利用質譜技術研究毛囊幹細胞的磷酸化蛋白質體

Abstract

對再生醫學而言，如何調控器官其內的幹細胞以維持器官的恆定或是快速修復受損的組織，是非常重要的課題。成體幹細胞廣泛分佈人體各個器官，這些幹細胞往往分佈於組織器官的某個特定環境或部位(niche)，以利接受外來環境所給予的訊號並做出反應。目前已知同一個器官內的幹細胞，可以是具備不同的性質，根據組織恆定或受傷後再生修補的需要，做出適當的反應。毛囊是一個迷你器官，在一生中不斷週期性再生，目前已知其內具有性質不同的幹細胞且具有其特殊的活化及再生能力。裝備態幹細胞(primed stem cell)以及靜態幹細胞(quiescent stem cell) ，分別座落於毛囊內的次級毛胚(secondary hair germ)及突部(bulge)。相較於靜態幹細胞，裝備態幹細胞較容易快速活化，以加速毛囊再生，並負責分化出生長期毛囊的結構。而靜態幹細胞則較不容易活化及分化，主要負責維持幹細胞數量上的恆定。這樣的特性使毛囊成為研究不同族群幹細胞調控的重要模型。而蛋白質分子的磷酸化是調控細胞活性的重要途徑，這種磷酸化的過程(phosphorylation cascade)最後將會影響到細胞核內表觀遺傳學(epigenetics)或是轉錄(transcription)上的變化。 在我們的研究當中，我們先利用毛囊幹細胞中特異性表現之表面抗原分選並純化出裝備態幹細胞以及靜態幹細胞，再透過蛋白質譜與磷酸化蛋白質譜的方法分析在休止期和生長期早期的毛囊幹細胞的表現量與磷酸化程度。透過這些蛋白質分子表現量與磷酸化程度之差異，我們可以得知這兩群細胞在休止及活化時，是否因其訊息調控的不同，而造成這兩群細胞在行為上的差異。我們發現裝備態幹細胞在從休止期到活化期的過程當中，蛋白質表現量與磷酸化差異的程度比靜態幹細胞大。藉由分析這些磷酸化分子之間的交互作用，我們發現促使毛囊生長的Wnt signaling其下游分子與mTORC1之間可能有所關聯。而從組織染色上來看，mTORC1的活化與毛囊幹細胞的動態在時間上之重疊性非常的高。因此我們假設mTORC1可能是調控毛囊幹細胞活化的重要途徑。 為了證實mTORC1在毛囊幹細胞活化之角色，我們利用mTORC1的小分子抑制劑進行功能性的分析，結果發現在毛囊幹細胞活化前抑制mTORC1的活性會延後毛囊幹細胞活化的時程，若是在毛囊幹細胞活化了之後再給予mTORC1的抑制劑則不會對於毛髮的生長產生結構性的影響。利用基因剃除鼠進行功能性分析時也可以得到延遲毛囊幹細胞活化的效果。因此我們證實mTORC1在活化毛囊幹細胞扮演一個很重要的角色，讓毛囊幹細胞可以在適當的時間上活化。這個功能性的分析也同時證實了磷酸化蛋白體可以為幹細胞研究提供更多有用的資訊。 雖然mTORC1並不會抑制進入生長期的毛髮生長，但我們發現mTORC1的抑制會增強γ射線對生長期毛囊所造成的輻射傷害。使得生長期內的毛囊發生更大量的細胞凋亡，並且對於修復性的增生也有抑制的作用。在這雙重影響之下，造成毛囊分化結構的破壞以及生長期落髮。因此mTORC1在毛囊中扮演著非常重要的角色: 1. 調控毛囊幹細胞的活化 2. 調控細胞對於輻射傷害的修復。

How an organ regulates its stem cells to maintain homeostasis or to quickly regenerate damaged tissues is vital to the maintenance of organ functions. Adult stem cells are often located in designated niches and their behavior is tightly regulated by the niche environment. It has been demonstrated that stem cells of an organ are not identical and can be composed of heterogeneous cellular populations of cells to meet to specific needs for homeostasis maintenance or for repair of damaged tissues. Hair follicle, a miniorgan with life-long cyclic regeneration, provides an excellent model to study the behaviors of stem cells during tissue regeneration. Specifically, it harbors primed stem cells and quiescent stem cells in secondary hair germ and bulge, respectively. Compared with quiescent stem cells, primed stem cells are activated faster and they regenerate the most part of the growing follicles. In contrast, quiescent stem cells are shown to be activated slowly and are responsible for the long-term maintenance of stem cells. There properties make HF as an important model for investigating the regulation of HFSC heterogeneity. The modulation of instructive or repressive signals is depended on protein phosphorylation toward downstream targets, and the phosphorylation cascade would finally affect the epigenetics or transcription. In our study, we sorted out the pSCs and qSCs by the specific expressed surface marker by FACS. Then, with the help of mass spectrometry technique, we can analyze the degree of protein abundance and protein phosphorylation in different HFSC populations and in different hair cycle stages. Via the proteome and phosphoproteome analysis, we can reveal if the signaling molecules could lead to the difference in behavior during the activation of the two HFSCs population. We found that the difference in protein abundance and phosphorylation were dramatically increased in pSCs, and the downstream of wnt signaling might have the correlation with mTORC1 signaling. From histology, the activation of mTORC1 is correlated with the dynamic of activation of HFSCs. Thus, we hypothesize mTORC1 might play an important role in regulating the activation of HFSCs. To address the possible role of mTORC1 in HFSCs, we use the mTORC1 inhibitor for further investigation. Activation of HFSCs could be hampered and delayed with mTORC1 inhibition. Once HFSCs were activated and the HF entered the growing phase, the mTORC1 inhibitor was unable to affect hair growth in this stage. The genetic ablation of raptor gene yielded to similar results. Thus, we can concluded that mTORC1 has an influence on activation of HFSCs in a timely dependent manner. Besides, our data showed that the proteome and phosphoproteome analysis could shed light on further stem cell research. Although the inactivation of mTORC1 did not perturb the hair growth in anagen, but we found that the mTORC1 inhibition increased the sensitivity of damage form r-ray irradiation, leading to more extensive apoptosis and less regenerative proliferation. Therefore, mTORC1 suppression aggravated radiation-induced anagen effluvium. Taken together, our study revealed that mTORC1 plays an important role in activating HFSCs and attenuating irradiation-induced follicular damage.