Cooperation between Classical and Alternative NF-κB Pathways Regulates Proinflammatory Responses in Epithelial Cells

Abstract

The transcription factor NF-kB has been causally linked to inflammatory lung diseases. Recent studies have unraveled the complexity of NF-kB activation by identifying two parallel activation pathways: the classical NF-kB pathway, which is controlled by IkB kinase complex-b (IKKb) and RelA/p50, and the alternative pathway, which is controlled by IKKa and RelB/p52. The alternative pathway regulates adaptive immune responses and lymphoid development, yet its role in the regulation of innate immune responses remains largely unknown. In this study, we determined the relevance of the alternative NF-kB pathway in proinflammatory responses in lung epithelial cells. The exposure of C10 murine alveolar lung epithelial cells to diverse stimuli, or primary murine tracheal epithelial cells to LPS, resulted in the activation of both NF-kB pathways, based on the nuclear translocation of RelA, p50, RelB, and p52. Increases in the nuclear content of RelA occurred rapidly, but transiently, whereas increases in nuclear RelB content were protracted. The small interfering (si) RNAmediated knockdown of IKKa, RelA, or RelB resulted in decreases of multiple LPS-induced proinflammatory cytokines. Surprisingly, the siRNA ablation of IKKa or RelB led to marked increases in the production of IL-6 in response to LPS. The simultaneous expression of constitutively active (CA)-IKKa and CA-IKKb caused synergistic increases in proinflammatory mediators. Lastly, the disruption of the IKK signalsome inhibited the activation of both NF-kB pathways. These results demonstrate that the coordinated activation of both NF-kB pathways regulates the magnitude and nature of proinflammatory responses in lung epithelial cells.Keywords: lung; IkB kinase-b; IkB kinase-a; RelA; RelB NF-kB is a transcription factor that plays a cardinal role in multiple cellular processes, including survival, proliferation, and inflammation. In unstimulated cells, NF-kB dimers RelA and p50 are sequestered in the cytosol by the inhibitor of kB (IkBa). The IkB kinase complex (IKK) consists of two catalytic subunits, IKKb and IKKa, and the regulatory protein, IKKg (also known as NF-kB essential modulator). Upon ligation by a variety of stimuli such as TNF-a or Toll-like receptor agonists, IKKb is phosphorylated and in turn phosphorylates IkBa, leading to its subsequent ubiquitination and degradation by the 26S proteasome (1, 2). The processing of IkBa promotes the nuclear translocation of RelA/p50, leading to the transcriptional activation of NF-kB-dependent genes. NF-kB activates the transcription of many proinflammatory cytokine and chemokine genes that initiate and propagate innate immune responses (1, 2).The airway epithelium, classically regarded as the first line of defense against inhaled agents, toxic factors, and physical trauma, is now recognized as a key component of the innate immune system, and plays an active role in the orchestration of acute inflammatory and adaptive immune responses (3, 4). Upon stimulation, epithelial cells secrete proinflammatory mediators such as...

p52 Overexpression Increases Epithelial Apoptosis, Enhances Lung Injury, and Reduces Survival after Lipopolysaccharide Treatment

“…Here, we show that over-expression of the non-canonical pathway effector p52 does not cause inflammation on its own, but in the setting of LPS treatment can enhance inflammatory cell influx into the lungs, although it is likely that the inflammatory cell infiltrate is an indirect effect of epithelial cell death and altered barrier function. While other studies have suggested that p52 cooperates in regulating inflammatory cytokines in airway epithelial cells (7), we observed no differences in inflammatory cytokine production. In contrast, RelB over-expression led to increased inflammatory cell recruitment, which was not enhanced further by p52 over-expression.…”

“…We have previously shown that NF-κB signaling in the lung epithelium regulates the inflammatory response after LPS stimulation (6), suggesting that epithelial NF-κB signaling is a critical component of ARDS pathogenesis. Although the role of the non-canonical NF-κB pathway in LPS-induced inflammation is unknown, studies with lung epithelial cells in vitro have shown that LPS stimulation induces non-canonical NF-κB activation with slower and more protracted kinetics compared to canonical NF-κB activation and that non-canonical NF-κB signaling may be important for regulation of pro-inflammatory cytokines (7). …”

NR4A Receptors Differentially Regulate NF-κB Signaling in Myeloid Cells

“…We reveal that NR4A depletion in THP-1 cells results in attenuated MIP-3α with enhanced IL-6 protein secretion, and overexpression of NR4A2 in MEF cells shows the inverse during LPS stimulation. Intriguingly, Tully et al revealed in lung epithelial cells that Relb depletion resulted in enhanced IL-6 production alongside attenuated MIP-3α production (32). Here, we show this phenomenon is paralleled in NR4A-depleted cells, and furthermore, we reveal Relb to be a novel NR4A target gene during LPS stimulation.…”

Anticancer effect of SZC017, a novel derivative of oleanolic acid, on human gastric cancer cells

“…In the classical pathway, stimulation induces IKK activation leading to phosphorylation of IκBα which subsequently is ubiquitinated and degradated. After posttranslational modifications, the NF-κB dimer then translocates into the nucleus and binds to κB sites to modulate specific gene expression (14,28,29). There are two IκBα-related mechanisms for inhibiting the NF-κB pathway.…”