Abstract
摘要:硫酸乙醯肝素醣胺聚醣乃是由尿羰基酸與葡萄糖胺所構成的長鏈聚合多醣,常見於細胞外基質及細胞表面之蛋白聚醣。硫酸乙醯肝素醣胺聚醣能夠調節多種細胞訊息,如Wnt、BMP、FGF、SDF-1 等細胞外分子之濃度梯度、協助配體與受體結合以及細胞內吞作用等。無論是將硫酸乙醯肝素醣胺聚醣合成的相關酵素剔除,或是過量表現硫酸乙醯肝素聚醣蛋白,都會干擾 Wnt 及 BMP 訊號的正常傳遞,表示細胞表面需要適量的硫酸乙醯肝素醣胺聚醣,Wnt 及 BMP 才能夠正常的與受體結合。此外,BMP 受體的內吞作用,也需要細胞表面的硫酸乙醯肝素醣胺聚醣。最近的研究指出,間質幹細胞的自我更新及分化等現象,受到 Wnt 及 BMP 的調節,而 SDF-1 則對間質幹細胞有強烈的趨化作用。由於細胞外微環境對於幹細胞的自我更新及分化具有十分重要的生理意義,而硫酸乙醯肝素醣胺聚醣則是常見的胞外基質成分,因此本計畫欲針對硫酸乙醯肝素醣胺聚醣對於間質幹細胞的自我更新以及分化作用的影響進行研究。
除了調節可溶性分子的訊息傳遞之外,也有證據表明,硫酸乙醯肝素聚醣蛋白會經由內吞作用進入細胞,然後被傳遞至細胞核內。細胞核內之硫酸乙醯肝素聚醣蛋白,會與DNA拓撲異構酶以及組蛋白乙醯轉移酵素產生交互作用而影響染色質之結構。因此,硫酸乙醯肝素聚醣蛋白也有可能透過此途徑,影響間質幹細胞的分化。
在之前的研究中,我們證明,脂締素可以藉由刺激脂肪細胞分化相關的轉錄因子(C/EBPα, PGC1α) 表現,進而提高骨髓間質幹細胞分化為脂肪細胞的效率。有研究指出,脂締素會與 SDF-1 結合,而且其結合位與硫酸乙醯肝素醣胺聚醣在 SDF-1 上的結合位重合。這個結果暗示脂締素可能與 SDF-1 及硫酸乙醯肝素醣胺聚醣發生交互作用,進而對間質幹細胞產生調節作用。
本計劃有以下三項重要目標:(1)硫酸乙醯肝素醣胺聚醣是否會透過其對胞外可溶性分子之作用,而對於間質幹細胞的自我更新以及分化產生影響;(2)硫酸乙醯肝素聚醣蛋白是否會透過內吞作用進入間質幹細胞的細胞核中,並藉此影響間質幹細胞的染色質結構;(3)硫酸乙醯肝素醣胺聚醣是否會與脂締素共同對間質幹細胞進行調節。本研究之結果,不但可以讓我們對幹細胞微環境對於幹細胞生理的調節作用有更多的了解,也能作為開發間質幹細胞組織工程技術的線索。
Abstract: Heparan sulfate glycosaminoglycans (HS-GAGs) are long, unbranched sulfated sugar chains covalently attached to the core proteins of heparan sulfate proteoglycans (HSPGs). HSPGs are abundantly existed in extracellular matrix (ECM) and cell surface and are able to mediate signaling of morphogens via HS-GAGs dependent gradient formation, ligand-receptor binding and competition and endocytosis. Recent studies showed that the lineage commitment and self-renewal of mesenchymal stem cells (MSCs) are largely dependent on Wnt and BMP signaling, while SDF-1 plays an important role in homing of MSCs. HS-GAGs are known to interact with various soluble factors such as Fgf, Wnt, BMP and SDF-1. The ligands of Wnt and BMP require appropriate amount of cell surface HS-GAGs to be able to present to the receptors. Knocking down the HS-GAGs synthesis enzymes perturbed both Wnt and BMP signaling, while overexpressed the core protein of the primary HSPGs also resulted in the insufficient signaling. Moreover, HSPGs also modulate BMP signaling via HS-GAGs mediated ligand/receptor endocytosis. Since extracellular microenvironment is important for stem cell self-renewal and lineage commitment and HSPGs are important composition of ECM, it is of interest to investigate the interactive effects of HS-GAGs metabolism and fate determination of MSCs.
In addition to morphogen regulation, other evidence lines indicated that HSPGs as well as extracellular HS catabolism enzyme, heparanase (HPSE), can be sorted into nucleus after being endocytosed and may play a role in modulating cellular behavior via chromatin regulation. Several studies reported that nuclear localized HSPG can bind to DNA topoisomerase I and HS-GAGs can inhibit the activities of histone acetyltransferase. The advancement in cell-free/serum-free culture condition implied that MSCs require basic fibroblast growth factor (bFGF) for self-renewal. HS-GAGs are necessary for the binding of bFGF and its receptor and may also contribute to bFGF nulear locolization of bFGF. Therefore, it is of interest to investigate the effects of nuclear localization of HS-GAGs in chromatin regulation of MSCs.
In the previous study, we demonstrated that adiponectin plays a role in adipogenic fate determination of MSCs by promoting adipocyte differentiation-related transcription factor (C/EBPα, PGC1α). Evidence showed that adiponectin share a common binding site on SDF-1 with HS-GAGs, implying a novel role of adiponectin in mediating MSCs behavior.
Here we proposed to investigate the roles of HS-GAGs in MSCs self-renewal and lineage commitment. In the first year, we will evaluate the effects of various HS moieties in MSCs proliferation and differentiation. In the second year, we will investigate the effects of nuclear localization of HS-GAGs/HSPGs during MSCs self-renewal and lineage commitment. In the third year, we will study the interactive effects of adiponectin and various HS-GAGs moieties in regulating MSCs fate and the potential implications of this network for understanding cellular aging. These results will lead us to know more about niche microenvironment for MSCs self-renewal and molecular machineries for MSCs lineage commitment through which we may develop novel strategies to modulate MSCs proliferation and differentiation in vivo.
Keyword(s)
硫酸乙醯肝素醣胺聚醣
硫酸乙醯肝素蛋白聚醣
骨髓間質幹細胞
脂締素
heparan sulfate glycosaminoglycans
heparan sulfate proteoglycan
bone marrow-derived mesenchymal stem cell
adiponectin