Activation of integrin α5β1 delays apoptosis of Ntera2 neuronal cells

www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 28 (2005) 588 – 598

Activation of integrin A5B1 delays apoptosis of Ntera2 neuronal cells Rosemary M. Gibson,* Susan E. Craig, Laura Heenan, Cathy Tournier, and Martin J. Humphries Faculty of Life Sciences, University of Manchester, 1.124 Stopford Building, Oxford Road, Manchester, M13 9PT, UK Received 25 May 2004; revised 28 October 2004; accepted 3 November 2004 Available online 20 January 2005 Integrins are dynamic membrane proteins that mediate adhesion of cells to the extracellular matrix. Integrins initiate signal transduction, alone and cooperatively with growth factor receptors, and regulate many aspects of cell behavior. We report here that a5B1-mediated adhesion of Ntera2 neuronal cells to fibronectin decreased apoptosis in response to serum withdrawal. Adhesion induced phosphorylation of FAK, and strongly increased the AKT phosphorylation induced by growth factors, demonstrating for the first time in neuronal cells that integrin-mediated adhesion and growth factors cooperate to regulate AKT activity. Integrins exist on cells in different activation states, and cell survival on fibronectin was enhanced by the antibody 12G10, that modulates the conformation of B1 in favor of its active form. The antibody 12G10 specifically delayed loss of phosphorylation of AKT on serine 473, and GSK-3B on serine 9, induced by serum withdrawal, suggesting that these kinases are critical sensors of integrin activation on neuronal cells. D 2004 Elsevier Inc. All rights reserved.

Introduction Integrins are transmembrane heterodimers of a and h subunits that provide dynamic, physical links between the extracellular matrix (ECM) and the cytoskeleton, and initiate signal transduction cascades, allowing the cell to sense and respond to its environment (reviewed by Hynes, 2002). In the periphery, integrins regulate many aspects of cell behavior including survival, proliferation, and differentiation. To date, 24 integrin subunits have been identified in mammals, and many of these have been detected in the central nervous system (CNS), with specific regional distributions (Pinkstaff et al., 1998, 1999). Integrins play important roles in development of the nervous system, notably in neuronal migration and formation of the cerebral cortex (reviewed by Milner and Campbell, 2002). Thus, conditional deletion of either h1 integrin or focal adhesion kinase (FAK), an enzyme implicated in many signal transduction pathways from integrins (Parsons, 2003), generates very similar phenotypes, with disorganization of the marginal zone of the cortex

* Corresponding author. Fax: +44 161 275 5948. E-mail address: [email protected] (R.M. Gibson). Available online on ScienceDirect (www.sciencedirect.com). 1044-7431/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2004.11.004

(Beggs et al., 2003; Graus-Porta et al., 2001). Integrins also participate in synaptic activity. Specifically, peptides bearing the integrin-binding RGD motif, found in many ECM proteins, prevent stabilization of long-term potentiation (Bahr et al., 1997; Chun et al., 2001; LeBaron et al., 2003; Staubli et al., 1990, 1998), increase fast excitatory postsynaptic potentials (Kramar et al., 2003), and inhibit pre- and postsynaptic maturation of synapses (Chavis and Westbrook, 2001). Different integrins in the hippocampus have been proposed to contribute to these effects: a3 (Kramar et al., 2002), a5h1 (Chun et al., 2001), a3, a5, a8 (Chan et al., 2003), and h3 (Chavis and Westbrook, 2001). Integrins have also been implicated, most recently, in the regulation of hyperalgesia (Dina et al., 2004). In the periphery, interactions of integrins with ECM have long been known to regulate cell viability (for reviews, see Damsky and Ilic, 2002; Frisch and Screaton, 2001; Stupack and Cheresh, 2002). Evidence is emerging that survival of neuronal cells is also regulated via integrin-mediated adhesion: blocking antibodies to h1 integrins can induce apoptosis of adherent neuroblastoma cells (Bonfoco et al., 2000); degradation of laminin sensitizes hippocampal neurons to cell death in vivo (Chen and Strickland, 1997), and adhesion to laminin can reduce excitotoxic death of hippocampal neurons in vitro (Gary and Mattson, 2001; Gary et al., 2003). Here, we have extended these studies by asking whether survival of neuronal cells, adherent to ECM, can be enhanced by activating integrin antibodies that modulate the conformation of the h1 integrin in favor of its active form (Humphries et al., 2003). We have employed human Ntera2 neuronal cells, which have a committed neuronal precursor phenotype (Pleasure and Lee, 1993), and are being evaluated for transplantation into the CNS as a replacement therapy for neurodegenerative disorders such as stroke and amyotrophic lateral sclerosis (Garbuzova-Davis et al., 2002; Lee et al., 2000; Nelson et al., 2002). We have previously shown that Ntera2 cells undergo apoptosis when starved of growth factors (Gibson, 1999), and that the cell death is accompanied by detachment from ECM (Lesay et al., 2001). Here, we report that the protection from apoptosis afforded by integrin-mediated adhesion to fibronectin is increased by the integrin activating antibody, 12G10. The key signaling molecules that are associated with these neuroprotective effects of 12G10 are the protein kinase AKT and its downstream substrate GSK-3h.

