8
1.3.2 Functional Role of the Rho GTPases, Cdc42 and Rac1,
and the Effectors, Pak2 and Pak4, in EC Tube
Morphogenesis
An important question raised by the above studies is which downstream signaling
pathways are activated by integrins to control these morphogenic processes
(Fig. 1.2). Integrins were known to activate Rho GTPases among other molecules
[49] such as a variety of kinases, including Src and focal adhesion kinase [34, 57,
89]. Our laboratory reported that Cdc42 was a critical GTPase controlling EC lumen
formation [8] (Fig. 1.2). This was also the first report from any system implicating
Cdc42 and tube formation. Subsequent studies have revealed that Cdc42 is a critical
regulator of lumen formation from both ECs and epithelial cells [8, 25, 66, 67, 75,
79, 83] (Fig. 1.2). Very recently, EC-specific knockout of Cdc42 in mice resulted in
embryonic lethality due to lack of EC lumen and tube formation. We reported a role
for Rac1 in EC tubulogenesis [8, 66], while RhoA had no ability to stimulate these
events. In contrast, expression of constitutively active RhoA leads to marked inhibition of EC lumen formation [8]. Both Cdc42 and Rac1 were shown to be activated
during the morphogenic cascade in 3D collagen matrices [66, 83]. To address the
question of downstream effectors that are responsible for the influence of Cdc42
and Rac1, we screened a series of known effectors using siRNA treatment of ECs.
Major blocking phenotypes were observed using siRNAs to p21-activated kinase
(Pak)-2 and Pak-4 [66]. Both EC tube formation and EC sprouting into 3D collagen
matrices were markedly inhibited by these siRNAs. We also demonstrated that a
time course of Pak-2 and Pak-4 activation, as indicated by phosphorylation, directly
correlated with the EC lumen formation process [66]. It was further demonstrated
that activated Pak-2 and Pak-4 could be demonstrated to be associated with activated Cdc42 during these events [66]. Expression of a dominant-negative mutant of
either Pak-2 or Pak-4 was shown to completely inhibit EC lumen formation [66].
Interestingly, both Cdc42 and Rac1 are able to activate Pak-2, while Cdc42 selectively activates Pak-4 [17, 44]. Recent experiments have revealed important roles
for both Pak-2 and Pak-4 during vascular development [44, 70, 98], which again
corroborate the in vitro findings.
1.3.3 Functional Role for PKCε and Src in EC Tube
Morphogenesis and Subsequent Pak Activation Events
Other known kinases that are activated by cell-ECM interactions include protein
kinase C isoforms and Src family kinases. In our studies of EC lumen formation in
3D collagen matrices, we have shown that PKCε, but not PKCα or PKCδ, is involved
in the process [66, 67]. siRNA suppression experiments or expression of
G. E. Davis
1.3.2 Functional Role of the Rho GTPases, Cdc42 and Rac1,
and the Effectors, Pak2 and Pak4, in EC Tube
Morphogenesis
An important question raised by the above studies is which downstream signaling
pathways are activated by integrins to control these morphogenic processes
(Fig. 1.2). Integrins were known to activate Rho GTPases among other molecules
[49] such as a variety of kinases, including Src and focal adhesion kinase [34, 57,
89]. Our laboratory reported that Cdc42 was a critical GTPase controlling EC lumen
formation [8] (Fig. 1.2). This was also the first report from any system implicating
Cdc42 and tube formation. Subsequent studies have revealed that Cdc42 is a critical
regulator of lumen formation from both ECs and epithelial cells [8, 25, 66, 67, 75,
79, 83] (Fig. 1.2). Very recently, EC-specific knockout of Cdc42 in mice resulted in
embryonic lethality due to lack of EC lumen and tube formation. We reported a role
for Rac1 in EC tubulogenesis [8, 66], while RhoA had no ability to stimulate these
events. In contrast, expression of constitutively active RhoA leads to marked inhibition of EC lumen formation [8]. Both Cdc42 and Rac1 were shown to be activated
during the morphogenic cascade in 3D collagen matrices [66, 83]. To address the
question of downstream effectors that are responsible for the influence of Cdc42
and Rac1, we screened a series of known effectors using siRNA treatment of ECs.
Major blocking phenotypes were observed using siRNAs to p21-activated kinase
(Pak)-2 and Pak-4 [66]. Both EC tube formation and EC sprouting into 3D collagen
matrices were markedly inhibited by these siRNAs. We also demonstrated that a
time course of Pak-2 and Pak-4 activation, as indicated by phosphorylation, directly
correlated with the EC lumen formation process [66]. It was further demonstrated
that activated Pak-2 and Pak-4 could be demonstrated to be associated with activated Cdc42 during these events [66]. Expression of a dominant-negative mutant of
either Pak-2 or Pak-4 was shown to completely inhibit EC lumen formation [66].
Interestingly, both Cdc42 and Rac1 are able to activate Pak-2, while Cdc42 selectively activates Pak-4 [17, 44]. Recent experiments have revealed important roles
for both Pak-2 and Pak-4 during vascular development [44, 70, 98], which again
corroborate the in vitro findings.
1.3.3 Functional Role for PKCε and Src in EC Tube
Morphogenesis and Subsequent Pak Activation Events
Other known kinases that are activated by cell-ECM interactions include protein
kinase C isoforms and Src family kinases. In our studies of EC lumen formation in
3D collagen matrices, we have shown that PKCε, but not PKCα or PKCδ, is involved
in the process [66, 67]. siRNA suppression experiments or expression of
G. E. Davis