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Regulation of NF-?B activation through a novel PI-3K-independent and PKA/Akt-dependent pathway in human umbilical vein endothelial cells.

ABSTRACT: The transcription factor NF-?B regulates numerous inflammatory diseases, and proteins involved in the NF-?B-activating signaling pathway are important therapeutic targets. In human umbilical vein endothelial cells (HUVECs), TNF-?-induced I?B? degradation and p65/RelA phosphorylation regulate NF-?B activation. These are mediated by IKKs (I?B kinases) viz. IKK?, ? and ? which receive activating signals from upstream kinases such as Akt. Akt is known to be positively regulated by PI-3K (phosphoinositide-3-kinase) and differentially regulated via Protein kinase A (PKA) in various cell types. However, the involvement of PKA/Akt cross talk in regulating NF-?B in HUVECs has not been explored yet. Here, we examined the involvement of PKA/Akt cross-talk in HUVECs using a novel compound, 2-methyl-pyran-4-one-3-O-?-D-2',3',4',6'-tetra-O-acetyl glucopyranoside (MPTAG). We observed that MPTAG does not directly inhibit IKK-? but prevents TNF-?-induced activation of IKK-? by blocking its association with Akt and thereby inhibits NF-?B activation. Interestingly, our results also revealed that inhibitory effect of MPTAG on Akt and NF-?B activation was unaffected by wortmannin, and was completely abolished by H-89 treatment in these cells. Thus, MPTAG-mediated inhibition of TNF-?-induced Akt activation was independent of PI-3K and dependent on PKA. Most importantly, MPTAG restores the otherwise repressed activity of PKA and inhibits the TNF-?-induced Akt phosphorylation at both Thr308 and Ser473 residues. Thus, we demonstrate for the first time the involvement of PKA/Akt cross talk in NF-?B activation in HUVECs. Also, MPTAG could be useful as a lead molecule for developing potent therapeutic molecules for diseases where NF-?B activation plays a key role.

SUBMITTER: Balwani S

PROVIDER: S-EPMC3465347 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Regulation of NF-κB activation through a novel PI-3K-independent and PKA/Akt-dependent pathway in human umbilical vein endothelial cells.

Balwani Sakshi S Chaudhuri Rituparna R Nandi Debkumar D Jaisankar Parasuraman P Agrawal Anurag A Ghosh Balaram B

PloS one 20121005 10

The transcription factor NF-κB regulates numerous inflammatory diseases, and proteins involved in the NF-κB-activating signaling pathway are important therapeutic targets. In human umbilical vein endothelial cells (HUVECs), TNF-α-induced IκBα degradation and p65/RelA phosphorylation regulate NF-κB activation. These are mediated by IKKs (IκB kinases) viz. IKKα, β and γ which receive activating signals from upstream kinases such as Akt. Akt is known to be positively regulated by PI-3K (phosphoinos ...[more]

PMID: 23071583

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Project description:The increasing burden of diabetes in low and middle‑income countries is attributable to both genetic and epigenetic factors. Environmental‑ and lifestyle‑associated changes are also considered to be important contributors to this disease. The resultant co‑morbidities arising from micro‑and macrovascular changes in diabetes are difficult to manage and are an economic burden. However, very little is known about the molecular mechanisms that drive this phenotype. The present study aimed to investigate the role of sirtuin 1 (SIRT1)‑ and transcription box‑3 (TBX‑3)‑mediated regulation of endothelial dysfunction, given the significance of SIRT1 in glucose metabolism and the role of TBX‑3 in the maintenance of cellular proliferation, senescence and apoptosis. Following the recruitment of adult patients with and without diabetes, both SIRT1 and TBX‑3 expression was confirmed to be present in the sera of the patients with diabetes and the patients without diabetes; however, both SIRT1 and TBX‑3 expression levels were higher in the sera of the patients with diabetes. Human umbilical vein endothelial cells (HUVECs) were further used for in vitro studies. Using TBX‑3 and SIRT1 knockdown models, the cellular responses to proliferation, migration, invasion and tube formation were investigated using an MTS, cell cycle analysis, wound healing, Transwell and tube formation assay, respectively. Western blotting was also used to determine the downstream signaling pathways involved. The genetic knockdown of TBX‑3 in hyperglycemic conditions significantly decreased the cellular proliferation, migration, invasion and angiogenesis of HUVECs. It was subsequently identified that TBX‑3 mediated its effects through the activation of AKT and vascular endothelial growth factor (VEGF) signaling. However, the genetic knockdown of SIRT1 in the presence of TBX‑3 overexpression and glucose failed to activate the AKT and VEGF signaling pathways. In conclusion, the results of the present study suggested that SIRT1 may positively regulate TBX‑3 in endothelial cells, therefore, SIRT1 and/or TBX‑3 may serve as potential novel biomarkers for disease progression.

Dataset Information

Regulation of NF-?B activation through a novel PI-3K-independent and PKA/Akt-dependent pathway in human umbilical vein endothelial cells.

Publications

Regulation of NF-κB activation through a novel PI-3K-independent and PKA/Akt-dependent pathway in human umbilical vein endothelial cells.

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