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Results Detachment of neuronal cells accelerates apoptosis Ntera2 neuronal cells undergo apoptosis when fetal bovine serum (FBS) is removed from their growth medium (Gibson, 1999). To examine the role of adhesion to the extracellular matrix (ECM) in modulating neuronal survival, we initially examined whether plating Ntera2 cells on the ECM protein fibronectin protected the cells from apoptosis compared to cells in suspension. At all the time points studied, the proportion of apoptotic cells in the suspension culture greatly exceeded the proportion of apoptotic cells in the culture that had been plated on fibronectin (Fig. 1A). To confirm this result, activation of the effector caspase, caspase-3, which is important for apoptotic execution, was evaluated by cleavage of its substrate poly(ADPribose) polymerase (PARP) (Lazebnik et al., 1994). Proteolysis of intact 116 kDa PARP into a characteristic 85 kDa fragment was increased in the cells in suspension at all time points compared to the cells on fibronectin. By 24 h, there was little intact PARP remaining in the cells in suspension, suggesting a small percentage of non-apoptotic cells (Fig. 1B); by this stage, the accumulated apoptotic cells will be undergoing secondary necrosis and becoming permeable, explaining the decrease of the cleaved PARP fragment (Gibson, 1999). These data suggest that survival of Ntera2 neuronal cells is increased by adhesion to fibronectin. Neuronal adhesion to fibronectin is mediated by the integrin a5b1 Adhesion to fibronectin can be mediated by several different integrin heterodimers including a3h1, a4h1, a5h1, a8h1, aIIbh3, avh1, avh3, avh6, and avh8 (van der Flier and Sonnenberg,

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2001). To determine which integrin(s) mediate adhesion of Ntera2 neuronal cells to fibronectin, the integrins expressed on the surface of the cells were characterized by flow cytometry with antibodies specific for the different subunits. Alpha 3, a5, a6, av, and h1 were expressed on the Ntera2 cells (Fig. 2A). To test the contribution of each integrin to adhesion to fibronectin, spreading of the cells was analyzed in the presence of antibodies that block the function of these integrins. The antibodies directed against a5 (mAb 16) and h1 (mAb 13) potently blocked spreading (Fig. 2B), suggesting that the a5h1 heterodimer mediates adhesion of Ntera2 cells to fibronectin. Integrin activation reduces neuronal apoptosis To investigate whether the neuroprotective effects of fibronectin could be increased by modulating the activation state of the a5h1 integrin on the surface of the cells, Ntera2 cells plated on fibronectin were starved of serum in the presence of 12G10, an activating antibody that recognizes a sequence in the A-domain of the h1 integrin subunit (Mould et al., 1995, 1998, 2002) or K-20, an antibody that recognizes, but neither activates nor inhibits the h1 integrin (Amiot et al., 1986). After both 6 and 9 h, 12G10 reduced the number of detached apoptotic cells compared to K-20 or no antibody (Fig. 3A), and decreased cleavage of PARP (Figs. 3B and C). To confirm that activation of h1 integrin on neuronal cells, adherent to fibronectin, could suppress apoptosis, the effects of different concentrations of 12G10 and a second activating antibody, TS2/16 (Hemler et al., 1987; Masumoto and Hemler, 1993) were compared with that of K-20. Both 12G10 and TS2/16 dose-dependently reduced neuronal cell death, relative to K-20 (Fig. 4A). Fab fragments of 12G10 also reduced the amount of cell death in response to serum withdrawal, relative to Fab

Fig. 1. Apoptosis of Ntera2 cells is reduced by adhesion to fibronectin. Ntera2 cells, adherent to fibronectin (FN; 1 Ag/cm2) or in suspension on poly-HEMA, were starved of FBS and harvested at the time intervals shown. (A) Cells were stained with Hoechst 33342 and those with brightly staining, condensed chromatin were scored as apoptotic. Results are the mean of three experiments (FSEM). (B) Caspase activation was analyzed by probing immunoblots of cell lysates with a monoclonal antibody to PARP that detects the intact 116 kDa protein and the 85-kDa fragment generated by caspase cleavage.

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Fig. 2. Integrin a5h1 mediates adhesion of Ntera2 cells to fibronectin. (A) Integrin expression on the surface of Ntera2 cells was analyzed by incubation of cells with monoclonal antibodies to the a and h integrin subunits shown or no antibody (bCQ). After incubation with FITCconjugated anti-rat or anti-mouse immunoglobulin secondary antibodies, the proportion of cells that gave a signal above that of the cells without primary antibody was determined by analysis on a Becton-Dickinson FACS Vantage flow cytometer. The bars represent the relative expression of each integrin on the cells. (B) The integrin heterodimer that mediates adhesion to fibronectin (1 Ag/cm2) was identified by plating cells in the presence of function blocking antibodies (10 Ag/ml) to h1, a3, a5, a6, or av integrins for 1 h, and scoring the number of fully spread cells by phase contrast microscopy. The results are the mean of triplicate wells (FSEM).

