Detailed mechanisms behind regeneration after nerve injury, in particular signal transduction and the fate of Schwann cells （SCs）, are poorly understood. Here, we investigated axotomy-induced activation of extracell...Detailed mechanisms behind regeneration after nerve injury, in particular signal transduction and the fate of Schwann cells （SCs）, are poorly understood. Here, we investigated axotomy-induced activation of extracellular- signal-regulated kinase-1/2 （ERK1/2; important for proliferation） and m-calpain in vitro, and the relation to Ca2＋ deletion and Schwann cell proliferation and death after rat sciatic nerve axotomy. Nerve segments were cultured for up to 72 hours with and without ethylene glycol-bis（β-aminoethyl ether）- N,N,N＇,N＇-tetraacetic acid （EGTA）. In some experiments, 5-bromo-2′-deoxyuridine （BrdU） was added during the last 24 hours to detect proliferating cells and propidium iodide （PI） was added at the last hour to detect dead and/or dying cells. Immunohistochemistry of sections of the cultured nerve segments was performed to label m-calpain and the phosphorylated and activated form of ERK1/2. The experiments revealed that immunoreactivity for p-ERK1/2 increased with time in organotypically cultured SCs. p-ERK1/2 and m-calpain were also observed in axons. A significant increase in the number of dead or dying SCs was observed in nerve segments cultured for 24 hours. When deprived of Ca2＋, activation of axonal m-calpain was reduced, whereas p-ERK1/2 was increased in SCs. Ca2＋ deprivation also significantly reduced the number of proliferating SCs, and instead increased the number of dead or dying SCs. Ca2＋ seems to play an important role in activation of ERK1/2 in SCs and in SC survival and proliferation. In addition, extracellular Ca2＋ levels are also required for m-calpain activation and up-regulation in axons. Thus, regulation of Ca2＋ levels is likely to be a useful method to promote SC proliferation.展开更多
Summary: In order to explore the role of activator protein-1 (AP-1) in the transcription of interleukin-5 (IL-5) gene regulated by protein kinase C (PKC) signal in peripheral blood T lymphocytes from asthmatic patient...Summary: In order to explore the role of activator protein-1 (AP-1) in the transcription of interleukin-5 (IL-5) gene regulated by protein kinase C (PKC) signal in peripheral blood T lymphocytes from asthmatic patient, T lymphocytes were isolated and purified from peripheral blood of each asthmatic patient. The T lymphocytes were randomly divided into 4 groups: group A (blank control), group B (treated with PKC agonist phorbol 12-myristate 13-acetate (PMA)), Group C (treated with PMA and AP-1 cis-element decoy oligodeoxynucleotides (decoy ODNs)), and group D (treated with PMA and AP-1 mutant decoy ODNs). The ODNs were transfected into the T cells of group C and D by cation liposome respectively. Reverse transcription-polymerase chain reaction (RT-PCR) was employed to assess IL-5 mRNA expression, and electrophoretic mobility shift assays (EMSA) for the activation of AP-1. The results showed that the activation of AP-1 (88 003.58±1 626.57) and the expression of IL-5 mRNA (0.8300±0.0294) in T lymphocytes stimulated with PMA were significantly higher than these in blank control (20 888.47±1103.56 and 0.3050±0.0208, respectively, P< 0.01), while the indexes (23 219.83±1 024.86 and 0.3425±0.0171 respectively) of T lymphocytes stimulated with PMA and AP-1 decoy ODNs were significantly inhibited, as compared with group B (P< 0.01). The indexes (87 107.41±1 342.92 and 0.8225±0.0222, respectively) in T lymphocytes stimulated with PMA and AP-1 mutant decoy ODNs did not exhibit significant changes, as compared with group B (P>0.05). The significant positive correlation was found between the activation of AP-1 and the expression of IL-5 mRNA (P< 0.01). It was concluded that AP-1 might participate in the signal transduction of PKC-triggered transcription of IL-5 gene in asthmatic T lymphocytes. This suggests the activation of PKC/AP-1 signal transduction cascade of T lymphocytes may play an important role in the pathogenesis of asthma.展开更多
