Integrin α4β1 (VLA-4) expression and activity in retinal and peripheral neurons

Abstract

The integrin α4β1 fulfills important roles in inflammation and hematopoesis, but its functions in neurons are not well understood. Here we show that the α4 subunit is expressed on mouse retinal ganglion cells (RGCs) and undifferentiated retinal neuroblasts during the period of axon extension and migration. To determine if α4 integrins expressed by retinal neurons were active, neurons were cultured on known α4 ligands in vitro. Recombinant soluble vascular cell adhesion molecule 1 (rsVCAM-1), fibronectin, and osteopontin (OPN) induced neurite outgrowth that was diminished by function blocking antibodies specific for α4. Neurite outgrowth on OPN was also blocked by antibodies to the integrin β1 subunit, implicating the α4β1 heterodimer as one integrin receptor mediating outgrowth on OPN. OPN immunoreactivity was detected in the RGC fiber layer and optic nerve, suggesting that it may act as an α4 ligand in vivo. Neurons from chick lumbar sympathetic ganglia, chick dorsal root ganglia, and mouse superior cervical ganglia also extended neurites on rsVCAM-1, suggesting that integrin α4β1 may play a role in the development of multiple neuronal cell types.

Introduction

Many adhesive and repulsive cell–cell and cell–extracellular matrix (ECM) contacts, in precise spatial and temporal windows, are crucial to the successful genesis of the nervous system. In the retina, adhesion–repulsion molecules known to be important in development include ephrins, netrins, proteoglycans, and other ECM molecules, receptor tyrosine phosphatases, cadherins, immunoglobulin (Ig) superfamily members, and integrins Hopker et al 1999, Wilkinson 2001; Stepanek et al 2001, Ranscht 2000; Crossin and Krushel 2000, Stuermer and Bastmeyer 2000.

Integrins in the β1 family have been implicated in retinal histogenesis, retinal neuroblast migration, and retinal ganglion cell (RGC) axon outgrowth, but functions of specific β1 heterodimers are less clear (Clegg et al., 2000). Immunological blockade of the β1 subunit was shown to drastically alter retinal patterning and growth (Svennevik and Linser, 1992) and inhibit RGC migration from the ventricular to vitreal retina (Cann et al., 1996). Expression of dominant negative β1 gene constructs in Xenopus inhibited outgrowth of RGC dendrites and axons (Lillienbaum et al, 1995), and inhibition of both β1 integrins and N-cadherin caused pathfinding errors (Stone and Sakaguchi, 1996). At least six integrin α subunits that form heterodimers with the β1 subunit are expressed in retina (α1,2,3,4,6,8,v) Bosco et al 1994, Cohen et al 1987, Hall et al 1987, Bossy et al 1991, de Curtis et al 1991, Duband et al 1992, de Curtis and Reichardt 1993; Sheppard et al 1994, Bradshaw et al 1995, Cann et al 1996. The α2β1 heterodimer has been shown to mediate neurite outgrowth and adhesion of retinal cells to collagens, and the α6β1 heterodimer is the primary laminin-1 (Ln1) receptor in the retina (deCurtis and Reichardt, 1993). Aberrant retinal lamination was observed in retinas of mice lacking the α6 gene, suggesting defective neuroblast migration (Georges-Labouesse et al., 1998). Integrin α4 subunit mRNA was detected throughout chick retinal development (Cann et al., 1996), and α4 immunoreactivity was observed in very early stages of mouse retinal development (Sheppard et al., 1994), but α4 function in the retina has not been investigated.

The α4 subunit can pair with either the β1 or β7 subunit. The α4β1 heterodimer (very late antigen-4, VLA4) is known to bind at least 11 ligands: vascular cell adhesion molecule-1 (VCAM-1), the alternatively spliced CS-1 region of fibronectin (Fn), thrombospondin-1, α4β1 itself, osteopontin (OPN), intercellular adhesion molecule-4 (ICAM-4), junctional cell adhesion molecule-2 (JAM-2), invasin, ADAM-28, the precursor to von Willebrand factor, and transglutaminase C Clegg et al 2002, Lobb and Hemler 1994, Elices et al 1990, Wayner et al 1989; Guan and Hynes 1990, Mould et al 1990, Qian et al 1994, Altevogt et al 1995, Yabkowitz et al 1993; Isobe et al 1997, Isobe et al 1999, Spring et al 2001, Bayless et al 1998, Cunningham et al 2002. In the immune system, the α4β1 receptor on leukocytes mediates interactions with blood vessel endothelial cells during inflammation, via binding to VCAM-1.

