Early studies have demonstrated the ability of dietary carotenoids to enhance immune response, but the mechanism underlying their influence on macrophage activity remains unclear. Here, we investigated the effects of carotenoids on macrophage activity. Carotenoids, including lutein and lycopene, enhanced MMP-9 activity in RAW264.7 macrophages. Lutein was chosen as a representative and analyzed further in this study. It increased the synthesis, activity, and release of MMP-9 in murine RAW264.7 and primary-cultured peritoneal macrophages. MMP-9 induction by lutein was through the transcriptional regulation of mmp-9. It was blunted by the MAPK inhibitors targeting ERK1/2 and p38 MAPK, the reagents that inhibit free radical signaling, and the inhibitors and siRNA targeting RARβ. Moreover, lutein induced Nox activation and intracellular ROS production at an early stage of treatment. This carotenoid also caused ERK1/2 and p38 MAPK activation, RARβ expression, and RAR interaction with its responsive element in the promoter region. These findings suggest the involvement of ROS, MAPKs, and RARβ activation in lutein-driven MMP-9 expression and release. Interestingly, lutein enhanced the phagocytic activity of macrophages, and the secreted MMP-9 appeared to be involved in this process. In summary, we provide evidence here for the first time that the carotenoid lutein induces intracellular ROS generation and MAPK and RARβ activation in macrophages, leading to an increase in MMP-9 release and macrophage phagocytosis. Our results demonstrate that lutein exerts an immunomodulatory effect on macrophages.
Background/Aims: The chemokine CXCL1 has been reported to be expressed in lung airway epithelium and non-small cell lung cancer biopsy specimens. In this study, we investigated the effects of TNF-α, an abundant cytokine detected in inflammation and various cancers, on CXCL1 release by human A549 lung carcinoma epithelial cells. Methods: CXCL1 expression was determined by ELISA and RT-PCR. TNF-α signaling was examined by western blotting. Monocyte migration was assayed by a Transwell migration system. Results: TNF-α stimulated CXCL1 release and mRNA expression, and this release was inhibited by inhibitors of JNK, p38 MAPK, PI-3K/Akt and AP-1 transcription factor. TNF-α treatment was followed by JNK, p38 MAPK and PI3K/Akt activation. However, only the JNK inhibitor could reduce the CXCL1 mRNA level, suggesting that JNK is required mainly for CXCL1 mRNA synthesis, whereas p38 MAPK and PI-3K/Akt might be responsible for CXCL1 secretion. Dexamethasone (dex) and TGF-β reduced CXCL1 secretion, with dex upregulating the expression of MAP kinase phosphatase-1 and TGF-β causing smad2/3 activation and nuclear translocation. A functional analysis showed that the released CXCL1 enhanced monocyte migration and could be abolished by a CXCL1 neutralizing antibody and CXCR antagonist. Conclusion: We demonstrate that TNF-α induces CXCL1 expression through the JNK, p38 MAPK and PI-3K/Akt signaling pathways in human pulmonary epithelial cells.
Lung cancer cells express different chemokines and chemokine receptors that modulate leukocyte infiltration within tumor microenvironment. In this study we screened several mediators/growth factors on CXCL1 release in human carcinoma epithelial cells. Of the tested mediators, VEGF was found to have a robust increase in causing CXCL1 release. VEGF stimulated CXCL1 release and mRNA expression in a time- and concentration-dependent manner. The release was inhibited by the VEGF receptor antagonists and the JNK, PI-3K, tyrosine kinase, and transcription inhibitors. In parallel, VEGF induced JNK, PI3K and Akt activation. Strikingly, among these inhibitors only the JNK inhibitor could reduce VEGF-induced CXCL1 mRNA expression, suggesting that JNK participated in VEGF-induced CXCL1 synthesis, whereas PI-3K was responsible for cellular CXCL1 secretory process. In addition, the steroid dexamethasone and TGF-β suppressed CXCL1 release through a transcriptional regulation. We also showed that cells stimulated with VEGF significantly attracted monocyte migration, which could be abolished by CXCL1 B/N Ab, CXC receptor 2 antagonist, TGF-β, and dexamethasone. In summary, we provide here evidence showing JNK activation for VEGF-induced CXCL1 DNA transcription and PI-3K pathway for extracellular CXCL1 release in human carcinoma epithelial cells. The released CXCL1 was functionally linked to recruiting monocytes into lung cancer cell microenvironment.
BackgroundPlatelet-derived growth factor (PDGF) is a potent stimulator of growth and motility of vascular smooth muscle cells (VSMCs). Abnormalities of PDGF/PDGF receptor (PDGFR) are thought to contribute to vascular diseases and malignancy. We previously showed that a carotenoid, lycopene, can directly bind to PDGF and affect its related functions in VSMCs. In this study we examined the effect of the other naturally occurring carotenoid, lutein, on PDGF signaling and migration in VSMCs.MethodsWestern blotting was performed to examine PDGF and H2O2 signaling. Flowcytometry was used to determine PDGF binding to VSMCs. Fluorescence microscopy was performed to examine intracellular ROS production. Modified Boyden chamber system (Transwell apparatus) was used for migration assay.ResultsLutein reduced PDGF signaling, including phosphorylation of PDGFR-β and its downstream protein kinases/enzymes such as phospholipase C-γ, Akt, and mitogen-activated protein kinases (MAPKs). Although lutein possesses a similar structure to lycopene, it was striking that lutein inhibited PDGF signaling through a different way from lycopene in VSMCs. Unlike lycopene, lutein not only interacted with (bound to) PDGF but also interfered with cellular components. This was evidenced that preincubation of PDGF with lutein and treatment of VSMCs with lutein followed by removing of lutein compromised PDGF-induced signaling. Lutein reduced PDGF-induced intracellular reactive oxygen species (ROS) production and attenuated ROS- (H2O2-) induced ERK1/2 and p38 MAPK activation. A further analysis indicated lutein could inhibit a higher concentration of H2O2-induced PDGFR signaling, which is known to act through an oxidative inhibition of protein tyrosine phosphatase. Finally, we showed that lutein functionally inhibited PDGF-induced VSMC migration, whereas its stereo-isomer zeaxanthin did not, revealing a special action of lutein on VSMCs.ConclusionsOur study reveals a differential action mechanism of lutein from other reported caroteinoids and suggests a possible beneficial effect of lutein but not zeaxanthin on prevention of vascular diseases.
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