前言︰內質網在細胞內部有合成及修飾分泌型蛋白及儲存大量鈣離子，並且利用鈣離子的調控來調整細胞功能。當內質網功能受到干擾時，內質網會產生壓力 (ER stress) 反應。Calpain 是中性蛋白?，由鈣離子濃度調控，當鈣離子失衡時會被活化。
研究目的：我們利用ER stress藥物 tunicamycin (TM)、thapsigargin (TG) 和會引發鈣離子上升的藥物ionomycin (Iono)來探討內質網壓力及鈣離子對calpain表現量的影響，以及其在內質網壓力所誘發細胞凋亡的角色。
實驗方法︰以RT-PCR分析ER stress相關基因及calpain mRNA表現；利用蛋白質電泳分析calpain蛋白表現量及Bid, pro-caspase 3, PARP切割；利用胞器分離技術分離細胞質、細胞核、粒線體分析calpain在細胞內分佈的情形; 利用染色體DNA萃取技術及洋菜膠水平電泳分析染色體DNA片段化的情形。
結果：TG及TM誘發ER stress 相關基因CHOP和Bip。calpain 1基礎表現量高，且表現不受藥物影響；calpain 2的基礎表現低，可被TG及TM所誘發。Iono並不會誘發calpain 2上升但其可加強TM所誘發的calpain 2。TM可抑制TPA誘發HL-60分化且導致calpain 2上升。Cycloheximide (CHX，抑制轉譯作用) 可抑制TG所誘發的calpain 2，但CHOP不受影響。藥物所誘發的calpain 2主要分佈在細胞質。TAT-BH4 (保護粒線體) 和PD150606 (calpain 抑制劑) 可降低TG所誘發的apoptosis現象，PD150606並可降低Bid的切割。
結論：本篇研究發現在HL-60細胞有內質網壓力加上細胞質鈣離子濃度增加時calpain 2會大量上升，此過程需要新生的蛋白作為轉錄因子，calpain 2可能參與ER的重組幫助細胞存活；或是經由切割Bid啟動粒線體的細胞凋亡路徑。未來可以研究calpain 2的基因調控區，以及探討calpain 2 與自體免疫疾病 (如僵直性脊椎炎、紅斑性狼瘡) 的相關性。
Background: ER (endoplasmic reticulum) is an organelle for the synthesis and modification of secretory proteins, as well as storage of calcium which plays important roles in the regulation of cellular function. Interference of ER functions may casuse unfolded protein responses (UPRs). Calpains are calcium-activated neutral protease and it may be induced by increased cytosolic [Ca2+].
Aims of study： By using ER stress drugs tunicamycin (TM) and thapsigargin (TG) and calcium ionophore ionomycin (Iono) we investigated the effects of ER stresses on calpain expression in HL-60 cells, and studied the role of calpain in ER stress-induced apoptosis.
Methods: RT-PCR was used to analyze mRNA expression of ER stress-associated genes and calpains. Western blot was used to analyze calpain, and cleavage of Bid, pro-caspase 3 and PARP. The distribution of calpain was analyzed by Western blot using cytosolic, nuclear and mitochondrial fractions of protein extracts. DNA fragmentation was detected by agarose electrophoresis.
Results: TG and TM induced ER stress-related CHOP and Bip expression. Calpain 1 was constitutively expressed and its expression was not affected by these drugs. Expression of calpain 2 was low at basal condition and was highly induced by TG and TM. Iono alone did not affect expression of calpain 2, but it enhanced that induced by TM. TPA-induced HL-60 differentiation was suppressed by the pretreatment of TM, while calpain 2 expression was induced. The protein synthesis inhibitor cycloheximide (CHX) suppressed drug-induced calpain 2 but not that of CHOP. Induced calpain 2 protein was mainly distributed at cytosolic fraction. TAT-BH4 (a protective peptide for mitochondria) and PD150606 (a calpain inhibitor) suppressed TG-induced apoptosis. PD150606 also suppressed TG-induced cleavage of Bid.
Conclusions: In this study we found that calpain 2 is induced by ER stress combined with increased cytosolic [Ca2+]. Its induction is dependent on de novo synthesized protein(s) and this protease may play roles in the reorganization of ER for cellular survival or apoptosis. Activation of calpain 2 causes cleavage of Bid, which elicits mitochondrial apoptotic pathway. The transcriptional regulation of this gene and its expression in autoimmune diseases such as ankylosing spondylitis (AS) and systemic lupus erythematosus (SLE) await further study.
