Fetuin-A (α2HS-Glycoprotein) Is a Major Serum Adhesive Protein That Mediates Growth Signaling in Breast Tumor Cells

Abstract

The identity of the cell adhesive factors in fetal bovine serum, commonly used to supplement growth media, remains a mystery due to the plethora of serum proteins. In the present analyses, we showed that fetuin-A, whose function in cellular attachment in tissue culture has been debated for many years, is indeed a major serum cell attachment factor particularly for tumor cells. We are able to report this because of a new purification strategy that has for the first time given us a homogeneous protein band in colloidal Coomassie-stained gels that retains biological activity. The tumor cells adhered to immobilized fetuin-A and not ␣ 2 -macroglobulin, its major contaminant. The interaction of cells with fetuin-A was driven mainly by Ca 2؉ ions, and cells growing in regular medium supplemented with fetal bovine serum were just as sensitive to loss of extracellular Ca 2؉ ions as cells growing in fetuin-A. Fractionation of human serum revealed that cell attachment was confined to the fractions that had fetuin-A. Interestingly, the tumor cells also took up fetuin-A and secreted it back to the medium using an unknown mechanism that can be observed in live cells. The attachment of tumor cells to fetuin-A was accompanied by phosphatidylinositol 3-kinase/Akt activation that was down-regulated in cells that lack annexin-A6, one of the cell surface receptors for fetuin-A. Taken together, our data show the significance of fetuin-A in tumor cell growth mechanisms in vitro and open new research vistas for this protein.Serum, particularly fetal bovine serum, is widely used as a supplement in culture media that is required for growth of most cells in culture (for a review, see Ref. 1). In the absence of serum, most cells fail to adhere properly, spread, and grow on culture dishes. Serum has a plethora of adhesion (2-4) and growth factors (5). The prevailing assumption over the past two decades has been that integrins are the major cellular receptors for adhesion in cell cultures (6). Cellular adhesion to extracellular adhesion molecules such as vitronectin, fibronectin, and laminin using integrins requires the divalent ions Mg 2ϩ /Mn 2ϩ (7). In addition, cell signaling cues for spreading and growth mechanisms in anchorage-dependent cells emanate from the interaction of cells with the adhesion molecules (8). Although it is easy to define such signals in situations where cells are allowed to adhere to known purified extracellular matrix proteins such as fibronectin and collagen (9), adhesion in the presence of fetal bovine serum is complex in that any one of the myriad attachment proteins and or growth factors has the potential to mediate adhesion and growth signals.Ever since it was first purified and described, fetuin-A/ ahsg 2 was suspected of being the principal cell adhesion molecule in serum (1, 10). Fetuin-A isolated and purified from serum by the Pedersen method (hereafter referred to as Pedersen fetuin-A) demonstrated cell adhesive properties in the presence of divalent ions (11). The Pedersen fetuin-A, however, is not cons...

Development of monoclonal antibodies for the identification of novel invasion associated targets in human cancer

“…Expression of Annexin A6 at the cell surface has been reported in numerous studies; as a receptor for chondroitin sulfate chains (Takagi et al, 2001), in association with S100A8/A9 in the cytoplasm (Bode et al, 2008) and the serum adhesive protein, fetuin-A (Sakwe et al, 2010). This is in agreement with the both immunofluorescence and immunohistochemistry results obtained in this study, showing strong 9E1 reactivity to the cell membrane.…”

Role of annexin A6 in cancer

“…Other studies indicated that AnxA6 acted as an adhesion receptor to fetuin-A (α2 HS-glycoprotein), a major serum adhesive protein on the surface of BC cells, by mediating growth signaling (43,44). AnxA6 has been observed to promote the invasiveness of BT-549 BC cells, which express higher levels of AnxA6 than MCF-10A cells, since the depletion of AnxA6 in BT-549 cells reduced their motility and abolished invasiveness (34,35).…”

Vascular Smooth Muscle Cell Calcification Is Mediated by Regulated Exosome Secretion

“…This mechanism was supported by data showing that dynosore, an inhibitor of the small GTPase dynamin, did not affect fetuin-A uptake, whereas uptake of a clathrin-dependent protein, transferrin, was inhibited by 50% (Online Figure IB) and no colocalization of internalized fetuin-A with clathrin was observed (Online Figure IC). Additionally, small interfering RNA knockdown of the putative fetuin-A endocytic receptors, annexin A2 and annexin A6, 18 had no effect on fetuin-A delivery to the early endosomes (Online Figure IIA-IIC).…”