Expression of Biologically Active Basic Fibroblast Growth Factor by Genetically Modified Rat Primary Skin Fibroblasts

Abstract

Basic fibroblast growth factor (FGF‐2) is normally expressed as a cell‐associated protein, and accordingly it is not clear how it exerts its action on target cells in vivo. It has been proposed that cells release, by death or other mechanisms, small amounts of FGF‐2 that then acts in an autocrine manner. To address the question of whether it is necessary that FGF‐2 remain cell associated or needs to be secreted from cells to have biological activity, we expressed the 18‐kDa form of FGF‐2 in primary fibroblasts as a cell‐associated (FGF‐2‐B) or as a secreted (FGF‐2‐S) protein. FGF‐2 protein is detected in cell lysates and membrane fractions of both cell types, whereas it is present in significant amounts only in the conditioned medium of FGF‐2‐S cells. No FGF‐2 is detected in control (untransfected) cells. FGF‐2‐S cells also grow faster than the control or FGF‐2‐B cells. Yet, when evaluated for their ability to promote the survival of embryonic hippocampal neurons in vitro, both the cell types are active, establishing the activity of the transgene product. We conclude that FGF‐2 is active when engineered to be expressed as a cell‐associated form or secreted from cells.

Delivery of FGF-2 but not VEGF by encapsulated genetically engineered myoblasts improves survival and vascularization in a model of acute skin flap ischemia

“…16,40 Primary cells modified by a retrovirus to produce FGF-2 using a signal peptide 40 and cell lines transduced with an adenovirus to secrete FGF-2 without a signal peptide 16 have both yielded secretion levels comparable with those observed in this study. Natural release of FGF-2 from cells in the absence of a signal peptide has been shown to occur using a unique export pathway which is independent of the ER/Golgi system.…”

Neurotrophic factor synergy is required for neuronal survival and disinhibited axon regeneration after CNS injury

“…Fibroblasts were prepared from Fischer 344 rats, engineered with either fgf2, nt-3 or bdnf (from Jaso Ray and Fred Gage, The Salk Institute, CA, USA) and characterized in detail elsewhere (Kawaja et al, 1992;Ray et al, 1995;Senut et al, 1995;Shults et al, 2000). Fibroblasts were grown to confluence in 75 cm flasks in Dulbecco's modified Eagle's medium with 1.0 g/l D-glucose, 10% foetal bovine serum, 2 mg/ml fungizone, 50 mg/ml gentamicin sulphate, 400 mg/ml G418, 1 mM L-glutamine (all from Invitrogen, Paisley, UK).…”

Intracellular fibroblast growth factor produces effects different from those of extracellular application on development of avian cochleovestibular ganglion cells in vitro

“…FGF‐2 could act through such a route. First, FGF‐2 does not have to be secreted to stimulate proliferation of fibroblasts (Bikfalvi et al, 1995; Ray et al, 1995). Second, extracellularly added FGF‐2 accumulates in the nucleus in a cell‐cycle‐dependent manner (Stachowiak et al, 1997).…”