fragments of the neutral antibody 8E3 or no antibody (Fig. 4B), ruling out integrin clustering as the main mechanism of action of 12G10. We also tested whether plating cells on different concentrations of fibronectin modulated the response to 12G10, since increased ECM concentrations might be expected to induce similar conformational changes to activating antibodies. When cells were plated on fibronectin at 0.1 Ag/cm2 (10-fold lower than used previously), 12G10 reduced cell death in a dose-dependent manner, whereas at 10 Ag/cm2 (10-fold higher), the dosedependent effects of 12G10 were lost (Fig. 4C). Signal transduction pathways downstream of 12G10 Integrin–ligand interactions have been shown to activate many different signaling cascades (reviewed by Hynes, 2002). To characterize the intracellular response to 12G10, changes in phosphorylation of key signaling proteins were analyzed by kinase assays and immunoblots with phospho-specific antibodies.

Activation of the c-jun N-terminal kinase (JNK) has been implicated in both pro-apoptotic and pro-survival signaling downstream of integrins (Almeida et al., 2000; Frisch et al., 1996). The activity of JNK was therefore investigated in Ntera2 cells treated with 12G10 in serum-free media. JNK activity was initially low in cells plated on fibronectin; JNK was activated upon serum starvation, but the response was similar in cells treated with 12G10 or K-20 (Fig. 5A). Integrin-mediated adhesion regulates both activation and nuclear translocation of the extracellular signal regulated kinases (ERK) (Howe et al., 2002). The phosphorylation state of ERK-1 and ERK-2 was therefore examined. Compared to the levels of total ERK, there was no change in phosphorylation of either isoform in response to serum starvation of Ntera2 cells in the presence of the integrin antibodies (Fig. 5B). Focal adhesion kinase (FAK) and protein kinase B/AKT play key roles in survival signaling from integrins (Stupack and Cheresh, 2002). Immunoblotting revealed that FAK was strongly phosphorylated on tyrosine 397 in Ntera2 cells growing on fibronectin in the presence of serum. Upon removal of FBS, FAK was rapidly dephosphorylated (Fig. 5C). The kinetics of dephosphorylation appeared slightly faster in the presence of K20 compared to 12G10, but densitometric analyses showed that the differences in FAK phosphorylation between cells treated with 12G10 versus K-20 were very small. The kinase AKT is activated by phosphorylation on threonine 308 and serine 473 (Alessi et al., 1996). Immunoblotting with antibodies that recognize AKT phosphorylated on these residues revealed that both sites were phosphorylated in Ntera2 cells growing in FBS on fibronectin and were rapidly dephosphorylated when serum was removed. Dephosphorylation of T308 occurred more rapidly than that of S473, and there was no detectable T308 phospho-AKT band at the first time point (15 min) studied (Fig. 5D). Loss of phosphorylation on S473 of AKT occurred more slowly, and most significantly, the decrease was delayed by 12G10 compared to K-20 (Fig. 5D). The lack of a response by FAK to 12G10 was somewhat surprising. The effects of adhesion of Ntera2 cells to fibronectin and exposure to the growth factors in FBS on phosphorylation of FAK and AKT were therefore analyzed. In detached cells in the absence of FBS, there was no detectable phosphorylation of FAK on Y397, and only very low levels of phosphorylation of AKT on S473 (Figs. 6A and B). Addition of FBS to detached cells did not increase the phosphorylation of FAK, but slightly increased that of AKT on S473. Adhesion of cells to fibronectin subsequently led to FAK phosphorylation and greatly increased AKT phosphorylation (Figs. 6A and B). These results show that Ntera2 cells respond to adhesion to fibronectin by phosphorylation of FAK as would be expected, and there is strong cooperative activation of AKT by growth factors and integrin-mediated adhesion to fibronectin. Since the kinetics of dephosphorylation of T308 and S473 of AKT were different, and maximal activity of the enzyme requires phosphorylation of both sites, the activity of AKT in response to serum starvation in the presence of the integrin antibodies was determined. The AKT kinase assay showed that treatment with 12G10 delayed the decrease in AKT kinase activity in response to serum starvation compared to K-20 (Fig. 7). Activation of AKT requires not only phosphorylation, but also interaction with phospholipids, generated by phosphatidylinositol 3-kinase (PI 3-K), that bind to its pleckstrin homology domain (Franke et al., 1995). Generation of these phospholipids by PI 3-K

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Fig. 3. Integrin activation by 12G10 reduces apoptosis of Ntera2 cells. Ntera2 cells growing on fibronectin (1 Ag/cm2) were transferred, at time 0, to serum-free medium with or without the integrin antibodies 12G10 or K-20 (10 Ag/ml). (A) After 6 and 9 h, the numbers of detached apoptotic cells were counted. The results are the mean of five experiments (FSEM), expressed relative to the number of apoptotic cells in the culture that had been starved of serum without integrin antibodies for 6 h. (B) Cleavage of PARP was analyzed in the Ntera2 cells after culture with FBS (lane 1) or without FBS in the absence (lane 2) or presence of 12G10 (lane 3) or K-20 (lane 4) for 9 h by Western blot analysis of 10 Ag total protein, and probing with a monoclonal antibody to PARP. A representative blot is shown. (C) The blots from three experiments were analyzed by densitometry, and the extent of PARP cleavage expressed relative to that for cells in FBS, where there was no detectable cleavage (FSEM).

