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( − )-Epigallocatechin Gallate Targets Notch to Attenuate the Inflammatory Response in the Immediate Early Stage in Human Macrophages

View Article: PubMed Central - PubMed

ABSTRACT

Inflammation plays important roles at different stages of diabetes mellitus, tumorigenesis, and cardiovascular diseases. (−)-Epigallocatechin gallate (EGCG) can attenuate inflammatory responses effectively. However, the immediate early mechanism of EGCG in inflammation remains unclear. Here, we showed that EGCG attenuated the inflammatory response in the immediate early stage of EGCG treatment by shutting off Notch signaling and that the effect did not involve the 67-kDa laminin receptor, the common receptor for EGCG. EGCG eliminated mature Notch from the cell membrane and the nuclear Notch intercellular domain, the active form of Notch, within 2 min by rapid degradation via the proteasome pathway. Transcription of the Notch target gene was downregulated simultaneously. Knockdown of Notch 1/2 expression by RNA interference impaired the downregulation of the inflammatory response elicited by EGCG. Further study showed that EGCG inhibited lipopolysaccharide-induced inflammation and turned off Notch signaling in human primary macrophages. Taken together, our results show that EGCG targets Notch to regulate the inflammatory response in the immediate early stage.

No MeSH data available.


(−)-Epigallocatechin gallate (EGCG) shuts off the Notch signal. (A) THP-1-derived macrophages were treated with lipopolysaccharide (LPS) (200 EU/mL) for 30, 60, and 90 min by pretreated EGCG (50 μg/mL, 30 min). (B) THP-1-derived macrophages were treated with EGCG (50, 25, 12.5, 6.3, 3.1, and 1.6 μg/mL) for 30 min. (C) THP-1-derived macrophages were treated with EGCG (50 μg/mL) from 2 to 120 min. (D) THP-1-derived macrophages were treated with EGCG (50 μg/mL) from 2 to 120 min. Notch intercellular domain (NICD) of Notch1 and Notch2 located in the nucleus was detected, with TATA-binding protein (TBP) used as the internal control. (E) THP-1-derived macrophages were treated with EGCG (50 μg/mL) for 30 min, and lysates were analyzed by a western blotting probe with an antibody recognizing the epitope specific for active Notch1. (F) THP-1-derived macrophages were pretreated with EGCG (50 μg/mL) for 30 min and exposed to LPS (200 EU/mL) for 1 h. mRNA expression was measured by real-time polymerase chain reaction. (G) Notch report assays: 293/16CSL alone (left) and 293/16CSL with HepG2 coculture (right). (H) THP-1-derived macrophages were treated with EGCG (50 μg/mL) for 30 min with or without MG132 (10, 20, and 40 μM). (I) Immunoprecipitation was used to pull-down Notch from macrophage cell lysates. The Notch eluate was digested with PNGase F (left) and ATX-3 (right) and probed with the appropriate antibody. (J) Immunoprecipitation undertaken with antibody against the C-terminal of Notch1. The western blotting membrane was probed with an antibody against Notch1 or ubiquitin separately. Data are represented as mean ± SD (n = 3). Differences between the two groups were assessed by two-way ANOVA using GraphPad. Representatives of three independent experiments with similar results are shown. Results are represented as mean ± SD (*p < 0.05, **p < 0.01, and ***p < 0.001). n.s., not significant.
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Figure 3: (−)-Epigallocatechin gallate (EGCG) shuts off the Notch signal. (A) THP-1-derived macrophages were treated with lipopolysaccharide (LPS) (200 EU/mL) for 30, 60, and 90 min by pretreated EGCG (50 μg/mL, 30 min). (B) THP-1-derived macrophages were treated with EGCG (50, 25, 12.5, 6.3, 3.1, and 1.6 μg/mL) for 30 min. (C) THP-1-derived macrophages were treated with EGCG (50 μg/mL) from 2 to 120 min. (D) THP-1-derived macrophages were treated with EGCG (50 μg/mL) from 2 to 120 min. Notch intercellular domain (NICD) of Notch1 and Notch2 located in the nucleus was detected, with TATA-binding protein (TBP) used as the internal control. (E) THP-1-derived macrophages were treated with EGCG (50 μg/mL) for 30 min, and lysates were analyzed by a western blotting probe with an antibody recognizing the epitope specific for active Notch1. (F) THP-1-derived macrophages were pretreated with EGCG (50 μg/mL) for 30 min and exposed to LPS (200 EU/mL) for 1 h. mRNA expression was measured by real-time polymerase chain reaction. (G) Notch report assays: 293/16CSL alone (left) and 293/16CSL with HepG2 coculture (right). (H) THP-1-derived macrophages were treated with EGCG (50 μg/mL) for 30 min with or without MG132 (10, 20, and 40 μM). (I) Immunoprecipitation was used to pull-down Notch from macrophage cell lysates. The Notch eluate was digested with PNGase F (left) and ATX-3 (right) and probed with the appropriate antibody. (J) Immunoprecipitation undertaken with antibody against the C-terminal of Notch1. The western blotting membrane was probed with an antibody against Notch1 or ubiquitin separately. Data are represented as mean ± SD (n = 3). Differences between the two groups were assessed by two-way ANOVA using GraphPad. Representatives of three independent experiments with similar results are shown. Results are represented as mean ± SD (*p < 0.05, **p < 0.01, and ***p < 0.001). n.s., not significant.

