Endolyn (CD164) Interacts with CXCR4 and Modulates the CXCL12-Mediated Homing and Migration of Cord Blood CD133⁺ Hematopoietic Precusor Cells.

The sialomucin, endolyn or CD164, has been shown to act as an important regulator in the adhesion of human haemopoietic stem/precursor cells (HPC) to stromal niche cells, while also controlling the entry of primitive human CD34⁺CD38^lo HPC into cycle. Here, we define a novel function for endolyn, by identifying its ability to modulate CD133⁺ cord blood HPC migration on fibronectin towards CXCL12 in vitro. Interestingly, CD133⁺ cell migration on fibronectin to CXCL12 was reduced 1) by engaging the functional class II glycosylation-dependent epitope on endolyn with the 103B2/9E10 class II but not N6B6 class III antibody; and 2) by RNAi knockdown of endolyn in both CD133⁺ HPC and Jurkat cells. The inhibition of migration was more pronounced in the more primitive CD34⁺CD38^lo/− HPC subset than in the CD38⁺ subset. We show a direct and temporal association of endolyn with the CXCR4 receptor, at the leading edge of CD133⁺ HPC. When CXCL12 is presented on fibronectin, we first see an upregulation in the association of CXCR4 with the a-4 and a-5 integrins that is closely followed by recruitment of endolyn to this complex. This was confirmed by co-immunoprecipitation studies. Knock-down of endolyn using siRNAs revealed that signaling through CXCR4 via PKC-zeta and Akt pathways was significantly dampened, while leaving MAPK phosphorylation unaffected. Our current studies are aimed at examining the in vivo importance of endolyn in HPC homing to the bone marrow of NOD/SCID mice. Our findings support a novel association between three distinct families of cell surface receptors that regulate both cell migratory and proliferative responses and identify endolyn as a key regulator of CXCR4/CXCL12 function.