Differential Regulation of Metabolic Pathways by S-nitrosation in Endothelial Cells

Background. Interleukin 6 (IL6) is a multifunctional cytokine produced by various cells, including vascular endothelial cells. IL6 has both pro- and non-/anti-inflammatory functions, and the response to IL6 is dependent on whether it acts via the membrane-bound IL6 receptor α (IL6Rα) (classic signaling) or the soluble form of the receptor (transsignaling). As human endothelial cells produce IL6 and at the same time express IL6Rα, we hypothesized that IL6 may have autocrine functions. Methods. Knockdown of IL6 in cultured human endothelial cells was performed using siRNA. Knockdown efficiency was evaluated using ELISA. RNA sequencing was employed to characterize the transcriptional consequence of IL6 knockdown, and Ingenuity Pathway Analysis was used to further explore the functional roles of IL6. Results. Knockdown of IL6 in cultured endothelial cells resulted in a 84-92% reduction in the release of IL6. Knockdown of IL6 resulted in dramatic changes in transcriptional pattern; knockdown of IL6 in the absence of soluble IL6Rα (sIL6Rα) led to differential regulation of 1915 genes, and knockdown of IL6 in the presence of sIL6Rα led to differential regulation of 1967 genes (fold change 1.5, false discovery rate<0.05). Pathway analysis revealed that the autocrine functions of IL6 in human endothelial cells are mainly related to basal cellular functions such as regulation of cell cycle, signaling, and cellular movement. Furthermore, we found that knockdown of IL6 activates functions related to adhesion, binding, and interaction of endothelial cells, which seem to be mediated mainly via STAT3. Conclusion. In this study, a large number of novel genes that are under autocrine regulation by IL6 in human endothelial cells were identified. Overall, our data indicate that IL6 acts in an autocrine manner to regulate basal cellular functions, such as cell cycle regulation, signaling, and cellular movement, and suggests that the autocrine functions of IL6 in human endothelial cells are mediated via IL6 classic signaling.

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Differential regulation of metabolism by nitric oxide andS-nitrosothiols in endothelial cells

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00210.2011 ◽

2011 ◽

Vol 301 (3) ◽

pp. H803-H812 ◽

Cited By ~ 21

Author(s):

Anne R. Diers ◽

Katarzyna A. Broniowska ◽

Victor M. Darley-Usmar ◽

Neil Hogg

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Oxidative Phosphorylation ◽

Metabolic Pathways ◽

Nitrosative Stress ◽

Surrogate Marker ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

No Donor

S-nitrosation of thiols in key proteins in cell signaling pathways is thought to be an important contributor to nitric oxide (NO)-dependent control of vascular (patho)physiology. Multiple metabolic enzymes are targets of both NO and S-nitrosation, including those involved in glycolysis and oxidative phosphorylation. Thus it is important to understand how these metabolic pathways are integrated by NO-dependent mechanisms. Here, we compared the effects of NO and S-nitrosation on both glycolysis and oxidative phosphorylation in bovine aortic endothelial cells using extracellular flux technology to determine common and unique points of regulation. The compound S-nitroso-l-cysteine (l-CysNO) was used to initiate intracellular S-nitrosation since it is transported into cells and results in stable S-nitrosation in vitro. Its effects were compared with the NO donor DetaNONOate (DetaNO). DetaNO treatment caused only a decrease in the reserve respiratory capacity; however, l-CysNO impaired both this parameter and basal respiration in a concentration-dependent manner. In addition, DetaNO stimulated extracellular acidification rate (ECAR), a surrogate marker of glycolysis, whereas l-CysNO stimulated ECAR at low concentrations and inhibited it at higher concentrations. Moreover, a temporal relationship between NO- and S-nitrosation-mediated effects on metabolism was identified, whereby NO caused a rapid impairment in mitochondrial function, which was eventually overwhelmed by S-nitrosation-dependent processes. Taken together, these results suggest that severe pharmacological nitrosative stress may differentially regulate metabolic pathways through both intracellular S-nitrosation and NO-dependent mechanisms. Moreover, these data provide insight into the role of NO and related compounds in vascular (patho)physiology.

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