半乳糖凝集素-3和-7於人類角質細胞移動中所扮演的角色

Abstract

半乳糖凝集素-3和-7是動物凝集素成員之一，共同具有可辨認beta型半乳糖修飾醣分子的醣類辨識區域(carbohydrate recognition domain, CRD)。許多研究指出半乳糖凝集素-3可參與細胞之間的交互作用、幫助格狀結構的形成、影響免疫細胞凋亡和分化過程、發炎反應以及腫瘤的發展，半乳糖凝集素-7的研究則著重於維持皮膚生理平衡的角色。先前我們實驗室發現，半乳糖凝集素-3可結合至一個蛋白質-Alix (ALG-2 linked protein-X)，Alix本身可以影響細胞凋亡、細胞膜內吞運輸和細胞骨架的重組，近期實驗結果更指出，半乳糖凝集素-3可以幫助Alix維持上皮細胞生長因子受體(epithelial growth factor receptor, EGFR)在細胞膜上的動態平衡和下游訊息傳遞，進而調控老鼠皮膚角質細胞的移動。 我的研究主題是探討半乳糖凝集素-3和-7於人類角質細胞移動的角色，和討論可能影響的細胞機制，內容包括半乳糖凝集素-3是否要透過影響Alix進而調控人類角質細胞的移動，以及半乳糖凝集素-7是否參與了組合蛋白(integrin)調控細胞移動的訊息傳遞。由實驗結果得知，半乳糖凝集素-3基因敲落和Alix基因敲落的人類角質細胞株-HaCaT細胞其傷口癒合能力均下降，並已證實是細胞移動性下降而非細胞增生或死亡因素所造成。在討論細胞移動中半乳糖凝集素-3和Alix相互關係時，發現部分半乳糖凝集素-3和Alix會聚集在移動細胞的前端，但不管是角質細胞在正常培養狀態抑或是脂多醣、alpha腫瘤壞死因子或gamma干擾素刺激下，都無法測到半乳糖凝集素-3和Alix的結合。藉由比較半乳糖凝集素-3和Alix共同敲落HaCaT細胞，其傷口癒合能力較單一基因敲落HaCaT細胞下降來得多，由此可推論在半乳糖凝集素-3和Alix協調人類角質細胞移動的方式也許不大相同。另外，我們也發現半乳糖凝集素-7基因敲落HaCaT細胞其移動性和傷口癒合能力下降，證實半乳糖凝集素-7會正向調控細胞的移動。而實驗室先前利用mRNA微陣列(microarray)技術，發現HaCaT細胞中許多基因的表現會受到半乳糖凝集素-7基因敲落的影響，其中包括許多組合蛋白，以及其下游調節與細胞移動相關的訊息分子。因此，在探討半乳糖凝集素-7基因敲落HaCaT細胞中組合蛋白調控細胞移動的訊息傳遞時，發現一些角質細胞主要表現的組合蛋白alpha2、alpha3、alpha6、beta4和beta6的mRNA有高量表現。在組合蛋白下游訊息傳遞分子中，儘管焦點黏結酵素(focal adhesion kinase)的早期活化並無受到影響，而vinculin做為焦點黏結的重要組成分子之一，其表現量也未受到影響，但我們發現，Rho結合酵素ROCK-1和ROCK-2在半乳糖凝集素-7基因敲落HaCaT細胞中有較高的蛋白質表現量。 總結我們在探討人類角質細胞中半乳糖凝集素-3和-7角色的實驗結果，發現內源性半乳糖凝集素-3確實可幫助人類角質細胞的移動，但不一定需要透過Alix的幫助；另外，內源性半乳糖凝集素-7也被證實可以幫助人類角質細胞的移動，且可能是透過影響組合蛋白的訊息傳遞中特定組合蛋白的表現，或是所引導的Rho酵素的表現，進而造成一個促進細胞移動的訊息。目前仍需更多實驗去探討半乳糖凝集素-7如何調控角質細胞的移動，我們希望透過此研究，能進一步了解半乳糖凝集素對於人類傷口癒合的影響。

Galectin-3 and galectin-7 are animal lectins that recognize beta-galactoside-containing glycoconjugates through their carbohydrate recognition domains (CRDs). Many studies have emphasized the multiple functions of galectin-3, including cell-cell interaction, lattice formation, apoptosis and differentiation of immune cells, inflammation, and tumor progression. The functions of galectin-7 are mainly emphasized in regulation of skin homeostasis. Our recent study showed galectin-3 modulates mouse keratinocytes migration by regulating epithelial growth factor receptor (EGFR) homeostasis through Alix (ALG-2 linked protein-X), which is involved in apoptosis, endocytic membrane trafficking, and cytoskeleton remodeling. In this study, we investigated the role of galectin-3 and galectin-7 in modulating human keratinocyte migration, and further examined the underlying mechanism, including whether Alix is indispensable in galectin-3-mediated cell migration and the involvement of galectin-7 in integrin-mediated migration. Our results suggested that both galectin-3-knockdown and Alix-knockdown HaCaT cells, a human keratinocyte cell line, have impaired wound healing abilities, which are verified to be a consequence of impaired single cell motility not proliferation rate. In the investigation of relationship between galectin-3 and Alix in regulating human keratinocyte migration, we found both galectin-3 and Alix can translocate to the leading edge of migrating human keratinocytes, but the interaction between galectin-3 and Alix could not be detected under both resting condition and LPS, TNF-alpha, or IFN-gamma stimulation. By comparison of the wound healing ability, galectin-3-Alix double knockdown HaCaT cells were found to have more impaired cell motilities compared with galectin-3-knockdown and Alix-knockdown HaCaT cells. Thus, galectin-3 may mediate human keratinocyte cell migration through an Alix-independent pathway. On the other hand, the positive role of galectin-7 in human keratinocyte migration was also confirmed by impaired wound-healing abilities and single cell motilities of galectin-7-knockdown HaCaT cells. According to the microarray data, expression of many genes was found to be changed after galectin-7 knockdown, including integrins and related downstream signal molecules. Therefore, we further examined the integrin-related cell migration in galecitn-7-knockdown HaCaT cells. Among the abundantly expressed integrins in keratinocytes, the amount of alpha2, alpha3, alpha6, beta4, and beta6 subunit mRNA transcripts are higher in galectin-7-knockdown HaCaT cells. In integrin downstream signaling, the early activation of focal adhesion kinase is not affected in galectin-7-knockdown HaCaT cells, so is the protein expression of focal contact component, vinculin. However, Rho-kinases, ROCK-1 and ROCK-2, protein expression are higher in galectin-7-knockdown HaCaT cells. In summary, endogenous galectin-3 has a pro-migratory effect in human keratinocytes and Alix may not be the major cellular effector of galectin-3-mediated human keratinocyte migration. Endogenous galectin-7 also has a pro-migratory effect in human keratinocytes. In integrin-related cell migration, galectin-7 can regulate certain integrins and Rho kinases expression, which may contribute to the pro-migratory function of galectin-7 in human keratinocytes. Additional studies are required to elucidate how endogenous galecitn-7 regulates keratinocyte migration, which may also hlp us understand its role in human wound repair.