The pleiotropic nature of FGF

The pleiotropic nature of FGF2 and its variety of downstream effects make generation of highly pure active protein essential. Heparin chromatography has been used for purification of 18kDa FGF2 isoform. However, subsequent heparin contamination in purified FGF2 preparations has been previously described to interfere with the stability and biological activity of FGF2 (Eiselleova et al., 2009; Gasparian et al., 2009). To avoid heparin contamination and achieve high protein purity after a single chromatographic step, we had DNA constructs synthesized for all FGF2 isoforms as 6xHis tag fusion proteins. Overexpressed 6xHis tagged FGF2 isoforms demonstrated high affinity for Ni-NTA, and the biological activity of purified HMW FGF2 isoforms was compared to commercially available 18kDa FGF2 by monitoring activation of downstream kinase pathways, in order to ensure that the pure protein activity was comparable to that of the commercial formulation. Furthermore, we examined the ability of each isoform to support self-renewal of human embryonic stem colony stimulating factor 1 receptor in vitro, promote mitogenic activity via activation of several FGFR variants, and increase the rate of proliferation of human dermal fibroblasts.
Materials and methods
Results
Discussion The study of FGF signaling is inherently important, as FGF signaling is ubiquitous and implicated in a myriad of important cellular and organismal processes, from embryogenesis to angiogenesis and beyond (Eiselleova et al., 2009; Ornitz et al., 1996; Zhang et al., 2006; Zoumaro-Djayoon et al., 2011). FGF signaling is mediated by specific ligand-receptor interactions, thus allowing for cell-specific tuning of FGF effects based on qualitative and quantitative expression patterns of FGFRs on the cell surface, and based on the presence of various FGFs in the milieu (Zhang et al., 2006). Better understanding of FGF ligand biology and their interactions with specific FGFR variants would allow us to better predict the effects of particular FGFs on specific cells based on their FGFR expression patterns, and better enable us to understand the cellular physiology of pathologies associated with aberrant FGF/FGFR signaling. The biological consequences of HMW FGF2 isoforms functioning as secreted ligands have not been extensively studied and are not well-understood. The data presented in this study demonstrate the ability of HMW FGF2 isoforms to act as extracellular ligands for select FGF receptor tyrosine kinases and to activate the canonical FGF2/FGFR/ERK pathway that is proven to mediate many different cellular responses in various cell types. We have demonstrated that HMW FGF2 isoforms can induce FGF receptor phosphorylation and lead to signal transduction at a level comparable to that induced by the 18kDa isoform. In addition to activation of the ERK/MAPK pathway (Fig. 3), the ability of HMW FGF2 isoforms to partake in canonical FGF2 signaling was also demonstrated by supporting hES cell growth in culture and maintenance of expression of stem cell-specific markers in embryonic stem cells (Fig. 4), mitogenic stimulation of fibroblasts (Fig. 5), and the ability to support proliferation of BaF3 cells expressing specific FGFR variants in the absence of exogenously supplemented IL-3, conditions under which they generally will not otherwise proliferate (Fig. 7). While all of the HMW FGF2 isoforms demonstrate the ability to recapitulate the activity of the 18kDa isoform qualitatively via the aforementioned metrics, activities differ between different FGF2 isoforms. The LMW 18kDa FGF2 isoform appears to be the most potent activator of receptor-mediated FGF2 signaling, which is unsurprising given that it is generally secreted from the cell and thus produces cellular responses via autocrine or paracrine signaling (Goetz and Mohammadi, 2013). Though all of the HMW isoforms appear to be less efficient than the 18kDa isoform at inducing canonical FGF2 signaling by at least one measure investigated in the work presented here, the least effective FGF2 isoforms by several measures appear to be the 22.5kDa and 34kDa isoforms.