基金supported by the Research School in Pharmaceutical Science in Lund,The Royal Physiographic Society in LundThe Swedish Research Council(Medicine)+1 种基金the Craaford’s and Thure Nilsson’s Funds for Medical ResearchFunds for diabetic research,Lund University and Region Skane
文摘Detailed mechanisms behind regeneration after nerve injury, in particular signal transduction and the fate of Schwann cells （SCs）, are poorly understood. Here, we investigated axotomy-induced activation of extracellular- signal-regulated kinase-1/2 （ERK1/2; important for proliferation） and m-calpain in vitro, and the relation to Ca2＋ deletion and Schwann cell proliferation and death after rat sciatic nerve axotomy. Nerve segments were cultured for up to 72 hours with and without ethylene glycol-bis（β-aminoethyl ether）- N,N,N＇,N＇-tetraacetic acid （EGTA）. In some experiments, 5-bromo-2′-deoxyuridine （BrdU） was added during the last 24 hours to detect proliferating cells and propidium iodide （PI） was added at the last hour to detect dead and/or dying cells. Immunohistochemistry of sections of the cultured nerve segments was performed to label m-calpain and the phosphorylated and activated form of ERK1/2. The experiments revealed that immunoreactivity for p-ERK1/2 increased with time in organotypically cultured SCs. p-ERK1/2 and m-calpain were also observed in axons. A significant increase in the number of dead or dying SCs was observed in nerve segments cultured for 24 hours. When deprived of Ca2＋, activation of axonal m-calpain was reduced, whereas p-ERK1/2 was increased in SCs. Ca2＋ deprivation also significantly reduced the number of proliferating SCs, and instead increased the number of dead or dying SCs. Ca2＋ seems to play an important role in activation of ERK1/2 in SCs and in SC survival and proliferation. In addition, extracellular Ca2＋ levels are also required for m-calpain activation and up-regulation in axons. Thus, regulation of Ca2＋ levels is likely to be a useful method to promote SC proliferation.
文摘Summary: In order to explore the role of activator protein-1 (AP-1) in the transcription of interleukin-5 (IL-5) gene regulated by protein kinase C (PKC) signal in peripheral blood T lymphocytes from asthmatic patient, T lymphocytes were isolated and purified from peripheral blood of each asthmatic patient. The T lymphocytes were randomly divided into 4 groups: group A (blank control), group B (treated with PKC agonist phorbol 12-myristate 13-acetate (PMA)), Group C (treated with PMA and AP-1 cis-element decoy oligodeoxynucleotides (decoy ODNs)), and group D (treated with PMA and AP-1 mutant decoy ODNs). The ODNs were transfected into the T cells of group C and D by cation liposome respectively. Reverse transcription-polymerase chain reaction (RT-PCR) was employed to assess IL-5 mRNA expression, and electrophoretic mobility shift assays (EMSA) for the activation of AP-1. The results showed that the activation of AP-1 (88 003.58±1 626.57) and the expression of IL-5 mRNA (0.8300±0.0294) in T lymphocytes stimulated with PMA were significantly higher than these in blank control (20 888.47±1103.56 and 0.3050±0.0208, respectively, P< 0.01), while the indexes (23 219.83±1 024.86 and 0.3425±0.0171 respectively) of T lymphocytes stimulated with PMA and AP-1 decoy ODNs were significantly inhibited, as compared with group B (P< 0.01). The indexes (87 107.41±1 342.92 and 0.8225±0.0222, respectively) in T lymphocytes stimulated with PMA and AP-1 mutant decoy ODNs did not exhibit significant changes, as compared with group B (P>0.05). The significant positive correlation was found between the activation of AP-1 and the expression of IL-5 mRNA (P< 0.01). It was concluded that AP-1 might participate in the signal transduction of PKC-triggered transcription of IL-5 gene in asthmatic T lymphocytes. This suggests the activation of PKC/AP-1 signal transduction cascade of T lymphocytes may play an important role in the pathogenesis of asthma.
