Cyclic AMP inhibitory effect and cAMP-responsive transcription factors in T lymphocytes

• Main Authors: Hsueh, Yi-Ping, 薛一蘋
• Other Authors: Lai, Ming-Zong
• Format: Others
• Language: zh-TW
• Published: 1995
• Online Access: http://ndltd.ncl.edu.tw/handle/13636316620689270936

博士 === 國立陽明大學 === 微生物暨免疫學研究所 === 83 === cAMP抑制T細胞核內活化反應的分子機制至今仍不清楚。因為cAMP刺激的基因表現需要CREB和ATFl兩種轉錄分子，因此本論文第一部分檢視cAMP對T細胞的抑制作用是否可能由CREB或ATF1媒介。其結果卻發現ATF1在統大多數轉形的淋巴細胞內有高量的表現。正常T細胞活化後ATFl表現也會上升到與轉形T細胞相當的量，而維持一段短暫時間。若活化轉形T細胞，則ATF1含量並不再增加。活化正常T細胞而刺激的ATFl表現需要TPA和A23187同時存在方可，單獨使用TPA、A23187、或dbCAMP均無法使ATF1表現量增加。在轉形及活化後的T細胞內的高量表現並不是由於ATF1轉錄起始速度的增加，而是源於ATF1 RNA穩定性增加。ATF1因RNA穩定性而在不同狀態細胞之差別性表現，顯示ATF1在細胞生長和分化過程中所扮演的角色。雖然CRB在T細胞內的表現量沒有明顯變化，但T細胞以TPA/A23187活化後，可引發CREB Ser133磷酸化，因而大幅度提高CREB轉錄能力。由於T細胞活化後能增加ATFl之表現及CREB轉錄能力，ATFl和CREB顯然不可能媒介cAMP對T細胞的抑制作用。 本論文第二部分檢視可能被cAMP所抑制的T細胞訊息傳導途徑。cANP在纖維原母細胞和肌肉細胞最近被發現可抑制MAPK訊息傳導途徑，類似的情形若在T細胞內可以解釋cAMP的抑制作用。而然在所有檢視過的T細胞內，cAMP均無法抑制ERK2活性。ERKl和ERK3也不受cAMP影響。此外，cAMP對Raf-l活性也幾乎沒有明顯的抑制。T細胞中，ERK及Raf-l對cAMP作用的耐受性很清楚地顯示T細胞與纖維原母細胞訊息傳遞之不同。相對的，MAPK家族的另一成員JNK，在T細胞中具則受cAMP抑制，且其受抑制的程度與介白素二分泌減少程度對等。更重要的是NK被cAMP抑制需一段遲緩期，這正是cAMP仰抑制T細胞活化的特 性。cAMP不會影響JNK蛋白質表現量，但是環己亞醯胺和放線菌素D可以阻止cAMP的抑制作用，顯示cAMP需藉由其一新合成的分子來抑制JNK活性。雖然ERK和JNK兩條訊息傳導途徑均為T細胞活化所必需，但cAMP會選擇性的抑制JNK活性，更支持了JNK在T細胞活化過程中可能具有特別的功能。 The molecular mechanism underlying the cAMP inhibition of nuclear activation events in T lymphocytes remains unknown. cAMP-activated transcription is mediated by cAMP-responsive element binding protein (CREB) and activation transcriptional factor-1 (ATF-l). In the first part, whether the inhibitory effect of cAMP on T cells is mediated by CREB or ATFl was examined. It was found that ATF-1 was overexpressed in most transformed lymphocytes. Furthermore, the activation of normal T lymphocytes induced a transient increase of ATF-1 expression to the level comparable to that of T lymphomas. Activation had no effect on the ATF-1 level of transformed T lymphocytes. The induction of ATF-1 required the costimulation of normal T lymphocytes with TPA and A23187. TPA, Ca++ ionophore, or cAMP alone did not stimulate ATF-l expression in normal lymphocytes. Nuclear runon assay indicates that the increased ATF-l expression in T cell lymphomas and in activated splenic T lymphocytes was not due to an enhanced t ranscription.Instead, an increase in ATF-1 mRNA stability was found in these lymphocytes. The regulation of ATF-1 expression through RNA stability in cells of different states suggests that ATF-1 may play an active role in cell growth and differentiation. Even though the level of CREB was little altered in various T lymphocytes, T cell activation led to phosphorylation of Ser-133, which confers the transcriptional activity of CREB. Both activation-induced expression of ATFl and activation-dependent phosphorylation of CREB clearly suggest that both ATFl and CREB do not transmit the inhibitory signal of cAMP. In the second part, the possible signaling pathway that is antagonized by cAMP was examined. Recently, the activation of fibroblasts and muscle cells are shown to be antagonized by cAMP through the inhibition of mitogen-activated protein (MAP) kinases signaling pathway. Whether a similar antagonism may account for the late inhibitory effect of cAMP in T cell was examined. Extracellular signal regulated kinase 2 (ERK2) activation was resistant to cAMP inhibition in all the T lymphocytes tested. Different isoforms (ERKl, ERK2 and ERK3) of MAP kinase were poorly inhibited by cAMP. In addition, cAMP had minimum effect on Raf-1 in T lymphocytes. The resistance of ERK and Raf-1 to cAMP clearly distinguishes T cells from fibroblasts. In contrast, another MAP kinase homologue c- Jun N-terminal kinase (JNK) was inhibited by cAMP in good correlation with that of IL-2 suppression. Moreover, JNK was antagonized by a delayed kinetics which is characteristic of cAMP inhibition. cAMP had no direct effecton the level of JNK. The cAMP inhibition on JNK can be prevented by cyclohexamide and actinomycin D, suggesting the involvement of de novo synthesis of inhibitory factor. Despite that both ERK and JNK are essential for T cell activation, selective inhibition by cAMP further supports the specific role of JNK in T cell activation.