Korean J Physiol Pharmacol.  2015 May;19(3):263-268. 10.4196/kjpp.2015.19.3.263.

LPS Increases 5-LO Expression on Monocytes via an Activation of Akt-Sp1/NF-kappaB Pathways

• Lee SJ
• Seo KW
• Kim CD

• KMID: 1791428
• DOI: http://doi.org/10.4196/kjpp.2015.19.3.263

Abstract

5-Lipoxygenase (5-LO) plays a pivotal role in the progression of atherosclerosis. Therefore, this study investigated the molecular mechanisms involved in 5-LO expression on monocytes induced by LPS. Stimulation of THP-1 monocytes with LPS (0~3 microg/ml) increased 5-LO promoter activity and 5-LO protein expression in a concentration-dependent manner. LPS-induced 5-LO expression was blocked by pharmacological inhibition of the Akt pathway, but not by inhibitors of MAPK pathways including the ERK, JNK, and p38 MAPK pathways. In line with these results, LPS increased the phosphorylation of Akt, suggesting a role for the Akt pathway in LPS-induced 5-LO expression. In a promoter activity assay conducted to identify transcription factors, both Sp1 and NF-kappaB were found to play central roles in 5-LO expression in LPS-treated monocytes. The LPS-enhanced activities of Sp1 and NF-kappaB were attenuated by an Akt inhibitor. Moreover, the LPS-enhanced phosphorylation of Akt was significantly attenuated in cells pretreated with an anti-TLR4 antibody. Taken together, 5-LO expression in LPS-stimulated monocytes is regulated at the transcriptional level via TLR4/Akt-mediated activations of Sp1 and NF-kappaB pathways in monocytes.

Keyword

Akt; Atherosclerosis; LPS; Monocytes; 5-Lipoxygenase

MeSH Terms

Arachidonate 5-Lipoxygenase
Atherosclerosis
Monocytes*
NF-kappa B
p38 Mitogen-Activated Protein Kinases
Phosphorylation
Transcription Factors
Arachidonate 5-Lipoxygenase
NF-kappa B
Transcription Factors
p38 Mitogen-Activated Protein Kinases

Figure

• 

  Fig. 1 Effects of LPS on 5-LO expression in monocytes. Monocytes were transiently cotransfected with the empty luciferase vector pRL CMV or 5-LO promoter constructs for 36 h, and then stimulated with the indicated concentrations of LPS for 4 h (A) or 1 µg/ml of LPS for the indicated time (B). The promoter activity of 5-LO was analyzed using a luciferase reporter assay. (C) Monocytes were stimulated with the indicated concentrations of LPS for 4 h, and the protein expression of 5-LO were analyzed by immunoblotting. Relative band intensity of 5-LO to β-actin was quantified, and the results were presented as the mean±SEM of 4-5 independent experiments performed in triplicate. *p< 0.05, **p<0.01 vs. value at concentration 0 or time 0.

• 

  Fig. 2 Effects of various signal pathway inhibitors on LPS-induced 5-LO expression. (A) Monocytes were pre-treated with MAPK inhibitors including PD98059 (PD, 30 mM), SP600125 (SP, 30 mM), SB203580 (SB, 30 mM), or AI (3 mM) for 30 min, and then stimulated with 1 µg/ml of LPS. 5-LO expressions were analyzed by immunoblotting, and data were presented as means±SEM from 4~6 independent experiments. **p<0.01 vs. value of control (Con), ##p<0.01 vs. value of vehicle (Veh). (B) Monocytes were stimulated with 1 µg/ml of LPS for the indicated times. The cell lysates were analyzed for phosphorylated (p-Akt) and total Akt (t-Akt) by Western blotting. Relative intensity of p-Akt to t-Akt was quantified, and data were presented as means±SEM from 6~7 independent experiments. *p<0.05, **p<0.01 vs. value at time 0. (C and D) Monocytes were transiently transfected with the Sp1 and NF-κB luciferase reporter constructs for 36 h, and then stimulated with LPS for the indicated times. Sp1 and NF-κB activities were analyzed using luciferase reporter assays. Data were presented as means±SEM of 4-5 independent experiments performed in triplicate. **p<0.01 vs. value at time 0.

• 

  Fig. 3 Role of Akt on LPS-induced 5-LO expression mediated by Sp1 and NF-κB signaling pathways. (A) Monocytes were transiently cotransfected with the empty luciferase vector pRL CMV or 5-LO promoter constructs for 36 h, and then stimulated with LPS (1 µg/ml) in the presence of AI (3 µm). 5-LO promoter activities were determined using a luciferase reporter assay. The data is presented as the mean±SEM from 5~6 independent experiments. (B) Monocytes were transiently transfected with the Sp1 and NF-κB luciferase reporter constructs for 36 h. After pretreatment with AI (3 µm) for 30 min, cells were treated with LPS (1 µg/ml). Sp1 and NF-κB activities were analyzed using luciferase reporter assays, and data were presented as the mean±SEM from 5~6 independent experiments. **p<0.01 vs. control (Con), ##p<0.01 vs. vehicle (Veh). (C) Monocytes were stimulated with LPS (1 µg/ml) for the indicated time in the presence or absence of AI (3 µm). The cell lysates were analysed for the phosphorylated levels of IKK (pIKK). Relative intensity to β-actin was presented as the mean±SEM from 6~7 independent experiments. **p<0.01 vs. value at time 0.

• 

  Fig. 4 Involvement of TLR4 pathway in Akt phosphorylation induced by LPS. Monocytes were pre-treated with a TLR4 functional blocking antibody (anti-TLR4) for 30 min, and then stimulated with LPS (1 µg/ml). Cell lysates were analyzed for the total (t-Akt) and phosphorylated levels of Akt (p-Akt) using Western blotting. Relative band intensity of p-Akt to t-Akt was quantified, and data were presented as the mean±SEM from 5~6 independent experiments. **p<0.01 vs. control (Con), #p<0.05, ##p<0.01 vs. vehicle (Veh).

• 

  Fig. 5 Schematic diagram showing the signal pathways involved in LPS-induced 5-LO expression in Monocytes.

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