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Identification and characterization of the direct interaction between methotrexate (MTX) and high-mobility group box 1 (HMGB1) protein.

Kuroiwa Y, Takakusagi Y, Kusayanagi T, Kuramochi K, Imai T, Hirayama T, Ito I, Yoshida M, Sakaguchi K, Sugawara F - PLoS ONE (2013)

Bottom Line: Although dihydrofolate reductase (DHFR) is a well-known target for the anti-tumor effect of MTX, the mode of action for the anti-inflammatory activity of MTX is not fully understood.These data suggested that binding of MTX to part of the RAGE-binding region (K149-V175) in HMGB1 might be significant for the anti-inflammatory effect of MTX.These data might explain the molecular basis underlying the mechanism of action for the anti-inflammatory effect of MTX.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Yamazaki, Noda, Chiba, Japan.

ABSTRACT

Background: Methotrexate (MTX) is an agent used in chemotherapy of tumors and autoimmune disease including rheumatoid arthritis (RA). In addition, MTX has some anti-inflammatory activity. Although dihydrofolate reductase (DHFR) is a well-known target for the anti-tumor effect of MTX, the mode of action for the anti-inflammatory activity of MTX is not fully understood. METHODOLOGY/RESULT: Here, we performed a screening of MTX-binding proteins using T7 phage display with a synthetic biotinylated MTX derivative. We then characterized the interactions using surface plasmon resonance (SPR) analysis and electrophoretic mobility shift assay (EMSA). Using a T7 phage display screen, we identified T7 phages that displayed part of high-mobility group box 1 (HMGB1) protein (K86-V175). Binding affinities as well as likely binding sites were characterized using genetically engineered truncated versions of HMGB1 protein (Al G1-K87, Bj: F88-K181), indicating that MTX binds to HMGB1 via two independent sites with a dissociation constants (KD) of 0.50±0.03 µM for Al and 0.24 ± 0.01 µM for Bj. Although MTX did not inhibit the binding of HMGB1 to DNA via these domains, HMGB1/RAGE association was impeded in the presence of MTX. These data suggested that binding of MTX to part of the RAGE-binding region (K149-V175) in HMGB1 might be significant for the anti-inflammatory effect of MTX. Indeed, in murine macrophage-like cells (RAW 264.7), TNF-α release and mitogenic activity elicited by specific RAGE stimulation with a truncated monomeric HMGB1 were inhibited in the presence of MTX.

Conclusions/significance: These data demonstrate that HMGB1 is a direct binding protein of MTX. Moreover, binding of MTX to RAGE-binding region in HMGB1 inhibited the HMGB1/RAGE interaction at the molecular and cellular levels. These data might explain the molecular basis underlying the mechanism of action for the anti-inflammatory effect of MTX.

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Effect of MTX on the truncated HMGB1 (Bj)-elicited TNF-α release and mitogenic activity in RAW 264.7 cells.(A) Bj protein-dependent TNF-α release. RAW 264.7 cells, in a 24-well culture dish format, were stimulated with the indicated concentrations of Bj protein for 6 h. The amount of TNF-α released into the conditioned medium was then determined by ELISA. N ≥2. (B) Influence of MTX alone for TNF-α release. RAW 264.7 cells were stimulated with 0–10 µM of MTX for 6 h. N ≥3. (C) Inhibition of Bj protein-elicited TNF-α release, in a 24-well culture dish format, stimulated with 0.5 µM of Bj protein for 6 h in the presence of 0–10 µM MTX. N ≥3. (D) Bj protein-elicited mitogenic activity for 10 h. Results are given in terms of relative cell growth. N ≥3. (E) Cell growth in the presence or absence of Bj protein (0.5 µM), or MTX (100 µM). N ≥3. (F) Time course of MTX cytotoxicity. RAW 264.7 cell growth was elucidated using the WST-8 cell proliferation assay and is shown as relative cell growth (%). Data represented as mean ± SE. *P<0.05, ****P<0.001.
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pone-0063073-g005: Effect of MTX on the truncated HMGB1 (Bj)-elicited TNF-α release and mitogenic activity in RAW 264.7 cells.(A) Bj protein-dependent TNF-α release. RAW 264.7 cells, in a 24-well culture dish format, were stimulated with the indicated concentrations of Bj protein for 6 h. The amount of TNF-α released into the conditioned medium was then determined by ELISA. N ≥2. (B) Influence of MTX alone for TNF-α release. RAW 264.7 cells were stimulated with 0–10 µM of MTX for 6 h. N ≥3. (C) Inhibition of Bj protein-elicited TNF-α release, in a 24-well culture dish format, stimulated with 0.5 µM of Bj protein for 6 h in the presence of 0–10 µM MTX. N ≥3. (D) Bj protein-elicited mitogenic activity for 10 h. Results are given in terms of relative cell growth. N ≥3. (E) Cell growth in the presence or absence of Bj protein (0.5 µM), or MTX (100 µM). N ≥3. (F) Time course of MTX cytotoxicity. RAW 264.7 cell growth was elucidated using the WST-8 cell proliferation assay and is shown as relative cell growth (%). Data represented as mean ± SE. *P<0.05, ****P<0.001.