Mentions: The Notch signaling pathway has a pivotal role in the development of multicellular organisms. Notch (specifically Notch1) is crucial for lymphocyte development (25). Emerging evidence has shown that the Notch pathway anticipates the immune response, including inflammation (26). Basal activation of Notch1 has been shown to be necessary for enhancement of the inflammatory response (27). To ascertain whether EGCG attenuates the inflammatory response via the Notch pathway immediately after its addition, we examined the Notch1 and Notch2 receptors, which are expressed on the surface of human macrophages at multiple time points shortly after EGCG treatment. We found that the number of mature Notch1 and Notch2 receptors was decreased from the cell surface at all time points after EGCG treatment and that this process was not dependent on LPS stimulation (Figure 3A). Further studies showed that mature Notch removed the effects of EGCG in a concentration-dependent manner (Figure 3B) and that this decreased response was rapid (2 min) (Figure 3C). Simultaneously, EGCG treatment accelerated the degradation of the nuclear Notch intercellular domain (NICD), the active form of Notch, in 2 min (Figure 3D). In accordance with this result, a western blotting probe with a specific antibody against the new epitope formed after Notch1 cleavage showed that degradation of the active form of Notch1 was promoted by EGCG (Figure 3E). These results suggested that the basal signaling of Notch was turned off immediately by EGCG treatment. In accordance with these results, expression of Hes1 (a typical Notch-regulated gene) was downregulated by EGCG (Figure 3A). To confirm this result, expression of Notch target genes was evaluated using real-time PCR. We found that expression of some typical Notch target genes was downregulated by EGCG (Figure 3F), but not all of them. This finding may have been due to the complexity of gene transcription, which can be influenced by many factors. To evaluate the effects of EGCG treatment on gene expression of Notch alone, a dual luciferase reporter assay was employed. This assay involves use of the luciferase gene, which is controlled by 16×CSL, which contains 16 major Notch-regulated elements (CSL) connected in a series. Results showed that only treatment with EGCG could decrease Notch-related luciferase signaling in 293T cells and HepG2/293T cocultures (Figure 3G).


