Role of podocalyxin in hematopoiesis and cell migration

• Main Author: Tan, Poh Choo
• Format: Others
• Language: English
• Published: University of British Columbia 2008
• Subjects: Podocalyxin; NHERF-1; Migration; Chemotaxis; CXVL 12; CXCR4
• Online Access: http://hdl.handle.net/2429/2860

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