Phosphorylation studies of eNOS, caveolin-1, and kinases, such as Src, phosphatidylinositol 3-kinase (PI3K), and Akt, were conducted in cells containing either functional or small-interfering RNA-silenced caveolin-1 protein. We also investigated caveolin-1-regulated mechanisms associated with PCB-induced markers of peroxynitrite formation and DNA binding of NF-kappaB. Cellular exposure to PCB77 increased eNOS phosphorylation and nitric oxide production, as well as peroxynitrite levels. A subsequent PCB-induced increase in NF-kappaB DNA binding may have implications in oxidative stress-mediated inflammatory mechanisms. The activation of eNOS by PCB77 treatment was blocked by inhibitors of the Src/PI3K/Akt pathway. PCB77 also increased phosphorylation of caveolin-1, indicating caveolae-dependent endocytosis. Caveolin-1 silencing abolished both the PCB-stimulated Akt and eNOS phosphorylation, suggesting a regulatory role of caveolae in PCB-induced eNOS signaling. These findings suggest that PCB77 induces eNOS phosphorylation in endothelial cells through a Src/PI3K/Akt-dependent mechanism, events regulated by functional caveolin-1. Our data provide evidence that caveolae may play a critical role in regulating vascular endothelial cell activation and toxicity induced by persistent environmental pollutants such as coplanar PCBs.

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    • "Porcine and human endothelial cells were treated with PCB 126 at 0.25 μM for 16 h or 4 h and mouse pulmonary endothelial cells were treated with 2.5 μM for 24 h, which are established concentrations used previously in our laboratory (Han et al., 2010, 2012). Porcine primary endothelial cells, Eahy.926 human endothelial cells, and isolated mouse endothelial cells contained Cav-1 protein levels (wildtype) and exhibited VCAM-1 induction due to PCB 126 treatment, as previously reported (Han et al., 2012; Lim et al., 2007, 2008). Endothelial cells isolated from wildtype and Cav-1−/− mice were identified by morphology , platelet endothelial cell adhesion molecule-1 (PECAM1) bead pulldown , and Von Willebrand Factor (VWF) protein expression. "
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    ABSTRACT: Environmental toxicants such as polychlorinated biphenyls (PCBs) have been implicated in the promotion of multiple inflammatory disorders including cardiovascular disease, but information regarding mechanisms of toxicity and cross-talk between relevant cell signaling pathways is lacking. To examine the hypothesis that cross-talk between membrane domains called caveolae and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathways alter PCB-induced inflammation, caveolin-1 was silenced in vascular endothelial cells, resulting in a decreased PCB-induced inflammatory response. Cav-1 silencing (siRNA treatment) also increased levels of Nrf2-ARE transcriptional binding, resulting in higher mRNA levels of the antioxidant genes glutathione s-transferase and NADPH dehydrogenase quinone-1 in both vehicle and PCB-treated systems. Along with this upregulated antioxidant response, Cav-1 siRNA treated cells exhibited decreased mRNA levels of the Nrf2 inhibitory protein Keap1 in both vehicle and PCB-treated samples. Silencing Cav-1 also decreased protein levels of Nrf2 inhibitory proteins Keap1 and Fyn kinase, especially in PCB-treated cells. Further, endothelial cells from wildtype and Cav-1-/- mice were isolated and treated with PCB to better elucidate the role of functional caveolae in PCB-induced endothelial inflammation. Cav-1-/- endothelial cells were protected from PCB-induced cellular dysfunction as evidenced by decreased vascular cell adhesion molecule (VCAM-1) protein induction. Compared to wildtype cells, Cav-1-/- endothelial cells also allowed for a more effective antioxidant response, as observed by higher levels of the antioxidant genes. These data demonstrate novel cross-talk mechanisms between Cav-1 and Nrf2 and implicate the reduction of Cav-1 as a protective mechanism for PCB-induced cellular dysfunction and inflammation.
