一氧化氮造成腸道上皮屏障功能缺損的分子機制:緊密連結蛋白之瓦解和肌凝蛋白輕鏈之磷酸化所扮演的角色

一氧化氮 (nitric oxide, NO)在腸道生理恆定功能中扮演重要角色，然而過去研究顯示黏膜中過量的一氧化氮可能與腸道發炎和上皮屏障缺損有關。腸道屏障主要由單層上皮細胞構成，並經由緊密連結蛋白 (tight junction, TJ)，例如ZO-1和occludin，和圍連結肌動肌凝蛋白環 (perijunctional actinomyosin ring, PAMR)將細胞緊密相連。緊密連結之破壞與PAMR之收縮會導致上皮通透性上升。腸道上皮細胞與平滑肌細胞相同，可透過兩種磷酸激酶，Rho蛋白激酶 (Rho kinase, ROCK)和肌凝蛋白輕鏈激酶 (myosin light chain kinase, MLCK)將肌凝蛋白輕鏈磷酸化而造成機動肌凝蛋白收縮。磷酸激酶C zeta (PKCzeta)(一種附膜的非典型磷酸激酶C)的活化對緊密連結蛋白的重組扮演重要角色，且被認為與Rho訊息傳遞有關。因此本篇研究的目的即是要確認肌凝蛋白輕鏈的磷酸化和緊密連結蛋白的瓦解是否參與一氧化氮所引起之腸道上皮屏障功能缺損的機制。將人類結腸癌上皮細胞株Caco-2細胞給予0.5到5 mM濃度的一氧化氮供體S-Nitroso-N-acetylpenicillamine (SNAP, a NO donor)刺激24小時後，透過測量跨上皮電阻值 (transepithelial resistance, TER)以及螢光探針dextran-FITC (分子量3000)從頂腔面到底側面的速率當作細胞間通透性的指標。正常控制組的Caco-2上皮細胞在培養14~21天達到過滿狀態，其TER可達250-300 Ω*cm2。給予SNAP刺激則造成TER的下降以及探針通透率的增加，並呈現濃度依賴性。SNAP (1 mM)可以在不引起細胞凋亡的情況下造成TER下降69%並使上皮通透性增加11倍。細胞經預處理pancaspase或caspase-3抑制劑後並不能反轉1 mM SNAP所造成的屏障缺損，證實此現象發生並非因為細胞凋亡之緣故。透過定量西方墨點法的結果可得知SNAP刺激15分鐘後會造成PKCzeta磷酸化程度上升。刺激24小時後會引起緊密連結蛋白occludin的切割、ZO-1表現量的下降以及肌凝蛋白輕鏈磷酸化的增加。利用免疫螢光染色也觀察到SNAP處理的細胞其緊密連結蛋白和細胞骨架之結構不規則化並有細胞圓型化之現象。ROCK抑制劑 (Y-27632, 20 μM)及MLCK抑制劑 (PIK, 125-175 μM)可以降低SNAP所造成肌凝蛋白輕鏈的磷酸化。預處理ROCK抑制劑Y-27632 (20-50 μM)可反轉SNAP所造成的上皮屏障功能缺損，但MLCK抑制劑如PIK (125-175 μM)或ML-7 (20 μM)則未見此效果。此外，將細胞預處理PKCzeta抑制劑PKCzeta pseudosubstrate (20 μM)也可反轉SNAP所造成通透性的增加和緊密連結的破壞，但並無法降低SNAP引起之肌凝輕鏈磷酸化。綜合以上結果，一氧化氮可以活化PKCzeta而促進緊密連結蛋白之瓦解，並經由ROCK造成肌凝蛋白輕鏈之磷酸化，進而導致腸道屏障功能的缺損。

