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Ursolic acid, a natural pentacyclic triterpenoid, inhibits intracellular trafficking of proteins and induces accumulation of intercellular adhesion molecule-1 linked to high-mannose-type glycans in the endoplasmic reticulum.

Mitsuda S, Yokomichi T, Yokoigawa J, Kataoka T - FEBS Open Bio (2014)

Bottom Line: By contrast, ursolic acid exerted weak inhibitory effects on the IL-1α-induced ICAM-1 expression at the protein level.Surprisingly, we found that ursolic acid decreased the apparent molecular weight of ICAM-1 and altered the structures of N-linked oligosaccharides bound to ICAM-1.Thus, our results reveal that ursolic acid inhibits intracellular trafficking of proteins and induces the accumulation of ICAM-1 linked to high-mannose-type glycans in the endoplasmic reticulum.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.

ABSTRACT
Ursolic acid (3β-hydroxy-urs-12-en-28-oic acid) is a natural pentacyclic triterpenoid that is present in many plants, including medicinal herbs, and foods. Ursolic acid was initially identified as an inhibitor of the expression of intercellular adhesion molecule-1 (ICAM-1) in response to interleukin-1α (IL-1α). We report here a novel biological activity: ursolic acid inhibits intracellular trafficking of proteins. Ursolic acid markedly inhibited the IL-1α-induced cell-surface ICAM-1 expression in human cancer cell lines and human umbilical vein endothelial cells. By contrast, ursolic acid exerted weak inhibitory effects on the IL-1α-induced ICAM-1 expression at the protein level. Surprisingly, we found that ursolic acid decreased the apparent molecular weight of ICAM-1 and altered the structures of N-linked oligosaccharides bound to ICAM-1. Ursolic acid induced the accumulation of ICAM-1 in the endoplasmic reticulum, which was linked mainly to high-mannose-type glycans. Moreover, in ursolic-acid-treated cells, the Golgi apparatus was fragmented into pieces and distributed over the cells. Thus, our results reveal that ursolic acid inhibits intracellular trafficking of proteins and induces the accumulation of ICAM-1 linked to high-mannose-type glycans in the endoplasmic reticulum.

No MeSH data available.


Related in: MedlinePlus

Ursolic acid affects N-linked oligosaccharide processing of ICAM-1. (A) A549 cells were treated with various concentrations of tunicamycin for 1 h and then incubated with (+) or without (−) IL-1α (0.25 ng/ml) for 6 h. Cell lysates were analyzed by Western blotting. Data are representative of three independent experiments. (B) A549 cells were preincubated with various concentrations of tunicamycin for 1 h and then incubated with (filled circles) or without (open circles) IL-1α (0.25 ng/ml) for 6 h. Cell-surface ICAM-1 expression was measured by the Cell-ELISA assay. ICAM-1 expression (%) is represented by the means ± S.D. of triplicate cultures. ∗∗P < 0.01, compared with control. Data are representative of four independent experiments. (C) A549 cells were preincubated with various concentrations of tunicamycin for 1 h and then incubated with (filled circles) or without (open circles) IL-1α (0.25 ng/ml) for 6 h. Cell viability (%) was measured by the MTT assay. Cell viability (%) is represented by the means ± S.D. of triplicate cultures. Data are representative of three independent experiments. (D) A549 cells were preincubated with (+) or without (−) ursolic acid (50 μM) for 1 h and then incubated with (+) IL-1α (0.25 ng/ml) for 6 h. Cell lysates were treated or not treated with PNGase F or Endo H, and analyzed by Western blotting. Data are representative of three independent experiments.
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f0015: Ursolic acid affects N-linked oligosaccharide processing of ICAM-1. (A) A549 cells were treated with various concentrations of tunicamycin for 1 h and then incubated with (+) or without (−) IL-1α (0.25 ng/ml) for 6 h. Cell lysates were analyzed by Western blotting. Data are representative of three independent experiments. (B) A549 cells were preincubated with various concentrations of tunicamycin for 1 h and then incubated with (filled circles) or without (open circles) IL-1α (0.25 ng/ml) for 6 h. Cell-surface ICAM-1 expression was measured by the Cell-ELISA assay. ICAM-1 expression (%) is represented by the means ± S.D. of triplicate cultures. ∗∗P < 0.01, compared with control. Data are representative of four independent experiments. (C) A549 cells were preincubated with various concentrations of tunicamycin for 1 h and then incubated with (filled circles) or without (open circles) IL-1α (0.25 ng/ml) for 6 h. Cell viability (%) was measured by the MTT assay. Cell viability (%) is represented by the means ± S.D. of triplicate cultures. Data are representative of three independent experiments. (D) A549 cells were preincubated with (+) or without (−) ursolic acid (50 μM) for 1 h and then incubated with (+) IL-1α (0.25 ng/ml) for 6 h. Cell lysates were treated or not treated with PNGase F or Endo H, and analyzed by Western blotting. Data are representative of three independent experiments.

