探討在嗜中性白血球 CYL-26z 抑制 phospholipase D 活化，artocarpol A 促進生成超氧自由基及formyl peptide 活化phosphoinositide 3-kinase的作用機制

Cellular mechanisms of CYL-26z inhibition of phospholipase D activation, artocarpol A stimulation of superoxide anion generation, and formyl peptide stimulation of phosphoinositide 3-kinase activation in neutrophils

關宇翔(Yu-Hsiang Kuan)

指導教授 ： 林瑞生

中山醫學大學/醫學院/醫學研究所/博士(2006年)

https://doi.org/10.6834/CSMU.2006.00054

摘要

嗜中性白血球扮演第一線防禦細菌感染的角色。防禦機制包括去顆粒作用（釋出蛋白分解酶）與呼吸爆發作用（生成超氧自由基及有毒活性氧代謝物）。這些釋出的物質也會造成周邊正常組織的傷害。尋找具影響嗜中性白血球去顆粒與呼吸爆發作用潛力的合成化合物或天然物，並研究其作用機轉，有助於抗發炎治療的發展。本研究在探討合嗜中性白血球扮演第一線防禦細菌感染的角色。防禦機制包括去顆粒作用（釋出蛋白分解酶）與呼吸爆發作用（生成超氧自由基及有毒活性氧代謝物）。這些釋出的物質也會造成周邊正常組織的傷害。尋找具影響嗜中性白血球去顆粒與呼吸爆發作用潛力的合成化合物或天然物，並研究其作用機轉，有助於抗發炎治療的發展。本研究在探討合成化合物 5-[4-acridin-9-ylamino] phenyl]-5-methyl-3-methylene- dihydrofuran-2-one (CYL-26z) 影響嗜中性白血球去顆粒的作用機轉，及來自猴面果 (Artocarpus rigida) 的酚類天然化合物 artocarpol A (ART) 影響嗜中性白血球呼吸爆發作用的細胞內訊息傳遞機轉。
CYL-26z 以濃度依存性的方式抑制 formyl-Met-Leu-Phe (fMLP) 刺激嗜中性白血球產生的 phospholipase D (PLD)活化，但不抑制 phorbol 12-myristate 13-acetate (PMA) 刺激 PLD 的活化。CYL-26z 僅微弱地抑制 fMLP引起的細胞蛋白質酪胺酸磷酸化。CYL-26z 抑制由 fMLP 刺激引起的 protein kinase C-a (PKC a) 及RhoA膜轉位作用，但不抑制 PKC-bI、-bII 及ADP-ribosylation factor (Arf)的膜轉位。CYL-26z 也可抑制 fMLP 引起的 RhoA 活化以及 RhoA-Rho guanine nucleotide dissociation inhibitor (GDI) 複合物的解離。在無細胞系統的反應中，CYL-26z抑制 GTPgS 引起的 RhoA 膜轉位，但不影響 Arf 的膜轉位。CYL-26z也會抑制 fMLP 引起的CD11b、CD45、CD63在細胞膜表現。上述結果顯示 CYL-26z抑制嗜中性白血球去顆粒作用可能經由阻止 RhoA 的活化來抑制 PLD 活性。
ART 刺激嗜中性白血球產生超氧自由基。此作用可被 p38 mitogen-activated protein kinase (MAPK) 抑制劑 4-(4-fluorophenyl) -2-(4-methyl-sulfinyl-phenyl)-5-(4-pyridyl)-1H-imidazole (SB 203580)、廣泛性PKC 抑制劑 2-[1-(3-dimethylaminopropyl)-1H- indol-3-yl]-3-(1H- indol-3-yl)-maleimide (GF 109203X)、phospholipase C 抑制劑 1-[6- ((17b-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)-hexyl]-1H-pyrrole-2,5-dione (U-73122)、inositol 1,4,5-trisphosphate (IP3) 接受器阻斷劑 2-aminoethyldiphenyl borate (2-APB)、phosphoinositide 3-kinase (PI3K) 抑制劑 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294002) 所抑制，但不會被MAP/ERK kinase (MKK) 抑制劑2'-amino- 3'-methoxyflavone (PD98059) 與 1,4-diamino-2,3-dicyano-1,4-bis(2- aminophenylthio)butadiene (U0126) 所抑制。ART 可引起 p38 MAPK 的磷酸化與活化作用，但不會造成 extracellular signal-regulated kinase (ERK) 的磷酸化。不論細胞外是否有鈣離子存在，ART皆可促進胞內鈣離子濃度的增加。ART 也會增加細胞內 IP3的含量。ART可引起PKC-a、-bI 與 bII 的膜轉位作用，也可以活化鈣離子依賴性 PKC 的活性。
LY 294002 可抑制ART引起 protein kinase B (PKB/Akt) 上 Thr308 與 Ser473 位置的磷酸化及phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) 的生成作用。大鼠嗜中性白血球中表現有 class IA PI3K (p85、p110a、p110b、和 p110d) 和 class IB PI3K (p110g) 蛋白質。在加入ART 刺激細胞1.5 分鐘、0.5 分鐘與 1.5 分鐘後，可分別見到明顯地促進 class IA PI3K、class IB PI3K 與 Ras 的膜轉位。ART 促進 Ras 活化的時序與促進 class IA PI3Ks 膜轉位的時序相同。ART可促進 Ras 與 class I PI3K 的蛋白質結合。ART 可活化 Src family kinase。使用Src family kinase 抑制劑4-amino-1-tert-butyl- 3-(1'-naphthyl)pyrazolo[3,4-d] pyrimidine (PP1 analog) 可抑制 ART 刺激細胞生成超氧自由基與PI(3,4,5)P3。ART 引起的 p47phox 磷酸化與膜轉位可被 GF109203X 所抑制。上述結果顯示ART 可經由活化 p38 MAPK、PLC/Ca2+、PKC 與 PI3K 的訊息傳遞路徑來促使中性白血球生成超氧自由基。
總結來說，CYL-26z 抑制 fMLP 刺激嗜中性白血球的去顆粒作用是經由阻止 RhoA 的活化來抑制 PLD 的活性。ART 可經由活化 PLC/Ca2+、PKC、p38 MAPK 與 PI3K 的訊息傳遞路徑來促使嗜中性白血球產生超氧自由基。

