敗血症時蛋白激酶C同功酶 alpha 調控肝衰竭之分子機制探討

Molecular mechanism of PKCalpha-mediated hepatic failure during sepsis

謝雅晶(Ya-Ching Hsieh)

指導教授 ： 許勝光 教授 ； 許 勤 教授

高雄醫學大學/醫學院/醫學研究所/博士(2003年)

摘要

肝臟已被認為在啟動及促進敗血症 (sepsis) 多重器官衰竭的發生為一重要器官。此疾病可引起肝細胞結構與功能的破壞，進而對體內能量之產生、代謝之維持及免疫反應的破壞，以致肝臟衰竭 (hepatic failure)。而且已知在敗血症引起的肝臟功能的破壞，例如：細胞凋亡 (apoptosis)、膽酸分泌及代謝失調、ATP產量下降等等，但相關之作用機制的瞭解仍然有限。
曾有報告指出在病理情況下，蛋白激酶C能使肝細胞免於凋亡，且在某些癌細胞蛋白激酶C同功酶alpha的減少常伴隨著細胞凋亡的增加。我們先前研究得知敗血症晚期肝臟衰竭時，細胞質蛋白激酶C明顯地不活化，而且蛋白激酶C同功酶中以同功酶alpha的減少最為顯著。所以，蛋白激酶C，特別是同功酶alpha，對於敗血症時調控肝細胞凋亡的瞭解，將可對敗血症導致肝臟衰竭的機制有進一步的了解。當然，敗血症造成肝臟衰竭的原因，除了細胞凋亡之外，仍有其它可能參與之機制。因細胞核蛋白激酶C同功酶alpha能直接磷酸化細胞核蛋白 (如轉錄因子)，故蛋白激酶C同功酶alpha可能透過轉錄因子調控基因的表現，所以我們希望能在分子機制層面找到蛋白激酶C同功酶alpha參與敗血症肝臟衰竭時之其它調控機制。
在我們探討敗血症過程中，肝臟蛋白激酶C同功酶alpha是否調控細胞凋亡及其可能調控之分子機制。結果顯示於敗血症早期和晚期時肝臟：(1) 細胞膜及細胞核蛋白激酶C同功酶alpha的表現減少；?(2) 轉錄因子 (transcription factor) CREB之磷酸化情形減弱且其結合活性下降；?(3) Bcl-2/Bax之比例減少；(4) 增加細胞凋亡的發生；而且這些變化於晚期敗血症時皆比敗血症早期更為明顯。由於蛋白激酶C同功酶alpha會磷酸化CREB且CREB會調控Bcl-2的表現，而Bcl-2是一種抗細胞凋亡蛋白，所以，由以上結果我們推測，敗血症肝臟衰竭時蛋白激酶C同功酶alpha的減少，可能參與CREB的磷酸化下降而導致Bcl-2的表現受抑制、Bcl-2/Bax之比例減少，因而增加細胞凋亡的發生。我們為了證實以上之推論，所以建構 (construction) 大白鼠 (rat) 的蛋白激酶C同功酶alpha反意質體 (antisense plasmid) (pAS-PKCalpha)，將此反意質體轉殖感染 (transfection) 至Clone-9肝細胞株，以抑制細胞內的蛋白激酶C同功酶alpha的表現。從本研究結果發現，我們建構之蛋白激酶C同功酶alpha反意質體成功的抑制細胞內的蛋白激酶C同功酶alpha的表現；且CREB之磷酸化的情形減弱及其結合活性下降、Bcl-2的基因及蛋白質表現亦隨之減少、Bcl-2/Bax的表現比例下降、PARP斷裂及細胞凋亡發生情形明顯增加，故我們推測：蛋白激酶C同功酶alpha表現受抑制確實會誘發肝細胞的凋亡且可能透過CREB調控Bcl-2所致。
蛋白激酶C同功酶alpha在敗血症時除了影響肝細胞凋亡外，是否有藉由調控其他基因的表現而導致肝臟衰竭？我們利用抑制相減雜交法 (suppression subtractive hybridization; SSH) 加上北方墨點法 (Northern blot analysis) 的證實，找出9個在敗血症晚期的肝臟中呈現差異之基因(differential genes)。分別為3alphaHSD、EST189895/mouse RNase4、IF1、alpha2u-G PGCL1、TII-kininogen、Spi2.2、kan-1/rBAT、albumin及alpha2M，而前5個基因是未曾被提及與敗血症有關。這些基因的功能顯示在敗血症晚期的肝臟呈現有膽酸的合成代謝障礙及ATP的減少等問題。於離體實驗，證實當蛋白激酶C同功酶 ? 表現下降時，與膽酸代謝合成有關之3alphaHSD及kan-1/rBAT的表現亦隨之下降，這顯示蛋白激酶C同功酶alpha會調控3alphaHSD及kan-1/rBAT的表現。故我們推測蛋白激酶C同功酶alpha經由調控kan-1/rBAT及3alphaHSD之表現而導致敗血症肝內膽汁鬱積 (intra-hepatic cholestasis) 之發生。
綜合上述之研究結果，我們得知在敗血症肝臟衰竭時，蛋白激酶C同功酶alpha的減少會促使細胞凋亡，可能是透過抑制CREB的磷酸化及影響下游基因Bcl-2的表現；此外，蛋白激酶C同功酶alpha會調控kan-1/rBAT及3alphaHSD的表現，故應與敗血症肝內膽汁鬱積之發生有關。此外，我們也發現5個未曾被前人提及與敗血症相關之基因，有待繼續的研發，期能對瞭解敗血症肝臟衰竭之病理機制有所貢獻。

