Integrin α9β1

Abstract

Integrins are transmembrane heterodimeric receptors responsible for transducing and modulating signals between the extracellular matrix and cytoskeleton, ultimately influencing cell functions such as adhesion and migration. Integrin α9β1 is classified within a two member sub-family of integrins highlighted in part by its specialized role in cell migration. The importance of this role is demon-strated by its regulation of numerous biological functions including lymphatic valve morphogenesis, lymphangiogenesis, angiogenesis and hematopoietic homeostasis. Compared to other integrins the signaling mechanisms that transduce α9β1-induced cell migration are not well described. We have recently shown that Src tyrosine kinase plays a key proximal role to control α9β1 signaling. Specifically it activates inducible nitric oxide synthase (iNOS) and in turn nitric oxide (NO) production as a means to transduce cell migration. Furthermore, we have also described a role for FAK, Erk and Rac1 in α9β1 signal transduction. Here we provide an over view of known integrin α9β1 signaling pathways and highlight its roles in diverse biological conditions.

Introduction

Integrins are transmembrane heterodimers that consist of an α and β subunit, and serve most commonly as receptors to extra cellular matrix (ECM) proteins.¹ In doing so, they promote numerous cellular functions including their prototypical roles of cell adhesion and migration.² Since integrins do not possess intrinsic enzymatic activity they transduce these complex functions through intricate, multi-protein and highly regulated intracellular signaling pathways.^3,4 A recent review outlined some 156 networks and 690 protein interactions that makeup just the integrin adhesome.⁵ Cell migration is a clear example of the complex interplay between extracellular matrix, integrin and intracellular proteins; and presumably the “migrasome” network of proteins mediating cell migration is at least as complex as the adhesome if not more so.

ECM proteins are the prototypical integrin ligands, and although many are integrin specific, they can also serve as common binding partners for more than one integrin.^1,2 The tri-amino acid sequence of arginine-glycine-aspartic acid (RGD), which serves as a cell attachment site in numerous ECM proteins is a common binding motif for several integrins⁶ and facilitates this integrin-ligand redundancy. Activation of the integrin and initiation of subsequent signaling cascades can be achieved by both intracellular “ligand” binding such as the actin adaptor protein talin (inside-out activation), and by binding a large array of extracellular ligands (outside-in activation).⁷ Likewise, the protein-protein signaling networks that transduce these communications are both common and unique to integrins, but in both cases the signaling network defines the nature of their biological effects.²

Integrin α9β1 is a relatively new addition to the β1 integrin subfamily⁷ and through homologous sequence it forms a unique subfamily with α4β1.⁹ Compared to other integrins, α9β1 has distinguished its functionality by facilitating accelerated cell migration.¹⁰ Despite an extensive description of its ligands^7,10–15 the signaling mechanisms that transduce its migratory effect are not well understood. Recently, we have shown that to transduce cell migration α9β1 utilizes a number of common integrin signaling intermediates, in particular Src; but also α9β1-specific intermediates, iNOS and nitric oxide.¹⁶ Herein we discuss these findings and highlight the unique nature of α9β1 as demonstrated through the nature of its ligands and diversity of its signal transduction mechanisms.

Integrin α9β1 Utilizes the Common Integrin Signaling Proteins: Src, FAK and Erk

FAK auto-phosphorylation at tyrosine-397 is an early event that prompts association of FAK and Src and in turn initiates a signaling cascade that may result in activation of the small GTPases rac-1 and cdc42.¹⁷ We found that specific ligation of integrin α9β1, as with other integrins activates Src, FAK and also promotes their association (Fig. 1A). Similar to its subfamily member α4β1 and in contrast to other β1 integrins, we found that FAK played a redundant role in α9β1-mediated cell migration.^16–18 Furthermore, we also found that Src activation was a proximal and dominant signaling protein regulating α9β1 led cell migration.¹⁶ Although FAK played a redundant role during cell migration, we postulate that it may serve to amplify migratory signaling; and may play a non-redundant role for non-migratory α9β1 functions such as cell survival or invasion.¹⁷

Figure 1.

Signaling mechanisms of integrin α9β1. (A) Selective ligation of integrin α9β1 activates proximal tyrosine kinase Src. Src coordinates subsequent signaling pathways; first through activation of FAK, tyrosine phosphorylation of the adaptor protein p130Cas and ultimately co-ordinates activation of small GTPase rac1, which translocates to the cell membrane and causes lamellipodial protrusion; second, Src activates iNOS resulting in increased NO production, activation of cGMP and protein kinase G signaling cascades to enhance cell migration (as reported by Gupta et al.¹⁶). (B) Integrin α9β1 can regionally recruit SSAT, an enzyme involved in the catabolism of higher order polyamines spermidine and spermine, which are potent blockers of inward rectifier K⁺ (Kir) channels. SSAT (along with PAO, polyamine oxidase) facilitates localized enzymatic processing of spermidine and spermine in to the smaller polyamine, putrescine and thus relieving outward efflux of K⁺ to co-ordinate cell migration (as reported by deHart et al.³⁷).

