| Summary: | CD34 and its relatives, Podocalyxin and Endoglycan, comprise of a family of
surface sialomucins expressed by hematopoietic stem/progenitor cells, and vascular
endothelia. Recent data suggest that they serve as either pro- or anti-adhesion molecules
depending on their cellular context and their post-translational modifications. We were
interested in identifying Podocalyxin ligands and their cellular distribution and
understanding the role of these factors in signaling, adhesion and migration. Using both a
lambda phage screen assay and mass spectrometry, we identified the Na⁺/H⁺ exchanger
regulatory factor-i (NHERF-l) as a selective ligand for Podocalyxin and Endoglycan but
not for the closely related CD34. Furthermore, we showed that NHERF-1 is expressed
by all, lineage⁻, Sca-1⁺ and c-kit⁺ (LSK) cells, which are known to express Podocalyxin
and have long-term repopulating characteristics of hematopoietic stem cells. In addition,
upon IL-3 stimulation of a factor dependent cell line (FDC-P 1) these proteins re-localize
and co-localize in an asymmetrical pattern. By using a lentiviral based shRNA system to
silence Podocalyxin and NHERF- i proteins, we observed that migration across stromal
monolayer towards a CXCL12 and SCF gradient is significantly impeded in cells that
lack Podocalyxin but not NHERF-1. Following in vitro stimulation with a combination
of CXCL12 and SCF we observed that Podocalyxin co-associates with CXCR4.
Furthermore, cells lacking Podocalyxin have decreased phospho-AKT, a key signaling
molecule downstream of c-kit and CXCR4 receptors. Taken together, our data supports
the conclusion that Podocalyxin co-association with CXCR4 modulates downstream
signaling to efficiently regulate HSC homing. === Medicine, Faculty of === Medicine, Department of === Experimental Medicine, Division of === Graduate
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