is antagonized by the lipid phosphatase PTEN (Leslie and Downes, 2002). We therefore examined phosphorylation of PTEN, since its membrane targeting and activation are regulated by phosphorylation (Das et al., 2003), and the role of PI 3-K in mediating the effects of 12G10 on survival of Ntera2 during serum starvation. Phosphorylation of PTEN on S380 did not alter with serum starvation in the presence of the integrin antibodies (Fig. 8A). To investigate the role of PI 3-K, the reversible inhibitor LY249002 and irreversible inhibitor wortmannin were used. Cells were pretreated with inhibitors, and serum starved in the presence of 12G10 or K-20 and the inhibitors (Fig. 8B). Inhibition of PI 3-K increased cell death under all the conditions, confirming the importance of this enzyme in modulating cell survival, but 12G10 still reduced the amount of apoptosis compared to either K-20 or the absence of either antibody (Fig. 8B). One target of AKT that is abundant in the nervous system, and has been implicated in neuronal apoptosis is glycogen synthase kinase-3h (GSK-3h) (reviewed by Kaytor and Orr, 2002). This kinase is negatively regulated by AKT; when phosphorylated on serine 9, it is inactive (Shaw et al., 1997). Similarly to AKT S473, phosphorylation of GSK-3h was greatly increased in Ntera2 cells grown on fibronectin in the presence of serum compared to cells either plated on fibronectin or treated with FBS alone (Fig. 9A). When the cells were starved of serum, GSK-3h was dephosphorylated more slowly than AKT itself, and the dephosphorylation was slowed by 12G10 compared to K-20 (Fig. 9B).

Discussion We have shown that survival of Ntera2 neuronal cells requires adhesion to fibronectin via the integrin a5h1, and that survival is enhanced by the integrin activating antibody 12G10. FAK is a central mediator of integrin signaling (reviewed by Parsons, 2003). Consistent with this role, FAK (Y397) was phosphorylated in Ntera2 cells in response to adhesion to fibronectin in the presence or absence of FBS, and was strongly phosphorylated following prolonged (24 h) exposure to fibronectin and growth factors. AKT is a downstream kinase that regulates growth factor-induced survival of cerebellar granule neurons (Dudek et al., 1997) and sympathetic neurons (Crowder and Freeman, 1998; Philpott et al., 1997), and that is activated in response to integrin-mediated adhesion (King et al., 1997; Tian et al., 2002). In Ntera2 cells, the low level of AKT phosphorylation on S473 in response to growth factors was strongly increased when cells were plated on fibronectin, suggesting that a5h1-dependent adhesion increases growth factor-induced AKT phosphorylation in neuronal cells. This agrees with previous results in epithelial cells (Lee and Juliano, 2000, 2002), and oligodendrocytes, in which integrin-dependent adhesion amplifies signal transduction from integrins and growth factor receptors colocalized in lipid rafts (Baron et al., 2003). AKT was more strongly regulated in response to the combination of integrin-mediated adhesion and growth factors than FAK.

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Fig. 4. Integrin activation by TS2/16, 12G10, Fab fragments or high concentrations of fibronectin reduces apoptosis of Ntera2 cells. (A) Ntera2 cells growing on fibronectin (1 Ag/cm2) were transferred to serum-free medium containing the integrin antibodies 12G10, TS2/16 or K-20 (0.1, 1.0, or 10 Ag/ml). After 9 h, the numbers of detached apoptotic cells were counted. The results are the mean of three experiments (FSEM). (B) Ntera2 cells growing on fibronectin (1 Ag/ cm2) were transferred to serum-free medium with or without Fab fragments of 12G10 or 8E3 (1 Ag/ml). After 9 h, the numbers of detached apoptotic cells were counted. The results are the mean of three experiments (FSEM). (C) Ntera2 cells plated on 0.1 Ag/cm2 or 10 Ag/cm2 fibronectin were transferred to serum-free medium containing 12G10 (0.1, 1.0, or 10 Ag/ml). After 9 h, the numbers of detached apoptotic cells were counted. The results are the mean of three experiments (FSEM).