Mentions: To investigate the inhibitory effect of MTX on the HMGB1/RAGE interaction at the cellular level, we measured HMGB1-elicited TNF-α release via RAGE in RAW 264.7 cells using ELISA [22], [33], [34]. Specific RAGE stimulation with noncomplexed Bj protein (0.01–0.5 µM) for 6 h increased the release of TNF in a concentration-dependent manner in RAW 264.7 cells (Figure 5A). By contrast, MTX alone had no such effect during this period (Figure 5B). However, incubation of RAW 264.7 cells with 0.5 µM Bj protein in the presence of 1 or 10 µM MTX (i.e., 2-fold and 20-fold excess over Bj, respectively) resulted in a reproducible suppression of TNF release (Figure 5C). Together with the T7 phage display and SPR data, these findings support the idea that binding of MTX to K149-V175 in HMGB1 might interfere with the HMGB1/RAGE interaction, thereby showing the anti-inflammatory activity of MTX.


Identification and characterization of the direct interaction between methotrexate (MTX) and high-mobility group box 1 (HMGB1) protein.

Kuroiwa Y, Takakusagi Y, Kusayanagi T, Kuramochi K, Imai T, Hirayama T, Ito I, Yoshida M, Sakaguchi K, Sugawara F - PLoS ONE (2013)

Effect of MTX on the truncated HMGB1 (Bj)-elicited TNF-α release and mitogenic activity in RAW 264.7 cells.(A) Bj protein-dependent TNF-α release. RAW 264.7 cells, in a 24-well culture dish format, were stimulated with the indicated concentrations of Bj protein for 6 h. The amount of TNF-α released into the conditioned medium was then determined by ELISA. N ≥2. (B) Influence of MTX alone for TNF-α release. RAW 264.7 cells were stimulated with 0–10 µM of MTX for 6 h. N ≥3. (C) Inhibition of Bj protein-elicited TNF-α release, in a 24-well culture dish format, stimulated with 0.5 µM of Bj protein for 6 h in the presence of 0–10 µM MTX. N ≥3. (D) Bj protein-elicited mitogenic activity for 10 h. Results are given in terms of relative cell growth. N ≥3. (E) Cell growth in the presence or absence of Bj protein (0.5 µM), or MTX (100 µM). N ≥3. (F) Time course of MTX cytotoxicity. RAW 264.7 cell growth was elucidated using the WST-8 cell proliferation assay and is shown as relative cell growth (%). Data represented as mean ± SE. *P<0.05, ****P<0.001.
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Related In: Results  -  Collection