關鍵字

腸道上皮細胞；屏障功能；一氧化氮；緊密連結；肌凝蛋白輕鏈； PAMR ； ROCK ； MLCK ； PKCzeta

並列摘要

Physiological level of nitric oxide (NO) is crucial for maintaining gastrointestinal homeostasis, yet excessive production of mucosal NO is associated with gut inflammation and barrier defects. Intestinal barrier is composed of a monolayer of enterocytes linked by tight junctional proteins, e.g. ZO-1 and occludin, and perijunctional actinomyosin ring (PAMR). Disruption of TJs or contraction of PAMR leads to the increase of epithelial permeability. The mechanism of actinomyosin contraction was similar between epithelial and smooth muscle cells, which involved the phosphorylation of myosin light chain (MLC) by kinases, e.g. myosin light chain kinase (MLCK) and Rho-associated kinase (ROCK). Activation of PKCzeta (a membrane-associated atypical PKC) played a role in tight junctional remodeling in epithelial cells and was found associated with Rho signaling in smooth muscle cells. The aim was to investigate whether MLC phosphorylation and TJ disruption were involved in NO-induced intestinal epithelial barrier defects. Human colonic carcinoma Caco-2 cells were exposed to 0.5-5 mM S-Nitroso-N-acetylpenicillamine (SNAP, a NO donor) for 24 hr, and the transepithelial resistance (TER) and apical-to-basolateral flux rate of dextran-FITC (MW3000) were used as indicators of paracellular permeability. Post-confluent Caco-2 cells grown on transwells for 14-21 days established TJ and TER of 250-300 Ω*cm2. Exposure to SNAP increased epithelial permeability in a dose-dependent manner. SNAP concentration at 1 mM induced a 69% drop of TER and an 11-fold increase of epithelial permeability that were apoptosis-independent. The loss of epithelial barrier triggered by 1 mM SNAP was not blocked by pretreatment with a pancaspase or caspase-3 inhibitor. Densitometric analysis of western blots showed that 1 mM SNAP caused PKCzeta phosphorylation at 15 min post-challenge, as well as MLC phosphorylation, occludin cleavage and ZO-1 decrease at 24 hr post-challenge. Disorganization of TJs and cytoskeleton was associated with cell rounding in SNAP-exposed cells. Inhibitors to ROCK (Y-27632, 20 μM) and to MLCK (a novel membrane-permeant inhibitor of MLCK (PIK), 125-175 μM) reduced the level of MLC phosphorylation caused by SNAP challenge. Pretreatment with a ROCK inhibitor (Y-27632, 20-50 μM) attenuated SNAP-induced barrier defects whereas MLCK inhibitors, i.e. PIK (125-175 μM) or ML-7 (20 μM) had no effect. Moreover, pretreatment with PKCzeta inhibitory pseudosubstrate (20 μM) blocked SNAP-induced permeability rise and TJ disruption, but did not decrease the MLC phosphorylation. These findings suggest that exposure to NO induced PKCzeta-dependent TJ disruption and ROCK-dependent MLC phosphorylation, resulting in an increase of epithelial permeability.

並列關鍵字

ntestinal epithelial cells ； barrier functions ； nitric oxide ； tight junctions ； myosin light chain ； PAMR ； ROCK ； MLCK ； PKCzeta

參考文獻

1. Rodriguez-Boulan, E. and W.J. Nelson, Morphogenesis of the polarized epithelial cell phenotype. Science, 1989. 245(4919): p. 718-25.

3. Madara, J.L., Intestinal absorptive cell tight junctions are linked to cytoskeleton. Am J Physiol, 1987. 253(1 Pt 1): p. C171-5.

4. Bresnick, A.R., Molecular mechanisms of nonmuscle myosin-II regulation. Curr Opin Cell Biol, 1999. 11(1): p. 26-33.

5. Horowitz, A., et al., Antibodies probe for folded monomeric myosin in relaxed and contracted smooth muscle. J Cell Biol, 1994. 126(5): p. 1195-200.

7. Citi, S. and J. Kendrick-Jones, Regulation in vitro of brush border myosin by light chain phosphorylation. J Mol Biol, 1986. 188(3): p. 369-82.

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一氧化氮造成腸道上皮屏障功能缺損的分子機制:緊密連結蛋白之瓦解和肌凝蛋白輕鏈之磷酸化所扮演的角色

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