Hyaluronan promotes signaling interaction between CD44 and the transforming growth factor β receptor I in metastatic breast tumor cells

Abstract

In this study we have examined the interaction between CD44 (a hyaluronan (HA) receptor) and the transforming growth factor β (TGF-β) receptors (a family of serine/threonine kinase membrane receptors) in human metastatic breast tumor cells (MDA-MB-231 cell line). Immunological data indicate that both CD44 and TGF-β receptors are expressed in MDA-MB-231 cells and that CD44 is physically linked to the TGF-β receptor I (TGF-βRI) (and to a lesser extent to the TGF-β receptor II (TGF-βRII)) as a complex in vivo. Scatchard plot analyses and in vitro binding experiments show that the cytoplasmic domain of CD44 binds to TGF-βRI at a single site with high affinity (an apparent dissociation constant (Kd) of ∼1.78 nM). These findings indicate that TGF-βRI contains a CD44-binding site. Furthermore, we have found that the binding of HA to CD44 in MDA-MB-231 cells stimulates TGF-βRI serine/threonine kinase activity which, in turn, increases Smad2/Smad3 phosphorylation and parathyroid hormone-related protein (PTH-rP) production (well known downstream effector functions of TGF-β signaling). Most importantly, TGF-βRI kinase activated by HA phosphorylates CD44, which enhances its binding interaction with the cytoskeletal protein, ankyrin, leading to HA-mediated breast tumor cell migration. Overexpression of TGF-βRI by transfection of MDA-MB-231 cells with TGF-βRIcDNA stimulates formation of the CD44·TGF-βRI complex, the association of ankyrin with membranes, and HA-dependent/CD44-specific breast tumor migration. Taken together, these findings strongly suggest that CD44 interaction with the TGF-βRI kinase promotes activation of multiple signaling pathways required for ankyrin-membrane interaction, tumor cell migration, and important oncogenic events (e.g. Smad2/Smad3 phosphorylation and PTH-rP production) during HA and TGF-β-mediated metastatic breast tumor progression.