Abstract
The myeloid 32D cell line, which grows in suspension and does not express FGF receptors or heparan sulfate proteoglycans, was transfected with the cDNA encoding FGF receptor-1 (32D-flg cells). When co-cultured with glutaraldehyde-fixed Chinese hamster ovary (CHO) cells, the 32D-flg cells remained in suspension in the absence of FGF-2 but attached to the CHO monolayer in the presence of 10 ng/ml FGF-2. In contrast, 32D cells transfected with the vector alone did not attach to the CHO monolayer in the presence of FGF-2. FGF-2-dependent attachment of 32D-flg cells was prevented by inclusion of 10 micrograms/ml heparin in the incubation medium and was diminished when CHO mutants in glycosaminoglycan synthesis or wild-type CHO cells treated with heparinase were used, indicating that the attachment occurred through FGF-2 interactions with heparan sulfates on the CHO cells. Attachment of 32D-flg cells to wild-type CHO cells was half-maximal at 0.4 ng/ml FGF-2 and was also observed with FGF-1 but not FGF-4. 32D-flg cells also attached to living CHO cells in a FGF-2-dependent manner, but attachment was transient at 37 degrees C. Induction of new proteins was not required for FGF-2-dependent attachment, since attachment occurred when the co-cultures were incubated at 4 degrees C and when the 32D-flg cells were preincubated with cycloheximide. FGF-2-dependent attachment of 32D-flg cells was also observed with Balb/C 3T3, NIH 3T3, and bovine capillary endothelial cells. We conclude that attachment is due to FGF-2 binding simultaneously to receptors on the 32D-flg cells and heparan sulfates on the CHO monolayers; thus, the FGF-2 acts as a bridge between receptor-expressing cells and heparan sulfate-bearing cells. In addition, induction of DNA synthesis in 32D-flg cells in response to FGF-2 was potentiated by the CHO-associated heparan sulfates to the same extent as by soluble heparin, indicating that this interaction has functional significance.
Highlights
The fibroblast growth factors (FGFs)1 are a family of nine polypeptides that share sequence homology and a high affinity for heparin [1, 2]
Preservation of heparan sulfates in the fixed cells was confirmed by the fact that the fixed cells bound 80% of the amount of 125I-FGF-2 on low affinity binding sites as parallel cultures of non-fixed cells
When the cells were co-cultured in the presence of 10 ng/ml FGF-2, approximately 80% of the 32D-flg cells attached to the fixed Chinese hamster ovary (CHO) cells (Fig. 1A)
Summary
Cells—CHO K-1 cells and mutants in glycosaminoglycan synthesis derived from them (pgsA-745, pgsB-618, pgsB-650, pgsD-677, and pgsE-606) were a generous gift of Dr Jeffrey Esko of the University of Alabama. 5 ϫ 105 32D-flg cells in serumfree Iscove medium containing no addition, 10 ng/ml FGF-2, or 10 ng/ml FGF-2 and 10 g/ml heparin were added to washed monolayers of CHO cells. CHO Attachment Assays—CHO-flg cells or nontransfected CHO-K1 cells were trypsinized and replated at subconfluent density Twentyfour hours later they were washed twice with Ca2ϩ- and Mg2ϩ-free PBS and detached from their dishes after incubation in Ca2ϩ- and Mg2ϩ-free PBS containing 10 mM EDTA. Five hundred thousand CHO cells in suspension were added to each 35-mm dish containing glutaraldehyde-fixed monolayers of wild type CHO-K1 cells or CHO-pgsB-618 mutants in heparan sulfate synthesis prepared as described above. The medium containing suspended 32D-flg cells was removed, and 32D-flg cells attached to the monolayer were harvested by washing the dishes with PBS containing heparin.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
