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Specific network perturbations for the analysis of S1P‐mediated cell migration
Author(s) -
Alford Shan K,
Longmore Gregory D,
Stewart Sheila A,
Elbert Donald L
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a631-b
Subject(s) - pten , microbiology and biotechnology , cdc42 , gene knockdown , phosphatase , biology , tensin , cell culture , topology (electrical circuits) , chemistry , signal transduction , pi3k/akt/mtor pathway , genetics , phosphorylation , mathematics , combinatorics
Endothelial cell (EC) migration is enhanced through the activation the sphingosine 1‐phosphate (S1P) receptor, S1P 1 . Uncertainty exists concerning network topology downstream of S1P 1 , which hinders prediction of EC response to the combinations of stimuli found in the blood. To address this question, we are employing a top‐down and modular systems approach to determine network topology. In our system, we will measure the steady‐state biochemical responses to genetically‐engineered perturbations in the signaling cascade. Using retrovirally delivered shRNA, telomerase‐immortalized human EC lines were produced with stable knockdowns of a highly specific Rac‐GAP (β 2 ‐chimaerin), a highly specific Akt phosphatase (PHLPP), the PIP3 phosphatase, PTEN, and the S1P 1 receptor. All cell lines exhibited a significant decrease in the mRNA of interest versus vector control, as measured by quantitative RT‐PCR. Protein expression was significantly decreased in PTEN and S1P 1 ‐targeted cells as measured by Western Blot. Functionally, the serum‐starved level of GTP‐bound Rac was increased by 39 ± 8% in β2‐chimaerin knockdown cells versus vector control. Finally, GFP‐vector control cells maintain a normal migration response when stimulated with 100 nM S1P, thus making these genetically‐modified cells important tools for the study of S1P‐mediated signaling networks.