Immune Netw.  2015 Dec;15(6):291-303. 10.4110/in.2015.15.6.291.

The Anti-inflammatory Effect of GV1001 Mediated by the Downregulation of ENO1-induced Pro-inflammatory Cytokine Production

• Choi J
• Kim H
• Kim Y
• Jang M
• Jeon J
• Hwang YI
• Shon WJ
• Song YW
• Kang JS
• Lee WJ

• KMID: 2132703
• DOI: http://doi.org/10.4110/in.2015.15.6.291

Abstract

GV1001 is a peptide derived from the human telomerase reverse transcriptase (hTERT) sequence that is reported to have anti-cancer and anti-inflammatory effects. Enolase1 (ENO1) is a glycolytic enzyme, and stimulation of this enzyme induces high levels of pro-inflammatory cytokines from concanavalin A (Con A)-activated peripheral blood mononuclear cells (PBMCs) and ENO1-expressing monocytes in healthy subjects, as well as from macrophages in rheumatoid arthritis (RA) patients. Therefore, this study investigated whether GV1001 downregulates ENO1-induced pro-inflammatory cytokines as an anti-inflammatory peptide. The results showed that GV1001 does not affect the expression of ENO1 in either Con A-activated PBMCs or RA PBMCs. However, ENO1 stimulation increased the production of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6, and these cytokines were downregulated by pretreatment with GV1001. Moreover, p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-kappaB were activated when ENO1, on the surface of Con A-activated PBMCs and RA PBMCs, was stimulated, and they were successfully suppressed by pre-treatment with GV1001. These results suggest that GV1001 may be an effective anti-inflammatory peptide that downregulates the production of pro-inflammatory cytokines through the suppression of p38 MAPK and NF-kappaB activation following ENO1 stimulation.

Keyword

GV1001; ENO1; Inflammation

MeSH Terms

Arthritis, Rheumatoid
Concanavalin A
Cytokines
Down-Regulation*
Humans
Inflammation
Interleukin-6
Interleukins
Macrophages
Monocytes
NF-kappa B
p38 Mitogen-Activated Protein Kinases
Protein Kinases
Telomerase
Tumor Necrosis Factor-alpha
Concanavalin A
Cytokines
Interleukin-6
Interleukins
NF-kappa B
Protein Kinases
Telomerase
Tumor Necrosis Factor-alpha
p38 Mitogen-Activated Protein Kinases

Figure

• 

  Figure 1 The effect of GV1001 on the increase in ENO1 expression following Con A stimulation. Con A-stimulated PBMCs were stained with (A) FITC-conjugated anti-ENO1 mAb. FITC-conjugated MOPC21 were used as the isotype controls. (B) PBMCs (1×106/ml) were pretreated with GV1001 (100 µM) or vehicle for 1 h and then cultured in the presence or absence of Con A (2 µg/ml) for 24 h. Then, the cells of each group were harvested, washed with PBS, and incubated for another 48 h. (C) PBMCs (1×106/ml) were stimulated with Con A (2 µg/ml) or vehicle for 24 h. Next, the cells were washed with PBS, treated with GV1001 (100 µM) for 1 h, and then incubated for another 48 h. ENO1 expression in these groups was analyzed by flow cytometry.

• 

  Figure 2 The inhibitory effect of GV1001 on ENO1-induced pro-inflammatory cytokine production in Con A-activated PBMCs. Activated PBMCs were pre-treated with GV1001 (100 µ M) for 1 h and then stimulated with anti-ENO1 mAb (1 µg) for 1 h. MOPC21 was used as an isotype control. Stimulated PBMCs were cultured in a 24-well plate (1×106/ml) for another 48 h. The culture supernatant of the stimulated PBMCs was collected, and (A) TNF-α, (B) IL-1β, and (C) IL-6 levels were measured by ELISA. Data are presented as the means±SD. *p<0.05, **p<0.01, ***p<0.001.

• 

  Figure 3 The regulation of p38 MAPK and NF-κ B activation by treatment with GV1001. Con A-activated PBMCs were pre-treated with GV1001 (100 µM), DMSO (vehicle), SB203580 (40 µM), and Bay11-7082 (2.5 µM) for 1 h and then stimulated with anti-ENO1 mAb for 1 h. After incubating for another 48 h, the supernatant was harvested, and pro-inflammatory cytokine levels (A) TNF-α, (B) IL-1β, and (C) IL-6 were determined by ELISA. Data are presented as the means±SD. ***p<0.001.

• 

  Figure 4 The suppression of p38 MAPK and NF-κB activation by treatment with GV1001. Con A-activated PBMCs were pre-treated with GV1001 (100 µM) for 1 h, and then stimulated with anti-ENO1 mAb for 120 min for p38 MAPK and 5 min for NF-κB. Then, the cells were collected and lysed with lysis buffer to extract proteins for immunoblotting. Levels of (A) the phosphorylated form of p38 (p-p38) and total p38 and (C) the phosphorylated form of p65 (p-p65) and total p65 were examined by immunoblotting. A densitometry analysis was performed and results are represented as the fold increase in the phosphorylated form over the total form of (B) p-p38/p38 and (D) p-p65/p65. Results are representative of three independent experiments. **p< 0.01, ***p<0.001.

• 

  Figure 5 The effect of GV1001 on ENO1 expression in RA PBMCs. (A) PBMCs (1×105) from RA patients (n=10) were stained with anti-ENO1 mAb or the isotype control, and ENO1 expression was examined by flow cytometry. RA PBMCs were incubated in the (B) absence or (C) presence of GV1001 (100 µM) for 48 h. After washing, the cells were double-stained with FITC conjugated anti-ENO1 mAb and PE-conjugated anti-CD14 Ab.

• 

  Figure 6 The effect of GV1001 on ENO1-induced pro-inflammatory cytokine production in RA PBMCs. RA PBMCs were pre-treated with GV1001 (100 µM) for 1 h and then incubated with anti-ENO1 mAb (1 µg) or MOPC21 isotype for 1 h. After the stimulated cells were seeded in a 24-well plate, they were further incubated for 12 h. The culture supernatant was collected, and the levels of (A) TNF-α, (B) IL-1β, and (C) IL-6 were measured by ELISA. Data are presented as the means±SD. **p<0.01, ***p<0.001.

• 

  Figure 7 The suppression of p38 MAPK and NF-κB activation in RA PBMCs following treatment with GV1001. RA PBMCs were stimulated with anti-ENO1 mAb for 120 min after pre-treatment with GV1001 (100 µM) for 1 h. (A) Phosphorylation of p38 and p65 was detected by immunoblotting. The relative expression is presented as the fold increase of (B) p-p38/p38 or (C) p-p65/p65 by densitometry analysis. Results are representative of three independent experiments. ***p<0.001.

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