Both kinases were dephosphorylated when the growth factors were removed from the Ntera2 cells, but the kinetics of dephosphorylation differed, both between the two enzymes and between T308 and S473 of AKT. These results point to a key role for AKT S473 in modulating neuronal survival signaling. Indeed, Gary and co-workers (2003) have shown that AKT and not FAK mediates survival signaling from laminin in hippocampal neurons, either when the cells are plated on laminin, or plated on poly-lysine and treated with a laminin-derived peptide (Gary and Mattson, 2001; Gary et al., 2003). The activating integrin antibody 12G10 delayed the loss of phosphorylation of AKT on S473 in response to serum withdrawal, but had no detectable effect on AKT T308 or FAK phosphorylation. This antibody recognizes a ligand and cation-induced binding site on the h1 integrin subunit (Mould et al., 1995, 2002), and shifts the equilibrium between inactive and active forms of the integrin in favor of the active conformation. K-20 is in contrast a neutral antibody, that binds to a distinct region of the h1 integrin from 12G10 since it does not compete with 12G10 for binding to a5h1 even when present in excess (Mould et al., 1995, 1998). K20 displays stronger reactivity to h1 integrins than activating antibodies such as TS2/16 in flow cytometry (Takada and Puzon, 1993), suggesting that the results presented herein are not simply explained by differences in affinity of the two antibodies for a5h1. Since the Ntera2 cells are already adherent to fibronectin, how might 12G10 modulate cell-surface integrins? Cell-surface integrins are not all active or bound to ligand; a significant proportion are in an inactive, unbound conformation (Cruz et al., 1997; Mould

et al., 1996). Treatment with 12G10 could therefore increase the proportion of integrins in the active conformation, which would enhance adhesiveness and increase the number of active signaling, adhesion complexes in the cell. Consistent with this hypothesis, when treated with 12G10 and viewed under light microscopy, Ntera2 cells had a more phase-dark, spread appearance than control cells (unpublished observations), and increasing the concentration of fibronectin on which the cells were plated, reduced the dosedependent response to 12G10. In addition, 12G10 induces surface protrusions and cell-to-cell adhesion in fibrosarcoma cells (Whittard and Akiyama, 2001). However, 12G10 did not increase the overall percentage of Ntera2 cells spread on fibronectin (data not shown), suggesting that it might increase integrin activation only in already spread cells. By decreasing the number of inactive, unligated integrins on the cells, 12G10 might suppress activation of caspase-8, which can be recruited to inactive integrins leading to apoptosis (Stupack et al., 2001). However, preliminary caspase assays on cells treated with 12G10 or control antibodies failed to detect differences in caspase-8 activity (unpublished observations). Since integrins and growth factor receptors interact, and integrins can potentiate signal transduction from growth factor receptors as well as initiate signal generation in the absence of growth factors (Moro et al., 2002), it is plausible that when serum is removed from the Ntera2 cells, 12G10 delays the loss of this cooperative signaling by increasing the proportion of active integrins in the complexes even in the absence of the growth factors. This hypothesis is supported by the observation that AKT phosphorylation, the major target of 12G10, is strongly dependent on both

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Fig. 5. Effects of activating integrin antibody 12G10 on activity of signaling enzymes JNK, ERK, FAK and AKT. Ntera2 cells growing on fibronectin (1 Ag/ cm2) were transferred, at time 0, to serum-free medium with the integrin antibodies 12G10 or K-20 (10 Ag/ml). The adherent cells were harvested after the incubation periods shown, lysed, and analyzed for (A) JNK activity as described in Experimental methods, and (B–D) phosphorylation of ERK, FAK, and AKT by immunoblot analysis of 10–20 Ag total protein, probing with antibodies specific for phospho-ERK-1/2 (ERK-P) and total ERK (B), phospho-Y397 FAK (FAK-P) and total FAK (C) or phospho-S473 AKT (AKT-S473P), phospho-T308 AKT (AKT-T308P) and total AKT (D). Densitometric analyses of FAK (phosphorylated versus total) and AKT (phosphorylated S473 versus total) expressed relative to time 0 values are shown beneath the blots (C and D). Vinculin served as a control to verify equal protein loading between samples (E). All the experiments were carried out at least in triplicate and representative immunoblots are shown.

adhesion and growth factors, in contrast to FAK which is phosphorylated only in response to adhesion to fibronectin (Fig. 6B). Consistent with this hypothesis, integrin activation by Mn2+ in the absence of growth factors, increases survival of oligodendrocytes to levels normally seen with high concentrations of platelet

Fig. 6. Phosphorylation of FAK and AKT in response to adhesion to fibronectin and treatment with serum. Ntera2 cells were maintained in suspension in the absence of FBS for 30 min (lane 1), and then either stimulated with 10% FBS for 1 h (lane 2) and then plated onto fibronectin (1 Ag/cm2) for 1 h (lane 3) or plated onto fibronectin in the absence of FBS (lane 4) and then stimulated with 10% FBS for 1 h (lane 5). After treatment, cells were lysed and phosphorylation of FAK and AKT analyzed by immunoblot of 10–20 Ag total protein, probing with antibodies specific for phospho-Y397 FAK (FAK-P) and total FAK (A), or phospho-S473 AKT (AKT-P) and total AKT (B). The immunoblots were carried out at least in triplicate and representative blots are shown.