Mentions: ICAM-1 is a transmembrane glycoprotein consisting of 505 amino acids and possesses multiple consensus sites for N-linked glycosylation [3,4]. Tunicamycin is known to block the synthesis of N-linked oligosaccharides on glycoproteins [33]. Consistent with a previous report [34], an unglycosylated form of ICAM-1 was detected as bands migrating to around 50–55 kDa in tunicamycin-treated cells (Fig. 3A). Indeed, the size of ICAM-1 in the tunicamycin-treated cells was apparently smaller than that in the ursolic-acid-treated cells (Figs. 2A, 3A). Moreover, tunicamycin at concentrations higher than 1 μM decreased the cell-surface ICAM-1 expression in A549 cells (Fig. 3B). By contrast, tunicamycin did not affect cell viability at concentrations up to 10 μM during 6 h incubation (Fig. 3C).


Ursolic acid, a natural pentacyclic triterpenoid, inhibits intracellular trafficking of proteins and induces accumulation of intercellular adhesion molecule-1 linked to high-mannose-type glycans in the endoplasmic reticulum.

Mitsuda S, Yokomichi T, Yokoigawa J, Kataoka T - FEBS Open Bio (2014)

Ursolic acid affects N-linked oligosaccharide processing of ICAM-1. (A) A549 cells were treated with various concentrations of tunicamycin for 1 h and then incubated with (+) or without (−) IL-1α (0.25 ng/ml) for 6 h. Cell lysates were analyzed by Western blotting. Data are representative of three independent experiments. (B) A549 cells were preincubated with various concentrations of tunicamycin for 1 h and then incubated with (filled circles) or without (open circles) IL-1α (0.25 ng/ml) for 6 h. Cell-surface ICAM-1 expression was measured by the Cell-ELISA assay. ICAM-1 expression (%) is represented by the means ± S.D. of triplicate cultures. ∗∗P < 0.01, compared with control. Data are representative of four independent experiments. (C) A549 cells were preincubated with various concentrations of tunicamycin for 1 h and then incubated with (filled circles) or without (open circles) IL-1α (0.25 ng/ml) for 6 h. Cell viability (%) was measured by the MTT assay. Cell viability (%) is represented by the means ± S.D. of triplicate cultures. Data are representative of three independent experiments. (D) A549 cells were preincubated with (+) or without (−) ursolic acid (50 μM) for 1 h and then incubated with (+) IL-1α (0.25 ng/ml) for 6 h. Cell lysates were treated or not treated with PNGase F or Endo H, and analyzed by Western blotting. Data are representative of three independent experiments.
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Related In: Results  -  Collection

