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Nanog regulation of endothelial asymmetric cell division (851.20)
Author(s) -
Baruah Jugajyoti,
Wary Kishore
Publication year - 2014
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.28.1_supplement.851.20
Subject(s) - gene knockdown , homeobox protein nanog , notch signaling pathway , wnt signaling pathway , microbiology and biotechnology , biology , numb , chemistry , signal transduction , cell culture , embryonic stem cell , induced pluripotent stem cell , biochemistry , genetics , gene
Objective: To test whether inhibition of Glycogen Synthase Kinase‐3 (GSK‐3) enzyme or GSK‐3 α/ β‐ knockdown, induces Asymmetric Cell Division ( ACD) in endothelial cells (ECs). Accordingly, we test the hypothesis that a subset of mature ECs retain the ability to divide unequally, a process called asymmetric cell division (ACD) and this event requires the activation of canonical Wnt signaling pathway. Methods and Results: Stimulation of cultured ECs with 6‐bromoindirubin‐3‐oxime (BIO) or shRNA mediated GSK‐3 α/ β ‐knockdown stabilized β‐catenin and increased expression of NANOG, thereby increased proliferation as well as migration of these cells in culture. Accordingly, BIO stimulation and 5‐bromo‐2'‐deoxyuridine (BrdU) pulse labeling of ECs followed by microscopy showed increased ACD in a subset of BrdU‐positive cells. The immunostaining of BrdU positive ECs (stalk‐cells) showed expression of NOTCH‐1 (NICD) and DLL4 (tip‐cells). Immunohistochemistry and biochemical analyses indicated altered expression of NOTCH and NUMB in asymmetrically dividing cells. Western blot analyses confirmed increased expression of NOTCH‐1, DLL4, NUMB and JAM‐A, protein differentially expressed during ACD, in response to BIO stimulation. In a converse experiment, NANOG ‐knockdown carried‐out in ECs after receiving BIO showed decreased ACD, while control knockdown had no effect. Interestingly, re‐expression of VEGFR‐2/Flk1 cDNA into NANOG‐depleted ECs partially restored the ability of these cells to divide asymmetrically. Conclusion: Thus, our data indicate that the activation of canonical Wnt signaling induces ACD in a subset of ECs, an event that may partly be responsible for mediating EC dedifferentiation and regeneration. Grant Funding Source : Supported by American Heart Association Grant to KKW (12GRNT12070159).