Abstract
血基質氧化酵素-1 (Heme oxygenase-1) 為血基質 (heme) 代謝上的關鍵酵素 (key enzyme), 同時也是細胞中的一種抗氧化分子 (antioxidant)。 先前的研究分別指出鎘 (cadmium) 會活化蛋白質激活酵素C(protein kinase C) 以及誘導血基質氧化酵素-1的表現, 但鎘處理所誘導血基質氧化酵素-1和鎘活化蛋白質激活酵素C之間的關係需要進一步的研究。 在本研究中發現在鎘處理的小鼠睪丸萊迪氏細胞中, 蛋白質激活酵素C的活性會隨著鎘劑量的增高而增加; 同時, 活化蛋白質激活酵素C會增加血基質氧化酵素的表現。 另外, 利用20-500 μM H-7 (蛋白質激活酵素C的抑制劑) 處理細胞5小時後, 鎘誘導的血基質氧化酵素-1的表現會被抑制。 但是鎘誘導的血基質氧化酵素的表現不會受到5小時 20-100μM的HA1004處理而受到抑制。 這些結果證實了在小鼠睪丸萊迪氏細胞中, 鎘所誘導的血基質氧化酵素-1的表現是受到蛋白質激活酵素C的活化所控制。 當細胞處理5小時的1 μg/mL actinomycin D 和 20μg/mLcycloheximide後, 鎘誘導的血基質氧化酵素-1的表現也會受到抑制, 這結果指出了鎘誘導的血基質氧化酵素-1的表現是被調控在基因轉錄的層次上。 進一步的研究中, 在無鈣 (不含血清) 的培養基中加入不同濃度的氯化鈣 (0-20 μM)來觀察鎘誘導的血基質氧化酵素-1的表現的情形, 結果發現鎘不能有效的誘導血基質氧化酵素-1的表現。 另外,細胞也用2μg/mL A23187 (鈣離子通道的通透劑) 處理1小時, 這會造成細胞內外鈣離子的流動, 但結果也顯示了改變細胞內外鈣離子濃度不會影響鎘誘導的血基質氧化酵素-1的表現。 這些的結果顯示了鎘誘導的血基質氧化酵素-1的表現可能是經由活化和鈣離子無關的蛋白激活酵素C (calcium-independent protein kinase C) 的路徑來達成。 最後, 我們發現當培養基中血清濃度為2.5%時, 血基質氧化酵素-1會受到40 μM氯化鎘的刺激而表現; 而且氯化鎘刺激血基質氧化酵素-1的表現是和血清濃度成正比的。,這些結果指出了, 血清為一種必需的因子, 而且包含在氯化鎘刺激血基質氧化酵素-1的路徑上。Heme oxygenase-1 (HO-1), which is involved in heme metabolism,plays a role of potent antioxidant. Since cadmium (Cd)administration was reported to activate protein kinase C (PKC)and HO-1 gene expression, the relationship between HO-1 geneexpression and PKC activation after Cd treatment needs to beexamined. In this study, we found that induction of HO-1protein accompanies PKC activation after CdCl2 treatment inmouse testicular Leydig TM3 cells. In addition, depression ofPKC activity by 20-500 μM H-7 for 5 h abolished Cd-inducedHO-1 gene expression. But Cd-induced HO-1 expression was notabolished by 20-100 μM HA1004 for 5 h. These resultssupported that Cd-stimulated HO-1 gene expression was drived byPKC activation in TM3 cells. Cd-induced HO-1 expression wasinhibited when cells were treated with 1 μg/mL actinomycin Dand 20 μg/mL cycloheximide for 5 h. The results indicatedthat stimulation of HO-1 expression by CdCl2 is controlledprimarily at the level of gene transcription. Furthermore,increase of extracellular calcium concentration by the additionof CaCl2 from 0 μM to 2 μM did not potentiate Cd-induced HO-1expression. In addition, cells pretreated with 2 μg/mL A23187for 1 h to induce calcium influx did not show any stumulatoryeffect on Cd-induced HO-1 expression. Those findings suggestthat stimulation of HO-1 expression by Cd may mediate bypathway of calcium-independent PKC pathway. In the last, HO-1expression was induced when cells were treated with 40 μMCdCl2 in medium of 2.5% serum. And the HO-1 induction by CdCl2was serum dose-dependent. The results were suggested thatserum was essential in Cd-induced HO-1 expression.