derived growth factor, and this is associated with increased phosphorylation of AKT (Decker and ffrench-Constant, 2004). AKT is activated by recruitment to the plasma membrane, via binding to phosphatidylinositol-3,4,5-triphosphate, and phosphorylation on T308 in the kinase domain and S473 in the C-terminal region. Activation of the kinase can therefore be regulated by the levels of phospholipids, and the activity of PI 3-K and phosphatases. We have asked whether 12G10 modulates AKT via PI 3-K or the phosphatase PTEN which can dephosphorylate the phospholipids generated by PI 3-K (Leslie and Downes, 2002). However, there was no change in phosphorylation of PTEN in response to 12G10, nor did the PI 3-K inhibitors LY294002 and wortmannin negate the effects of 12G10 on neuronal survival. These results suggest that PTEN and PI 3-K are not direct targets of increased activation of a5h1. Recently however, several studies have linked integrin h1 with the phosphatase PP2A which can dephosphorylate both T308 and S473 in AKT (Ivaska et al., 2002; Pankov et al., 2003). Pankov et al. (2003) identified a specific residue in the cytoplasmic tail of human h1 subunit (W775) that regulates AKT via PP2A. However, the differential effects of 12G10 on phosphorylation of S473 versus T308 in Ntera2, and the observation by these workers that the activating antibody TS2/16 but not 12G10 could activate the h1 W775A mutant, whereas both antibodies suppressed Ntera2 apoptosis suggest that regulation of AKT may be different in neuronal cells. The details of the mechanism whereby 12G10 modulates AKT and neuronal survival have therefore yet to be defined. To

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Fig. 7. The activating integrin antibody 12G10 delays loss of AKT kinase activity during serum starvation. Ntera2 cells growing on fibronectin (1 Ag/cm2) were transferred, at time 0, to serum-free medium with the integrin antibodies 12G10 or K-20 (10 Ag/ml). The adherent cells were harvested after the incubation periods shown. The amount of GSK-3 fusion protein phosphorylated by immunoprecipitated AKT from each cell lysate was analyzed by immunoblotting, and the efficiency of the immunoprecipitation checked by probing the immunoblots for AKT. Densitometric analysis of the immunoblots (GSK-3 phosphorylation versus total AKT) is shown beneath the blots. The experiment was carried out at least in triplicate and a representative assay is shown.

characterize the effects of 12G10 further, we sought to elucidate the downstream substrates for AKT in Ntera2 cells, in particular GSK-3h because it is proposed to play an important role in neurodegeneration (reviewed by Kaytor and Orr, 2002), specifically contributing to apoptosis in PC12 cells and primary neurons (Cross et al., 2001; Hetman et al., 2000; Pap and Cooper, 1998), but to our knowledge, has yet to be linked with integrin signaling in neuronal cells. Phosphorylation of GSK-3h on S9 was greatly increased by adhesion to fibronectin and growth factors, similarly to AKT. In addition, the loss of phosphorylation of GSK-3h following serum withdrawal was slower than that of AKT S473, and was delayed by 12G10. These results suggest that AKT and

GSK-3h are key effectors of altered integrin activation state on neuronal cells. In conclusion, we have shown that a5h1-mediated adhesion to fibronectin provides survival signals to neuronal cells, and potentiates the signals from growth factors. The activating integrin antibody 12G10 increases the integrin-dependent survival signals, and increased survival is associated with slowed dephosphorylation of AKT and its substrate GSK-3h. These results have therapeutic implications, since both a5 and h1 have been detected in the CNS, associated with neurons and glia (King et al., 2001; Pinkstaff et al., 1998, 1999), and the ligand, fibronectin, enhances neuronal survival following cerebral ischemia (Sakai et al., 2001)

Fig. 8. Effects of 12G10 on PTEN and PI 3-K. (A) Ntera2 cells cultured on fibronectin (1 Ag/cm2) were transferred, at time 0, to serum-free medium with the integrin antibodies 12G10 or K-20 (10 Ag/ml). The adherent cells were harvested after the incubation periods shown, lysed and the level of phosphorylation of PTEN analyzed by immunoblotting 20 Ag total protein, and probing with antibodies specific for phospho-S380 PTEN (PTEN-P) and total PTEN. (B) Ntera2 cells growing on fibronectin (1 Ag/cm2) were transferred, at time 0, to serum-free medium with or without the integrin antibodies 12G10 or K-20 (10 Ag/ml), and the PI 3-K inhibitors (bLYQ): LY294002 (20 AM) and Wortmannin (1 AM). After 9 h, the numbers of detached apoptotic cells were counted. The results are the mean of four experiments (FSEM), expressed relative to the number of apoptotic cells in the culture that had been starved of serum without integrin antibodies or inhibitors.