The molecular details of α9β1-induced Src activation remains to be elucidated, but it appears that unlike β3, its β1 cytoplasmic tail does not associate with Src.¹⁹ We postulate that Src may directly interact with the cytoplasmic tail of α9, subsequently recruiting other signaling proteins to form an associated multimeric signaling complex. Normally, Src is maintained in an inactive state by tyrosine phosphorylation (pY527) at its C-terminus, and can be reversibly activated by auto-tyrosine phosphorylation (pY416) in the kinase domain by phosphatases that dephosphorylate pY-527.²⁰ Genetic mutations at the C-terminus that uncouple Src from the PDZ domain of certain membrane proteins have also been shown to result in Src activation.²¹ Many human cancers especially those at advanced stages express constitutively active Src tyrosine kinase;²² whether or not α9β1, which is expressed in a number of cancer cell lines,^9,23–25 is involved in a similar mode of Src activation is also yet to be determined.

It has been clearly shown that extra cellular signal-regulated kinase (Erk) is a central component of integrin signaling and can be activated in a number of ways including through FAK association with adaptor proteins such as Shc and Grb2 and is involved in tumorigenesis and cancer cell proliferation.^26,27 In preliminary studies we found that specific ligation of α9β1-induced Erk phosphorylation was Src dependent and contributed to cancer cell migration. However, compared to other α9β1-induced (non-Erk) signaling, Erk-induced cancer cell migration was not robust. This highlights another important aspect of integrin signaling; its cell type variability and that defining integrin-specific signaling within cancer cell types will serve as a means to develop targeted pharmacotherapy. Further studies will also need to define the role of Erk in α9β1 regulation of other tumorogenic processes such as cancer cell proliferation or survival.

Integrin α9β1 Signals in a Unique Fashion through iNOS and NO

In novel findings, we recently showed that integrin α9β1 regulates iNOS activity via Src tyrosine kinase resulting in increased NO production and NO-induced cell migration¹⁶ (Fig. 1A). Of note, Erk activation in our in vitro system remains unaffected by inhibition of iNOS activity. Like Src, iNOS is another cytoplasmic protein maintained at low levels in almost all mammalian cells which can be either transcriptionaly upregulated or activated by various stimuli including cytokines and growth factors.^16,28–31 Of note, increased iNOS expression also serves as a cancer associated prognostic factor in addition to regulating several other biological processes.^28,32 The specialized role of integrin α9β1 in cell migration, its expression in cancer cells and iNOS signaling identifies this integrin as potentially playing a key role in carcinogenesis.

Activation of iNOS in malignant (and non-malignant) cells can promote cell migration via multiple mechanisms.^33,34 NO generated from iNOS activation can act as a cofactor to guanylyl cyclase (GC) to promote synthesis of the second messenger cGMP, which regulates cell migration in both a PKG dependent and independent fashion.^33,34 Furthermore, NO can regulate both the expression of MMP9 and its activation through cGMP dependent or independent mechanisms.^33,34 Relevant to integrin function NO released in to the cellular microenvironment can impact assembly of focal adhesions. NO-induced delay of focal adhesion assembly or their premature destabilization has significant effects on cell migratory responses. Furthermore, interactions between iNOS and rac-GTPase,³⁵ an important mediator of cell migration are now known to affect Rac-GTPase activation or its membrane translocation. We found that α9β1 induces activation of rac1 in cancer cells but there was no significant modulatory effect of iNOS.¹⁶ Despite no significant impact on rac1 activation we found a robust interaction between iNOS and rac1 (unpublished findings). Inhibition of iNOS activity however, delayed the spreading of cells on a α9 specific ligand which may be due to a slower accumulation of rac1 at the cell membrane and thus affecting the gener-ation of lamellipodia or actin assembly required for cell spreading. Whether or not iNOS has any role in membrane translocation of activated rac1 is yet to be determined. Regardless of molecular mechanism, it appears that α9β1-induced iNOS activation and NO production may modulate the mechanisms of cancer cell migration and invasion.