關鍵字

CYL-26z ； Artocarpol A ； 嗜中性白血球 ； 呼吸爆發作用 ； 去顆粒作用 ； 訊息傳遞

並列摘要

Neutrophils play a critical role in the first-line defense against invading bacteria via degranulation and respiratory burst. However, the released proteases and the generated reactive oxygen species may also responsible for tissue destruction in inflammatory conditions. The search for potential synthetic compounds and natural products and study of their molecular mechanisms to regulate degranulation and respiratory burst in neutrophils would be useful for anti-inflammatory chemotherapy. In the present study, the inhibition of degranulation by a synthetic compound, 5-[4-acridin-9-ylamino]phenyl]-5-methyl-3-methylenedihydrofuran-2-one (CYL-26z), and the stimulation of respiratory burst by artocarpol A (ART), a natural phenolic compound isolated from Artocarpus rigida, were examined in rat neutrophils.
CYL-26Z showed concentration-dependent inhibition of formyl-Met-Leu-Phe (fMLP)-stimulated phospholipase D (PLD) activity in neutrophils but did not affect the phorbol 12-myristate 13-acetate (PMA)-induced response. CYL-26z caused a slight but significant attenuation of the fMLP-induced global protein tyrosine phosphorylation. CYL-26z blocked the membrane recruitment of protein kinase C-a (PKC-a) but failed to affect the membrane association of PKC-bI and -bII. CYL-26z inhibited the translocation of RhoA, but not ADP-ribosylation factor (Arf), to the membrane, the dissociation of RhoA-Rho guanine nucleotide dissociation inhibitor (GDI) complex and the RhoA activation in fMLP-stimulated neutrophils. CYL-26z effectively attenuated the membrane association of RhoA but not Arf in response to GTPgS in a cell-free system. CYL-26z inhibited the membrane expression of CD11b, CD45 and CD63 in response to fMLP stimulation. These results indicate that the CYL-26z inhibition of fMLP-stimulated neutrophil degranulation is, at lest partly, attributable to the attenuation of PLD activity via the blockade of RhoA activation.
ART stimulated superoxide anion generation in neutrophils. Pretreatment of cells with 4-(4-fluorophenyl)-2-(4-methyl-sulfinyl-phenyl)-5-(4-pyridyl)-1H-imidazole (SB 203580), a p38 mitogen-activated protein kinase (MAPK) inhibitor, 2-[1- (3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF 109203X), a broad PKC inhibitor, 1-[6-((17b-3-Methoxyestra-1,3,5(10)-trien-17-yl)amino)- hexyl]-1H-pyrrole-2,5-dione (U-73122), a phospholipase C (PLC) inhibitor, 2- aminoethyldiphenyl borate (2-APB), an inositol 1,4,5-trisphosphate (IP3) receptor blocker, and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294002), a phosphoinositide 3-kinase (PI3K) inhibitor, but not with 2'-amino-3'-methoxyflavone (PD98059) and 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126), both MKK (MAP/ERK kinase) inhibitors, attenuated ART-induced response. ART induced the phosphorylation and activation of p38 MAPK but not the phosphorylation of extracellular signal-regulated kinase (ERK). ART stimulated the [Ca2+]i elevation in the presence or absence of external Ca2+, and increased the cellular IP3 levels. ART increased the recruitment of PKC-a, -bI, and -bII to the plasma membrane of neutrophils, and stimulated Ca2+-dependent PKC activation in the cytosol preparation.
ART stimulated the dual phosphorylation (Thr308 and Ser473) of protein kinase B (PKB/Akt) and the formation of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3), whereas LY 294002 inhibited these effects in neutrophils. Rat neutrophils expressed both class IA PI3K (p85, p110a, p110b, and p110d) and class IB PI3K (p110g) proteins. Detectable membrane-association of class IA PI3Ks, class IB PI3K and Ras was seen as early as 1.5 min, 0.5 min and 1.5 min, respectively, after stimulation with ART. The kinetics of ART-induced Ras activation paralleled the kinetics of class IA PI3Ks recruitment to membrane caused by ART, and the p85 and p110g immunoprecipitates contained Ras. ART stimulated Src family kinase activation. Both Src kinase activity and PI(3,4,5)P3 formation in ART-stimulated neutrophils were inhibited by 4-amino-1-tert-butyl-3-(1'-naphthyl)pyrrazolo [3,4-d]pyrimidine (PP1 analog), a Src family kinase inhibitor. PP1 analog also attenuated the ART-stimulated superoxide anions generation in neutrophils. ART induced the serine phosphorylation and the membrane association of p47phox, whereas GF 109203X inhibited both responses. These results indicate that the ART stimulation of superoxide anion generation involved the activation of p38 MAPK, PLC/Ca2+, PKC and PI3K signaling pathways in neutrophils.
In summary, CYL-26z inhibition of PLD activation, through the blockade of RhoA activation, is attributed to the fMLP-induced degranulation. ART stimulation of superoxide anion generation is attributable to the activation of PLC/Ca2+, PKC, p38 MAPK and PI3K signaling pathways in rat neutrophils.

並列關鍵字

CYL-26z ； Arotocarpol A ； Neutrophil ； Respiratory burst ； Degranulation ； Signal pathway

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