Sustained Induction of ERK, Protein Kinase B, and p70 S6 Kinase Regulates Cell Spreading and Formation of F-actin Microspikes Upon Ligation of Integrins by Galectin-8, a Mammalian Lectin*

Galectin-8, a member of the galectin family of mammalian lectins, is a secreted protein that promotes cell adhesion and migration upon binding to a subset of integrins through sugar-protein interactions. Ligation of integrins by galectin-8 triggers a distinct pattern of cytoskeletal organization, including formation of F-actin-containing microspikes. This is associated with activation of integrin-mediated signaling cascades (ERK and phosphatidylinositol 3 kinase (PI3K)) that are much more robust and are of longer duration than those induced upon cell adhesion to fibronectin. Indeed, formation of microspikes is enhanced 40% in cells that overexpress protein kinase B, the downstream effector of PI3K. Inhibition of PI3K activity induced by wortmannin partially inhibits cell adhesion and spreading while largely inhibiting microspike formation in cells adherent to galectin-8. Furthermore, the inhibitory effects of wortmannin are markedly accentuated in cells overexpressing PKB or p70S6K (CHOPKB and CHOp70S6Kcells), whose adhesion and spreading on galectin-8 (but not on fibronectin) is inhibited ∼25–35% in the presence of wortmannin. The above results suggest that galectin-8 is an extracellular matrix protein that triggers a unique repertoire of integrin-mediated signals, which leads to a distinctive cytoskeletal organization and microspike formation. They further suggest that downstream effectors of PI3K, including PKB and p70 S6 kinase, in part mediate cell adhesion, spreading, and microspike formation induced by galectin-8.

Extracellular matrix (ECM) 1 proteins have important functions in providing structural integrity to tissues and in presenting proper environmental cues for cell adhesion, migration, growth, and differentiation (1)(2)(3)(4)(5). These functions rely on spatio-temporal expression of adhesive as well as anti-adhesive components of the ECM proteins (6). ECM proteins like fibronectin (7)(8)(9), collagen (10), and laminin (11) are best characterized, though other types of proteins, including mammalian lectins, also function as modulators of cell adhesion. Selectins mediate cell-cell interactions (12) through calciumdependent recognition of sialylated glycans (13,14), whereas galectins, animal lectins that specifically bind ␤-galactoside residues (15), were implicated as modulators of cell-matrix interactions. Although lacking a signal peptide and found mainly in the cytosol, galectins are externalized by an atypical secretory mechanism (16) to regulate cell growth, cell transformation, embryogenesis, and apoptosis (reviewed in Refs. 17 and 18). In accordance with their proposed functions, galectins enhance or inhibit cell-matrix interactions (reviewed in Ref. 19).
Cellular adhesion to extracellular matrix proteins is mediated by a diverse class of cell surface ␣␤ heterodimeric receptors known as integrins (2,20,21). In addition to mediating cell adhesion, integrins induce multiple signal transduction pathways that regulate cytoskeletal rearrangements, cell spreading, migration, differentiation, survival, and cell growth. These processes are associated with activation of a number of signaling elements, (4), most prominent of which is focal adhesion kinase (FAK), which undergoes integrin-stimulated autophosphorylation. Tyr-phosphorylated FAK recruits Grb2-Sos complexes, which activate the Ras-MAPK signaling pathway. FAK also phosphorylates p130 Cas , which binds Crk and generates further signals through c-Jun NH 2 -terminal kinase. P-Tyr 397 of FAK serves as a binding site for the SH2 domain of p85␣, the regulatory subunit of PI3K that propagates signals to protein kinase B (PKB) and p70 S6 kinase (p70S6K) (reviewed in Refs. 4 and 22). Stimulation of integrins also activates the Rhofamily GTPases Rho, Rac, and Cdc42, which mediate the formation of stress fibers, lamellipodia, and filopodia, respectively (23).
