趨化激素CXCL14於乳腺上皮細胞之醣基化研究

The study of CXCL14 glycosylation in mammary epithelial cell

曾亭育(Ting-Yu Tseng)

指導教授 ： 姜延年 ； 朱有田

國立臺灣大學/生物資源暨農學院/動物科學技術學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.02754

摘要

乳腺組織隨著每一次的懷孕分娩過程而經歷細胞快速增生、分化、生長休止及細胞凋亡等過程，若此一平衡在發育過程遭受破壞可能造成乳腺發育異常，而導致乳癌的發生，故乳腺提供了我們一個良好的系統去研究細胞週期、細胞分化及癌症形成機制等模式。隨著泌乳的停止，退乳時期的乳腺上皮經歷了大規模的細胞凋亡，並且回復到近似於未懷孕時的狀態。而在此過程中，有許多重要的基因參與調控乳腺細胞凋亡或重建。利用RNA扣除法篩選小鼠退乳第4天及泌乳第15天的乳腺組織，發現數個有差異表現的基因，而趨化激素CXCL14即為其中一個，此結果與英國研究團隊利用微陣列方式所得到者相同。
CXCL14為CXC趨化激素的一員，先前研究發現其於B細胞和單核球的運輸扮演一重要角色，且CXCL14會過量表現於乳腺上皮細胞，並且與其接受器結合以促進細胞的增生。根據先前的發現，推測CXCL14可能在乳腺退乳過程中參與免疫調控及乳腺的重建，故本研究目的為探討CXCL14於乳腺上皮細胞中可能所扮演之角色。
為了得知CXCL14在乳腺上皮細胞表現後是否會經歷轉譯後修飾作用，首先由乳腺上皮細胞選殖到CXCL14的cDNA後再進一步架接至可於哺乳動物細胞表現的載體上，最後將其轉染至乳腺上皮細胞中，分別抽取細胞內蛋白質並收集細胞培養液再以西方吸漬分析，均可在約16 kDa的位置偵測到與預期分子量大小相符的重組CXCL14蛋白質。此外，在接近20 kDa的位置也偵測到一片段，顯示CXCL14可能在分泌至細胞外的過程中被修飾。為了確認此修飾作用是否為醣基化，使用O-linked抑制劑BAG處理已轉染重組CXCL14的乳腺上皮細胞，相較於未處理的組別，其分子量較大的訊號確實有減弱的現象。在O-linked醣基化中，O-glycan會與serine或threonine的hydroxyl group連接，因此為了更進一步研究CXCL14蛋白質序列上可能被醣基化的位置，利用點突變CXCL14的方式來進行確認。將CXCL14蛋白質序列上的serine或threonine突變成為alanine後，得到六種突變的CXCL14。再分別轉染至乳腺上皮細胞後，發現在T59A/T60A-CXCL14及S79A/T80A-CXCL14兩個突變型相較於野生型的CXCL14，其分子量較大的訊號減弱的現象相似於經由BAG處理過後的情形。為了能夠進一步瞭解CXCL14於乳腺上皮細胞所扮演的角色，建立了一個可表達重組CXCL14的腺病毒表現系統，所得到的病毒液均經由Plaque Assay測定力價，並且能夠成功地感染乳腺上皮細胞。
由以上結果可得知，推測CXCL14為一醣蛋白，並且在其T59/T60和S79/T80可能為其醣基化的位置。除此之外，本研究所建立的腺病毒表現系統在未來能夠使用於探討CXCL14於乳腺發育過程中的分子機制。

關鍵字

趨化激素CXCL14 ； 乳腺上皮細胞 ； 醣基化

並列摘要

Mammary epithelial cells periodically undergo proliferation, differentiation, growth arrest, and apoptosis during each pregnancy-weaning cycle. The abnormal in controlling cell proliferation and apoptosis of developing mammary gland might result in breast tumor formation. Hence, the mammary gland provides an ideal model system for studying the molecular mechanisms of cell cycle progression, differentiation and tumorigenesis. Following the cessation of lactation, the mammary gland undergoes apoptosis on an extensive scale, leading to a remodelled gland that resembles that of a nulliparous mammary gland. Several important genes involved in the involution process that operate in specific signaling pathway of remodeling or apoptosis. By using PCR-select cDNA subtraction to select genes differentially expressed in lactating day 15 and involution day 4 mouse mammary gland, a chemokines called CXCL14 was identified. Similarly, a British research group identified CXCL14 was significantly upregulated in mouse mammary gland at involution day 4 by microarray technology.
CXCL14, a member of Cys-X-Cys chemokine, has initially been described to play a role in trafficking B cell and monocyte. Besides, previous study suggested CXCL14 overexpressed in tumor epithelial cells via bind to receptors to enhance their proliferation. Based on these findings, CXCL14 might involve in regulating cell growth and immune response during involution. The characteristics of CXCL14 in mammary gland are still unknown. Therefore, the aim of this study was to reveal the characteristics of CXCL14 in mammary epithelial cells. To investigate whether CXCL14 is modified after translation in mammary epithelial cells, CXCL14 cDNA from mammary epithelial cells was subcloned into a mammalian expression vector, then transiently expressed in mammary epithelial cells. Total protein and medium from transfected cells were collected for detecting the molecular weight (MW) of recombinant CXCL14 by western blotting. The recombinant CXCL14 of predicted size was observed both in medium and cell extract. A larger shift in MW was also observed, suggesting that CXCL14 might be modified before secreting. To determine whether the CXCL14 was glycosylated, the recombinant CXCL14-expressing mammary epithelial cells was treated with O-linked glycosylation inhibitor BAG. A decreased shift of CXCL14 in electrophoretic mobility was observed compared to that of untreated cells. To further confirm the possible glycosylation site, a site-directed mutagenesis that serine and/or threonine were mutated to alanine to generate six CXCL14 mutants. At T59A/T60A-CXCL14 and S79A/T80A-CXCL14 mutants, the resultant MW was similar to that of BAG treated cells. In order to investigate the functional role of CXCL14 in future, an adenoviral expression system encoding CXCL14 was constructed. Viral titers were determined by the plaque-forming assay with 293A cells which proved its ability to successfully infect mammary epithelial cells.
Taken all together, these data suggested that CXCL14 might be a glycoprotein and the site of T59/T60 were necessary for their glycosylations. In addition, the adenoviral expression system could be used to explore the possibly molecular mechanism that CXCL14 in regulating mammary gland development.

並列關鍵字

CXCL14 ； mammary epithelial cell ； glycosylation

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