Role of functional domains of disabled-2 gene in hTERT-immortalized caprine mammary epithelial cells
Date Issued
2011
Date
2011
Author(s)
Ke, Meng-Wei
Abstract
Finite lifespan limited the application of primary cultured mammary epithelial cells (MECs), whereas immortal cell lines retaining major characteristics of primary cultured MECs were more desirable. For the purpose of obtaining immortal caprine mammary epithelial cells (CMCs), human telomerase reverse transcriptase (hTERT) gene was introduced into primary cultured caprine-MECs (CMECs). Both luminal and myoepithelial cells were successfully immortalized and expressed their cell-lineage specific cytoskeleton markers. Activated telomerase in obtained immortal CMCs was confirmed by telomeric repeat amplification protocol (TRAP). The integrity of chromosomal structure and Capra hircus origin of CMCs was examined by karyotypic analysis. For morphologic differentiation, CMCs of luminal group, but not myoepithelial group, formed well-organized alveolar structures (acinus) when grown in Matrigel extracellular matrix (ECM). Furthermore, one luminal CMC clone expressed αs1- or β-casein gene in response to lactohormone stimulation. These results demonstrated that hTERT-immortalized CMCs do reserve important functional characteristics of primary cultured CMECs.
As well-organized acinus is essential to full differentiation of MECs, the function of a cytosolic adaptor protein disabled-2 (Dab2), which was measured with a moderate level in pregnancy, followed by evident decrease during lactation and peaked in wean of normal caprine mammary epithelium, was explored. Immunohistochemistry (IHC) further showed Dab2 majorly distributed in alveolar structures. Thus, Dab2 was hypothesized to engage in process of alveologenesis, which was supported by evidence of elevated level of Dab2 during in vitro acinus formation of primary cultured CMECs. Dab2 molecule hold two functional domains, the amino-terminus phosphotyrosine-binding domain (PTB) and the carboxyl-terminus proline-rich domain (PRD), CMC clones stably expressed each domain were established for exploring their contributions on alveologenesis. The effect of PTB or PRD domain in CMC clones was confirmed by significant reduction of Erk phosphorylation after growth factor stimulation. As alveologenesis was associated with cell migration, wound healing assay showed enhanced cell motility of both PTB and PRD CMC clones. To examine the effect of PTB or PRD domain on alveolar structure formation, in vitro acinus culture exhibited CMC clone bearing PRD domain, but not PTB domain, represented obvious protrusions of the acinus. These data indicated both PTB and PRD domains of Dab2 were involved in regulating alveologenesis of MECs, but different mechanisms might be employed by different domains.
In this study, the establishment and elaborate characterization of hTERT-immortalized CMC cell lines was demonstrated. These immortal CMC cell lines were valuable models for exploring the molecular mechanisms regulating the development of caprine mammary gland and provided a platform to examine the expression of recombined plasmids used to generate transgenic livestock. In addition, the findings of PTB and PRD domains in mediating alveologenesis of immortal CMC cells, which might claim the importance of Dab2 during the development of caprine mammary gland.
Subjects
caprine
mammary epithelial cells
telomerase
immortalization
Dab2
Type
thesis
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