In this study we found that neurotrophins such as NGF, BDNF and NT3 contain a third common receptor, α9β1 integrin. Currently characterized receptors for these molecules belong to the highly specific tyrosine kinase family (Trks), and common to all the neurotrophins, the low affinity receptor p75NTR
. Very specific interaction of this integrin with these three molecules was observed in adhesion assays of cells transfected with the α9 integrin subunit, verifying the expectation that α9β1 integrin is another common, low affinity receptor for neurotrophins similar as p75NTR
. However, these two cell surface receptors, following binding to NGF appeared to show completely opposite functions. Integrin induces pro-proliferative and pro-migratory activity of cells, whereas p75NTR
is involved in transferring pro-apoptotic signals. Interaction of NGF with α9β1 does not require any integrin stimulators or binding enhancers such as monoclonal antibodies (e.g. TS2/16) or high concentrations of Mn2+
(Bazzoni et al., 1995
; Byzova and Plow, 1998
). This observation increases the relevancy of α9β1-neurotrophin interaction in the physiology and pathology of various organs.
The deficiency of this integrin on brain cells () (Staniszewska et al., 2007
) is a little intriguing in the context of our major discovery that it is a receptor for neurotrophins. This suggests that the interaction of NGF with α9β1 integrin does not have a regulatory effect on the central nervous system, however, it may influence the physiology of other organs affecting the peripheral nervous system. The binding of neurotrophins to α9β1 integrin may be important in the development of pathology in the brain. A majority of malignant gliomas express this integrin and NGF potently induces α9β1-dependent proliferation and invasion of these cancer cells (our unpublished data).
The binding of NGF to α9β1 integrin is promoted similarly to other natural ligands such as VCAM1 or ECM proteins. The necessity of divalent cations for this interaction indicates that subunits of α9β1 need to form an active conformation of their ligand binding pocket. This requirement is strictly essential for specific integrin-ligand interactions in cell physiology. Moreover, the ability of NGF to induce the LIBS epitope on the β subunit of α9β1 exhibits a similar mechanism of binding to integrins as endogenous (ECM) and exogenous (disintegrins) ligands, which in association with their receptor express a neo-epitope that is recognized by an anti-LIBS antibody (Marcinkiewicz et al., 1997
). Finally, the ELISA assay confirmed the direct binding of hrNGF to α9β1 integrin in a manner opposite to that of immobilized mNGF and integrin in solution. Interestingly, we found species selectivity for soluble neurotrophin in ELISA. Experiment with hrNGF showed two orders of magnitude of increasing binding affinity in comparison with mNGF (Kd
4.5 vs 440 nM). By contrast, NGF in the immobilized form appears to have species-independent pro-adhesive properties for cells expressing α9β1 integrin, suggesting that ligand-receptor interaction in the solid phase may have different conformational requirements.
We found in cell migration and proliferation assays, the importance of α9β1-NGF interaction in cell physiology. Young et al. (Young et al., 2001
) reported that the cytoplasmic domain of the α9 integrin subunit may be important in enhancing the rate of cell migration, which agrees with the α9β1 integrin-dependent pro-migratory activity of NGF. However, the mechanism that involves α9β1 integrin in the promotion of cell migration is still unclear. Our results confirmed that α9β1 integrin, following binding to its ligand NGF, induces activation of a focal adhesion adaptor protein, paxillin (). Binding of paxillin to the α9 subunit is sufficient to modulate cell spreading, but it has no effect on the regulation of cell migration. However, using two different inhibitors of this integrin, we observed a correlation between phosphorylation of paxillin and cell migration. The NGF-dependent chemoattraction of α9SW480 cells was potently inhibited by blocking with an anti-α9β1 monoclonal antibody, whereas the effect of the MLD-disintegrin, VLO5 was not significant. The same activity of α9β1 integrin inhibitors was obtained in the transmigration assay of human neutrophils. Accordingly, inhibition of NGF-induced paxillin phosphorylation was more potently inhibited by Y9A2 than by VLO5. These data may suggest that growth factor-induced α9β1 integrin-dependent cell migration is associated with paxillin activation, whereas extracellular matrix (Tenascin-C)-induced cell migration is paxillin independent (Young et al., 2001
). Further work with paxillin-deficient cells will verify this hypothesis.
