776-2 Constitutive Expression of an Intracellular Form of Fibroblast Growth Factor Receptor Triggers Myogenic Differentiation

Abstract

Fibroblast growth factors (FGFs) have been demonstrated to regulate myogenic differentiation. Recently, forms of FGF have been generated that reside in the cytoplasm and appear to be biologically active. suggesting that an intracellular form of FGF may be able to transduce its signal through signaling pathways independent of its interaction with cell surface (transmembrane) FGF receptors (FGFR). We report in this study a potential mechanism by which such signaling can be modulated by an intracellular form of the receptor (I-FGFR). To generate this form of the receptor, the cDNA encoding the 2 IgG-like domain form of the murine transmembrane FGFR was mutated to remove its signal sequence and acidic box by PCR mutagenesis. This construct was then epitope-tagged to allow it to be distinguished from the endogenous surface FGF receptor. When the I-FGFR was transientlytransfected into COS cells, a diffuse and reticular pattern was observed by indirect immunofluorescence microscopy, indicating cytoplasmic localization with association to organized intracellular structures, such as cytoskeleton or organellar membrane. To test for the biologic effect of the I-FGFR, stable cell lines containing the I-FGFR were generated in Sol 8 cells. Quadruplicate samples of four cell lines expressing high levels of the I-FGFR, and four control cell lines transfected with vector alone, were grown in mitogen poor medium (5% horse serum). Their ability to differentiate was assessed by their levels of creatine kinase activity normalized to total soluble protein (nmol NADPH/min/mg protein): Days 0 1 2 3 4 Control 53 ± 71 55 ± 30 236 ± 60 759 ± 143 1295 ± 205 I-FGFR 47 ± 12 104 ± 32 391 ± 30 * 1338± 312 * 2143 ± 677 * * p < 0.05 for I-FGFR versus control The results demonstrate that the intracellular form of the FGF receptor localizes to the cytoplasm of myocytes, and the increased expression of this receptor isoform triggers myogenic differentiation. We propose that this receptor promotes differentiation by binding endogenous FGF, and thus inhibiting the activity of FGF that normally preserves the undifferentiated state. The ability to generate and express the intracellular FGF receptor, therefore, may be a generally useful method of modulating FGF dependent regulation of proliferation and differentiation of other cell types such as smooth muscle and transformed cells.