Compared to treatments with other FGF isoforms

Compared to treatments with other FGF2 isoforms, stimulation with 22.5kDa FGF2 results in lower stem cell gene expression in hESCs (Fig. 4d), weaker proliferative activity in human dermal fibroblasts (Fig. 5), and lower relative mitogenic activity in BaF3 continine manufacturer ectopically expressing FGFR1 IIIb, one of the primary receptor variants that is known to be activated by 18kDa FGF2 (Fig. 7d,e) (Ornitz et al., 1996). Similarly, stimulation with 34kDa FGF2 results in a lower level of ERK1/2 phosphorylation and weaker proliferative activity in human dermal fibroblasts (Figs. 3b, 5), and lower relative mitogenic activity in BaF3 cells ectopically expressing FGFR1 IIIb (Fig. 7d, e). Despite their lower activities by several measures, both 22.5kDa and 34kDa FGF2 still maintain the ability to support several FGF2-related phenotypes. In a manner qualitatively similar to other FGF2 isoforms, 22.5kDa and 34kDa FGF2 are able to induce phosphorylation of FGFR1, FRS2α, and ERK (Fig. 3a, b), support embryonic stem cell gene expression and self-renewal in culture (Fig. 4c, d), stimulate proliferation in human dermal fibroblasts (Fig. 5), and induce mitogenesis to some degree in BaF3 cells transduced with FGFR1 IIIb (Fig. 7). There are several possible explanations for the differential efficiencies among the HMW isoforms, in particular for the 22.5kDa and 34kDa isoforms, to maintain canonical FGFR-mediated phenotypes compared to each other and to the 18kDa isoform. Since the HMW FGF2 isoforms are all co-linear, N-terminal extensions of the 18kDa isoform, it is possible that some portions of the N-terminal extensions of the HMW FGF2 isoforms affect the interaction of FGF2 with its receptor, resulting in quantitative differences in stimulation of downstream signaling cascades. Additionally, we cannot discount the possibility that the differential activity of FGF2 isoforms may be due to differences in proteolytic degradation or protein stability conferred by the N-terminal extensions to the 18kDa isoform, or due to differences in subcellular protein localization upon internalization of the isoform after interaction with its receptor (Więdłocha and Sørensen, 2004). Further study into the biochemical details of canonical FGFR-mediated signal transduction by HMW FGF2 isoforms should be performed keeping in mind these paradigms as possibilities. Though localization of HMW isoforms of FGF2 is primarily intracellular and generally considered to be contained to the nucleus (Arnaud et al., 1999; Quarto et al., 1991), it has also been proposed that HMW FGF2 isoforms can be released from FGF2-expressing cells by several processes including vesicle shedding, cell death, or wounding (McNeil et al., 1989; Schweigerer et al., 1987; Taverna et al., 2003). Since we have determined that extracellular, HMW isoforms of FGF2 can cause many of the same phenotypic effects of 18kDa, it is plausible to suspect that release of these HMW FGF2 isoforms may potentiate canonical FGF2 signaling via FGFRs, allowing for them to act via transmembrane receptors in a similar manner to secreted, 18kDa FGF2. Thus, conditions under which HMW FGF2 isoforms are released into the extracellular space could lead to potentiation of canonical autocrine or paracrine FGF2 signaling. Improved understanding of the ability of different FGF2 isoforms to interact with different receptors will lead to a better understanding of the different functions of FGF2 not only on various cell types, which express different patterns of FGFs and FGFRs (Hughes, 1997), but also in different cellular processes, such as angiogenesis, embryogenesis, differentiation, proliferation, self-renewal, and others involved in normal or pathological physiologies (Eswarakumar et al., 2005). Future studies may reveal differences among FGF2 isoforms in binding kinetics or in ability to activate particular FGFR-mediated pathways, thus explaining some of the differences we see in cell phenotypes induced by treatment with different FGF2 isoforms.