探討NRIP在骨骼肌再生扮演的角色

Abstract

核受體交互作用蛋白(Nuclear receptor interaction protein, NRIP)，是一種可以藉由Ca2+ 和攜鈣素 (calmodulin, CaM)結合的蛋白質，其可以活化下游蛋白鈣調磷酸酶( calcineurin)，進而活化calcineurin-NFAT 和 CaMKII 訊號傳遞路徑，使得特定基因可以在肌肉修復時表現。此外，我們實驗室之前發現骨骼肌中含有大量的NRIP 蛋白，因此我們生產出NRIP 剔除小鼠來進行實驗;目前已知缺乏NRIP蛋白會延遲肌肉修復的速度。 因此，我們假設NRIP可能會藉由活化下游的calcineurin-NFAT 和 CaMKII 訊號傳遞路徑，使得衛星細胞(satellite cell)活化，參與肌肉損傷修復。衛星細胞是一種肌肉前驅細胞，在肌肉發生損傷時會被活化來修復受損的區域。在一般狀態下，大部分的衛星細胞會處於靜置時期，但是當肌肉發損傷時，這群靜置的細胞就會被活化並且進行增生，之後移動到受傷區域；接著這群細胞會分化成成肌細胞(myoblast)，最終變成肌小管(myotube)，並且合併到舊的肌纖維裡完成整個肌肉修復。在本次實驗中，我們將探討NRIP是否會影響衛星細胞的活化、增生和分化，因此我們檢視了MyoD和Pax7這兩個衛星細胞的指標蛋白，探討其在正常小鼠和NRIP敲除小鼠有何差異。由免疫螢光染色的實驗結果得知，NRIP敲除小鼠Pax7+細胞的數量在損傷後第三天較正常小鼠要來得少;此外正常小鼠的Pax7+BrdU+ 細胞也較NRIP敲除小鼠多。有鑑於衛星細胞會同時表現Pax7和MyoD兩種蛋白，我們也想探討缺乏NRIP蛋白是否亦會影響MyoD蛋白表現。實驗結果顯示在正常小鼠上的MyoD 和MyoD+BrdU+ 細胞在損傷後第三天都會增加，並且在損傷後第六天下降;然而，NRIP敲除小鼠的細胞則是到了損傷後第六天才增加;此外，正常小鼠的MyoD+Pax7+ 細胞也較NRIP敲除小鼠高。一言以蔽之，缺乏NRIP蛋白會影響衛星細胞的Pax7和MyoD蛋白表現。藉由西方點墨法 (Western blot)的實驗結果使我們得知NRIP蛋白的表現量會在損傷後第三天開始攀升並在第六天達到高峰，之後逐漸下降。此外，正常小鼠Pax7和MyoD兩種蛋白的表現量會在損傷後第三天達到峰值，之後逐漸回復水平;然而，NRIP敲除小鼠的蛋白表現則是到了第六天才達到頂峰。藉由所有實驗結果可得知，缺乏NRIP蛋白會延遲整個肌肉修復的進行。

NRIP (nuclear receptor interaction protein) is a Ca2+ dependent calmodulin binding protein that can activate its downstream protein, calcineurin. Due to calmodulin can activate calcineurin-NFAT and CaMKII pathways those involve in muscle specific gene expression during muscle regeneration. Besides our previous finding demonstrates that NRIP protein is abundant in skeletal muscle. We therefore generated NRIP conventional knock out mice; and currently characterized that NRIP deficiency would delay muscle regeneration. Therefore, we hypothesize that NRIP may involve in muscle regeneration through activating downstream calcineurin-NFAT pathway or CaMKII to activate satellite cells. Satellite cell is a kind of muscle progenitor cell that would be activated by muscle injury to repair the injury lesions. In resting muscle, the majority of satellite cells are quiescent, but when the muscle injury is occurred, the quiescent satellite cells will be activated and proliferated, and migrate to the injury lesions. Then these cells will be differentiated into myoblast to become myotube. Finally, they will incorporate into the pre-existing myofiber, or fused together to finish the muscle repair. In this study, we focus on whether NRIP would affect satellite cell activation, proliferation and differentiation. Therefore, we examined MyoD and Pax7, two satellite cell markers between WT and NRIP KO mice. First we demonstrate the amount of Pax7 cells would be reduced in NRIP KO mice at day 3 post injury by immunofluorescence assay to compare with WT. The amount of Pax7+BrdU+ cells is higher in WT mice compared with NRIP KO mice. Since satellite cell would coexpress Pax7 and MyoD, we tend to investigate whether NRIP deficiency would also have effect on MyoD expression. Both the number MyoD and MyoD+BrdU+ cells in WT mice are enhanced at day 3 and then fell down at day 6. However, NRIP KO mice is increased at day 6 post injury. Besides, the amount of MyoD+Pax7+ cells in WT mice is higher than NRIP KO mice. In sum, NRIP deficiency would affect Pax7 and MyoD expression in satellite cell; the result of western blot further support that NRIP expression level would increase at day 3 and reach to the peak at day 6 post injury, then gradually fell down. In addition, The Pax7 and MyoD expression in WT mice would get to the maximum at day 3, than gradually return to normal level. However, it is until day 6 that the Pax7 and MyoD expression level reach to the maximum in NRIP KO mice. Taken together, NRIP deficiency would postpone the speed of muscle regeneration.