關鍵字

敗血症 ； 蛋白激酶C同功酶alpha ； 肝衰竭

並列摘要

Liver is recognized as a key organ in the initiation and/or promotion of multiple organ failure during sepsis for its major role in regulation of the immune, anti-inflammatory, and metabolic responses. Hepatic failure during sepsis may be caused by cholestasis, apoptosis, ATP-deprivation, etc. However, the underlying molecular mechanisms for heaptic failure during sepsis have not yet been fully understood.
Protein kinase C (PKC) has been implicated to play an important role in intracellular signal transduction and cell survival. In addition, inhibition of PKCalpha is accompanied by the increase of apoptosis in various cancer cells. Our previous studies showed that the cytosolic PKC activity in the liver was inactivated and apoptosis also occurred during late sepsis. Moreover, we demonstrated that PKCalpha is the most dominant isoform, which decreased significantly during sepsis. Inactivation of PKC, especially PKCalpha, during sepsis may be crucial in exploring the mechanism of hepatic failure in sepsis. In addition to apoptosis, there are some other mechanisms involved in hepatic failure in sepsis. The nuclear PKCalpha?has been reported to directly phosphorylate a putative transcriptional factor, suggesting that PKCalpha may regulate gene expression through certain transcriptional factors. It prompts us we expect to find out the genes involved in the down-regulation of PKCalpha in hepatic failure during sepsis.
Because the apoptosis of hepatocytes was observed during the progression of sepsis, the correlation between PKC? and apoptosis in hepatic failure during sepsis was investigated. The results showed that (1) the expression of membrane-associated and cytosolic PKCalpha was significantly decreased at both early and late sepsis; (2) the binding activity and expression of phospho-CREB were decreased in early and late phase of sepsis; and (3) the decrease of the Bcl-2/Bax ratio associated with significant increase of apoptotic incidence was observed at both early and late sepsis. Accordingly, the decrease of PKCalpha maybe involved in the phosphorylation of CREB and the Bcl-2 expression during sepsis and coincides with the appearance of apoptotic cell death.
To determine whether and how PKCalpha-inactivation mediated the hepatic apoptosis, the rat PKCalpha antisense plasmid (pAS-PKCalpha) was constructed and transfected into Clone-9 rat hepatocytes to suppress the PKCalpha gene expression per se. The results showed that the transfection of antisense PKCalpha lead to (1) a decrease of the cellular contents of PKCalpha; (2) a significant reduction in binding activity and expression of phospho-CREB; (3) a decrease of Bcl-2 mRNA and protein levels; (4) a decrease of Bcl-2/Bax protein expression; and (5) an increase of the apoptotic cell death and PARP cleavage. These results suggested that the decrease of PKCalpha may suppress the DNA-binding activity of CREB and the Bcl-2 expression through, which lead to the apoptosis of hepatocytes.
To further explore the genes involved in the down-regulation of PKCalpha in hepatic failure during sepsis, the differentially expressed genes in the late septic livers were identified by suppression subtractive hybridization (SSH) and reconfirmed by Northern blot analysis. Three up-regulated genes (TII-kininogen, Spi2.2, and alpha2M) and 6 down-regulated genes (3alphaHSD, EST189895/mouse RNase4, kan-1/rBAT, IF1, albumin, and alpha2u-G PGCL1), which may play a role in the fatal consequence of the late septic shock, have been identified. Analysis of their gene expression profiles in the late septic liver indicated that the pathophysiology for liver failure may include a disrupted metabolism and synthesis of bile acid and abolishment of ATP production. To study the role of PKCalpha in gene transcription, the levels of differentially expressed genes during sepsis were further analyzed in pAS-PKCalpha stable clone cells by RT-PCR. The results showed that the suppression of PKCalpha resulted in the decreases of 3alphaHSD and kan-1/rBAT, which have been indicated to be responsible for bile acid synthesis and metabolism. These findings suggested that PKCalpha suppression may reduce the expression of 3alphaHSD and kan-1/rBAT. However, the causal relationship between PKCalpha and bile acid metabolism needs further investigation.
In conclusion, the present studies provide evidence that PKCalpha plays important roles in both intrahepatic cholestasis and apoptosis during sepsis. Therefore, PKCalpha may be a therapeutic target for prevention of hepatic failure during sepsis. Besides, further studies of the 5 differentially expressed genes 3alphaHSD, EST189895/mouse RNase4, IF1, alpha2u-G PGCL1, and TII-kininogen, which have not been reported in sepsis, may shed light into the understanding of the pathogenesis of hepatic failure.

並列關鍵字

sepsis ； PKC alpha ； hepatic failure

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