This study shows that interactions between the PPRARI sequence in the HepII domain and a co-signaling pathway involving α4β1 integrin regulate the contractile properties of TM cells. This co-signaling pathway occurs in the presence of both type I and IV collagens in the extracellular matrix, as activation of this HepII-mediated signaling pathway decreased the contractility of TM cells in collagen gels, of cell plated on type IV collagen, and in cultured anterior segments where both collagens are prevalent [47
]. Taken together, these results suggest that the α4β1 integrin may be part of a signaling mechanism along with an α1/α2β1 collagen integrin signaling pathway that regulates TM contractility in the eye. This finding has special significance for the TM of the eye where changes in the composition of the extracellular matrix are often associated with increased intraocular pressure and glaucoma. Furthermore, it demonstrates how critical the microenvironment of the cell is for the modulation of cell behavior via integrins.
These findings reflect a novel function for the PPRARI site in the HepII domain of fibronectin and for α4β1 integrin in contractile tissues. α4β1 integrin is usually found on non-adherent cells such as lymphocytes and melanomas where it plays a major role in regulating contractility during migration [48
]. Although α4β1 integrin has been reported to be expressed on adherent cells including TM cells, retinal ganglion cells, interstitial fibroblasts in the kidney, and smooth muscle cells [31
], relatively little is known about the role of α4β1 integrins outside of the immune system. These studies suggest α4β1 signaling in adherent cells is similar to that which occurs in non-adherent cells and that α4β1 signaling regulates contractile processes in tissues like the TM.
Our cell adhesion studies showed that TM cells used both α1 and a2 integrins to mediate adhesion to the collagen matrix which would suggest involvement of an α4β1 and α1/α2β1 integrin co-signaling pathway. To the best of our knowledge, this is the first time that that this integrin co-signaling pathway has been observed. Such a co-signaling pathway would explain the apparent discrepancy with the previously reported actions of HepII. In the current study, the HepII domain decreased actin polymerization and contractility of TM cells on collagen substrates, whereas in previous studies the cells were plated on fibronectin or the RGD cell binding domain of fibronectin which interacts with α5β1 integrin [31
]. In those earlier studies,. Thus, the increase in actin stress fiber formation and contractility in the earlier studies involved an α5β1/α4β1 co-signaling pathway and not a distinct α4β1 and α1/α2β1 pathway as indicated by this study.
Interestingly, the PPRARI peptide, but not the IDAPS peptide, triggered the α4β1 signaling pathway in TM cells and increased outflow facility in cultured anterior segments [29
]. IDAPS is a RGD homologue located at the III13
junction of the HepII domain and was originally identified as the α4β1 binding site in the HepII domain [53
]. More recent structural studies suggest that the PPRARI sequence within the III14
repeat is the main α4β1 integrin binding site and that IDAPS contributes indirectly to α4β1 binding by forming a stabilizing hydrogen bond with the PPRARI site [22
]. Alternatively, ligand-specific conformational differences in the α4β1 active state may account for varying activities of the two peptides. PPRARI, which binds the α4 subunit, most likely induces an α4β1 conformation that is unique from IDAPS, which binds to the β1 subunit [22
]. Such differences in the conformation of α4β1 have been reported to regulate integrin function [54
] and are consistent with our observations that the 12G10 induced conformation of α4β1, but not the TS2/16 induced conformer, significantly blocked binding to the HepII domain. It is also consistent with previous studies in which the 12G10 antibody produced a conformation of α4β1 that lead to the disruption of the cytoskeleton [12
]. Furthermore, other α4β1 ligands such as the QIDSP peptide from VCAM and the IIICS domain of fibronectin did not have a significant effect on TM cellular contractility (data not shown), suggesting that there is something unique about the activation state of α4β1 integrins and their interactions with the PPRARI sequence.
Based on the observations with the 12G10 antibodies and the enhanced binding of TM-1 cells to HepII in the presence of Mn2+, we propose that the PPRARI sequence in the HepII domain interacts with an activated conformation of α4β1 integrins. We further suggest that this conformation of α4β1 integrin only exists when a collagen signaling pathway is activated. Whether, this collagen signaling pathway is regulating the conformation of α4β1 is unknown. This co-signaling pathway is likely to play a significant role in regulating intraocular pressure where the remodeling of the extracellular matrix that occurs in the eye under conditions of increased intraocular pressure would result in the release of fibronectin fragments.
The outflow study supports previous findings in which the HepII domain and the PPRARI peptide increased outflow facility in cultured human and monkey anterior segments [29
]. Response to the peptides and the HepII domain were not uniform and some of the donor anterior segments did not respond to the HepII treatment. In the study by Gonzalez et al
] only five of nine monkey anterior segments responded to treatment with the HepII domain, and in cultured human anterior segments, 9 out of the 10 responded [36
]. Once again, this raises the questions whether cooperative integrin signaling events are involved in regulating contractility and whether outflow facility in the eye and cultured anterior segments that did not respond are lacking a necessary co-signaling factor.
Activation of α4β1 integrin by the HepII domain did not require HSPGs or the heparan sulfate binding activity of the HepII domain. Neither removal of heparan sulfates from the cell surface of TM cells nor treatment of TM cells with a mutant HepII domain that lacks the major heparan sulfate binding site affected the activity of HepII. In addition, siRNA specific knockdown of syndecan-4 expression did not abolish the actin disrupting activity of the HepII domain. Therefore, it appears that HepII acts strictly through an α4β1-dependent mechanism and does not require participation by syndecans or other cell surface HSPGs. This supports previous studies which showed that α4β1 integrin, unlike α5β1 integrin, does not require a proteoglycan as a co-receptor [55
]. It also supports previous studies utilizing human skin fibroblasts and TM cells in which heparan sulfate interactions were shown to play a minor role in HepII-mediated cell spreading [56
] and stress fiber formation [31
Although HSPGs are not co-receptors for α4β1 integrin in the TM, other protein-protein interactions may co-direct α4β1 signaling. For example, it has been shown that the core protein of chondroitin sulfate glycosaminoglycan interacts directly with α4β1 integrins [57
]. Additionally, cooperative signaling in TM cells between α4β1 and αvβ3 has been reported to enhance the formation of cross-linked actin networks [58
], and cross-talk between α4β1 and α5β1 in sub-confluent proliferating TM cells plated on fibronectin, augments cell attachment by increasing actin stress fiber formation and focal adhesion kinase (FAK) phosphorylation [31
]. Thus, the α4β1 pathway in confluent cultures is likely to involve cooperative signaling events with cell adhesion receptors which trigger the down-regulation of actin.
The finding that α4β1 is involved in the down-regulation of actin assembly and contractility in TM cells was not completely unexpected. Studies using migrating lymphocytes, neutrophils, and melanoma cells have shown that the α4 subunit plays a key role in regulating contractility [44
]. Protein kinase A phosphorylation of Ser988
on the α4 subunit blocks binding of the adaptor protein paxillin to the cytoplasmic tail of the α4 integrin subunit thereby preventing the establishment of connections with the actin cytoskeleton and activation of small GTPases such as Rac, CDC42, and Rho [59
]. While these studies suggest that an α4β1 integrin mediated signaling pathway down-regulates contractility in the TM and plays a role maintaining intraocular pressure, further studies examining how a collagen mediated signaling pathway affects α4β1 integrin signaling in the TM are warranted.