Determine how the different concentrations of FGF-2 for stimulating the differen

Question

Determine how the different concentrations of FGF-2 for stimulating the different effects (growth stimulation, uPA induction, and the morphology shift) in NIH 3T3 cells relate to the known dissociation constants for cellular binding.
Maximal growth stimulation is seen at 0.5 ng/ml FGF-2, while uPA induction and the morphology shift require 5-10 ng/ml for maximal induction. For FGF-2 binding to FGFR, Kd=2x10^-11M (same for FGFR 1-4); and for binding to cell-surface heparin sulfate alone (not as part of FGF:FGFR:heparin sulfate complex), Kd=8x10^-9M.
One other piece of information you will need is that we are working with the “Low M.W. Isoform” of FGF-2.
Show me how you did the calculations, and be able to discuss what this means from a biological standpoint.

Explanation / Answer

Addition of exogenous FGF2 to the group of NIH 3T3 cells is found to be influencing the Ras-MAP kinases and Phospholipase C signal transduction pathways. These pathways on activation are responsible for downstream effects in NIH 3T3 cells that include growth stimulation, morphology shifts and uPA gene expression induction.

After western blot and ELISA analysis, it was concluded that FGF-2 upregulation of Erk-MAP kinase and PLC pathways is dependent on certain doses. The highest upregulation was observed at 5 ng/ml concentration of FGF-2. Greater than 1 ng/ml concentration decreased the cell growth. At concentrations of 10 and 20 ng/ml, FGF-2 concentration could influence the cell morphology shift.

Maximum urokinase plasminogen activator gene expression in NIH 3T3 cells occurred at FGF-2 concentrations of 5ng/ml and 10 ng/ml.

A significant decrease in the tritium –thymidine incorporation and band intensity in northern blotting for uPA RNA transcription was observed when the NIH 3T3 samples are subjected to FGF-2 and Suramin (inhibitor of binding between FGF-2 and FGFR).

The band intensity of northern blots of phosphorylated MAP kinase proteins cells exposed to suramin and FGF-2 was not different from when only FGF-2 was used.

Binding of heparin to FGF-2 protects the protein from getting proteolysed with trypsin. FGF-2 gets protection from circulating proteases. The binding of FGF-2 with heparin also creates a local reservoir of growth factors. Spatial regulation of FGF signaling occurs very clearly.

Binding of FGF-2 at the high affinity sites of FGFR will stimulate the uPA expression. Low affinity binding is usually competed by heparin molecules. Binding of heparin molecules to the low affinity sites of FGFR will make the protein to be physiologically irrelevant for FGF signaling. If FGF-2 is saturated with heparin, the FGF-2 will not attack the low affinity sites of FGFR and hence the stimulation of uPA gene expression does not occur.

Clear understanding of the actual mechanism by which FGF signaling is regulated will have applications in wound healing, embryonic development and tumor growth investigations. These investigations will help in the development of cancer therapy that involves targeting these signaling pathways.

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