α4 integrins play important roles in development as well. Knockout mice lacking α4 die at E11 due to failures in formation of the heart and placenta (Yang et al., 1995; Hynes, 1996). This embryonic lethality has made α4 function later in development more difficult to study, but analysis of α4 null cells in chimeric mice has shown that α4 is required for bone marrow hematopoesis (Arroyo et al., 1996), which is consistent with earlier studies Miyake et al 1991a, Miyake et al 1991b. α4 integrins are also abundantly expressed on neural crest cells, where they have been implicated in migration by antibody and peptide perturbation experiments Stepp et al 1994, Kil et al 1998. However, genetically modified neural crest cells lacking α4 appear to migrate normally (Haack and Hynes, 2001). α4 null glial cells derived from the neural crest showed increased apoptosis in grafts and in culture, suggesting a role for α4 in glial cell survival, rather than migration. Previous investigators have speculated that neuronal α4β1 integrins may mediate neurite outgrowth on Fn, since the Fn fragment containing the α4β1-binding site (CS-1) supports outgrowth of both central and peripheral neurons Rodgers et al 1987, Hall et al 1987, Carri et al 1988, Sparrow et al 1990, Reichardt et al 1990. Recently, it was shown that α4 integrins are expressed on dorsal root ganglion growth cones in regenerating sciatic nerve, suggesting a role in nerve regeneration. The interaction with alternatively spliced isoforms of recombinant fibronectins containing the α4-binding site enhanced neurite outgrowth by these neurons (Vogelezang et al., 2001).

OPN is a highly acidic, negatively charged, phosphorylated glycoprotein with diverse functions that include cell adhesion, chemoattraction, immunomodulation, wound healing, and tissue remodeling (Denhardt and Guo, 1993; Denhardt and Noda, 1998; Denhardt et al 2001a, Denhardt et al 2001b, O’Regan and Berman 2000, Rodan 1995. Mice lacking OPN appear normal, but show increased susceptibility to bacterial and viral infection Liaw et al 1998, Ashkar et al 2000. OPN contains an RGD (arginine–glycine–aspartate) motif, which is recognized by the integrins αvβ3, αvβ5, αvβ1, α8β1, and α5β1 Katagiri et al 1996, Barry et al 2000, 1999. Other integrins, such as α4β1 Bayless and Davis 2001, Green et al 2001 and α9β1 Smith et al 1996, Taooka et al 1999 appear to bind to alternative, non-RGD sites on OPN.

Although OPN was first purified from bone (Oldberg et al., 1986), it has now been detected in many tissues, including kidney (Denda et al., 1998), arterial smooth muscles Giachelli et al 1995, Takano et al 2000, Weintraub et al 2000, inner ear, gallbladder, urinary/reproductive tracts Bobe and Goetz 2001, Cancel et al 1997, Cancel et al 1999, gastrointestinal tracts, mammary glands, bronchi, salivary glands, sweat ducts, and the nervous system (Mark et al., 1998). OPN expression has been documented in various parts of the nervous system, including various regions in the brain (Shin et al., 1999), trigeminal ganglion (Ichikawa et al., 2001), Schwann cells (Jander et al., 2002), and the retina, where immunoreactivity was detected in adult rat retinal ganglion cells (Ju et al., 2000). However, a neuronal function for OPN has not been documented.

VCAM-1 is a member of the immunoglobulin superfamily that is widely expressed in developing mesenchyme, bone marrow, heart, muscle, and on inflamed endothelium in response to several inflammatory cytokines Osborn et al 1989, Sheppard et al 1994. The predominant form consists of a single-transmembrane domain and contains seven IgG domains. VCAM-1 promotes cell–cell adhesion and migration of a variety of cell types via α4β1–VCAM-1 interactions Rosen et al 1992, Miyake et al 1991a, Qian et al 1994, Lobb and Hemler 1994, Yang et al 1996. These interactions are essential for normal development since VCAM-1 knockout mice show defective heart formation and (mostly) die at E11 Kwee et al 1995, Gurtner et al 1995.

In this report, we have investigated the distribution and function of the integrin α4β1 in developing neurons. We show that α4 integrin is expressed by both retinal neuroepithelial cells and RGCs and that it functions to mediate neurite outgrowth on VCAM-1, Fn, and OPN in vitro. OPN is expressed in a pattern that suggests that it may serve as a ligand for α4 integrins in vivo. We also show that recombinant, soluble VCAM-1 (rsVCAM-1) elicits robust neurite outgrowth from peripheral neurons, suggesting multiple neuronal functions for α4 integrins.