Fibroblast Growth Factor-based Signaling through Synthetic Heparan Sulfate Blocks Copolymers Studied Using High Cell Density Three-dimensional Cell Printing*

Background: Fibroblast growth factor-receptor-heparan sulfate (FGF-HS-FGFR) signals cell proliferation. Results: HS synthesized with sulfated domains at its non-reducing ends actively promotes cellular proliferation in a three-dimensional cell microarray. Conclusion: A symmetric 2:2:2 FGF-HS-FGFR complex is preferred over an asymmetric 2:1:2 model by these data. Significance: This paper suggests a preference for symmetry in the signal transduction complex having two FGF-FGFR on the non-reducing end of two HS chains. Four well-defined heparan sulfate (HS) block copolymers containing S-domains (high sulfo group content) placed adjacent to N-domains (low sulfo group content) were chemoenzymatically synthesized and characterized. The domain lengths in these HS block co-polymers were approximate to 40 saccharide units. Microtiter 96-well and three-dimensional cell-based microarray assays utilizing murine immortalized bone marrow (BaF3) cells were developed to evaluate the activity of these HS block co-polymers. Each recombinant BaF3 cell line expresses only a single type of fibroblast growth factor receptor (FGFR) but produces neither HS nor fibroblast growth factors (FGFs). In the presence of different FGFs, BaF3 cell proliferation showed clear differences for the four HS block co-polymers examined. These data were used to examine the two proposed signaling models, the symmetric FGF(2)-HS2-FGFR(2) ternary complex model and the asymmetric FGF(2)-HS1-FGFR(2) ternary complex model. In the symmetric FGF(2)-HS2-FGFR(2) model, two acidic HS chains bind in a basic canyon located on the top face of the FGF(2)-FGFR(2) protein complex. In this model the S-domains at the non-reducing ends of the two HS proteoglycan chains are proposed to interact with the FGF(2)-FGFR(2) protein complex. In contrast, in the asymmetric FGF(2)-HS1-FGFR(2) model, a single HS chain interacts with the FGF(2)-FGFR(2) protein complex through a single S-domain that can be located at any position within an HS chain. Our data comparing a series of synthetically prepared HS block copolymers support a preference for the symmetric FGF(2)-HS2-FGFR(2) ternary complex model.