We have recently shown that different cell types adhere and spread when cultured on immobilized galectin-8, a mammalian ␤-galactoside-binding protein (24). Galectin-8 (25)(26)(27), a member of the galectin family, is a secreted protein that is widely expressed. It is made of two homologous carbohydrate-recognition domains linked by a short (ϳ26 amino acids) peptide. Upon secretion, galectin-8 binds to a subset of cell surface integrins, which include integrin ␣ 3 ␤ 1 or ␣ 6 ␤ 1 but not ␣ 4 ␤ 1 (27). Immobilized galectin-8 is equipotent to fibronectin in promoting cell adhesion and spreading, effects that involve sugar-protein interactions of integrins with galectin-8 (24). Accordingly, cell adhesion to galectin-8 is potentiated in the presence of Mn 2ϩ , whereas adhesion is interrupted in the presence of soluble galectin-8, integrin ␤ 1 inhibitory antibodies, EDTA, or thiodigalactoside but not RGD peptides (24). Whereas immobilized galecin-8 promotes cell adhesion, soluble galectin-8 interacts This article has been withdrawn by Yehiel Zick and Alexander D. Bershadsky. The withdrawing authors have become aware of several errors in the way images were presented in this manuscript. Because the original data are no longer available, the authors wish to withdraw the article in the interests of maintaining their publication standards and those of the journal. In Fig. 3B, lanes 1 and 5 of the Ras-GTP immunoblot and lanes 4 and 8 of the Ras immunoblot were duplicated. In Fig.  5A, lanes 1,4,7,10, and 11 of the pp70S6K were duplicated. The withdrawing authors state that these presentational errors do not impact the underlying scientific findings of the article, which are also presented as quantitative line/bar graphs that essentially summarize data of a number of experiments. Therefore, the withdrawing authors stand by the original scientific results as described which in the authors' opinion has been confirmed in other laboratories (Norambuena, A., Metz, C., Vicuña, L., Silva, A., Pardo, E., Oyanadel, C., Massardo, L., González, A., and Soza, A. (2009) Galectin-8 induces apoptosis in Jurkat T cells by phosphatidic acid-mediated ERK1/2 activation supported by protein kinase A down-regulation. J. Biol. Chem. 284, 12670-21679 and Oyanadel, C., Holmes, C., Pardo, E., Retamal, C., Shaughnessy, R., Smith, P., Cortés, P., Bravo-Zehnder, M., Metz, C., Feuerhake, T., Romero, D., 5th, Roa, J. C., Montecinos, V., Soza, A., and González, A. (2018) Galectin-8 induces partial epithelial-mesenchymal transition with invasive tumorigenic capabilities involving a FAK/EGFR/proteasome pathway in Madin-Darby canine kidney cells. Mol. Biol. Cell. 29, 557-574).
both with cell surface integrins and other soluble ECM proteins and inhibits cell-matrix interactions (27). These observations suggest that galectin-8 is a matrix protein that can positively or negatively modulate cell adhesion (24).
In the present study we undertook to characterize the signaling cascades downstream of FAK, which are activated by galectin-8 and confer upon cells adherent to this lectin a unique cytoskeletal organization. Our results indicate that ligation of integrins by galectin-8 is associated with GTP loading onto Ras as well as sustained and potent activation of ERK, PKB, and p70S6K. These downstream effectors of PI3K modulate cell adhesion and spreading on galectin-8 and account for the extensive F-actin-containing microspikes that are formed when cells adhere onto galectin-8. Our findings therefore implicate galectin-8 as an ECM protein that triggers a unique repertoire of integrin-mediated signals, leading to a distinctive cell adhesion, spreading, and cytoskeletal organization.

EXPERIMENTAL PROCEDURES
Materials-Bacterially expressed recombinant galectin-8 was generated as previously described (25). Isopropyl-␤-D-thiogalactopyranoside was purchased from MBI Fermentas (Amherst, NY). Bovine fibronectin, puromycin, wortmannin, glutathione-agarose beads, cycloheximide, and crystal violet were purchased from Sigma. Protein G-PLUS-agarose beads were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). LipofectAMINE reagent was from Invitrogen. A construct of wildtype (WT) PKB in the pCIS2 expression vector was kindly provided by M. Quon (NHLBI, National Institutes of Health, Bethesda, MD). A construct of WT p70S6K in the p2B4 expression vector was kindly provided by G. Thomas, (Friedrich Meischer Institute, Basel, Switzerland). GST⅐RBD (Ras-binding domain of Raf), coupled to glutathione-agarose beads was kindly provided by R. Seger (Weizmann institute, Rehovot, Israel).
Generation of Stable Clones of CHO PKB and CHO p70S6K Cells Overexpressing PKB or p70S6K, Respectively-CHO-P cells were stably transfected using LipofectAMINE as previously described (30). The plasmids used for overexpression of WT-PKB or WT-p70S6K were pCIS2 or p2B4, respectively (31,32). The cells were co-transfected with pBabe-Puro plasmid, which encodes a puromycin resistance gene. Following transfection, 10 g/ml puromycin was added for selection of stable colonies. Puromycin-resistant clones that overexpress WT-PKB or WT-p70S6K were isolated and further propagated.
Cell Adhesion Assay-Bacterial or tissue-culture plates were precoated for 2 h at 22°C with galectin-8 or fibronectin in PBS. Cells, grown on tissue-culture plates, were detached from the plates with 5 mM EDTA, washed with PBS, resuspended in serum-free medium, and re-seeded on the coated plates. At the indicated times, cells were washed, and the adherent cells were stained with 0.2% crystal violet and 20% methanol in H 2 O for 10 min at 22°C. Excess dye was removed by washes with water, and cells were solubilized in 1% SDS for 1 h at 22°C. The amount of the adherent cells was quantified by measuring the absorbance at 540 nm in an enzyme-linked immunosorbent assay plate reader-TECAN (Spectra, Austria). Specific binding was defined as the difference between the absorbance of cells bound to ligand-coated wells less the absorbance of cells bound to bovine serum albumin-coated wells. All assays were performed in quadruplicate.
