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Lipopolysaccharide-induced CCN1 production enhances interleukin-6 secretion in bronchial epithelial cells.

ABSTRACT: Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is a clinical complication caused by primary or secondary lung injury, as well as by systemic inflammation. Researches regarding molecular pathophysiology of ALI/ARDS are immerging with an ultimate aim towards developing prognostic molecular biomarkers and molecule-based therapy. However, the molecular mechanisms concerning ALI/ARDS are still not completely understood. The purpose of the present study was to identify a crucial role of CCN1 in inflammatory microenvironment during ALI/ARDS and focus on a potential communication between CCN1 and interleukin-6 (IL-6) in the airway epithelial cells. Our data illustrated that the expression levels of CCN1 and IL-6 in bronchoalveolar lavage fluid (BALF) in a lipopolysaccharide (LPS)-induced ALI mouse model were significantly elevated and the pulmonary expression of CCN1 was restricted to bronchial epithelial cells. Interestingly, both endogenous and exogenous CCN1 stimulated IL-6 production in vitro. Furthermore, LPS-induced IL-6 production in a bronchial epithelial cell line was blocked by CCN siRNA whereas CCN1 induced by LPS was sensitive to PI3K inhibition. Together, our data indicate a linear signal pathway, LPS-CCN1-IL-6, existing in bronchial epithelial cells after LPS exposure. This finding may represent an additional mechanism and a novel target for development of therapy and biomarker on ALI/ARDS.

SUBMITTER: Shi L

PROVIDER: S-EPMC5775366 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Lipopolysaccharide-induced CCN1 production enhances interleukin-6 secretion in bronchial epithelial cells.

Shi Lin L Dong Nian N Ji Dongxiang D Huang Xiaomin X Ying Zhaojian Z Wang Xiangdong X Chen Chengshui C

Cell biology and toxicology 20170621 1

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is a clinical complication caused by primary or secondary lung injury, as well as by systemic inflammation. Researches regarding molecular pathophysiology of ALI/ARDS are immerging with an ultimate aim towards developing prognostic molecular biomarkers and molecule-based therapy. However, the molecular mechanisms concerning ALI/ARDS are still not completely understood. The purpose of the present study was to identify a crucial role ...[more]

PMID: 28638955

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Project description:Toll-like receptor (TLR) 4 was originally thought to be the sole pattern recognition receptor for lipopolysaccharide (LPS). Transient receptor potential ankyrin 1 (TRPA1), a Ca2+-permeant channel, has been suggested as a non-TLR receptor membrane-bound sensor of LPS. We recently reported that TRPA1 is expressed in lung epithelial cells (LECs) and mediates lung inflammation induced by cigarette smoke. However, the role of TRPA1 in LPS-induced lung inflammation has not been conclusively defined, and its underlying cellular mechanisms remain unclear. In this study, our in vitro results showed that LPS sequentially produced a cascade of events, including the elevation of intracellular Ca2+, the activation of NADPH oxidase, increase in intracellular reactive oxygen species (ROS), the activation of mitogen-activated protein kinase (MAPK)/nuclear factor-kB (NF-κB) signaling, and the induction of IL-8. The increase in intracellular Ca2+ was inhibited by HC030031 (a TRPA1 antagonist) but was unaffected by TAK-242 (a TLR-4 inhibitor). The activation of NADPH oxidase was prevented by its inhibitor apocynin, EGTA (an extracellular Ca2+ chelator), and HC030031. The increase in intracellular ROS was attenuated by apocynin, N-acetyl-cysteine (NAC, a ROS scavenger), EGTA, and HC030031. The activation of the MAPK/NF-κB signaling was halted by NAC, EGTA, and HC030031. IL-8 induction was suppressed by HC030031 and TRPA1 siRNA, and further reduced by the combination of HC030031 and TAK-242. Our in vivo studies showed that trpa1-/- mice exhibited a reduced level of LPS-induced lung inflammation compared with wild-type mice as evidenced by the alleviations of increases in vascular permeability, inflammatory cell infiltration, inflammatory cytokine levels, oxidative stress, and MAPK signaling activation. Thus, in LECs, LPS may activate TRPA1 resulting in an increase in Ca2+ influx. The increased intracellular Ca2+ leads to NADPH oxidase activation, which causes an increase in intracellular ROS. The intracellular ROS activates the MAPK/NF-κB signaling resulting in IL-8 induction. This mechanism may possibly be at work to induce lung inflammation in mice.