The major difference between α9β1 and the other common receptor for neurotrophins, p75NTR
, is an effect on cell proliferation. p75NTR
without association with TrkA is an inhibitor of this process and an inducer of pro-apoptotic signals (Lee et al., 2001
). Following binding to α9β1, NGF induces the activation of MAPK Erk1/2, which is generally characterized as a signaling molecule mediating pro-proliferative and pro-survival signals. Interestingly, this growth factor caused increased phosphorylation of Erk1/2 only in transfected cells expressing α9β1 integrin, whereas no effect was observed in control cells. The α9β1-dependent mediation of cell signaling upon binding to NGF was also confirmed by specific integrin inhibitors, as well as by interaction with GD10 cells transfected with α9 subunit and hybrid β1/β3 subunits. VLO5 and Y9A2 significantly decreased Erk1/2 phosphorylation in α9SW480 cell adhesion to immobilized NGF, whereas soluble NGF increased signal transduction in GD10 cells transfected with wild-type α9β1 integrin. This effect was not observed in the cells that were transfected with a hybrid β subunit containing extracellular and transmembrane domains from β1 and the cytoplasmic domain from β3 subunit. It suggests that NGF, similarly to ECM proteins (e.g. fibronectin) (Danen et al., 2002
), induces cell signaling pathways through the β1 subunit of the integrin.
The signal transduction activity links α9β1 integrin with the high affinity NGF receptor, TrkA, which is also involved in transferring signals inside the cells utilizing the Erk1/2 pathway (Edsjo et al., 2001
; Slack et al., 2005
). In this context, expression of p75NTR
appears to be less important, because this is a pro-apoptotic receptor that is not involved in the stimulation of cell proliferation and Erk1/2 signaling. Although RT-PCR and western blot results showed a very low expression of TrkA on α9SW480 cells, and a radioisotope study confirmed an almost entire lack of high affinity-dependent interaction of these cells with NGF, we are not able to exclude the possibility that both receptors may cooperate in signal transduction. In this context, the observation that cells transfected with α9 integrin subunit significantly decrease expression of TrkA is very interesting. It may suggest that both NGF receptors are in a functional relationship and the deficiency of one is compensated by the other. Although blocking experiments with anti-TrkA serum and TrkA siRNA (data not shown) proved that α9β1 integrin is independent in transferring pro-survival signals, the possibility of participation of TrkA in the cross-talk with this integrin may occur under certain physiological conditions.
Previously published reports showed a high and selective expression of α9β1 integrin on neutrophils and the involvement of this integrin in chemotaxis of these granulocytes across an activated endothelial monolayer (Taooka et al., 1999
; Marcinkiewicz et al., 2000
). Our findings may contribute to the explanation of mechanisms that are involved in NGF-dependent interaction of neutrophils in the progression of certain autoimmune diseases. Increased NGF levels have been observed in various inflammatory states including asthma (Braun et al., 1999
) and arthritis (Aloe et al., 1992
) and this upregulation was associated with a significant influx of neutrophils. Gee et al. (Gee et al., 1983
) also observed activity of NGF as an inducer of polymorphonuclear leukocyte chemotaxis at nanomolar concentrations. However, the mechanism of this pro-migratory effect was not evaluated. The interaction of NGF with integrin as proposed by us may contribute to the explanation of this phenomenon. Another very important pathology induced by NGF is thermal hyperalgesia. Injection of NGF into experimental animals induced neutrophil accumulation and hyperalgesia in skin (Bennett et al., 1998
). A recent report showed that the involvement of certain integrins, especially the β1 subunit, is important in developing mechanical hyperalgesia induced by NGF (Malik-Hall et al., 2005
). However, NGF protected murine neutrophils from apoptosis and enhanced their survival (Kannan et al., 1991
). Based on the data presented in this work the mechanism of neutrophil accumulation and induction of pro-survival signals may be dependent on the interaction of NGF with α9β1 integrin.
NGF has been detected in body fluids as well as in a majority of tissues, but in different concentrations (Katoh-Semba et al., 1989
). Our finding that α9β1 integrin binds this growth factor in immobilized and soluble form suggests the availability of this receptor-ligand interaction in circulatory systems and solid tissues of the body. Published studies show that the effective concentration of NGF required to stimulate neurite outgrowth in vitro is in the range of 10-100 ng/ml (Levi-Montalcini, 1982
), whereas the NGF level in blood plasma was estimated to be approximately 100 pg/ml. Therefore, this in vitro activity that is dependent on a high affinity NGF receptor, TrkA, requires a concentration of NGF at least a two orders of magnitude higher than those observed in vivo. The affinity interaction of isolated human recombinant α9β1 integrin with hrNGF in ELISA (Kd
=4.5 nM) is in the same range as for p75NTR
, which was Kd
=1.4 nM in a Scatchard analysis of 125
I-NGF binding to cells expressing this receptor (Hempstead et al., 1991
). The same radioactive analysis showed Kd
=25 pM for a high affinity complex of TrkA. Therefore, the concentrations of NGF that we used in our in vitro experiments corresponds to this ratio, because α9β1 integrin is a low affinity receptor and in all cell signaling and proliferation experiments its effective doses did not exceed 1 μg/ml. Higher amounts of mNGF used in the cell migration and adhesion assays resulted from specificity of assays. Immobilization of protein on the plastic is not 100%, especially for proteins of low molecular mass, such as NGF.