Binding of Activated Ras to its Effector-Activation of Ras was determined by binding assay of the activated (GTP-loaded) forms of Ras to a minimal RBD of Raf1 (33), coupled to GST (GST⅐RBD). GST⅐RBD, coupled to glutathione-agarose beads, was washed three times in buffer C (50 mM Tris-HCl, pH 7.5, 10% glycerol, 2.5 mM MgCl 2 , 200 mM NaCl, 1.0% Nonidet P-40, 1 g/ml aprotinin, 1 g/ml leupeptin, 0.1 g/ml trypsin inhibitor, 250 M phenylmethylsulfonyl difluoride, 10 mM NaF, 1 mM sodium orthovanadate). CHO-P cells were washed, and extracts were prepared in buffer C. Insoluble material was removed by 15 min of centrifugation (20,000 ϫ g) at 4°C. Then, 10 -20 g of fusion proteins coupled to glutathione-agarose beads were incubated with the cell extract (0.5 mg) for 45 min at 4°C. The Ras⅐GTP-bound beads were further washed (four times) with buffer F (20 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 0.1% Triton X-100). The bound proteins were suspended in Laemmli's sample buffer, resolved by 15% SDS-PAGE, and Western-immunoblotted with the indicated antibodies.
Immunofluorescence Microscopy-Cultured cells, plated on glass coverslips, were washed and fixed with paraformaldehyde (3%) containing 0.5% Triton X-100 (for phalloidin staining) or cold 100% methanol (for tubulin staining). Following several washes with PBS, cells were incubated for 1 h at 22°C with TRITC-labeled phalloidin or with antitubulin monoclonal antibodies. Cells were washed with PBS and further incubated for 1 h at 22°C with secondary Cy3-conjugated goat anti-mouse antibody in PBS. The specimens were washed, mounted onto glass microscope slides, and examined on Zeiss fluorescence microscope.

Cell Adhesion onto Galectin-8 Induces a Characteristic Pattern of Cytoskeletal
Organization-Immobilized galectin-8, similar to other ECM proteins, promotes cell adhesion and spreading (24). Still, there is a marked difference in cytoskeletal organization between cells adherent to galectin-8 versus fibronectin (24). Time-dependent formation of prominent stress fibers that traverse the cell body are readily observed in HE ( Fig. 1) and CHO-P cells (Fig. 2) adherent onto fibronectin but are less abundant in cells adherent to galectin-8. Instead, adhesion and spreading onto galectin-8 were characterized by initial formation (within 10 min) of sheet-like projections of the membrane, known as lamellipodia (34), that were followed by the appearance of short individual projections (Ͻ 2 microns long) at the cellular perimeter, known as F-actin microspikes (35)(36)(37). Microspikes were readily detected 30 -60 min following cell adhesion on galectin-8, and they continued to develop for at least the first 120 min (Figs. 1 and 2). Microspike formation induced upon cell adhesion onto galectin-8 is rather a general phenomenon. It was observed in a number of cell lines, derived from different tissues and species such as HE cells (Fig.  1), CHO-P (Fig. 2), and NIH-hIR fibroblasts. Still, it is not a ubiquitous phenomenon because certain cell types, such as B16 murine melanoma cells, fail to produce microspikes upon adhesion to galectin-8. 2 Additional features characterize cells adherent to galectin-8; whereas vinculin and paxillin are associated with large focal contacts in cells adherent to fibronectin, the number and size of vinculin-and paxillin-containing focal contacts is reduced in cells attached to galectin-8 (24). The differences in actin-microfilament organization were not accompanied by differences in microtubules organization, and a similar microtubular network developed when cells adhered to galectin-8 or fibronectin (Fig. 2B).
Phosphorylation of p130 Cas Is Induced upon Cell Adhesion to Galectin-8 -The differences in cytoskeletal organization between cells adherent to galectin-8 versus fibronectin could be attributed to different signaling readouts elicited upon ligation of cell surface integrins by these two ECM molecules. We have already demonstrated that FAK phosphorylation, which occurs at early stages of cell-matrix interactions, is a common signal emitted upon cell adhesion to fibronectin or galectin-8 (24). Hence, bifurcation of the signals mediated by fibronectin and galectin-8 presumably takes place downstream of FAK. To address this possibility, Tyr phosphorylation of p130 Cas , a downstream effector of FAK, was studied. As shown in Fig. 3, the P-Tyr content of p130 Cas increased to a similar extent when CHO-P cells were allowed to adhere for 60 min onto fibronectin-or galectin-8-coated plates. The effect was rapid, and maximal levels of P-Tyr p130 Cas were already reached after 15 min of cell adhesion onto galectin-8 (Fig. 3A). These findings suggest that galectin-8, similar to fibronectin, triggers Tyr phos-2 Y. Levy and Y. Zick, unpublished data. phorylation of p130 Cas , presumably through activation of FAK.
Cell Adhesion to Galectin-8 Activates Ras and the MAPK Cascade-Next, the effects of galectin-8 on the activation of Ras and the MAPK cascade were examined. Consistent with previous studies, activation of Ras (Fig. 3B) and phosphorylation of ERK1 and -2 ( Fig. 4) were largely diminished upon detachment of CHO-P cells from culture plates (e.g. Fig. 3B, time zero and 15-60 min without galectin-8 or fibronectin). Re-adhesion of the cells onto fibronectin-or galectin-8-coated plates lead to activation of Ras (Fig. 3B) and phosphorylation of ERK1 and -2 (Fig. 4). Maximal GTP loading onto Ras and phosphorylation of ERK1 and -2 was achieved by 15 min. However, whereas phosphorylation of ERK1 and -2 in cells adherent to galectin-8 was sustained for at least 60 min, ERK phosphorylation in cells adherent to fibronectin was transient and rapidly declined (Fig. 4B). Addition of wortmannin, a PI3K inhibitor, diminished ERK phosphorylation induced either by fibronectin or galectin-8 (Fig. 4), indicating that PI3K is an upstream regulator of ERK1 and -2 phosphorylation, which occurs upon integrin ligation by galectin-8.
Galectin-8 Induces Robust and Sustained Activation of PKB and p70S6K-The above results have indicated that activation of PI3K triggers the MAPK cascade when cells adhere onto galectin-8. To explore whether other downstream effectors of PI3K are being activated, the effects of galectin-8 on PKB and p70S6K were studied. Phosphorylation of both proteins was largely diminished upon detachment of CHO-P cells from culture plates (Fig. 5, A and B, time zero), whereas re-adhesion of the cells onto fibronectin or galectin-8 lead to a time-dependent phosphorylation of PKB and p70S6K (Fig. 5A). Phosphorylation of both proteins was significantly higher upon cell adhesion to galectin-8 than fibronectin at all time points tested. For example, at 60 min, the extent of phosphorylation of PKB and p70S6K was 8-and 3-fold higher, respectively, in cells adherent to galectin-8. Pretreatment with wortmannin largely diminished the phosphorylation of PKB and p70S6K (Fig. 5, A and  B), indicating that these proteins are indeed downstream effectors of PI3K.
Overexpression of PKB and p70S6K Diverts Adhesion of CHO Cells into a PI3K-dependent Pathway-To determine whether downstream effectors of PI3K affect cell adhesion to galectin-8, PKB and p70S6K were introduced into CHO-P cells, and stable clones that overexpress either PKB (CHO PKB ) or p70S6K (CHO p70S6K ) were generated. These clones expressed 1.7-and 2.5-fold higher levels of PKB or p70S6K, respectively, than the in serum-free medium, and seeded in serum-free medium on the coated plates. Following incubation at 37°C for the indicated times, cells were washed and extracted using buffer I. Cell extracts (1.0 mg) were subjected to immunoprecipitation with anti-p130 Cas antibody, resolved by 10% SDS-PAGE, transferred to nitrocellulose membranes, and Western immunoblotted with anti-P-Tyr antibody (a). Alternatively, proteins (100 g) were resolved by 10% SDS-PAGE, transferred to nitrocellulose membranes, and Western immunoblotted with anti-p130 Cas antibody (b). B, CHO-P cells were grown and treated as indicated in panel A. Cells were washed and extracted using buffer C. Cell extracts (0.5 mg) were incubated with GST⅐RBD. The bound proteins, corresponding to Ras-GTP, were resolved by 15% SDS-PAGE, transferred to nitrocellulose membranes, and Western immunoblotted with anti-Ras antibody (c). Alternatively, proteins (100 g) from total cell extracts were resolved by 15% SDS-PAGE, transferred to nitrocellulose membranes, and Western immunoblotted with anti-Ras antibody (d). Quantitation of the intensity of the bands, corresponding to Ras⅐GTP is presented as a bar graph. The results are mean Ϯ S.D. of four experiments. endogenous expression levels of these proteins (Fig. 6A). As shown in Fig. 6B, adhesion of CHO PKB and CHO p70S6K cells to fibronectin or galectin-8 triggered similar signaling responses seen in wild-type CHO-P cells. Phosphorylation of PKB and p70S6K was largely diminished upon detachment of the cells from culture plates, whereas re-adhesion stimulated the phosphorylation of the overexpressed PKB and p70S6K (Fig. 6, B  and C). Again, the signals elicited upon cell adhesion to galectin-8 were stronger and more sustained when compared with the signals emitted upon cell adhesion onto fibronectin, and pretreatment with wortmannin largely diminished the phosphorylation of PKB and p70S6K (Fig. 6, B and C). Next, the effects of wortmannin on cell adhesion were evaluated. As shown in Fig. 7, wortmannin had a slight (ϳ15%) albeit significant inhibitory effect on adhesion of CHO-P cells to galectin-8. However, this inhibitory effect was markedly accentuated in CHO PKB and CHO p70S6K cells, whose adhesion onto galectin-8 was inhibited 35 and 28%, respectively, in the presence of wortmannin. This inhibitory effect was specific, because cell adhesion to fibronectin was unaffected by the drug. Thus, overexpression of PKB and p70S6K generates wortmannin-sensitive signals that promote cell adhesion onto galectin-8, although the overall