The more the better: not always for growth factors
Most growth factors produce a sigmoidal dose-response curve. However, biphasic responses can also be generated, even from a single receptor. The specific reasons underlying a biphasic dose response caused by FGF-2 binding to FGFR1 have been elucidated.
One of the biggest cell culture input variables is growth factor concentration. Researchers typically use unformulated growth factors in cell culture at excess concentrations which overstimulate cells but then degrade rapidly and therefore need to be frequently replenished. This cycling of availability invariably contributes to drug assay variability as the stimulation of receptors varies throughout the experiment. Complex interactions underlie these effects of growth factor concentration.
Usually, growth factor response curves are sigmoidal, like the curve shown above in A above. These sigmoidal curves eventually flatten out when the receptors become saturated and the excess growth factor has no place to bind. However, there are many published assays in which a biphasic (Gaussian) curve (Figure B) is produced. Examples of this include FGF-2, SHH and NGF.
In some cases, a biphasic curve can be produced when a growth factor binds to more than one receptor, with different affinities, that affect interfering pathways. For example, IL-2 binds to different receptors with different affinities leading to distinct pharmacological effects at low and high doses. However, a biphasic effect can also be seen for some growth factors interacting with a single receptor. A good example of this is provided by FGF-2.
FGF-2 is part of a family of 18 fibroblast growth factors which bind to a family of seven main FGF receptors. FGF-2’s primary receptor dimerizes when bound by FGF-2. This causes autophosphorylation of the receptor dimer and activation of a signalling cascade, which results in ERK phosphorylation. As well as binding to its receptor, FGF2 binds to an extracellular matrix molecule called heparan sulphate glycosaminoglycan (HSGAG).
FGF-2 receptor with its cognate receptor, FGFR1. See the text for an explanation. Image Cell Guidance Systems after Kanodia et al
The mechanism behind the biphasic curve of FGF was elucidated by researchers at Merrimack Pharmaceuticals who, based on our understanding of FGF-2 stimulated pathways, produced mathematical models and experimental data which support the following explanation:
The strength of binding of FGF2 to its receptor is increased if FGF2 is first bound to HSGAG. HSGAG molecules are present on the cell surface at much higher concentrations than FGF-2, such that FGF2 normally binds HSGAG and they then bind the FGF-2 receptor together to form a trimeric complex.
FGF-2 can bind the receptor without first binding HSGAG, but this interaction is weak and only occurs at high concentrations of FGF-2. This weak binding does not lead to high levels of receptor dimerization and autophosphorylation. Moreover, once free FGF-2 is attached to the receptor, it is unable to bind to HSGAG. Consequently, at high concentrations, weakly binding FGF-2 blocks the binding of FGF2-HSGAG complexes and the activity of FGF2 is reduced. The experimental data produced by the researchers show that the peak of FGF2’s biphasic curve is about 10 ng/ml.
How does this understanding affect now we use growth factors in cell culture? It is self-evident that reducing input variables improves the reliability and value of cell-based assays. Similarly, assays performed under physiological conditions are more likely to be physiologically relevant. However, the traditional, unformulated recombinant growth factors most widely used in research are used at unphysiologically high concentrations which gyrate through cycles of degradation and replenishment with media replacement. This creates cells in constant flux which impacts data outputs using those cells.
Cell Guidance Systems is addressing this issue by providing formulated sustained-release growth factors. PODS are nature-mimetic, in-cell, protein crystals which, under the action of proteases, provide a steady stream of growth factor availability from a polyhedrin scaffold.
IMAGE Sigmoidal and biphasic response curves. CREDIT Cell Guidance Systems
Learn more about powerful technologies that are enabling research:
