Research ArticleHeparin II domain of fibronectin mediates contractility through an α4β1 co-signaling pathway
Introduction
The actin cytoskeleton is a dynamic structure and modulates tissue function by altering its contractile properties. For example, reorganization of the actin cytoskeleton within the trabecular meshwork (TM) of the eye leads to changes in intraocular pressure. The TM is a specialized tissue located within the anterior segment of the eye that regulates intraocular pressure by mediating the flow of aqueous humor through the anterior segment. A decrease in cell contractility or disruption of an assembled actin network in the TM facilitates aqueous humor outflow and consequently decreases intraocular pressure [1], [2], [3], [4]. As with other contractile tissues, contractility in the TM is regulated by the activation of Rho-kinase, protein kinase C, or myosin light chain kinase which modulate myosin light chain (MLC) phosphorylation and the subsequent contraction of the TM [5], [6]. Inhibition of MLC phosphorylation decreases contractility by disrupting actin polymerization and formation of focal adhesions [7], [8]. However, the exact mechanisms by which external stimuli trigger contractile responses in the TM require further study.
Integrins are ubiquitously expressed heterodimeric α/β transmembrane receptors that bind extracellular matrix (ECM) proteins. They establish a direct link between the ECM and the actin cytoskeleton, transmitting signals that regulate adhesion, actin organization, and contractility [9]. Integrins control contractility and the organization of the actin cytoskeleton by modulating Rho GTPases. Of all the integrins, α4β1 integrin is most recognized for its role in decreasing cell contractility by disrupting focal adhesion formation and actin organization [10], [11], [12]. α4β1 integrin binds a wide range of cell surface and extracellular matrix ligands including vascular cell adhesion molecule-1 (VCAM-1), thrombospondin, mucosal addressin cell adhesion molecule-1 (MAdCAM-1), osteopontin, CD14, and the LDV and REDV sequences in the alternatively spliced V region of fibronectin [13], [14], [15], [16], [17], [18], [19]. α4β1 integrin also binds other regions of fibronectin, including the KLDAPT sequence in the III5 repeat, the EDGIHEL sequence in the alternatively spliced EDA segment, and the PPRARI/IDAPS sequence in the III14 repeat of the heparin II (HepII) domain [20], [21], [22]. The interaction between the PPRARI/IDAPS sequence in the HepII domain and α4β1 integrin, however, has never been shown to produce any physiological response.
The HepII domain of fibronectin comprises the type III12 through III14 repeats. It contains a high-affinity heparin binding domain within the III13 repeat, as well as a lower affinity heparin binding site within the PPRARI sequence of the III14 repeat [23], [24]. Although, PPRARI has been reported to serve as a ligand for α4β1 [22], it is best known as a ligand for syndecan-4, a member of the heparan sulfate proteoglycan (HSPG) family of transmembrane receptors [25]. The interaction between PPRARI and syndecan-4 mediates the formation of focal adhesions and actin stress fibers by triggering the clustering of the syndecan-4 core protein and the subsequent activation of protein kinase Cα and RhoA [26], [27].
A peptide containing the PPRARI sequence of the HepII domain in fibronectin has recently been shown to down-regulate the organization of the actin cytoskeleton in confluent cultures of TM cells [28] as well as lower intraocular pressure when perfused through cultured human and monkey anterior segments [29]. Presumably, the decrease in intraocular pressure is due to the PPRARI site in the HepII domain activating a signaling pathway that triggers a decrease in contractility. Because both syndecan-4 and α4β1 integrins have been found in TM cell cultures and in vivo [30], [31], the purpose of this study was to identify the signaling pathway utilized by the HepII domain to regulate contractility in TM cells and potentially increase aqueous humor movement in cultured anterior segments.
Using a line of human TM cells (TM-1), we showed that the HepII domain of fibronectin uses a co-signaling pathway involving α4β1 integrin and collagen to trigger the disruption of the actin cytoskeleton and decrease cellular contractility. These data further suggest that it is the PPRARI sequence within the HepII domain which interacts with an activated α4β1 integrin. The activation occurs independently of syndecan-4 indicating that PPRARI is an α4β1 ligand as previously proposed [22]. This study demonstrates, for the first time, that interactions between the HepII domain and α4β1 integrin co-signaling pathway produce a physiological consequence, especially in the regulation of intraocular pressure.
Section snippets
Cell culture
The immortalized human TM-1 cell line was maintained in low-glucose Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, St. Louis, MO), 10% fetal bovine serum (Atlanta Biologicals, Atlanta, GA), 2 mM l-glutamine (Sigma-Aldrich), 1% amphotericin B (Mediatech, Herndon, VA), and 0.05% gentamicin (Mediatech) [32]. In some experiments, cells were plated on 10 μg/ml plasma fibronectin or type IV collagen (Millipore, Billerica, MA) and treated at confluence with serum free medium in the absence or
Knockdown of syndecan-4 does not block the activity of the HepII domain
It has been reported that the PPRARI sequence of the HepII domain interacts with the glycosaminoglycan chains of syndecan-4 to promote the formation of actin stress fibers and focal adhesions [37]. PPRARI has also been identified as the active site in the HepII domain that triggers a disruption in the actin cytoskeleton in TM cells [29]. To determine if interactions of the HepII domain with syndecan-4 [38] could contribute to a disruption of the actin cytoskeleton, syndecan-4 expression in TM-1
Discussion
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 cells plated on type IV collagen, and in cultured anterior segments where both collagens
Acknowledgments
This work was supported in part by National Eye Institute grants EY018274 (MKS), EY012515, EY017006 (DMP), EY02698 (PLK), EY016995, EY018179 (NS), Research Resources Grant RR000167 to the Wisconsin National Primate Research Center, and a core grant EY016665 to the Department of Ophthalmology and Visual Sciences. NS is also a recipient of a research award from the Retina Research Foundation.
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