adhesion rates (in the absence of wortmannin) were not affected by the overexpressed PKB and p70S6K (Fig. 7). Cellular Spreading over Galectin-8 -An immediate consequence of cell adhesion is cell spreading. Cells spread much faster on galectin-8, compared with fibronectin. Although most cells were already spread on galectin-8 by 10 min, cells adherent to fibronectin were still round and began to spread only after ϳ20 min (compare Figs. 1 and 2). Cell spreading (measured as increased cell area) on either galectin-8 or fibronectin was comparable by 2 h (Fig. 8). This was evident by the similar distribution of cells areas, with a mean of 875 Ϯ 175 arbitrary units following 2 h of adhesion on either galectin-8 or fibronectin, (Fig. 8). Still, the rate of cellular spreading over galectin-8 was faster, and comparable cell areas were observed in cells adherent to galectin-8 or fibronectin for 10 and 20 min, respectively. Interestingly, although the area distribution of cells adherent to fibronectin by 20 min was rather limited (Fig. 8), a much wider area distribution (Ͼ 700 arbitrary units) characterized cells adherent to galectin-8 by 10 min (Fig. 8). This phenomenon was not unique to CHO-P cells (Fig. 8), because a wide area distribution was also observed when B16, HE, or

FIG. 6. Activation of PKB and p70S6K induced upon adhesion of CHO PKB and CHO p70S6K cells to galectin-8 or fibronectin. A,
puromycin-resistant cells that overexpress PKB or p70S6K (CHO PKB or CHO p70S6K cells, respectively) were isolated and extracted using buffer I. Proteins (100 g) were resolved by 10% SDS-PAGE, transferred to nitrocellulose membranes, and Western immunoblotted with anti-PKB or anti-p70S6K antibodies. B, CHO PKB and CHO p70S6K cells were incubated in the absence or presence of wortmannin and seeded on galectin-8-or fibronectin-coated plates as described in Fig. 4A. Cells were washed and extracted using buffer I. Proteins (100 g) were resolved by 10% SDS-PAGE, transferred to nitrocellulose membranes, and Western immunoblotted with (a) anti-phospho-PKB (␣-pPKB); (b) anti-PKB (␣-PKB); (c) anti-phospho-p70S6K (␣-pp70S6K); or (d) anti-70S6K antibodies (␣-p70S6K). C, quantitation of the intensity of the bands, corresponding to the phosphorylated PKB (e) or the phosphorylated p70S6K (f) is presented.
NIH cells adhered and spread over galectin-8 for 10 -20 min (Fig. 9, a-c) as compared with the limited area distribution of these cells when spread on fibronectin (Fig. 9, d-f). The reason for this variability is presently unknown, but as shown in Fig.  10, the variance cannot be attributed to variable induction of protein synthesis because inclusion of cycloheximide did not alter the rate or phenotype of cells adherent to galectin-8. In contrast, cellular attachment on galectin-8 was inhibited ϳ50%, whereas spreading hardly took place at 4°C (Fig. 10), indicating that energy-consuming processes and active protein trafficking are required to mediate these events.
Wortmannin Inhibits Cell Spreading on Galectin-8 -The faster initial rate of cell spreading on galectin-8 occurred despite the fact that the rates of cell adhesion onto galectin-8 or fibronectin were comparable (compare Fig. 7 and Fig. 8, inset). These results suggest that the initial affinity of integrins to either galectin-8 or fibronectin is comparable, although subsequent cytoskeletal organization associated with cell spreading occurs faster in cells adherent to galectin-8. To assess the contribution of PKB and p70S6K to this process, cellular spreading was studied in CHO-P, CHO PKB , and CHO p70S6K cells. As shown in Fig. 11A, the three cell types spread to a similar extent following 10 or 20 min of adhesion over galectin-8 or fibronectin, respectively. Under these conditions about 60% of all populations had areas of Ͻ 350 arbitrary units (Fig.  11B). These results indicate that the rate of cell spreading, as well as cell adhesion (Fig. 7), is independent of the overexpressed PKB and p70S6K. However, similar to its inhibitory effects on cell adhesion, wortmannin selectively inhibited cell spreading on galectin-8 (Fig. 11, A and B), but not on fibronectin, and its inhibitory effects were more pronounced in cells overexpressing PKB or p70S6K. Indeed, wortmannin increased the percentage of CHO-P, CHO PKB , and CHO p70S6K cells having areas of Ͻ 350 arbitrary units (following 10 min of adhesion) by 20, 30, and 40%, respectively (Fig. 11A). Hence, overexpression of downstream effectors of PI3K sensitizes the cells to the inhibitory effects of wortmannin, implicating the overexpressed PKB and p70S6K as potential mediators of cell spreading on galectin-8. The inhibitory effects of wortmannin on cell spreading were not restricted to CHO-P cells. As shown in Fig. 9, wortmannin effectively inhibited spreading of HE,  Fig. 7. Following incubation at 37°C for the indicated time periods, cells were fixed and incubated with TRITC-labeled phalloidin for actin staining. Quantitation of the cell areas was determined using the NIH Image program. The number of cells (n) whose area was calculated under each experimental condition is indicated.

