The study of CXCL14 glycosylation in mammary epithelial cell
Date Issued
2008
Date
2008
Author(s)
Tseng, Ting-Yu
Abstract
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. XCL14, 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.
Subjects
CXCL14
mammary epithelial cell
glycosylation
Type
thesis
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