( − )-Epigallocatechin Gallate Targets Notch to Attenuate the Inflammatory Response in the Immediate Early Stage in Human Macrophages
(−)-Epigallocatechin gallate (EGCG) shuts off the Notch signal. (A) THP-1-derived macrophages were treated with lipopolysaccharide (LPS) (200 EU/mL) for 30, 60, and 90 min by pretreated EGCG (50 μg/mL, 30 min). (B) THP-1-derived macrophages were treated with EGCG (50, 25, 12.5, 6.3, 3.1, and 1.6 μg/mL) for 30 min. (C) THP-1-derived macrophages were treated with EGCG (50 μg/mL) from 2 to 120 min. (D) THP-1-derived macrophages were treated with EGCG (50 μg/mL) from 2 to 120 min. Notch intercellular domain (NICD) of Notch1 and Notch2 located in the nucleus was detected, with TATA-binding protein (TBP) used as the internal control. (E) THP-1-derived macrophages were treated with EGCG (50 μg/mL) for 30 min, and lysates were analyzed by a western blotting probe with an antibody recognizing the epitope specific for active Notch1. (F) THP-1-derived macrophages were pretreated with EGCG (50 μg/mL) for 30 min and exposed to LPS (200 EU/mL) for 1 h. mRNA expression was measured by real-time polymerase chain reaction. (G) Notch report assays: 293/16CSL alone (left) and 293/16CSL with HepG2 coculture (right). (H) THP-1-derived macrophages were treated with EGCG (50 μg/mL) for 30 min with or without MG132 (10, 20, and 40 μM). (I) Immunoprecipitation was used to pull-down Notch from macrophage cell lysates. The Notch eluate was digested with PNGase F (left) and ATX-3 (right) and probed with the appropriate antibody. (J) Immunoprecipitation undertaken with antibody against the C-terminal of Notch1. The western blotting membrane was probed with an antibody against Notch1 or ubiquitin separately. Data are represented as mean ± SD (n = 3). Differences between the two groups were assessed by two-way ANOVA using GraphPad. Representatives of three independent experiments with similar results are shown. Results are represented as mean ± SD (*p < 0.05, **p < 0.01, and ***p < 0.001). n.s., not significant.
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Figure 3: (−)-Epigallocatechin gallate (EGCG) shuts off the Notch signal. (A) THP-1-derived macrophages were treated with lipopolysaccharide (LPS) (200 EU/mL) for 30, 60, and 90 min by pretreated EGCG (50 μg/mL, 30 min). (B) THP-1-derived macrophages were treated with EGCG (50, 25, 12.5, 6.3, 3.1, and 1.6 μg/mL) for 30 min. (C) THP-1-derived macrophages were treated with EGCG (50 μg/mL) from 2 to 120 min. (D) THP-1-derived macrophages were treated with EGCG (50 μg/mL) from 2 to 120 min. Notch intercellular domain (NICD) of Notch1 and Notch2 located in the nucleus was detected, with TATA-binding protein (TBP) used as the internal control. (E) THP-1-derived macrophages were treated with EGCG (50 μg/mL) for 30 min, and lysates were analyzed by a western blotting probe with an antibody recognizing the epitope specific for active Notch1. (F) THP-1-derived macrophages were pretreated with EGCG (50 μg/mL) for 30 min and exposed to LPS (200 EU/mL) for 1 h. mRNA expression was measured by real-time polymerase chain reaction. (G) Notch report assays: 293/16CSL alone (left) and 293/16CSL with HepG2 coculture (right). (H) THP-1-derived macrophages were treated with EGCG (50 μg/mL) for 30 min with or without MG132 (10, 20, and 40 μM). (I) Immunoprecipitation was used to pull-down Notch from macrophage cell lysates. The Notch eluate was digested with PNGase F (left) and ATX-3 (right) and probed with the appropriate antibody. (J) Immunoprecipitation undertaken with antibody against the C-terminal of Notch1. The western blotting membrane was probed with an antibody against Notch1 or ubiquitin separately. Data are represented as mean ± SD (n = 3). Differences between the two groups were assessed by two-way ANOVA using GraphPad. Representatives of three independent experiments with similar results are shown. Results are represented as mean ± SD (*p < 0.05, **p < 0.01, and ***p < 0.001). n.s., not significant.
Mentions: The Notch signaling pathway has a pivotal role in the development of multicellular organisms. Notch (specifically Notch1) is crucial for lymphocyte development (25). Emerging evidence has shown that the Notch pathway anticipates the immune response, including inflammation (26). Basal activation of Notch1 has been shown to be necessary for enhancement of the inflammatory response (27). To ascertain whether EGCG attenuates the inflammatory response via the Notch pathway immediately after its addition, we examined the Notch1 and Notch2 receptors, which are expressed on the surface of human macrophages at multiple time points shortly after EGCG treatment. We found that the number of mature Notch1 and Notch2 receptors was decreased from the cell surface at all time points after EGCG treatment and that this process was not dependent on LPS stimulation (Figure 3A). Further studies showed that mature Notch removed the effects of EGCG in a concentration-dependent manner (Figure 3B) and that this decreased response was rapid (2 min) (Figure 3C). Simultaneously, EGCG treatment accelerated the degradation of the nuclear Notch intercellular domain (NICD), the active form of Notch, in 2 min (Figure 3D). In accordance with this result, a western blotting probe with a specific antibody against the new epitope formed after Notch1 cleavage showed that degradation of the active form of Notch1 was promoted by EGCG (Figure 3E). These results suggested that the basal signaling of Notch was turned off immediately by EGCG treatment. In accordance with these results, expression of Hes1 (a typical Notch-regulated gene) was downregulated by EGCG (Figure 3A). To confirm this result, expression of Notch target genes was evaluated using real-time PCR. We found that expression of some typical Notch target genes was downregulated by EGCG (Figure 3F), but not all of them. This finding may have been due to the complexity of gene transcription, which can be influenced by many factors. To evaluate the effects of EGCG treatment on gene expression of Notch alone, a dual luciferase reporter assay was employed. This assay involves use of the luciferase gene, which is controlled by 16×CSL, which contains 16 major Notch-regulated elements (CSL) connected in a series. Results showed that only treatment with EGCG could decrease Notch-related luciferase signaling in 293T cells and HepG2/293T cocultures (Figure 3G).

View Article: PubMed Central - PubMed

ABSTRACT

Inflammation plays important roles at different stages of diabetes mellitus, tumorigenesis, and cardiovascular diseases. (&minus;)-Epigallocatechin gallate (EGCG) can attenuate inflammatory responses effectively. However, the immediate early mechanism of EGCG in inflammation remains unclear. Here, we showed that EGCG attenuated the inflammatory response in the immediate early stage of EGCG treatment by shutting off Notch signaling and that the effect did not involve the 67-kDa laminin receptor, the common receptor for EGCG. EGCG eliminated mature Notch from the cell membrane and the nuclear Notch intercellular domain, the active form of Notch, within 2&thinsp;min by rapid degradation via the proteasome pathway. Transcription of the Notch target gene was downregulated simultaneously. Knockdown of Notch 1/2 expression by RNA interference impaired the downregulation of the inflammatory response elicited by EGCG. Further study showed that EGCG inhibited lipopolysaccharide-induced inflammation and turned off Notch signaling in human primary macrophages. Taken together, our results show that EGCG targets Notch to regulate the inflammatory response in the immediate early stage.

No MeSH data available.