Abstract 13365: ABI Gene Family, Member 3 (NESH) Binding Protein (Abi3bp) Regulates MSC Proliferation in Autocrine/Paracrine Fashion
Introduction: Mesenchymal stem cells overexpressing Akt (Akt-MSCs) release various paracrine factors which promote cardiac repair following acute myocardial infarction. We have identified Abi3bp, a relatively novel secreted protein involved in cancer pathogenesis, as highly expressed in Akt-MSCs and aimed to determine its relevance in MSC biology.
Methods: Stable knockdown of Abi3bp in Akt-MSCs was achieved by shRNA. Two different shRNA constructs were employed and both reduced Abi3bp mRNA levels by ∼90%.Cell growth and proliferation were assessed by Elisa, FACS and/or immunocytochemistry. Activation of the Ras-ERK pathway was assessed by western blot analysis.
Results: Abi3bp knockdown increased Akt-MSC proliferation by ∼2-fold as determined by cell counting (n=3, p<0.001), Ki67 immunostaining (n=3, p<0.05), and BrdU ELISA (n=3, p<0.01). Subsequent analysis of the cell cycle using a BrdU FACS method indicated that Abi3bp knockdown increased the number of Akt-MSCs in S-phase (n=3, p<0.001) and increased levels of the cell cycle regulator cyclin D1. Conditioned media from HEK293 cells over-expressing a myc-tagged Abi3bp inhibited proliferation in both Akt-MSCs and MSCs when compared to control conditioned media, indicating that Abi3bp was acting in an autocrine/paracrine fashion. To address the mechanism underlying the proliferative effect of Abi3bp, we examined several cell-signaling pathways known to play a vital role in proliferation. ERK1/2 phosphorylation was also found to be substantially increased in the Abi3bp knockdown Akt-MSCs, indicating a possible role for this pathway. Accordingly chemical inhibition of Ras, Raf, and MEK1/2 in Akt-MSCs with Abi3bp knockdown reduced ERK phosphorylation, cyclin D1 expression, and BrdU incorporation. The Src inhibitor PP2 had the same effect indicating a role for this oncogene in mediating the Abi3bp effects.
Conclusion: In short, we have identified Abi3bp as novel secreted suppressor of proliferation that modulated MSC survival in a paracrine and autocrine fashion.
- © 2011 by American Heart Association, Inc.