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pone-0063073-g005: Effect of MTX on the truncated HMGB1 (Bj)-elicited TNF-α release and mitogenic activity in RAW 264.7 cells.(A) Bj protein-dependent TNF-α release. RAW 264.7 cells, in a 24-well culture dish format, were stimulated with the indicated concentrations of Bj protein for 6 h. The amount of TNF-α released into the conditioned medium was then determined by ELISA. N ≥2. (B) Influence of MTX alone for TNF-α release. RAW 264.7 cells were stimulated with 0–10 µM of MTX for 6 h. N ≥3. (C) Inhibition of Bj protein-elicited TNF-α release, in a 24-well culture dish format, stimulated with 0.5 µM of Bj protein for 6 h in the presence of 0–10 µM MTX. N ≥3. (D) Bj protein-elicited mitogenic activity for 10 h. Results are given in terms of relative cell growth. N ≥3. (E) Cell growth in the presence or absence of Bj protein (0.5 µM), or MTX (100 µM). N ≥3. (F) Time course of MTX cytotoxicity. RAW 264.7 cell growth was elucidated using the WST-8 cell proliferation assay and is shown as relative cell growth (%). Data represented as mean ± SE. *P<0.05, ****P<0.001.
Mentions: To investigate the inhibitory effect of MTX on the HMGB1/RAGE interaction at the cellular level, we measured HMGB1-elicited TNF-α release via RAGE in RAW 264.7 cells using ELISA [22], [33], [34]. Specific RAGE stimulation with noncomplexed Bj protein (0.01–0.5 µM) for 6 h increased the release of TNF in a concentration-dependent manner in RAW 264.7 cells (Figure 5A). By contrast, MTX alone had no such effect during this period (Figure 5B). However, incubation of RAW 264.7 cells with 0.5 µM Bj protein in the presence of 1 or 10 µM MTX (i.e., 2-fold and 20-fold excess over Bj, respectively) resulted in a reproducible suppression of TNF release (Figure 5C). Together with the T7 phage display and SPR data, these findings support the idea that binding of MTX to K149-V175 in HMGB1 might interfere with the HMGB1/RAGE interaction, thereby showing the anti-inflammatory activity of MTX.

Bottom Line: Although dihydrofolate reductase (DHFR) is a well-known target for the anti-tumor effect of MTX, the mode of action for the anti-inflammatory activity of MTX is not fully understood.These data suggested that binding of MTX to part of the RAGE-binding region (K149-V175) in HMGB1 might be significant for the anti-inflammatory effect of MTX.These data might explain the molecular basis underlying the mechanism of action for the anti-inflammatory effect of MTX.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Yamazaki, Noda, Chiba, Japan.

ABSTRACT

Background: Methotrexate (MTX) is an agent used in chemotherapy of tumors and autoimmune disease including rheumatoid arthritis (RA). In addition, MTX has some anti-inflammatory activity. Although dihydrofolate reductase (DHFR) is a well-known target for the anti-tumor effect of MTX, the mode of action for the anti-inflammatory activity of MTX is not fully understood. METHODOLOGY/RESULT: Here, we performed a screening of MTX-binding proteins using T7 phage display with a synthetic biotinylated MTX derivative. We then characterized the interactions using surface plasmon resonance (SPR) analysis and electrophoretic mobility shift assay (EMSA). Using a T7 phage display screen, we identified T7 phages that displayed part of high-mobility group box 1 (HMGB1) protein (K86-V175). Binding affinities as well as likely binding sites were characterized using genetically engineered truncated versions of HMGB1 protein (Al G1-K87, Bj: F88-K181), indicating that MTX binds to HMGB1 via two independent sites with a dissociation constants (KD) of 0.50±0.03 µM for Al and 0.24 ± 0.01 µM for Bj. Although MTX did not inhibit the binding of HMGB1 to DNA via these domains, HMGB1/RAGE association was impeded in the presence of MTX. These data suggested that binding of MTX to part of the RAGE-binding region (K149-V175) in HMGB1 might be significant for the anti-inflammatory effect of MTX. Indeed, in murine macrophage-like cells (RAW 264.7), TNF-α release and mitogenic activity elicited by specific RAGE stimulation with a truncated monomeric HMGB1 were inhibited in the presence of MTX.

Conclusions/significance: These data demonstrate that HMGB1 is a direct binding protein of MTX. Moreover, binding of MTX to RAGE-binding region in HMGB1 inhibited the HMGB1/RAGE interaction at the molecular and cellular levels. These data might explain the molecular basis underlying the mechanism of action for the anti-inflammatory effect of MTX.

Show MeSH
Related in: MedlinePlus