Project description:HBEpCs (human bronchial epithelial cells) contribute to airway inflammation by secreting a variety of cytokines and chemokines in response to allergens, pathogens, viruses and environmental toxins and pollutants. The potent neutrophil chemoattractant, IL-8 (interleukin-8), is a major cytokine secreted by HBEpCs. We have recently demonstrated that LPA (lysophosphatidic acid) stimulated IL-8 production in HBEpCs via protein kinase C delta dependent signal transduction. However, mechanisms of IL-8 expression and secretion are complex and involve multiple protein kinases and transcriptional factors. The present study was undertaken to investigate MAPK (mitogen-activated protein kinase) signalling in the transcriptional regulation of IL-8 expression and secretion in HBEpCs. Exposure of HBEpCs to LPA (1 microM) enhanced expression and secretion of IL-8 by 5-8-fold and stimulated threonine/tyrosine phosphorylation of ERK (extracellular-signal-regulated kinase), p38 MAPK and JNK (c-Jun N-terminal kinase). The LPA-induced secretion of IL-8 was blocked by the p38 MAPK inhibitor SB203580, by p38 MAPK siRNA (small interfering RNA), and by the JNK inhibitor JNK(i) II, but not by the MEK (MAPK/ERK kinase) inhibitor, PD98059. LPA enhanced the transcriptional activity of the IL-8 gene; that effect relied on activation of the transcriptional factors NF-kappaB (nuclear factor kappaB) and AP-1 (activator protein-1). Furthermore, SB203580 attenuated LPA-dependent phosphorylation of IkappaB (inhibitory kappaB), NF-kappaB and phospho-p38 translocation to the nucleus, NF-kappaB transcription and IL-8 promoter-mediated luciferase reporter activity, without affecting the JNK pathway and AP-1 transcription. Similarly, JNK(i) II only blocked LPA-mediated phosphorylation of JNK and c-Jun, AP-1 transcription and IL-8 promoter-mediated luciferase reporter activity, without blocking p38 MAPK-dependent NF-kappaB transcription. Additionally, siRNA for LPA(1-3) receptors partially blocked LPA-induced IL-8 production and activation of MAPKs. The LPA1 and LPA3 receptors, as compared with LPA2, were most efficient in transducing LPA-mediated IL-8 production. These results show an independent role for p38 MAPK and JNK in LPA-induced IL-8 expression and secretion via NF-kappaB and AP-1 transcription respectively in HBEpCs.

Project description:Acute lung injury (ALI) is mainly caused by inflammation and is associated with high mortality rates. Emerging evidence has suggested that microRNAs (miRNAs or miRs) serve a significant function in ALI. However, the fundamental mechanism underlying ALI remain to be fully elucidated. Although miR-16 has been reported to be involved in the occurrence and development of a number of diseases its association with ALI has not been previously investigated. Therefore, the present study aimed to explore the role of miR-16 in the lipopolysaccharide (LPS)-induced ALI model. The expression levels of tumor necrosis factor α (TNF-α), interleukin (IL)-1β and IL-6 were measured by ELISA in the blood samples of rats with ALI and in the normal human bronchial epithelial (NHBE) cell line. The role of miR-16 in inflammation was evaluated using gene overexpression and silencing experiments in NHBE cells by reverse transcription-quantitative PCR. In addition, the expression levels of inflammatory factors TNF-α, IL-1β and IL-6 were also determined using ELISA. The potential interaction between miR-16 and TLR4 was assessed using bioinformatics analysis by the TargetScan database and then verified in 293T cells using luciferase reporter assay. The expression of miR-16 was notably decreased in the lung tissues of rats with LPS-induced ALI compared with the PBS treated-group. Additionally, the levels of the proinflammatory cytokines TNF-α, IL-1β and IL-6 were reduced following transfection of NHBE cells with miR-16 mimics compared with those in the miR-negative control group. Western blot analysis revealed that miR-16 overexpression could downregulate TLR4 expression in NHBE cells compared with that in the miR-NC group. Luciferase reporter assay confirmed that TLR4 may be directly targeted by miR-16. The effect of miR-16 on TLR4 was rescued in NHBE cells following treatment with LPS. Overall, these aforementioned findings suggest that miR-16 may serve a protective role against LPS-mediated inflammatory responses in NHBE cells by regulating TLR4, where this mechanism may be considered to be a novel approach for treating ALI in the future.

Dataset Information

Lipopolysaccharide-induced CCN1 production enhances interleukin-6 secretion in bronchial epithelial cells.

Publications

Lipopolysaccharide-induced CCN1 production enhances interleukin-6 secretion in bronchial epithelial cells.

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