The prolactin signal transduction cascade in immortal caprine mammary epithelial cell lines
Date Issued
2009
Date
2009
Author(s)
Wang, Chien-Yu
Abstract
Development of the adult female mammary gland encounters four distinct stages: virgin, pregnancy, lactation, and involution. During mammary gland development, prolactin (PRL), a pituitary hormone, mediates mammary epithelial cell growth, differentiation and lactation. The act of prolactin in regulating the activity of b-casein promoter via the activation of prolactin tyrosine kinase associated receptor, and its associated protein kinase, Jak-2. Signal transducer and activator of transcription 5a (STAT5a) is the key signaling molecule transfers the prolactin signal from Jak-2 to b-casein promoter. Once STAT5a phosphorylated by Jak-2, STAT5a translocates from the cytoplasm to the nucleus, and activates b-casein promoter during lactation. Besides, based on the study of Caveolin-1 (Cav-1), its null mice accelerated the development of the labuloalveolar compartment, premature milk production, and hyperphosphorylation of STAT5a. In addition, Cav-1 is identified as a negative regulator of Jak-2/STAT5a signaling pathway. Prolactin down-regulates Cav-1 expression via Ras-MAP Kinase pathway also been identified in mouse mammary epithelial cells.he Cav-1 promoter sequence identity between goat versus human and goat versus mouse was 74% and 67%, respectively. The comparison of 1683 nucleotides upstream the transcription start site of b-casein gene between goat versus human and goat versus mouse the sequence similarity is 46% and 49%, respectively. Because diversity among those sequences, the goal in present study is to understand whether the function of goat Cav-1 in caprine mammary epithelial cell (CMEC) is similar to that in mouse and human.irst of all, stable expression of PRL receptor CMEC cells (CMEC/PRLR) lines were established via puromycin selection. In order to measure the b-casein promoter activity, a plasmid possessed goat b-casein promoter (-1683 – -1) drove firefly luciferase (Fluc) reporter protein was constructed. For equivalent the transfection efficiency, another plasmid possessed TK (thymidine kinase) promoter drove the renilla luciferase (Rluc) reporter protein also been constructed. A system that prolactin mediated b-casein promoter in CMEC/PRLR cells was established by co-expression of b-casein promoter Fluc plasmid and TK promoter Rluc plasmid in the CMEC/PRLR cells in response to the stimulation of PRL. The activity of goat b-casein promoter activity was enhanced 36.1 fold after PRL treatment. To understand the dosage effect of PRL to the endogenous Cav-1 expression, 6 final PRL concentrations (range from 0 to 15 ug/ml) were used to treat the CMEC/PRLR cells, then the cell proteins were harvested at 3 time courses (24, 48, 72 hr post-treatment). The results show that endogenous Cav-1 down-regulated in response to PRL treatment at 48, and 72 hr and dose dependent. To study whether goat Cav-1 could down-regulate the activity (phosphorylation) of STAT5a via PRL signal cascade, the phsphorylayion state of STAT5a were determined with or without exogenous goat Cav-1 protein expression in CMEC/PRLR cells after PRL stimulation. The phosphorylation of STAT5a was decreased after PRL stimulation, furthermore expression of the control protein EGFP (Enhanced Green Fluorescent Proteins) did not alter STAT5a phosphorylation. Finally, to study whether goat Cav-1 could down-regulate the b-casein promoter activity by down-regulate the phosphorylation of STAT5a. We transient transfect gCav-1 in the PRL induce b-casein promoter system. The gCav-1 also down-regulate the b-casein promoter activity.n conclusion, these results showed that PRL down-regulated the endogenous Cav-1 expression, and elevated expression of exogenous Cav-1 decreased the phosphorylation of STAT5a proteins and b-casein promoter activity. Therefore, PRL down-regulated endogenous Cav-1 expression and up-regulated STAT5a phosphorylation is important for improving b-casein promoter activity in CMEC.
Subjects
mammary epithelial cell
prolactin
casein promoter
lactation
Type
thesis
File(s)![Thumbnail Image]()
Loading...
Name
ntu-98-R96626014-1.pdf
Size
23.53 KB
Format
Adobe PDF
Checksum
(MD5):3202b05fa808a72e72fa1c1e4cfcfb11