Abstract 619: Blockage Of Toll-Like Receptor 4 Inhibits Renin Stimulation Of NF-KB-Mediated Cytokine Signaling In Brain Neurons

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Toll-like receptor 4 (TLR4) has been shown to play an important role in inflammation and initiation and progression of cardiovascular pathologies. This, coupled with the observation that activation of the brain (pro)renin receptor (PRR) initiates NF-Kappa B (KB) -cytokine signaling to induce neurogenic hypertension has led us to hypothesize that brain PRR activation, in part, stimulates TLR4 to induce NF-KB-cytokine signal pathway. Thus, blockage of TLR4 should attenuate the expression of NF-KB and proinflammatory cytokines induced by PRR activation. To test this hypothesis, we harvested neuronal cells from neonatal Sprague Dawley rat brain that abundantly express PRR, and incubated with 20 nM of renin for 6 hours. Real time PCR was performed to analyze expression of NF-KB and other inflammatory mediators. Renin treatment resulted in significant increases in interleukin-1 β (IL-1β, 1200-fold), IL-6 (33-fold), tumor necrosis factor (TNF-α, 17-fold), leukemia inhibitory factor (LIF, 18-fold) and C-C ligand 5 (CCL5, 330-fold). In addition, the mRNA levels of P50, a subunit of NF-KB was increased by 5-fold, and nfkbia, a subunit of I-kappa B kinase (IKK), was increased by 7-fold. Furthermore, mRNA levels of inducible nitric oxide synthase (iNOS) was increased by 8000-fold, and a neuronal activation maker, c-FOS was increased by 28 fold as compared with control neurons. However, Quinazoline (10μM), a NF-KB activation inhibitor, effectively blocked renin-induced increases in IL-1β, IL-6, TNF-α, CCL5, iNOS and P50 by more than 85%, but failed to inhibit LIF, nfkbia and c-FOS. In contrast, renin stimulation of all the above genes (IL-1β, IL-6, TNF-α, CCL5, iNOS, LIF, P50, nfkbia and c-FOS) was almost entirely blocked (>90%) by the TLR4 inhibitor, TAK-242 (2 μM), indicating a novel PRR mediated activation of TLR4 signaling. This PRR regulation of neuronal inflammatory mediators and neuronal activity was not affected by AT1 receptor antagonist losartan, demonstrating that the effect is not mediated by PRR-induced Ang-II generation. Our studies suggest that PRR plays a central role in the development of inflammatory mediated neurogenic hypertension through activation of TLR4.

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