- 學位論文
NRIP調控小鼠骨骼肌功能，運動神經存活以及前列腺發育之研究
NRIP regulates skeletal muscle functions, motor neuron survival and prostate development
摘要
第一部分: 核受體結合蛋白 (Nuclear receptor interaction protein, NRIP)，也可稱作DCAF6及IQWD1，是一個鈣離子 (Calcium) 依賴鈣素 (calmodulin) 結合蛋白。本篇研究中，我們發現 NRIP 是一個新發現的骨骼肌Z-盤(Z-disc)蛋白。經由NRIP基因剔除鼠的產生，我們發現在缺少NRIP後，小鼠會呈現肌力降低，肌肉易疲乏及被動式運動能力降低之性狀。NRIP調控肌肉收縮之機轉主要是藉由位在鈣離子下游之NRIP與鈣素結合後，活化了鈣調神經磷酸酶 (calineurin) 及鈣調蛋白依賴性蛋白質激酶第二型 (calmodulin-dependent protein kinase II ,CaMKII)，進而誘導粒線體活性及慢速肌凝蛋白(slow myosin)的產生，也穩定肌漿網 (sarcoplasmic reticulum, SR) 內鈣離子存量之恆定。另外，NRIP基因剔除鼠也表現出肌肉再生延遲之現象。在肌肉受損後 NRIP 的表現量會上升，NRIP的表現上升與肌細胞生成素 (myogenin)，肌間線蛋白 (desmin) ，胚胎肌球蛋白重鏈 (embryonic myosin heavy chain, eMyHC) 的表現量上升及肌纖維生成有關。總結，NRIP 是一個新發現的骨骼肌Z-盤 (Z-disc) 蛋白，對肌肉力量的產生及肌肉再生是重要的。 第二部分: 先前結果顯示 NRIP 可透過鈣離子鈣素 (Ca2+/CaM) 之訊息傳遞，進而調控骨骼肌之收縮。在元駿論文中，他發現 NRIP 可維持運動神經元之存活，在冠渝論文中，她進一步利用肌肉專一 NRIP 基因剔除鼠證明運動神經元的退化是因為肌肉缺乏NRIP 蛋白所導致。在本研究中，我們對於NRIP 調控運動神經元存活之機制感到興趣。之前我們已證明，NRIP 基因剔除鼠再生肌肉的肌細胞生成素(myogenin)表現量降低。因此我們推測 NRIP 藉由活化myogenin的表現來維持運動神經元之存活。在基因表現晶片的結果中我們發現，NRIP 缺失再生肌細胞的肌細胞生成素基因的表現下降。在 C2C12 細胞分化過程中，NRIP 和 myogenin 的表現會同時上升並表現在細胞核中。進一步，在 C2C12 細胞分化過程中將 NRIP敲落後，肌纖維的生成與 myogenin 的表現會下降。反之，將 NRIP 大量表現在 C2C12 細胞後，myogenin 的表現會上降。總括上述結果，myogenin 的表現可能需要 NRIP的表現。運動神經元與相連接之肌肉細胞可互相維持其結構及功能之完整，因此，藉由建立治療肌肉退化之藥物篩選模式，對搜尋可能治療肌肉萎縮與肌肉萎縮所導致之神經退化有幫助。我們藉由 CRISPR 方法建構了 NRIP 基因剔除 C2C12 細胞，此細胞具與NRIP敲落C2C12細胞一樣的表現出肌纖維生成能力喪失。因此，NRIP 基因剔除之 C2C12 細胞可能可做為篩選治療肌肉退化藥物之工具。 第三部分: 我們先前已經證明了 NRIP 是一個轉錄輔助因子,可促進雄激素受體 (Androgen receptor, AR) 驅動基因的表現,並且保護 AR 蛋白質使更加穩定。前列腺是一個AR 所影響的器官。因此,在本研究中我們利用 NRIP 基因剃除鼠探討 NRIP 在前列腺發育過程中所扮演的角色。我們的結果發現,在 8 週大的 NRIP 基因剃除鼠的 anterior prostate (AP) 有發育遲緩現象,然而在 ventral prostate (VP) 及 dorsolateral prostate (DLP) 卻沒有此現象。並且AP在 12 週後其發育程度就與正常鼠相同。由於 NRIP 可影響 AR 蛋白分解，因此在 8 周大 NRIP 基因剔除鼠中 AR 的表現量降低到使 AP 無法形成分支。 然而，VP 與 DLP 則可維持一定量的 AR 使其正常發育。我們也發現，在發育遲緩的 AP，K5, K8, 及TUNEL 的表現量增加，顯現出此時期的 AP 正走向分化。 此外, DDB2 是一個 AR 結合蛋白,並且可藉由 CUL4A-DDB1 E3 泛素接連酶複合物分解 AR。 NRIP 像 DDB2 一樣是一個 DCAF 蛋白，擁有可與 DDB1 結合的 DxR 序列。因此NRIP 可能藉由與 DDB2 競爭與 AR 的結合，使得 AR 不被分解。總結，NRIP 可穩定 AR 蛋白質並且在前列腺發育扮演角色。
並列摘要
Part 1: Nuclear receptor interaction protein (NRIP, also known as DCAF6 and IQWD1) is a calcium-dependent calmodulin binding protein (Ca2+/CaM). In this study, we found that NRIP is a novel Z-disc protein in skeletal muscle. NRIP knockout mice (NRIP KO) were generated and found to have reduced muscle strength, susceptibility to fatigue and impaired adaptive exercise performance. The mechanisms of NRIP-regulated muscle contraction depend on NRIP being downstream of calcium signaling, where it stimulates activation of both calcineurin-nuclear factor of activated T-cells, cytoplasmic 1 (CaN-NFATc1) and calmodulin-dependent protein kinase II (CaMKII) through interaction with CaM, resulting in the induction of slow myosin gene expression and mitochondrial activity, and balancing of Ca2+ homeostasis of the internally stored Ca2+ of the sarcoplasmic reticulum. Moreover, NRIP KO mice have delayed regenerative capacity. The amount of NRIP can be enhanced after muscle injury and is responsible for muscle regeneration, coupled with the increased expression of myogenin, desmin and embryonic myosin heavy chain for myogenesis, as well as myotube formation. In conclusion, NRIP is a novel Z-disc protein important for skeletal muscle strength and regenerative capacity. Part 2: Previous results showed that NRIP activates Ca2+/CaM signaling to regulate muscle contraction. In Yuan-Chun thesis, NRIP can support motor-neuron survival. In Kuan-Yu thesis, the muscle-specific NRIP KO mice was generated and further demonstrated that motor neuron degeneration is resulted from NRIP-deleted skeletal muscle. Thereof in this study, we were interested in investigation of what is the mechanism of muscle NRIP-regulating motor neuron survival. We previously demonstrated the myogenin expression is dwonregulated in regenerative muscles of NRIP KO mice. Thus, we hypothesized that NRIP can support motor neuron survival through activating myogenin expression. In C2C12 cells, NRIP and myogenin expression were consistently upregulated during differentiation colocalized in neucleus of C2C12 myotubes. Furthermore, the expression of myogenin and myotube formation was disrupted in NRIP knockdown C2C12 during differentiation; in contrast, force expressing NRIP into C2C12 myoblasts could activate expression of myogenin. Taken together, NRIP may be required for myogenenin expression. Motor neurons and skeletal muscles are requiring each other for maintaining normal structure and functions. To establish drug screening for muscle degeneration could be help for searching the potential therapeutic factors either to muscle dysfunction or muscle-derived motor neuron degeneration. We generated the NRIP KO C2C12 myoblasts by RNA-guided clustered, regularly interspaced, short palindromic repeat (CRISPR)-mediated method. The myogenesis was disrupted in NRIP KO C2C12 myoblasts that is also observed in NRIP silenced C2C12 myoblasts. Thus, the NRIP-deleted C2C12 myoblasts could be an in vitro model for searching the therapeutic factors on muscle degeneration. Part 3: Previously we demonstrated that NRIP is a transcription coactivator to enhance androgen receptor (AR)-driven gene expression and protects AR protein stability. The prostate development is dependent on AR expression. In this part, we investigate NRIP role in prostate development by NRIP KO mice. Our results show that anterior prostate (AP) of NRIP KO mice develops delay at 8-week but becomes normal at 12 -week in comparison with WT mice; but not ventral lateral prostate (VLP) and dorsal lateral prostate (DLP). Due to NRIP role for AR protein stability, the decreased AR level of AP in NRIP KO mice causes the delay branch formation at 8-week that may reach the threshold for branch differentiation. As to VLP and DLP of NRIP KO mice developed normal as wild type may come from the crucial amount AR in NRIP KO mice is enough for branch formation. K5, K8, TUNEL markers, and Ki67 are highly expressed in delayed-development of AP from NRIP KO mice at 8-week; indicating that the luminal glandular is processing to formation. Additionally, we previously found that DDB2 is AR-interacting protein and can degrade AR via CUL4A-DDB1 E3 ligase complex (Chang et al., 2012). NRIP, like DDB2, is a DCAF and therefore contains the classical double DxR box that is responsible for DDB1 binding to the CUL4A-DDB1 complex. We suggest NRIP protects AR stability by competing with DDB2 for AR binding, but not by competing with DDB1 for recruitment to the AR-DDB2-DDB1-CUL4A complex. In sum, NRIP can stabilize AR protein and play a role in prostate development.