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The Role of Receptor for Advanced Glycation End Products (RAGE) in Fibroblast Chemotaxis
Description
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A5797 - The Role of Receptor for Advanced Glycation End Products (RAGE) in Fibroblast Chemotaxis
Author Block: N. Tanaka, J. Ikari, R. Anazawa, M. Suzuki, Y. Katsumata, A. Shimada, Y. Matsuura, N. Kawata, Y. Tada, K. Tatsumi; Respirology, Graduate School of Medicine, Chiba University, Chiba, Japan.
Rational:
Lung fibroblasts are the major mesenchymal cells that migrate into inflamed tissue and mediate lung maintenance and repair. In chronic obstructive pulmonary disease (COPD), chemotaxis of lung fibroblasts is impaired. Although receptor for advanced glycation end products (RAGE) expression in the lung of patients with COPD is higher than that of healthy subjects, the function of RAGE in the lung, including lung fibroblasts, has yet to be elucidated. Thus, we sought to examine the role of RAGE in lung fibroblast chemotaxis.
Methods:
Human fetal lung fibroblast 1 (HFL-1) cells were cultured in 100 mm tissue culture dishes in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS at 37℃ in a humidified 5% CO2 incubator. HFL-1 cells migration was assessed using a 48 well micro chemotaxis chamber (Neuro Probe). HFL-1 cells (5×105 cells/ml) were loaded into upper well of the chamber with and without RAGE agonists (HMGB1, S100A12). Human fibronectin (5 μg/ml) was placed in the bottom of chamber as a chemoattractant. The lower and upper compartments were separated by a polycarbonate filter with 8 μm pores. The chamber was incubated for 6 hours. The number of migrated cells was counted in five high-power fields with an optical microscope. The sum of migrated cells in the five separate fields was used as the measure of chemotaxis for that well.
Results:
The number of migrated cells was increased in the presence of HMGB1 in a concentration-dependent manner. In contrast, S100A12 inhibited chemotaxis in a concentration-dependent manner. In the presence of anti-RAGE antibody, the effect of S100A12 was hampered, while that of HMGB1 was not affected, suggesting that the reduced chemotaxis by S100A12 is RAGE dependent. Furthermore, TLR4 inhibitor did not alter the effect of S100A12 nor HMGB1 on fibroblasts chemotaxis. In the presence of MAPK (p38) inhibitor, but not of NFκB, PKA, PTEN, nor COX inhibitor, the reduced chemotaxis by S100A12 was significantly attenuated.
Conclusions:
RAGE agonists differently affect the lung fibroblasts migration. HMGB1 augments lung fibroblasts chemotaxis in a RAGE-independent manner. S100A12 inhibits lung fibroblast chemotaxis acting through RAGE and p38 signaling pathway. Through these actions, RAGE signaling could modulate lung maintenance and repair.
Author Block: N. Tanaka, J. Ikari, R. Anazawa, M. Suzuki, Y. Katsumata, A. Shimada, Y. Matsuura, N. Kawata, Y. Tada, K. Tatsumi; Respirology, Graduate School of Medicine, Chiba University, Chiba, Japan.
Rational:
Lung fibroblasts are the major mesenchymal cells that migrate into inflamed tissue and mediate lung maintenance and repair. In chronic obstructive pulmonary disease (COPD), chemotaxis of lung fibroblasts is impaired. Although receptor for advanced glycation end products (RAGE) expression in the lung of patients with COPD is higher than that of healthy subjects, the function of RAGE in the lung, including lung fibroblasts, has yet to be elucidated. Thus, we sought to examine the role of RAGE in lung fibroblast chemotaxis.
Methods:
Human fetal lung fibroblast 1 (HFL-1) cells were cultured in 100 mm tissue culture dishes in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS at 37℃ in a humidified 5% CO2 incubator. HFL-1 cells migration was assessed using a 48 well micro chemotaxis chamber (Neuro Probe). HFL-1 cells (5×105 cells/ml) were loaded into upper well of the chamber with and without RAGE agonists (HMGB1, S100A12). Human fibronectin (5 μg/ml) was placed in the bottom of chamber as a chemoattractant. The lower and upper compartments were separated by a polycarbonate filter with 8 μm pores. The chamber was incubated for 6 hours. The number of migrated cells was counted in five high-power fields with an optical microscope. The sum of migrated cells in the five separate fields was used as the measure of chemotaxis for that well.
Results:
The number of migrated cells was increased in the presence of HMGB1 in a concentration-dependent manner. In contrast, S100A12 inhibited chemotaxis in a concentration-dependent manner. In the presence of anti-RAGE antibody, the effect of S100A12 was hampered, while that of HMGB1 was not affected, suggesting that the reduced chemotaxis by S100A12 is RAGE dependent. Furthermore, TLR4 inhibitor did not alter the effect of S100A12 nor HMGB1 on fibroblasts chemotaxis. In the presence of MAPK (p38) inhibitor, but not of NFκB, PKA, PTEN, nor COX inhibitor, the reduced chemotaxis by S100A12 was significantly attenuated.
Conclusions:
RAGE agonists differently affect the lung fibroblasts migration. HMGB1 augments lung fibroblasts chemotaxis in a RAGE-independent manner. S100A12 inhibits lung fibroblast chemotaxis acting through RAGE and p38 signaling pathway. Through these actions, RAGE signaling could modulate lung maintenance and repair.
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