16
present within unfolded collagen molecules following proteolysis [24, 26] (an event
which controls the generation of the tunnel spaces).
Another important finding from this work is that inhibitors of EC lumen and tube
formation, including anti-α2 and anti-β1 integrin blocking antibodies, chemical
inhibitors of PKC and Src, as well as MT1-MMP inhibitors, completely abrogate
the formation of vascular guidance tunnels [95]. Thus, the formation of EC tubes is
an obligate step in the formation of vascular guidance tunnels, and thus, these processes are directly linked in some fundamental manner. Several critical questions
arise from these studies, including how the lumen and tube formation processes are
functionally connected with the cell surface proteolytic machinery to create vascular guidance tunnels. Very recent work, described below, provides some insights
into these questions.
1.3.9 Cdc42 and MT1-MMP Are Functionally Interdependent
Signaling Molecules Which Are Components of an EC
Lumen Signaling Complex that Controls EC
Tubulogenesis in 3D Extracellular Matrices
Several newer findings begin to shed light into how Cdc42-dependent signaling
events, which activate kinase cascades and interact with cell polarity machinery
(i.e., Par3, Par6, atypical PKC), intersect with MT1-MMP proteolysis to create EC
lumens, tubes, and vascular guidance tunnels [66, 67, 83, 95]. One important point
is that the EC lumen and tube formation is a 3D matrix-specific process [27], in that
tubulogenesis does not occur on a 2D matrix surface. In contrast, EC motility can
occur quite readily on a 2D matrix surface, while it also occurs in 3D matrices in a
manner that depends on MT1-MMP proteolytic events. Importantly, MT1-MMP
activity is not required for EC motility on a 2D matrix surface, as discussed above.
With this introduction, our findings show that blockade of MT1-MMP activity using
siRNA suppression or MT1-MMP inhibitors leads to marked interference with
Cdc42 activation (a critical step necessary for activation of effectors such as Pak2,
Pak4, and Par6 that leads to EC tubulogenesis) in 3D collagen matrices [83].
However, this blockade of MT1-MMP does not affect Cdc42 activation of ECs
when they are seeded on 2D collagen surfaces, and coincidently, their motility is
also not affected [83]. Expression of the dominant-negative MT1-MMP construct
also markedly blocks lumen formation and Cdc42 activation [83]. Interestingly, the
activation of RhoA, which is not involved in EC lumen and tube formation, is not
affected by blockade of Cdc42 or MT1-MMP, nor is it affected by 2D vs. 3D collagen matrices. This data shows that MT1-MMP activity is directly coupled to
Cdc42 activation in 3D, but not 2D, matrices to control the tube formation process
[83]. The reverse is also true, in that blockade of Cdc42 using siRNA suppression
leads to marked decreases in vascular guidance tunnel formation, a consequence of
inactivation of MT1-MMP-dependent proteolysis [83]. Thus, this work suggests a
G. E. Davis
present within unfolded collagen molecules following proteolysis [24, 26] (an event
which controls the generation of the tunnel spaces).
Another important finding from this work is that inhibitors of EC lumen and tube
formation, including anti-α2 and anti-β1 integrin blocking antibodies, chemical
inhibitors of PKC and Src, as well as MT1-MMP inhibitors, completely abrogate
the formation of vascular guidance tunnels [95]. Thus, the formation of EC tubes is
an obligate step in the formation of vascular guidance tunnels, and thus, these processes are directly linked in some fundamental manner. Several critical questions
arise from these studies, including how the lumen and tube formation processes are
functionally connected with the cell surface proteolytic machinery to create vascular guidance tunnels. Very recent work, described below, provides some insights
into these questions.
1.3.9 Cdc42 and MT1-MMP Are Functionally Interdependent
Signaling Molecules Which Are Components of an EC
Lumen Signaling Complex that Controls EC
Tubulogenesis in 3D Extracellular Matrices
Several newer findings begin to shed light into how Cdc42-dependent signaling
events, which activate kinase cascades and interact with cell polarity machinery
(i.e., Par3, Par6, atypical PKC), intersect with MT1-MMP proteolysis to create EC
lumens, tubes, and vascular guidance tunnels [66, 67, 83, 95]. One important point
is that the EC lumen and tube formation is a 3D matrix-specific process [27], in that
tubulogenesis does not occur on a 2D matrix surface. In contrast, EC motility can
occur quite readily on a 2D matrix surface, while it also occurs in 3D matrices in a
manner that depends on MT1-MMP proteolytic events. Importantly, MT1-MMP
activity is not required for EC motility on a 2D matrix surface, as discussed above.
With this introduction, our findings show that blockade of MT1-MMP activity using
siRNA suppression or MT1-MMP inhibitors leads to marked interference with
Cdc42 activation (a critical step necessary for activation of effectors such as Pak2,
Pak4, and Par6 that leads to EC tubulogenesis) in 3D collagen matrices [83].
However, this blockade of MT1-MMP does not affect Cdc42 activation of ECs
when they are seeded on 2D collagen surfaces, and coincidently, their motility is
also not affected [83]. Expression of the dominant-negative MT1-MMP construct
also markedly blocks lumen formation and Cdc42 activation [83]. Interestingly, the
activation of RhoA, which is not involved in EC lumen and tube formation, is not
affected by blockade of Cdc42 or MT1-MMP, nor is it affected by 2D vs. 3D collagen matrices. This data shows that MT1-MMP activity is directly coupled to
Cdc42 activation in 3D, but not 2D, matrices to control the tube formation process
[83]. The reverse is also true, in that blockade of Cdc42 using siRNA suppression
leads to marked decreases in vascular guidance tunnel formation, a consequence of
inactivation of MT1-MMP-dependent proteolysis [83]. Thus, this work suggests a
G. E. Davis