In this study, we found that distinct signaling pathways activated by β1 and β3 integrins converge and cooperate to promote CLAN formation (). The basal level of CLAN formation in HTM cells mediated by β1 integrins involved a SFK/PI3-K-dependent signaling pathway. Although it is likely that the SFK is Src, we have not yet ruled out that two other SFK members, Yes, and Lyn57, 58
, are involved given that all SFK members can be inhibited by PP247–50
and EPA only inhibits Fyn and Lck51, 52
. This basal level of CLAN formation induced via β1-integrin could be further enhanced by co-activating a β3 integrin signaling pathway that utilized distinct and separate signaling components. β3 integrins enhanced CLAN formation via a Src dependent, PI3-K independent pathway that also involved Rac1-Trio. Furthermore this β3 integrin pathway could be activated by the G-protein coupled receptor CD47 via the TSP1-derived peptide 4N1K. These data suggest β1-and β3-mediated signaling pathways converge downstream of PI3-K and Rac1-Trio to promote CLAN formation ().
Schematic diagram showing that β1- and β3-mediated CLAN formation is the result of distinct signaling pathways
CD47 induced CLAN formation was most likely mediated through interactions with αvβ3 integrins rather than αIIbβ3 or α2β1 integrins59, 60
. The expression of αIIbβ3 integrins is restricted to megakaryocyte-derived cells and previous studies showed that direct activation of α2β1 integrin did not induce significant CLAN formation17
. Thus, the contribution of CD47-mediated activation of αIIbβ3 and/or α2β1 integrins to TM cell's CLAN formation is minimal. Furthermore, the CD47 function blocking antibody B6H12.2 blocked CLAN formation induced by the β3 integrin activating antibody mAb AP-5 further suggesting an interaction between a β3 integrin and CD47.
The involvement of CD47 in β3-induced CLAN formation raises some very interesting questions about how this might possibly relate to the induction of CLAN formation by DEX in cultured anterior segments and cells3, 9–11
. CD47 is a receptor for TSP1, the expression of which is up-regulated by both TGF-β1 and DEX treatment in human and mouse TM cells, and increased TSP1 deposition has been found in the ECM of the meshwork in cases of POAG and SIG30
. Additionally, Liu et al23
showed that DEX caused a decrease in TSP1 released into culture medium, consistent with the idea that DEX treatment results in an increase in matrix- or cell-associated TSP1, similar to what has been demonstrated for other ECM components44, 61–63
. Increased TSP1 within the ECM could lead to increased CD47 activation thereby contributing to αvβ3-integrin-mediated CLAN formation in DEX-treated HTM cells. The suppression of TSP1 production and subsequent decreased CLAN formation in HTM cells in response to the actin-disrupting drug LAT-A would further support the notion that TSP1 and CLAN formation are connected23
The point where these two pathways converge to enhance CLAN formation could not be determined from these studies. Clearly, these signaling pathways must converge since activation β3 integrin signaling alone is not sufficient to induce CLAN formation in TM cells17
. One point may be Src, since PP2 blocked both β1- and β3-mediated CLAN formation. However, Src is generally thought of as an early upstream signaling event before Rac1, so this would not explain the differential inhibition of β1- and β3-mediated CLAN formation by LY94002 and NSC23766, respectively. The most likely explanation is that the pathways converge at Rac1 or downstream of Rac1 activation at one of the steps involved in the formation of a branched actin network since Rac1 is known to regulate the formation of these structures55
. However, only αvβ3 integrin-mediated CLAN formation was blocked by the Rac1 inhibitor, NSC23766 which is somewhat puzzling because β1-mediated CLAN formation is regulated by PI3-K and there are numerous studies that show Rac1 activation being dependent upon PI3-K64, 65
. The NSC23766 inhibitor that was used, however, specifically blocks Rac1 signaling mediated by the GEFs Tiam1 or Trio. Hence. it is possible that β1-mediated CLAN formation involves activation of Rac1 by another Rac1 GEF such as Vav166
. The fact that gene-silencing experiments indicated that β3-mediated CLAN formation involved Trio rather than Tiam1 supports the idea that CLAN formation in HTM cells is regulated by specific GEFs.
It is not surprising that Trio was found to be the active GEF in HTM cultures. Trio is highly expressed in neuronal tissues67
, and HTM cells have a neural crest origin68
while Tiam1 expression appears to be more widespread69
. It is interesting though that the β3 integrin signaling was linked to Trio-Rac1. Integrin signaling has previously been associated with Tiam170, 71
while Trio has been associated with heterotrimeric G proteins72
. To our knowledge, this is the first time that any integrin signaling has been linked to Trio-Rac1. However, the fact that CD47 is considered an atypical GPCR that can activate the Gαi
subfamily of heterotrimeric G proteins35
and is also a co-receptor for β3 integrin, supports the idea that the β3 integrin/CD47 complex activates Trio-Rac1 signaling.
Additional data suggest that CLAN formation may involve Gαi
signaling. A recent study in Schwann cells73
showed that CLANs (“geodesic actin networks”) were formed in response to lysophosphatidic acid (LPA) or sphingosine 1-phosphate (S1P). Both LPA and S1P signal via GPCRs and activate the Gαi
signaling pathway74, 75
. Interestingly, LPA- and S1P- induced CLAN formation in Schwann cells was also found to be dependent upon Rac1, rather than RhoA, activation. Clearly, future studies will be needed to explore possible links among CD47, Rac1 and Trio in CLAN formation.
Although the function of CLAN formation in HTM cells is still unknown, CLANs do not appear to be necessary for cell spreading, since the assembly of actin filaments into a CLAN appears to involve a distinct process that is not involved in cell spreading. Under conditions when β1-mediated CLAN formation was inhibited by the Src kinase inhibitor, PP2, cell spreading was only partially inhibited. Also, activation of β3-mediated CLAN formation in the presence of PP2 induced a partial recovery of cell spreading, but not CLAN formation. Thus, we were able to de-couple cell spreading from CLAN formation and show that CLAN formation is not needed for cell spreading. We can only speculate as to why CLAN formation, but not cell spreading was dependent on Src. However, Src-independent cell spreading has previously been observed in osteoclasts76
and it is possible that this can occur in TM cells.
Interestingly, it took a higher concentration of PP2 to inhibit β3 integrin-mediated CLAN formation than β1-mediated CLAN formation. It is possible that αvβ3-dependent activation of Src is a later event in the formation of CLANs than in β1-mediated CLAN formation and that activation of αvβ3 could lead to the additional recruitment of activated Src to the cell membrane. It has been shown in platelets77
that there is a pool of inactive Src that is bound to β3 integrins in the absence of any ligand binding. This Src pool becomes activated afterβ3 integrins are activated. Thus, the recruitment of this pool of Src upon activation of αvβ3 via mAb AP-5 could have been sufficient to partially overcome the effects of PP2 at relatively low concentrations and lead to a partial recovery of CLAN formation.
In summary, these studies suggest that increases in CLAN formation might be due to a significant up-regulation in one or the other integrin signaling pathways. Clearly additional studies examining the physiological role of these signaling molecules in CLAN formation are warranted and should be useful in furthering our understanding of the organization of the actin cytoskeleton in the TM and its role in outflow facility and the pathophysiology of glaucoma.