摘要
我們實驗室發現了一個新的基因命名為核受體交互作用蛋白(Nuclear receptor interaction protein, NRIP)，發現其為一種男性荷爾蒙接受器(Androgen receptor, AR)和質固醇受體(Glucocorticoid receptor, GR)的互動蛋白。NRIP的基因表現會受到AR所調控，在功能上NRIP可加強AR下游所調控基因的表現，並增加AR蛋白質的穩定性。除此之外，NRIP亦是攜鈣素(calmodulin, CaM)的結合蛋白。在鈣離子存在時，NRIP可藉由它結構上的IQ 模組直接與CaM結合進而活化鈣調磷酸酶(calcineurin)。活化後的calcineurin會對下游的調控蛋白質NFAT (nuclear factor of activated T cells)進行去磷酸化,使NFAT進入細胞核內，開啟一系列氧化能力高的慢收縮肌纖維基因的表現。現今已有文獻證實細胞內的鈣離子增加會活化calcineurin-NFAT訊息傳遞路徑，促進肌凝蛋白重鏈(myosin heavy chain)亞型之間的基因表現由快縮肌轉變成慢縮肌; 然而，現今對於調控快縮肌的基因表現機轉尚未明瞭。先前實驗室發現NRIP基因在骨骼肌中大量表現，此外NRIP剔除鼠在滾輪測試和跑步機的運動試驗上表現能力下降。根據2006年的報導指出，臨床上利用微陣分析(microarray assay)發現肢帶型肌肉萎縮症患者(Limb Girdle Muscular Dystrophy, 簡稱LGMD)缺乏NRIP基因的表現。因此我們利用NRIP剔除鼠探討NRIP在骨骼肌功能中所扮演的角色，並進一步探討NRIP引起肌肉無力的機轉。 首先，我們證明NRIP和calcineurin共同位於橫紋肌肌節的Z盤(Z-disc)上。此外，在離體肌肉收縮實驗中，當存有神經肌肉阻斷劑箭毒素(d-Tubocurarine)的條件下，NRIP剔除的公鼠和母鼠其比目魚肌(soleus，屬慢縮肌)的肌肉收縮力較各自的正常鼠弱；並且在連續電擊刺激下，公和母的NRIP剔除鼠肌肉的耐受力較正常鼠差。這些結果表示NRIP剔除的公母鼠其肌肉在功能表現上有缺陷。除此之外，無論NRIP剔除與否，母鼠維持肌耐力的能力皆比公鼠高。 根據2008年Stupka等人的報導，活化calcineurin會使氧化能力高的慢縮肌表現上升，進而增加肌耐力。因此接著分析在剔除鼠中calcineurin活性與肌凝蛋白重鏈的組成。結果顯示NRIP剔除鼠中的calcineurin-NFAT訊息傳遞的報告基因modulatory calcineurin interacting protein 1.4 (MCIP1.4)和慢縮肌凝重鏈蛋白的表現皆下降。同時，藉由小干擾RNA (siRNA)在細胞中抑制NRIP的表現也進一步驗證在老鼠骨骼肌肉組織中觀察到的結果。因此NRIP可能藉由calcineurin-NFAT的訊息傳遞路徑而影響肌肉功能的表現。
並列摘要
NRIP, nuclear receptor interacting protein, is a novel gene that we identified previously. NRIP is a ligand-dependent coactivator of androgen receptor and glucocorticoid receptor. Also, NRIP is a Ca2+-dependent calmodulin binding protein (Ca+2/CaM), and it activates calcineurin phosphatase activity. Calcineurin is a serine/threonine protein phosphatase and localizes at sarcomeric Z-disc. The calcineurin-NFAT signaling pathway plays an essential role in fiber type transition promoting oxidative slow type myofibers expression in response to calcium waves. We previously found that the mRNA transcripts (Tsai et al., 2005) and protein expression of NRIP are abundant in skeletal muscle (Hsing-Hsung, thesis). Moreover, NRIP deficient mice exhibit decreased exercise performance on rotarod and treadmill tests (Hsing-Hsung thesis). Therefore, we hypothesize NRIP may play an important role in muscle strength and endurance performance. In this study, we first demonstrated NRIP co-localizes with calcineurin at sarcomeric Z-disc by immunofluorescence assay. Specifically, slow-twitch soleus muscles of male and female NRIP-null mice significantly display impaired muscle function by in vitro muscle contraction assay, which the results show: 1) a reduced contractile force output in the present of neuromuscular blocker d-Tubocurarine to exclude the nerve activity, and 2) lower fatigue resistance compared to their counterpart NRIP+/+ controls. However, female soleus muscles exhibit higher fatigue resistance compared to males regardless of NRIP expression, suggesting muscle fatigue is gender-related. Based on Stupka et al (2008), calcineurin activation enhances hindlimb muscles endurance by promoting slow oxidative phenotype. Hence, calcineurin activity and fiber type composition were further examined. NRIP-/- muscles show a decrease in mRNA expression of calcineurin-NFATc1 reporter gene, MCIP1.4, and a reduced number of type I slow myosin. To further verify whether NRIP modulates slow myosin expression, mouse C2C12 myoblasts with siNRIP knockdown confirmed a reduced expression of MCIP1.4 and type I slow myosin, indicating a downregulation of calcineurin- NFATc1 signaling in vivo. Taken together, these results suggest that NRIP involves in skeletal muscle exercise performance through regulation of calcineurin-NFAT signaling pathway.
參考文獻
延伸閱讀
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