    Full-text · Article · Apr 2014 · Toxicology and Applied Pharmacology
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    • "Porcine and human endothelial cells were treated with PCB 126 at 0.25 μM for 16 h or 4 h and mouse pulmonary endothelial cells were treated with 2.5 μM for 24 h, which are established concentrations used previously in our laboratory (Han et al., 2010, 2012). Porcine primary endothelial cells, Eahy.926 human endothelial cells, and isolated mouse endothelial cells contained Cav-1 protein levels (wildtype) and exhibited VCAM-1 induction due to PCB 126 treatment, as previously reported (Han et al., 2012; Lim et al., 2007, 2008). Endothelial cells isolated from wildtype and Cav-1−/− mice were identified by morphology , platelet endothelial cell adhesion molecule-1 (PECAM1) bead pulldown , and Von Willebrand Factor (VWF) protein expression. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Environmental toxicants such as polychlorinated biphenyls (PCBs) have been implicated in the promotion of multiple inflammatory disorders including cardiovascular disease, but information regarding mechanisms of toxicity and cross-talk between relevant cell signaling pathways is lacking. To examine the hypothesis that cross-talk between membrane domains called caveolae and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathways alter PCB-induced inflammation, caveolin-1 was silenced in vascular endothelial cells, resulting in a decreased PCB-induced inflammatory response. Cav-1 silencing (siRNA treatment) also increased levels of Nrf2-ARE transcriptional binding, resulting in higher mRNA levels of the antioxidant genes glutathione s-transferase and NADPH dehydrogenase quinone-1 in both vehicle and PCB-treated systems. Along with this upregulated antioxidant response, Cav-1 siRNA treated cells exhibited decreased mRNA levels of the Nrf2 inhibitory protein Keap1 in both vehicle and PCB-treated samples. Silencing Cav-1 also decreased protein levels of Nrf2 inhibitory proteins Keap1 and Fyn kinase, especially in PCB-treated cells. Further, endothelial cells from wildtype and Cav-1-/- mice were isolated and treated with PCB to better elucidate the role of functional caveolae in PCB-induced endothelial inflammation. Cav-1-/- endothelial cells were protected from PCB-induced cellular dysfunction as evidenced by decreased vascular cell adhesion molecule (VCAM-1) protein induction. Compared to wildtype cells, Cav-1-/- endothelial cells also allowed for a more effective antioxidant response, as observed by higher levels of the antioxidant genes. These data demonstrate novel cross-talk mechanisms between Cav-1 and Nrf2 and implicate the reduction of Cav-1 as a protective mechanism for PCB-induced cellular dysfunction and inflammation.
    Full-text · Article · Jan 2014 · Toxicology and Applied Pharmacology
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    • "Caveolin-1 (Cav-1), the major structural protein of caveolae, contains the " Cav-1 binding domain " that is known to bind multiple proteins including endothelial nitric oxide synthase (eNOS), v-src sarcoma, protein kinase C (PKC), extracellular signal-regulated kinase, and protein kinase B; many of which are involved in inflammatory pathways (Sowa 2012). For example, downregulation of Cav- 1 can lead to eNOS activation and subsequent increases in diffusible nitric oxide, which has been shown to play a major role in healthy blood pressure and vessel tone (Lim et al. 2007). Many publications from our laboratory and others show downregulation and elimination of Cav-1 to be antiatherosclerotic (Lim et al. 2008; Majkova et al. 2009). "
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    ABSTRACT: The nutritional profile of an individual can influence the toxicity of persistent environmental toxicants. Polychlorinated biphenyls (PCBs), prevalent environmental pollutants, are highly lipid-soluble toxic compounds that biomagnify through trophic levels and pose cancer, neurocognitive, and atherosclerotic risk to human populations. There is a growing body of knowledge that PCBs can initiate inflammatory responses in vivo, and this inflammation can be either exacerbated or ameliorated by nutrition. Data indicate that diets high in certain dietary lipids such as omega-6 fatty acids can worsen PCB-induced vascular toxicity while diets enriched with bioactive food components such as polyphenols and omega-3 polyunsaturated fatty acids can improve the toxicant-induced inflammation. There is evidence that bioactive nutrients protect through multiple cell signaling pathways, but we have shown that lipid raft caveolae and the antioxidant defense controller nuclear factor (erythroid-derived 2)-like 2 (Nrf2) both play a predominant role in nutritional modulation of PCB-induced vascular toxicity. Interestingly, there appears to be an intimate cross-talk between caveolae-related proteins and cellular Nrf2, and focusing on the use of specific bioactive food components that simultaneously alter both pathways may produce a more effective and efficient cytoprotective response to toxicant exposure. The use of nutrition as a protective tool is an economically beneficial means to address the toxicity of persistent environmental toxicants and may become a sensible means to protect human populations from PCB-induced vascular inflammation and associated chronic diseases.
    Full-text · Article · Feb 2013 · Environmental Science and Pollution Research
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