FGF family members are among the most potent angiogenesis inducers and actively participate in tumor angiogenesis. In vitro, both FGF2 and FGF8b stimulate EC proliferation, migration, invasion and tube formation. We show here that both FGFs stimulate STAT transcription factors, in particular STAT5. Importantly, we report the novel finding that FGF-induced STAT5 activation is necessary and sufficient for brain EC migration, invasion and tube formation, all of which are critical components of the angiogenic cascade. Conversely, STAT5 activation is dispensible for FGF-induced EC mitogenesis.
A considerable wealth of information has accumulated on signaling events downstream of the FGFR. Most attention has been directed toward the classical ras-raf-MEK-MAPK pathway [reviewed in (10
)]. This signaling cascade appears to be primarily responsible for the regulation of cell proliferation. The PI3K-Akt pathway has been linked to FGF induced cell survival although Akt does not appear to be involved in FGF signaling in our cell model (9
). In agreement with our findings, a limited number of reports support a role for members of the STAT family in FGF signaling. For example, a constitutively active mutant FGFR3, which is responsible for thanatophoric dysplasia type II dwarfism, specifically activates STAT1, resulting in chondrocyte growth arrest (15
). The FGFR-STAT1 axis also negatively regulates bone growth pre- and post-natally under physiological conditions (38
). Deo et al. (16
) observed STAT3 phosphorylation in response to FGF2 in human umbilical vein ECs, which required platelet activating factor. The biologic consequence of STAT3 activation in this cell type was not further examined.
The multiplicity of the intracellular signaling pathways downstream of FGFR raises questions about their regulation and biologic purpose. It is conceivable that different FGFRs preferentially activate distinct signaling pathways (e.g. MAPK, Akt, STATs, or individual STAT family members). This hypothesis appears plausible as ECs isolated from diverse organ sites express different combinations of FGFRs (unpublished observation) and show distinct patterns of STAT activation. In bone ECs, FGF2 primarily stimulates STAT3, whereas FGF8b activates STAT5. Since FGF2 and FGF8b preferentially bind to and activate different FGFRs, this observation supports a model of FGFR-specific STAT activation. An attractive physiologic role for a network of divergent FGF signaling pathways might be that it generates numerous opportunities for crosstalk and integration with other signaling systems. For example, EGFR-mediated activation of ERK1/2 can diminish FGFR signaling via a negative feedback loop involving FRS2 (40
). Interleukin-3 induced EC proliferation and migration is dependent on STAT5, which provides a mechanism for this cytokine (and probably other factors) to modulate FGF-mediated angiogenesis (41
STAT proteins are activated by tyrosine phosphorylation through members of the Janus or Src kinase families (30
). A direct activation by receptor tyrosine kinases has also been proposed (42
). Our data point toward FGFR-induced STAT activation via Jak2 and Src intermediaries. The involvement of Jak2 downstream of a receptor tyrosine kinase indicates an unorthodox transduction mechanism for which there is some precedent. Dudley and co-workers described VEGF-induced STAT5 stimulation that required Jak2 activity (43
). A role for Src family kinases in FGFR signaling has been described more frequently (20
). Similar to our findings in ECs, Maciag’s group (20
) attributed FGF1-induced fibroblast migration to Src activation, whereas mitogenesis was associated with the ERK1/2 pathway. Claesson-Welsh’s group (45
) observed that Src inhibition abolishes FGF2-induced EC tube formation, consistent with the FGFR-Src-STAT5 signal cascade proposed here.
Our central observation is that STAT5 is required for FGF2 and FGF8b induced assembly of brain ECs into vascular tubes. This raises the question whether other pro-angiogenic factors can activate STATs, thus elevating STATs to the rank of general regulators of vascular endothelial morphogenesis. Indeed, the potent angiogenic stimulator VEGF induces tyrosine phosphorylation of several STAT family members, including STAT1, 3, 5 and 6 (43
). Yahata and colleagues (48
) found that VEGF-mediated STAT3 activation is essential for migration and tube formation in human dermal microvascular ECs. Consistent with the concept of EC heterogeneity, different STAT family members may be responsible for regulating the differentiation of ECs in different organ sites. Alternatively, both STAT3 and STAT5 may be required. The central role of STAT transcription factors in angiogenesis is further supported by the observation that the angiopoietin receptor, Tie-2, can activate STAT1, 3 and 5 (49
). Interestingly, activated STAT3 was detected in tumor vessel ECs of gliomas, suggesting roles in tumor angiogenesis (17
). Together, these studies point to an integration of several pro-angiogenic signaling pathways by STATs to orchestrate EC migration and organization into vascular channels.
The critical position of STAT family members in multiple pro-angiogenic signaling pathways renders them attractive targets for anti-angiogenic therapeutic approaches. Naturally, the involvement of STATs in cytokine signaling and their role in regulating the immune system and hematopoiesis may require very selective inhibition and/or targeted delivery of therapeutic agents. However, a recent preclinical study using a transcription factor decoy approach targeting STAT3 provided promising preliminary support for such a treatment strategy (50
). Additional work is needed to better define the FGFR-STAT signaling pathway and evaluate STATs as therapeutic targets.