Molecular Control of Intracellular Signaling

Cell transplantation, or cellular cardiomyoplasty, has shown therapeutic promise for repair of myocardial infarcts and improvement of cardiac function. Experimental studies have evaluated the efficacy of transplanted cardiomyocytes, skeletal myoblasts, and, more recently, adult and embryonic stem cells, to replace injured myocardium with viable tissue to improve cardiac function following myocardial infarction. Previous studies in the Murry Lab focused on injecting skeletal myoblasts into the injured heart due to their resistance to ischemia and improved cell survival compared with cardiomyocytes. Skeletal myoblasts can be obtained autologously from muscle biopsies, thus eliminating immune rejection concerns, however, they are also susceptible to cell death, possess limited proliferative capacity in the infarcted heart, and grafts are isolated from host myocardium due to encapsulation within scar tissue. To increase skeletal muscle graft size, viability, and interaction with host myocardium, we developed a method to selectively expand grafted myoblasts in situ.

Although treatment with the skeletal myoblast mitogen basic fibroblast growth factor (bFGF) may be envisioned as a means of stimulating grafted myoblast expansion, its use in vivo is confounded by its proliferative effect on fibroblasts, which can lead to exaggerated fibrosis in infarcted myocardium. To circumvent this limitation, we designed a system in which the intracellular domain of the fibroblast growth factor receptor (FGFR-1) is fused with a drug binding protein (F36V), followed by expression of this chimeric receptor (F36Vfgfr-1) in the target cell type. Treatment with a bivalent drug (AP20187, ARIAD Pharmaceuticals) results in chemically induced dimerization of the drug binding protein domains, with concomitant activation of the receptor signaling domains (See Figure 1). In a bFGF-dependent murine myoblast line, MM14, the effects of signaling through FGFR-1, including stimulation of proliferation, inhibition of differentiation, and phosphorylation of extracellular regulated kinase (ERK), were activated in the absence of bFGF in cells expressing the F36Vfgfr-1 chimera (Whitney et al., 2001).

Ongoing studies of dimerizer-mediated myoblast proliferation will include evaluation of the effects of AP20187 on mouse and human primary myoblasts, as well as its in vivo effects on grafted myoblast proliferation and cardiac function in normal and infarcted hearts. In addition, the dimerizable FGFR-1 system is being adapted to endothelial cell proliferation for therapeutic angiogenesis and tissue engineering applications, as well as studies of FGFR-1 signaling in mouse embryonic stem (ES) cells to promote enrichment and expansion of ES-derived cell types, such as cardiomyocytes.

References - Chemical Inducers of Dimerization

Spencer, D.M., Wandless, T.J., Schreiber, S.L., Crabtree, G.R. (1993) Controlling signal transduction with synthetic ligands. Science 262, 1019-24.

Clackson, T., Yang, W., Rozamus, L.W., Hatada, M., Amara, J.F., Rollins, C.T., Stevenson, L.F., Magari, S.R., Wood, S.A., Courage, N.L., Lu, X., Cerasoli, F., Jr., Gilman, M., Holt, D.A. (1998) Redesigning an FKBP-ligand interface to generate chemical dimerizers with novel specificity. Proc Natl Acad Sci U S A 95, 10437-42.

Jin, L., Zeng, H., Chien, S., Otto, K.G., Richard, R.E., Emery, D.W., Blau, C.A. (2000) In vivo selection using a cell-growth switch. Nat Genet 26, 64-6.

Whitney, M.L., Otto, K.G., Blau, C.A., Reinecke, H., Murry, C.E. (2001) Control of myoblast proliferation with a synthetic ligand. J Biol Chem 276, 41191-6.

http://www.ariad.com/regulationkits

Figure 1. Schematic illustration of endogenous (left) and chimeric (right) FGFR-1 activation. Basic FGF binds to heparin sulfate proteoglycans (HSPG) at the cell surface to form multimeric complexes that present more than one molecule of bFGF to its receptor, FGFR-1. FGFR-1 binding to bFGF multimers (left) brings receptor molecules into proximity through dimerization, thereby allowing transphosphorylation by receptor tyrosine kinases, and activation of receptor signal transduction. Alternatively, chemically-induced dimerization (right) may be achieved by fusing the FGFR-1 cytoplasmic domain with a dimerizable drug binding domain, F36V. Treatment with the dimeric F36V ligand, AP20187, leads to dimerization of the chimeric receptor, F36Vfgfr-1, and activation of signal transduction, independent of bFGF binding.