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Fig. 9. GSK-3h is phosphorylated in response to integrin-mediated adhesion and growth factors, and 12G10 slows dephosphorylation after serum withdrawal. (A) Ntera2 cells were maintained in suspension in the absence of FBS for 30 min (lane 1), and then either stimulated with 10% FBS for 1 h (lane 2) and then plated onto fibronectin (1 Ag/cm2) for 1 h (lane 3) or plated onto fibronectin in the absence of FBS (lane 4) and then stimulated with 10% FBS for 1 h (lane 5). After treatment, cells were lysed and phosphorylation of GSK-3h analyzed by immunoblot of 15 or 25 Ag total protein, probing with antibodies specific for phospho-S9 GSK-3h (GSK-3h-P) and total GSK-3h. (B) Ntera2 cells cultured on fibronectin (1 Ag/cm2) were transferred, at time 0, to serum-free medium with the integrin antibodies 12G10 or K-20 (10 Ag/ml). The adherent cells were harvested after the incubation periods shown, lysed and the level of phosphorylation of GSK-3h analyzed by immunoblotting 25 Ag total protein, probing with antibodies specific for phospho-S9 GSK3h and for vinculin. All immunoblots were carried out at least in triplicate and representative blots are shown.

All general reagents were obtained from Sigma-Aldrich Co. (UK), and all cell culture reagents were from Gibco-Invitrogen (UK), unless stated otherwise.

Sanchez Madrid] or K-20 [Immunotech, France]), or Fab fragments generated from 12G10 or 8E3 as described by Whittard and Akiyama (2001). Cells were harvested after 15 min, 30 min, 1, 3, 6, or 9 h, depending on the individual experiment. For treatment with the PI 3-K inhibitors, cells were pre-incubated with LY294002 (20 AM) and wortmannin (1 AM), both diluted in DMEM, for 30 min prior to incubation in FBS-free DMEM with or without integrin antibodies as above. Preliminary experiments established that these concentrations of inhibitors were required to inhibit AKT phosphorylation. By the criteria established previously (Gibson, 1999), apoptosis was scored by counting the number of detached apoptotic cells, since in adherent cultures of Ntera2 cells, the attached cells are morphologically normal, while all of the detached cells are apoptotic.

Cell culture

Flow cytometry

Ntera-2 cells were maintained as described previously (Gibson, 1999). For comparison of viability when cells were grown on fibronectin or in suspension, cells were plated at a density of 20,000/cm2 either on fibronectin (1 Ag/cm2; Fred Baker Scientific, UK) and incubated for 24 h at 378C, 5% CO2 prior to experimentation, or on poly (2-hydroxylethyl methacrylate) (poly-HEMA, 5 mg/cm2; Sigma) at the time of the experiment. Cells were rinsed in phosphate-buffered saline (PBS) and incubated in Dulbecco’s modified Eagle’s medium (DMEM; with 2 mM glutamine) without fetal bovine serum (FBS) for 3, 6, 9, 24, 48, or 72 h. To score the amount of apoptosis, the cultures were harvested (pooling adherent and detached cells from the culture on fibronectin), fixed in 1% formaldehyde and stained with Hoechst 33342 (10 Ag/ml). Cells were scored as healthy if they had diffuse chromatin, or apoptotic if they had condensed, brightly stained chromatin (see Gibson, 1999). For treatment with integrin antibodies, Ntera2 cells were plated on fibronectin (0.1, 1.0 or 10 Ag/cm2) at a cell density of 40,000/ cm2 and incubated for 24 h at 378C, 5% CO2. The cells were rinsed with PBS, and then incubated in DMEM with or without 1–10 Ag/ ml purified integrin antibody (12G10, TS2/16 [a kind gift from

Cells were harvested using 3 mM EDTA in Hanks balanced salt solution, and diluted in DMEM containing 1% FBS. Cells (5.105) were incubated with anti-integrin antibodies, diluted in PBS with 0.02% sodium azide: mouse anti-human a1 (5E8D9; Upstate Biotechnology, UK), mouse anti-human a2 (10A4), mouse antihuman a3 (11G5), mouse anti-human a4 (HP21; Serotec, UK), mouse anti-human av (LM142; Chemicon, UK), rat anti-human a5 (mAb16, a kind gift of Ken Yamada), rat anti-mouse a6 (GoH3, a kind gift of Arnoud Sonnenberg), that cross reacts with human a6 integrin, rat anti-human h1 (mAb13, a kind gift of Ken Yamada), and mouse anti-human h3 (LM609; Chemicon). Following incubation on ice for 1 h, cells were washed and incubated with fluorescein-conjugated anti-mouse or anti-rat secondary antibody (in PBS with 10% FBS) for 1 h on ice. Cells were washed and analyzed on a Becton-Dickinson FACS Vantage flow cytometer.

and transplantation of neural stem cells (Tate et al., 2002). Furthermore, Ntera2 cells are of interest for transplantation into the CNS to replace cells lost in stroke or chronic neurodegenerative disorders (Kleppner et al., 1995; Nelson et al., 2002; Philips et al., 1999; Watson et al., 2003) and the results reported here suggest that their viability might be significantly manipulated by integrinmediated adhesion to the ECM in their microenvironment.