NIH, and B16 cells on galectin-8 but had trivial effects on spreading of these cells on fibronectin.
Effects of PI3K and Its Downstream Effectors on Formation of Microspikes in Cells Adherent to Galectin-8 -Because formation of microspikes appeared to be a characteristic feature of cells adherent onto galectin-8, the effects of PI3K and its downstream effectors on microspike formation were evaluated. Cells were considered as possessing microspikes when at least 35% of the cellular perimeter (not in contact with other cells) contained these structures. As shown in Fig. 12, A and B, formation of microspikes in cells adherent onto galectin-8 was significantly (Ͼ 60%) inhibited when cells were treated with wortmannin, implicating PI3K and its downstream effectors as mediators of microspike formation. This conclusion was supported by the fact that there was a ϳ40% increase in formation of microspikes in CHO PKB cells compared with CHO-P or CHO p70S6K cells (Fig. 12A), implicating PKB as being actively involved in signals mediating microspike formation upon cell adhesion to galectin-8. Microspikes could not be detected in cells adherent to fibronectin. DISCUSSION In the present study we provide evidence that galectin-8, a mammalian lectin (24,27), functions as an ECM protein that triggers a unique spectrum of signaling events upon ligation of sugar moieties of integrins. Although cell adhesion onto galectin-8 or fibronectin activates to the same extent FAK and p130 Cas , the signaling cascades triggered upon adhesion to FIG. 10. Effects of low temperature and cycloheximide on adhesion and spreading of CHO-P cells on galectin-8. Cover glasses were coated for 2 h at 22°C with galectin-8 (0.7 M) in PBS. CHO-P cells were incubated for 16 h in serum-free medium. Following incubation the cells were further incubated in the absence or presence of 50 g/ml cycloheximide for 3 h in serum-free medium. Next, the cells were detached from the culture plates with 5 mM EDTA, washed, and further incubated in suspension for 30 min at 37°C in serum-free medium in the absence or presence of cycloheximide. The cells were seeded on galectin-8-coated cover glasses at 37 or 4°C for the indicated times. Cells were fixed and incubated with TRITC-labeled phalloidin for actin staining.  and e), and CHO p70S6K (c and f) cells were incubated in the absence or presence of 1 M wortmannin and were seeded (1 ϫ 10 6 ) on galectin-8-or fibronectin-coated cover glasses as described in Fig. 7. Following incubation at 37°C for 10 min with galectin-8 or 20 min with fibronectin, cells were fixed and incubated with TRITC-labeled phalloidin for actin staining (A). The distribution of the cell areas was determined using the NIH Image program as described in the legend to Fig. 8. B, the number of cells (n) whose area was calculated under each experimental condition is indicated.
galectin-8 are characterized by a robust and sustained activation of ERK-1 and -2, which contrasts with the transient nature of ERK activation upon cell adhesion to fibronectin. Similarly, activation of PKB and p70S6K, which serve as downstream effectors of PI3K, is several -folds more intense and sustained when cells adhere to galectin-8 than fibronectin. The unique signaling pattern triggered upon cell adhesion to galectin-8 is associated with faster cell spreading and with a distinctive organization of cytoskeletal elements. Prominent stress fibers that traverse the cell body are readily observed in cells adherent to fibronectin, but they are less abundant in cells adherent to galectin-8. Similarly, formation of focal contacts is limited when cells adhere onto galectin-8 (24). Instead, adhesion to galectin-8 triggers sustained formation of F-actin microspikes. This is rather a general phenomenon observed in a number of cell lines; still, it is not a ubiquitous phenomenon because certain cell types, such as B16 murine melanoma cells, fail to produce microspikes upon adhesion to galectin-8.
Inhibitors of PI3K impair cell adhesion and spreading on galectin-8 and the formation of microspikes but have no effects on cells adherent to fibronectin, indicating that downstream effectors of PI3K selectively regulate cytoskeletal rearrangements that occur when cells adhere to and spread on galectin-8. Indeed, overexpression of PKB potentiates the formation of microspikes, whereas overexpression of PKB or p70S6K accentuates the sensitivity of cells adherent to and spread on galectin-8 to inhibitors of PI3K. Hence, the differences in cytoskeletal organization observed when cells adhere to galectin-8 or fibronectin can be attributed to differences in the robustness and duration of the PI3K-mediated signals emitted upon adhesion to the two matrices.
