Richard C, Liuzzo J P, Moscatelli D
Department of Cell Biology, New York University Medical Center, New York 10016, USA.
J Biol Chem. 1995 Oct 13;270(41):24188-96. doi: 10.1074/jbc.270.41.24188.
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.
髓系32D细胞系生长于悬浮状态，不表达FGF受体或硫酸乙酰肝素蛋白聚糖，用编码FGF受体-1的cDNA转染该细胞系（32D-flg细胞）。当与戊二醛固定的中国仓鼠卵巢（CHO）细胞共培养时，在无FGF-2的情况下，32D-flg细胞保持悬浮状态，但在存在10 ng/ml FGF-2时，细胞会附着于CHO单层细胞。相反，仅用载体转染的32D细胞在有FGF-2存在时不附着于CHO单层细胞。在孵育培养基中加入10微克/毫升肝素可阻止32D-flg细胞依赖FGF-2的附着，当使用糖胺聚糖合成缺陷的CHO突变体或用肝素酶处理的野生型CHO细胞时，这种附着会减弱，这表明附着是通过FGF-2与CHO细胞上的硫酸乙酰肝素相互作用而发生的。32D-flg细胞与野生型CHO细胞的附着在FGF-2浓度为0.4 ng/ml时达到半数最大效应，FGF-1也能引起这种附着，但FGF-4不能。32D-flg细胞也以FGF-2依赖的方式附着于活的CHO细胞，但在37℃时这种附着是短暂的。FGF-2依赖的附着不需要诱导新蛋白，因为当共培养物在4℃孵育时以及32D-flg细胞预先用放线菌酮孵育时，附着仍会发生。在Balb/C 3T3、NIH 3T3和牛毛细血管内皮细胞中也观察到32D-flg细胞依赖FGF-2的附着。我们得出结论，附着是由于FGF-2同时与32D-flg细胞上的受体以及CHO单层细胞上的硫酸乙酰肝素结合；因此，FGF-2充当了表达受体的细胞与携带硫酸乙酰肝素的细胞之间的桥梁。此外，CHO相关的硫酸乙酰肝素增强32D-flg细胞对FGF-2应答的DNA合成的程度与可溶性肝素相同，这表明这种相互作用具有功能意义。