Experimental methods

Spreading assay Spreading assays were carried out as described previously (Gibson, 1999). Anti-integrin antibodies were added to 96-well plates coated with fibronectin prior to adding Ntera2 cells. All the

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antibodies were function-blocking: rat anti-human h1 integrin (mAb13), mouse anti-human a3 integrin (P1B5, Invitrogen), rat anti-human a5 integrin (mAb16), rat anti-mouse a6 integrin (GoH3), and mouse anti-human av integrin (14D9.F8, Merck, UK). Immunoblotting Cells were lysed on ice in 1% Triton X-100 (20 mM Tris–HCl pH 7.4, 237 mM NaCl, 2 mM EDTA, 25 mM h glycerophosphate, 5% glycerol), containing protease inhibitors (1 mM phenylmethyl sulfonyl fluoride and 10 Ag/ml aprotinin) and the phosphatase inhibitor sodium vanadate (1 mM). The concentration of proteins in the lysates was determined using the BCA assay (Pierce, UK). The proteins (10–30 Ag) were separated by SDS-PAGE and transferred onto Immobilon P membrane (Millipore, UK). The membranes were incubated for 1–2 h at room temperature (RT) in 10% dried skimmed milk powder in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T) to block non-specific sites. The membranes were incubated at 48C overnight in primary antibodies, diluted as follows: mouse anti-poly(ADP-ribose) polymerase (PARP; Serotec), diluted 1:1000 in TBS-T; rabbit anti-p44/42 (ERK) phosphorylated on T202/Y204 (Cell Signaling, UK), diluted 1:1000 in 5% bovine serum albumin (BSA) in TBS-T; anti-ERK (Santa Cruz, USA) diluted 1:5000 in 1% skimmed milk powder in TBS-T; rabbit anti-focal adhesion kinase (FAK) phosphorylated on Y397 (BioSource, UK), diluted 1:1000 in 3% BSA in TBS-T; rabbit anti-FAK (Pharmingen, UK), diluted 1:1000 in 5% skimmed milk powder in TBS-T; rabbit anti-AKT phosphorylated on S473 (Cell Signaling), diluted 1:1000 in 5% BSA in TBS-T; rabbit anti-AKT phosphorylated on T308 (Cell Signaling), diluted 1:1000 in 5% BSA in TBS-T; rabbit anti-AKT (Cell Signaling), diluted 1:1000 in 5% BSA in TBS-T; rabbit antiPTEN phosphorylated on S380 (Cell Signaling), diluted 1:1000 in 1% skimmed milk in TBS-T; rabbit anti-PTEN (Upstate Biotechnology), diluted 1:2000 in 1% skimmed milk in TBS-T; rabbit anti-GSK-3h phosphorylated on S9 (Cell Signaling), diluted 1:1000 in 5% BSA in TBS-T; rabbit anti-GSK-3h (Cell Signaling), diluted 1:1000 in 5% BSA in TBS-T; or mouse anti-vinculin (Serotec) diluted 1:1000 in TBS-T. The membranes were washed in TBS-T, and incubated for 1–2 h with horseradish peroxidase conjugated secondary anti-mouse or anti-rabbit antibody (Dako, UK), diluted 1:4000 in 5% skimmed milk in TBS-T. The membranes were washed again in TBS-T, and the proteins detected with a chemiluminescent substrate (Amersham, UK). Films were analyzed by densitometry using Northern Eclipse software (Empix Imaging Inc., Canada). Kinase assays Cells were lysed as described above. The AKT kinase assay was carried out with a kit from Cell Signaling according to the manufacturer’s instructions. In brief, cell lysate (100 Ag) was incubated with immobilized AKT antibody for 3–6 h at 48C, and then the beads were washed in lysis buffer followed by kinase buffer (25 mM Tris–HCl pH 7.5, 5 mM h-glycerophosphate, 10 mM MgCl2, 2 mM DTT, 0.1 mM sodium vanadate). The beads were incubated with ATP (200 AM) and GSK-3 (0.5 Ag per reaction) in kinase buffer at 308C for 30 min, and heated to 958C for 5 min in SDS-PAGE loading buffer. Equal volumes were subject to immunoblotting, probing the membranes with rabbit

antibody to GSK-3h, phosphorylated on S21/9, and rabbit antibody to AKT. The JNK assay was carried out as described previously (Raingeaud et al., 1995). Briefly, lysate (100 Ag) was incubated with glutathione Sepharose (Pharmacia, UK) and c-jun-GST for 5– 6 h at 48C, and the beads were washed in lysis buffer followed by kinase buffer (25 mM HEPES pH 7.4, 25 mM h glycerophosphate, 25 mM MgCl2, 2 mM DTT, 0.1 mM sodium vanadate). The beads were then incubated with 1 ACi g32P-ATP and 0.5 mM ATP in kinase buffer at 308C for 15 min, and heated to 958C for 5 min in SDS-PAGE loading buffer. Equal volumes were subject to SDSPAGE, and the gels dried and exposed to autoradiographic film.

Acknowledgments We thank Ken Yamada for his gifts of mAb13 and mAb16 antibodies, Sanchez Madrid for TS2/16 and Arnoud Sonnenberg for GoH3 antibody. We also thank Caroline Dive for her assistance with the FACS analysis, and Lorraine Schmidt for technical assistance. This work was supported by the Medical Research Council (RMG, LH), The Royal Society (RMG), The Wellcome Trust (MJH, SEC) and the Lister Institute (CT).

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