Hyaluronate binding and CD44 expression in human glioblastoma cells and astrocytes

CD44 is an integral membrane glycoprotein of approximately 90 kDa which has been implicated in the binding of hyaluronate to the cell surface. The expression of CD44 in astrocytes was investigated by means of indirect immunofluorescence on cultured cells. The vast majority of these cells were found to express CD44. Western blot analysis of these cells revealed a highly polydisperse species having an M(r) corresponding to 74-86 kDa. In order to visualize hyaluronate-binding cells, living cultures were probed with fluorescein-conjugated hyaluronate (FI-HA). Some astrocytes were able to bind FI-HA, provided that they were first treated with hyaluronidase. Streptomyces hyaluronidase, which is hyaluronate-specific, was effective in exposing the hyaluronate-binding capacity of these cells. This leads one to conclude that hyaluronate is bound to the surface of these cells and that it masks their capacity to bind hyaluronate. Provided that they were first treated with hyaluronidase, the U-87 MG (glioblastoma-astrocytoma), U-373 MG (glioblastoma), and Hs 683 (glioma) cell lines were also able to bind FI-HA. The U-138 MG (glioblastoma) cell line was unable to bind FI-HA, with or without prior hyaluronidase treatment. A quantitative assay was developed with the use of [3H]hyaluronate ([3H]HA). This revealed the binding to be highly specific, inasmuch as the addition of unlabeled hyaluronate, but not other glycosaminoglycans, was effective in inhibiting the binding of the [3H]HA. An anti-CD44 monoclonal antibody, 50B4, was able to inhibit the binding of the [3H]HA to the U-373 MG cell line. In this cell line, then, CD44 functions as a hyaluronate receptor and one may infer that this is also the case in some astrocytes.