Vascular Development
Initially it had been expected that picking out direct relationships between MAPK function and development would be complicated by extensive cross-talk between the different MKK pathways, as well as by overlapping and redundant functions of MAPK isoforms. However, studies of MKK pathway knock-out mice, though pointing to a common role in regulating vascularization and blood vessel development during early embryogenesis, have revealed a surprising level of independence between these pathways (Table 1).
Gene targeting of BRAF causes embryonic death between E10.5 and 12.5 [99]. These embryos displayed extensive vascular defects including irregularly shaped large blood vessels that were incompletely lined with endothelial cells. Raf-1 knock-out mice die in utero at E10 with diminished vascularization of the embryo and placenta [100,101]. Targeted deletion of MEKK3—an MKKK that can activate MEK5 and ERK5 as well as MKK3 and p38MAPK—causes embryonic death at E11 that is characterized by extensive defects in blood vessel formation in the embryo, placenta, and yolk sac while not affecting VEGF expression [102].
MEK1 knock-out mice die in utero at E10.5 due to defects in placental vascularization [103]. MKK4 knock-out mice die between E10.5 and 12.5. Although MKK4–/– embryos appear anemic, their deaths have been linked not to defects in vasculogenesis or hematopoiesis but to impaired hepatogenesis [104,105,106]. MEK5 knock-out mice die at E10.5. Though the reasons for their inviability have not been extensively characterized, MEK5–/– mice display cardiovascular defects prior to death [107]. By contrast, MEK2, MKK3, and MKK6 null embryos are viable, but the latter two show defects in T-cell signaling [108,109,110], indicating that these kinases do not function in developmental vascularization or that their loss can be compensated for by the other MKKs.
Whereas ERK1-deficient mice are viable and fertile, demonstrating only defects in T-cell maturation [111], ERK2 knock-out mice are embryonic-lethal and die at E7.5-E11 as a result of defects in trophoblast development, leading to decreased placental vascularization [112,113]. Knock-out of p38alpha MAPK causes embryonic lethality starting at E10.5 that is characterized by cardiovascular defects in the embryo that arise secondarily to defects in placental vascularization [114,115] and defective hematopoiesis [116]. By contrast, p38beta MAPK knockouts are viable and apparently healthy. Targeted inactivation of either JNK1 nor JNK2 does not impact embryonic vascularization but instead causes defects in T-cell development [117,118]. ERK5 knock-outs die at E10-E11 not due to an inability to form primary vasculature, but from an inability to remodel that vasculature to form embryonic blood vessels [119,120]. Notably, ERK5 deficiency is accompanied by increased expression of VEGF [120], indicating that ERK5 can regulate expression of angiogenic factors during development. In addition to its role in blood vessel formation during development, ERK5 also plays an important role in maintaining endothelial function and vascular integrity in adults; its conditional inactivation causes increased vascular permeability and hemorrhage in multiple organs [121].
Further evidence for a role for MKK signaling in vascularization comes from studies of growth factor–induced angiogenesis in chick chorioallantoic membrane. In this system, fibroblast growth factor–induced angiogenesis causes sustained ERK activity in endothelial cells and can be blocked by inhibition of Raf-1 [122] or MEK1 and MEK2 activity [75]. Interestingly, although fibroblast growth factor also causes sustained activation of p38 MAPK, p38 MAPK inhibition with SB202190 does not inhibit neovascularization but rather enhances it [123], indicating that balanced p38 MAPK activity may be essential for neovascularization of chick chorioallantoic membrane.
Collectively, the preceding studies indicate that the in vivo activities of B-Raf (likely acting through MEK1 and ERK2) as well as Mekk3 (acting through MEK5, ERK5, and p38alpha MAPK) are essential for vascularization and blood vessel patterning of the placenta (MEK1, ERK2) and embryo proper (Mekk3, ERK5, p38alpha MAPK) during early (E10-11) development. However, despite in vitro evidence supporting a role for MKK signaling in VEGF regulation, the loss of neither MEK2, MKK3, MKK4, MKK6, Erk1, p38beta MAPK, JNK1, nor JNK2 has a significant impact upon developmental vascularization.