9
dominant- negative mutants of PKCε block lumen formation and downstream Src
and Pak activation [67]. Interestingly, increased expression of PKCε strongly stimulates EC lumen and tube formation, and both increased Src, Pak-2, and Pak-4 phosphorylation events that directly correlate with its morphogenic influence [67]
(Fig. 1.2). Our studies indicate that PKCε is upstream of Src activation, while Src
activation is upstream of Pak activation [67]. Blockade of Src kinases by siRNA
suppression; increased expression of the Src inhibitor, CSK (i.e., C-terminal Src
kinase); or treatment with chemical inhibitors (e.g., PP2) completely interferes with
EC tube formation. Expression of a dominant-negative Csk construct strongly
increased lumen formation, again suggesting a positive role for Src in EC lumen
formation [67]. Interestingly, Src and Pak kinases are known to active Raf kinases
to affect processes such as cell survival which has previously been shown to influence angiogenesis in vivo [2], and we have recently shown that they are required for
EC lumen formation [67]. Mouse knockout of B-Raf shows an embryonic lethal
phenotype that is due to vascular abnormalities [43]. Of great interest is that we
have shown that Raf kinase activation (of both C-Raf and B-Raf) occurs downstream of Src and Pak activation and controls EC tube morphogenic events along
with survival [67] (Fig. 1.2). This is accompanied by Erk1/Erk2 activation which
also directly correlates with the ability of these ECs to form tube networks.
Interestingly, expression of a phosphatase, MKP-3, with selectivity for phosphoErk1/Erk2, markedly decreases Erk phosphorylation and strongly blocks lumen formation [67]. A dominant- negative MEK kinase inhibitor also abrogates lumen
formation and Erk1/Erk2 phosphorylation events. What is interesting about these
results is that a known pathway to regulate both proliferation and survival is utilized
by ECs to regulate a separate tubulogenic pathway in 3D matrices. In our systems
there is little to no evidence for proliferation during these processes so the signaling
cascade appears particularly focused on tube morphogenesis [12, 30]. Overall, this
morphogenic pathway is coupled to cytoskeletal signaling (i.e., PKC, Src, Pak),
survival (i.e., Raf), and transcriptional events (i.e., Erk) to coordinately control this
process [30, 66, 67] (Fig. 1.2). Also, it is likely that these kinases are not limited to
affecting only one of the critical functions during these events.
We have recently described a novel function for Src family kinases during EC
lumen and tube assembly, which is to target and control the development of the apical membrane surface [64]. Marked intracellular vacuole membrane labeling with
activated phospho-Src is observed which traffic along acetylated tubulin tracks to
fuse in a subapical domain to create an apical membrane also decorated with activated Src isoforms [64]. Blockade of Src isoforms with PP2 completely blocks this
process and siRNA suppression of Src, Fyn, and Yes, but not Lyn, interfere with EC
lumen formation (Fig. 1.3). PP2 blocks lumen formation whether it is occurring
during vasculogenic or angiogenic sprouting events, and interestingly, the addition
of PP2 appears to markedly increase the number of EC tip cells as a result of this
strong reduction in lumen formation [64].
1 Molecular Control of Capillary Tube Morphogenesis and Maturation Through…
dominant- negative mutants of PKCε block lumen formation and downstream Src
and Pak activation [67]. Interestingly, increased expression of PKCε strongly stimulates EC lumen and tube formation, and both increased Src, Pak-2, and Pak-4 phosphorylation events that directly correlate with its morphogenic influence [67]
(Fig. 1.2). Our studies indicate that PKCε is upstream of Src activation, while Src
activation is upstream of Pak activation [67]. Blockade of Src kinases by siRNA
suppression; increased expression of the Src inhibitor, CSK (i.e., C-terminal Src
kinase); or treatment with chemical inhibitors (e.g., PP2) completely interferes with
EC tube formation. Expression of a dominant-negative Csk construct strongly
increased lumen formation, again suggesting a positive role for Src in EC lumen
formation [67]. Interestingly, Src and Pak kinases are known to active Raf kinases
to affect processes such as cell survival which has previously been shown to influence angiogenesis in vivo [2], and we have recently shown that they are required for
EC lumen formation [67]. Mouse knockout of B-Raf shows an embryonic lethal
phenotype that is due to vascular abnormalities [43]. Of great interest is that we
have shown that Raf kinase activation (of both C-Raf and B-Raf) occurs downstream of Src and Pak activation and controls EC tube morphogenic events along
with survival [67] (Fig. 1.2). This is accompanied by Erk1/Erk2 activation which
also directly correlates with the ability of these ECs to form tube networks.
Interestingly, expression of a phosphatase, MKP-3, with selectivity for phosphoErk1/Erk2, markedly decreases Erk phosphorylation and strongly blocks lumen formation [67]. A dominant- negative MEK kinase inhibitor also abrogates lumen
formation and Erk1/Erk2 phosphorylation events. What is interesting about these
results is that a known pathway to regulate both proliferation and survival is utilized
by ECs to regulate a separate tubulogenic pathway in 3D matrices. In our systems
there is little to no evidence for proliferation during these processes so the signaling
cascade appears particularly focused on tube morphogenesis [12, 30]. Overall, this
morphogenic pathway is coupled to cytoskeletal signaling (i.e., PKC, Src, Pak),
survival (i.e., Raf), and transcriptional events (i.e., Erk) to coordinately control this
process [30, 66, 67] (Fig. 1.2). Also, it is likely that these kinases are not limited to
affecting only one of the critical functions during these events.
We have recently described a novel function for Src family kinases during EC
lumen and tube assembly, which is to target and control the development of the apical membrane surface [64]. Marked intracellular vacuole membrane labeling with
activated phospho-Src is observed which traffic along acetylated tubulin tracks to
fuse in a subapical domain to create an apical membrane also decorated with activated Src isoforms [64]. Blockade of Src isoforms with PP2 completely blocks this
process and siRNA suppression of Src, Fyn, and Yes, but not Lyn, interfere with EC
lumen formation (Fig. 1.3). PP2 blocks lumen formation whether it is occurring
during vasculogenic or angiogenic sprouting events, and interestingly, the addition
of PP2 appears to markedly increase the number of EC tip cells as a result of this
strong reduction in lumen formation [64].
1 Molecular Control of Capillary Tube Morphogenesis and Maturation Through…