Several lines of evidence support these conclusions. First, we could demonstrate that cell adhesion onto galectin-8 induces signaling cascades that are being utilized by integrins upon ligation by other ECM proteins. Common signaling elements, activated to about the same extent by galectin-8 and fibronectin, include FAK (24) and p130 Cas (Fig. 3), indicating that upstream elements of integrin signal transduction, such as FAK and p130 Cas , are activated irrespective of the mode of ligation and clustering of integrins, which might involve either protein-protein interactions, in the case of fibronectin, or protein-sugar interactions, in the case of galectin-8. Hence, the restricted lateral mobility of integrins at the plane of the membrane upon binding of a bivalent lectin to their extracellular domains is sufficient to induce a conformational change that is conveyed to the cytoplasmic domains of integrins and triggers the recruitment and activation of FAK. However, the overall ligand-induced conformational change of integrins differs, depending on whether integrin clustering is induced upon protein-protein or protein-sugar interactions, because the cytoskeletal organization and the nature of the distal signals emitted downstream of FAK and p130 Cas differ when cells adhere onto galectin-8 or fibronectin. Whereas cell adhesion to fibronectin leads to transient activation of MAPK, engagement of integrins by galectin-8 leads to sustained activation of ERK-1 and -2. Similarly, ligation of integrins by galectin-8 results in more robust and sustained activation of PKB and p70S6K. How is a similar extent of activation of FAK and p130 Cas by galectin-8 or fibronectin translated into differences in the state of activation of their downstream effectors (Ras, ERK-1,2, PKB, and p70S6K)? One possibility is that a different set of integrins is ligated by galectin-8 or fibronectin. We have already shown that galectin-8 preferentially ligates ␣ 3 ␤ 1 or ␣ 6 ␤ 1 , but not ␣ 4 ␤ 1 integrins (27), whereas the repertoire of integrins ligated by fibronectin is much broader (38). As a result, the composition of signaling complexes formed between the different cytoplasmic tails of integrins and their downstream effectors might differ, depending on whether integrins were clustered by galectin-8 or fibronectin.
The robustness and duration of the activation of a given signaling pathway has far reaching biological consequences. For example, it is well established that transient activation of  Fig. 7. B, following 2 h of incubation at 37°C, cells were fixed and incubated with TRITC-labeled phalloidin for actin staining. Cells having microspikes were counted (A). Values are the mean Ϯ S.D. of the indicated n measurements of a representative experiment. The reduction in the number of cells harboring microspikes upon wortmannin treatment is highly significant (***, p Ͻ 0.01). Similarly, the number of CHO PKB cells harboring microspikes is significantly higher (***, p Ͻ 0.01) compared with CHO-P cells. the MAPK cascade (e.g. by epidermal growth factor) leads to enhanced growth of PC-12 cells, whereas stimulation of these cells with nerve growth factor induces sustained activation of the MAPK cascade, which leads to cellular differentiation (39). Accordingly, the sustained and robust activation of the MAPK and PI3K signaling pathway upon cell adhesion to galectin-8 might account for the unique cytoskeletal organization and biological functions of cells adherent to this lectin. Activation of ERK was inhibited in the presence of wortmannin, suggesting that PI3K is an upstream regulator of ERK signaling, triggered upon cell adhesion to galectin-8 or fibronectin. This conclusion is in accordance with previous studies that have demonstrated that PI3K may function upstream of Raf-1 but downstream of Ras upon integrin ligation by fibronectin (40). These results further support the role of integrins as ligands for galectin-8 because, unlike integrin signaling, activation of the ERK pathway by other means, such as ligation of growth factor receptors, is most often insensitive to inhibitors of PI3K (41).