Further Evidence of the Unique and Diverse Nature of α9β1 Signaling Mechanisms

Spermidine, spermine acetyl transferase (SSAT) is an enzyme that acetylates its polyamine substrates spermidine and spermine, ultimately resulting in the generation of the lower order polyamine, putrescine. Spermidine and spermine are potent blockers of outward potassium currents from inward rectifier K⁺ (Kir) channels.³⁶ Catabolism of spermidine and spermine into putrescine is thus considered to result in rapid efflux of K⁺ ions, which can modulate cell migration.³⁷ The cytoplasmic tail of α9 interacts with SSAT and facilitates a localized enzymatic process-ing of spermine and spermidine in to putrescine, thereby creating a spatially restricted impairment of K+ ion channel rectification leading to increased K+ ion efflux, and enhanced cell migration^37,38 (Fig. 1B). Inwardly rectifying K⁺ (Kir) channels significantly contribute towards maintaining membrane potential and to cause blood vessel dilation in response to extracellular K⁺ or shear stress.³⁹ Although the role of α9β1 mediated K⁺ channel rectification in tumor cells is not yet clear; gene expression profiling has shown ∼3-fold increase in SSAT expression in a late stage (metastatic) colon carcinoma cell line, compared to early stage carcinomas.⁴⁰ Whether the interaction of α9β1 and SSAT plays a significant role in carcino-genesis remains to be determined.

Integrin α9β1 also associates with and modulates signaling from several growth factor receptors.^7,10,41 Evidence suggests that integrins and growth factors can synergize signaling for proliferation, survival and migration, since both are able to utilize common signaling mediators such as Src, FAK and MAP kinase.^42,43 The molecular basis of functional synergy between integrins and growth factor receptors continues to be defined but appears to fit two separate paradigms (Fig. 2): first, that both integrin and growth factor receptor bind their respective ligands, inducing their clustering within distinct membrane micro domains, and synergistic activation of downstream signaling; second, that ligand binding to integrins alone can trans-activate growth factor receptors to induce downstream signaling.⁴³ Integrin α9β1 plays an essential role in granulopoiesis, wherein it synergizes signaling through the receptor for granulocyte-colony stimulating factor.⁴⁰ Furthermore, endothelial cell α9β1 appears to play an important role in both lymphangiogenesis and angiogenesis.^7,10,44,45 The physiological role of α9β1 was clearly evident in α9-deficient mice, as described by Huang et al.⁴⁶ α9-deficient mice die 7-10 d post-partum from chylous pleural effusions and demonstrate defective lymphatic valve morphogenesis and function.^46,47 In addition, it is now well established that expression of α9β1 is part of the protein signature of lymphatic endothelium.^44,48 Similar to its ability to directly bind the lymphangiogenic proteins VEGF-C and D,⁷ α9β1 also binds VEGF-A and promotes the angiogenic response of VEGF in part through synergy with the VEGF-R2 receptor.¹⁰ Through these biological functions, α9β1 positions itself as an important regulatory factor in patho-physiological processes such as wound healing and carcinogenesis.

Figure 2.

Integrin crosstalk with growth factors and their cognate receptors. Left: shows direct ligation of integrin α9β1 by growth factors such as VEGF-A, C, D and NGF triggering the necessary signaling pathways to transduce cell attachment and migration (reported by Vlahakis et al.⁷,¹⁰ and Staniszewska et al.¹⁵). Right: shows an alternative mechanism, whereby growth factor stimulation (with or without) integrin ligation can promote co-association of growth factor receptor with integrins, where they synergize their respective effects. Specifically integrin α9β1 associates with VEGF-R2 or GCSF-R following co-stimulation of the integrin and the growth factor receptors with VEGF-A or GCSF, respectively to co-ordinate signaling for cell migration, proliferation, angiogenesis and granulopoiesis (as reported by Vlahakis et al.¹⁰ and Chen et al.⁴¹).

Summary

As described here integrin α9β1 has been implicated in diverse biological functions including angiogenesis, lymphangiogenesis, lymphatic valve morphogenesis, granulopoiesis and tumorigenesis. Like other integrins α9β1 ligation can activate signaling through Src and FAK mediated tyrosine phosphorylation of multiple proteins including p130Cas¹⁶ and paxillin.⁴⁹ Redundancy of FAK and paxillin^16,49 and a less robust migratory stimulus through Erk MAP kinase demonstrates the complexity of α9β1’s signaling network involving several parallel pathways. Recent reports showing activation of outward potassium ion channels via localized modulation of polyamines³⁷ and nitric oxide16 production through modulating iNOS activity are two specific signaling pathways assigned to α9β1 thus far. The potential role of these α9β1 specific signaling pathways and their significance in such processes as carcinogenesis or wound healing are unclear at this point.

In human cancers, integrins have been shown to contribute to tumor growth and metastasis, and thus have been identified as legitimate therapeutic targets with anti-integrin αv targeted therapies already evaluated.^50,51 It is worth noting that due to the redundancy in expression and function of integrins, inhibiting a single integrin may not result in a clinically significant effect. Therefore, strategies to target multiple integrins and/or growth factor receptors will be more useful. Since integrin α9β1 not only orchestrates biologically significant signaling in isolation, but also associates with growth factor receptors to modulate their function, it represents a novel potential pharmacotherapeutic target.