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f0015: Ursolic acid affects N-linked oligosaccharide processing of ICAM-1. (A) A549 cells were treated with various concentrations of tunicamycin for 1 h and then incubated with (+) or without (−) IL-1α (0.25 ng/ml) for 6 h. Cell lysates were analyzed by Western blotting. Data are representative of three independent experiments. (B) A549 cells were preincubated with various concentrations of tunicamycin for 1 h and then incubated with (filled circles) or without (open circles) IL-1α (0.25 ng/ml) for 6 h. Cell-surface ICAM-1 expression was measured by the Cell-ELISA assay. ICAM-1 expression (%) is represented by the means ± S.D. of triplicate cultures. ∗∗P < 0.01, compared with control. Data are representative of four independent experiments. (C) A549 cells were preincubated with various concentrations of tunicamycin for 1 h and then incubated with (filled circles) or without (open circles) IL-1α (0.25 ng/ml) for 6 h. Cell viability (%) was measured by the MTT assay. Cell viability (%) is represented by the means ± S.D. of triplicate cultures. Data are representative of three independent experiments. (D) A549 cells were preincubated with (+) or without (−) ursolic acid (50 μM) for 1 h and then incubated with (+) IL-1α (0.25 ng/ml) for 6 h. Cell lysates were treated or not treated with PNGase F or Endo H, and analyzed by Western blotting. Data are representative of three independent experiments.
Mentions: ICAM-1 is a transmembrane glycoprotein consisting of 505 amino acids and possesses multiple consensus sites for N-linked glycosylation [3,4]. Tunicamycin is known to block the synthesis of N-linked oligosaccharides on glycoproteins [33]. Consistent with a previous report [34], an unglycosylated form of ICAM-1 was detected as bands migrating to around 50–55 kDa in tunicamycin-treated cells (Fig. 3A). Indeed, the size of ICAM-1 in the tunicamycin-treated cells was apparently smaller than that in the ursolic-acid-treated cells (Figs. 2A, 3A). Moreover, tunicamycin at concentrations higher than 1 μM decreased the cell-surface ICAM-1 expression in A549 cells (Fig. 3B). By contrast, tunicamycin did not affect cell viability at concentrations up to 10 μM during 6 h incubation (Fig. 3C).

Bottom Line: By contrast, ursolic acid exerted weak inhibitory effects on the IL-1α-induced ICAM-1 expression at the protein level.Surprisingly, we found that ursolic acid decreased the apparent molecular weight of ICAM-1 and altered the structures of N-linked oligosaccharides bound to ICAM-1.Thus, our results reveal that ursolic acid inhibits intracellular trafficking of proteins and induces the accumulation of ICAM-1 linked to high-mannose-type glycans in the endoplasmic reticulum.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.

ABSTRACT
Ursolic acid (3β-hydroxy-urs-12-en-28-oic acid) is a natural pentacyclic triterpenoid that is present in many plants, including medicinal herbs, and foods. Ursolic acid was initially identified as an inhibitor of the expression of intercellular adhesion molecule-1 (ICAM-1) in response to interleukin-1α (IL-1α). We report here a novel biological activity: ursolic acid inhibits intracellular trafficking of proteins. Ursolic acid markedly inhibited the IL-1α-induced cell-surface ICAM-1 expression in human cancer cell lines and human umbilical vein endothelial cells. By contrast, ursolic acid exerted weak inhibitory effects on the IL-1α-induced ICAM-1 expression at the protein level. Surprisingly, we found that ursolic acid decreased the apparent molecular weight of ICAM-1 and altered the structures of N-linked oligosaccharides bound to ICAM-1. Ursolic acid induced the accumulation of ICAM-1 in the endoplasmic reticulum, which was linked mainly to high-mannose-type glycans. Moreover, in ursolic-acid-treated cells, the Golgi apparatus was fragmented into pieces and distributed over the cells. Thus, our results reveal that ursolic acid inhibits intracellular trafficking of proteins and induces the accumulation of ICAM-1 linked to high-mannose-type glycans in the endoplasmic reticulum.

No MeSH data available.


Related in: MedlinePlus