An interesting outcome of the present study is the finding that PKB and p70S6K are actively involved in mediating cell adhesion and spreading on galectin-8. Their positive role is particularly evident in cells overexpressing PKB or p70S6K (CHO PKB and CHO p70S6K cells), whose adhesion and spreading is inhibited ϳ30% in the presence of PI3K inhibitors. The role of PKB or p70S6K as positive regulators of cell adhesion is in accordance with the fact that ligation of growth factor receptors, which stimulates the activity of PKB or p70S6K (42), potentiates the adhesive process in a number of cell types (43). At present, the signaling pathways regulating cell adhesion and spreading are not fully understood. Still, our results suggest that inhibition of the PI3K activity, induced by wortmannin, does not directly affect the active conformation of integrins because wortmannin does not inhibit cell adhesion to fibronectin. Instead, our results suggest that protein substrates for PKB or p70S6K are phosphorylated to a higher extent in cells adherent to galectin-8, in particular CHO PKB and CHO p70S6K cells, and this enables them to replace other signaling molecules that promote cell adhesion and spreading on galectin-8. The displacement of the native signaling elements with downstream effectors of PI3K is an irreversible process, because inhibition of PI3K activity does not enable the original participants to resume their positions within the integrin signaling complex and to turn the adhesive process less sensitive to PI3K inhibitors, as in non-transfected CHO cells. PKB has already been implicated as a mediator of cell adhesion (44), but the role of p70S6K in this process is less obvious. There is little evidence that p70S6K is required for the processes of cell adhesion, and activation of p70S6K was shown to be independent of pathways that regulate formation of focal adhesions (45). Our results suggest that p70S6K under certain conditions might modulate integrin activation by selective ECM proteins such as galectin-8, although the direct targets of this kinase within cell adhesion complexes remain to be determined. Our findings further suggest that cells that overexpress specific signaling elements, such as downstream effectors of PI3K, are bound to utilize these new elements not only for the promotion of cellular growth but also for remodeling of their "inside out" signaling elements and integrins function.
An immediate consequence of cell adhesion is cell spreading. In the present study we provide evidence that cells spread much faster on galectin-8, compared with fibronectin. Interestingly, although the variance of areas of cells adherent to fibronectin at early time points is rather limited, a much wider variance characterizes cells adherent to galectin-8. The reason for this variability is presently unknown, but it cannot be attributed to variable induction of protein synthesis because inclusion of inhibitors of protein synthesis did not alter the rate or phenotype of cells adherent to galectin-8. In contrast, cellular attachment to galectin-8 is inhibited ϳ50%, whereas spreading hardly takes place at 4°C, indicating that energyconsuming processes and active protein trafficking are required to mediate these events. In that respect, cell adhesion to galectin-8 resembles cell adhesion to other ECM proteins, which is largely inhibited at low temperatures (compare Ref. 46). Cellular attachment and spreading on fibronectin involves an initial requirement for Cdc42 in the formation of filopodial protrusions and the subsequent involvement of both Cdc42 and Rac during cell spreading and organization of the actin cytoskeleton (47). In galectin-8-adherent cells, focal contacts poorly assemble (24) and microspikes containing F-actin are formed instead. These structures have been functionally implicated in cell migration (37), which is readily induced by galectin-8 (24). Microspikes are readily formed when cells adhere to a variety of other ECM proteins such as thrombospondin-I (37), laminin-5 (48), and Tenascin-C splice variants (49). On fibronectin, Cdc42-and Rac-dependent formation of microspikes is involved in early steps of cell adhesion, but these events are transient and microspikes are rapidly replaced by focal contacts (37). In contrast, microspikes are stabilized when cells adhere to galectin-8, and the cells do not proceed to form highly developed focal contacts (24). Whereas Cdc42 leads to the formation of elongated projections containing F-actin, Rac leads to the formation of ribbons of short spikes (37). The microspikes formed when cells adhere to galectin-8 are short and radial and in that respect resemble microspikes formed when C2C12 cells, overexpressing a constitutively active Rac, adhere onto thrombospondin-I (36). We can therefore suggest that formation of microspikes upon cell adhesion to galectin-8 presumably involves Rac activation. Still, additional signaling elements, induced by galectin-8, are likely to be involved. Potential candidates are elements of the PI3K/PKB pathway, which are activated to a much greater extent by galectin-8 than fibronectin. In accordance with this idea, addition of wortmannin, a potent inhibitor of PI3K, effectively inhibits PKB activity and formation of microspikes when cells adhere onto galectin-8. Furthermore, overexpression of PKB promotes formation of microspikes in cells adherent onto galectin-8. The possible involvement of PI3K and its downstream effectors in galectin-8mediated formation of microspikes is supported by recent findings implicating the signaling pathway from the insulin-like growth factor-I receptor through PI3K in the rapid organization of microspikes at cell-cell junctions (50,51). PKB, the downstream effector of PI3K, activates a number of kinases, including the p21-activated kinase (PAK) (52) that has been implicated as playing a role in actin organization. PAK inhibits the activity of coffilin (reviewed in Refs. 53 and 54) and in such a way may inhibit actin depolymerization and promote formation of F-actin microspikes induced by galectin-8.
Finally, it should be noted that prostate carcinoma tumor antigen-1, the human isoform of galectin-8, is highly expressed in certain forms of prostate carcinomas (55) and other tumors (56). In contrast, galectin-8 expression decreases in human colon cancer when compared with normal and dysplastic colon tissues (57). This is associated with reduced migration of the colon cancer cells on immobilized galectin-8. Because interactions of soluble galectin-8 with cell surface integrins inhibit cell adhesion (27), whereas immobilized galectin-8 has the potential to promote cell attachment and spreading (24), galectin-8 may modulate cell-matrix interactions under a variety of physiological and pathological conditions, depending on the reper-toire, duration, and robustness of signals emitted when cells interact with this lectin.