Mike P Williamson
email: williamson@sheffield.ac.uk

Yu Suzuki

Nathan T Bourne

Tetsuo Asakura

Binding of Amyloid β to Ganglioside Micelles is dependent on Histidine 13

Keywords: HSQC, heteronuclear single-quantum correlation, TOCSY, total correlation spectroscopy amyloid, ganglioside, sialic acid, fibril, NMR 2

The amyloid β-peptide Aβ is a major component of plaques in Alzheimer's disease, and formation of senile plaques has been suggested to originate from regions of neuronal membrane rich in gangliosides. Here we demonstrate using NMR on 15 Nlabelled Aβ(1-40) and Aβ(1-42) that the interaction with ganglioside GM1 micelles is localised to the N-terminal region of the peptide, particularly residues His13 to Leu17, which become more helical when bound. The key interaction is with His13, which undergoes a GM1-specific conformational change. The sialic acid residue of the ganglioside headgroup is important for determining the nature of the conformational change. The isolated pentasaccharide headgroup of GM1 is not bound, suggesting the need for a polyanionic surface. Binding to heparin confirms this suggestion, since binding is of similar affinity but does not produce the same conformational changes in the peptide. A comparison of Aβ(1-40) and Aβ(1-42) indicates that binding to GM1 micelles is not related to oligomerisation, which occurs at the C-terminal end. These results imply that binding to ganglioside micelles causes a transition from random coil to helical in the N-terminal region, leaving the C-terminal region unstructured.

INTRODUCTION

The brains of Alzheimer's patients are characterised by amyloid plaques, whose main constituent is the amyloid β-peptide Aβ, which forms 'cross-β' fibrils [START_REF] Kirschner | X-Ray diffraction from intraneuronal paired helical filaments and extraneuronal amyloid fibers in Alzheimer disease indicates cross-β conformation[END_REF]. This peptide ranges from 40 to 43 residues in length, with the difference being at the Cterminal end. Longer peptides are much more fibrillogenic [START_REF] Jarrett | The carboxy terminus of the β-amyloid protein is critical for the seeding of amyloid formation[END_REF]. All adult brains contain amyloid plaques, but in most individuals these are 'diffuse' and not apparently harmful; by contrast, in Alzheimer's sufferers, the plaques are fibrillar and are associated with dystrophic neurons. This, together with many other results, has suggested that it is the conversion from diffuse to fibrillar form that dictates disease progression, and has focussed efforts on identifying the 'seed' from which fibrils are propagated in what appears to be a nucleation-dependent process that involves a change in the conformation of the peptide [START_REF] Huang | Alternate aggregation pathways of the Alzheimer β-amyloid peptide[END_REF]. Interestingly, the diffuse plaques are predominantly Aβ and the neuritic plaques predominantly Aβ(1-40) [START_REF] Esiri | The neuropathology of Alzheimer's disease[END_REF].

A major debate has focused on whether the seed for fibril formation is formed in solution (possibly as an oligomer [START_REF] Cleary | Natural oligomers of the amyloidprotein specifically disrupt cognitive function[END_REF]) or on membrane surfaces. In recent years, much attention has focussed on interactions between Aβ and gangliosides, particularly GM1 (Figure 1), which is one of the most abundant gangliosides in the brain, constituting approximately 20% of brain gangliosides [START_REF] Kracun | Topographical atlas of the gangliosides of the adult human brain[END_REF]. Gangliosides are sialic acid-containing glycosphingolipids with a role in synaptic transmission and signalling, and are found in high concentration in neural cell membrane, particularly in synaptic membrane [START_REF] Nagai | Functional roles of gangliosides in bio-signaling[END_REF]. The finding that GM1-bound Aβ is generated in human brain [START_REF] Yanagisawa | GM1 ganglosidebound amyloid β-protein (Aβ): a possible form of preamyloid in Alzheimer's disease[END_REF] has stimulated further studies in this area, including the recent results that ganglioside micelles stimulate aggregation and fibrillisation of Aβ [START_REF] Yamamoto | Environment-and mutation-dependent aggregation behavior of Alzheimer amyloid β-protein[END_REF], that regional deposition of Aβ in the brain is induced by the local gangliosides [10], and that Aβ-GM1 binding in living cells takes place in a seed-dependent manner and directly induces cytotoxicity [11], and may be a mechanism common to many amyloidoses [START_REF] Gellermann | Raft lipids as common components of human extracellular amyloid fibrils[END_REF]. A particularly interesting line of research locates GM1-rich membranes in cholesterol-rich lipid rafts [START_REF] Kakio | Interactions of amyloid β-protein with various gangliosides in raft-like membranes: Importance of GM1 ganglioside-bound form as an endogenous seed for Alzheimer amyloid[END_REF][START_REF] Kakio | Cholesterol-dependent formation of GM1 ganglioside-bound amyloid β-protein, an endogenous seed for Alzheimer amyloid[END_REF], thereby suggesting how the amyloid deposits could affect neuronal membranes and signalling, and also why cholesterol and apolipoprotein E (which redistributes cholesterol in the brain) might be linked to Alzheimer's disease [START_REF] Wolozin | A fluid connection: Cholesterol and Aβ[END_REF].

In this paper, we describe NMR experiments aimed at characterising the interactions between Aβ and ganglioside micelles. We identify a small part of the peptide, residues 13-17, that we demonstrate to be most crucial for substrate-specific interaction. A comparison of Aβ(1-40) and Aβ suggests that Aβ(1-42) forms oligomers in solution by interactions at the C-terminus, but that these are not related to GM1 binding. The relationship to seeding of amyloid plaques is discussed.

EXPERIMENTAL

Uniformly 15 N-labelled Aβ(1-40) and Aβ were purchased from rPeptide (Athens, GA), and gangliosides were purchased from Alexis Biochemicals (now Axxora (UK) Ltd), Nottingham, UK. The purity of the gangliosides is quoted as >98% by the manufacturer, but was not checked. All other reagents were from Sigma-Aldrich. The heparin used was the sodium salt (H4784).

Aβ(1-40) solutions were prepared following [START_REF] Hou | Solution NMR studies of the Aβ(1-40) and Aβ(1-42) peptides establish that the Met35 oxidation state affects the mechanism of amyloid formation[END_REF]: the peptide was dissolved at a concentration of 200 µM in 10 mM NaOH with 1 min sonication, and immediately frozen if required. Subsequently, the pH was adjusted to 7.2 with a minimal amount of 0.1 M HCl, and D 2 O was added to make approximately 200 µM peptide solution containing 10% D 2 O. Solutions prepared in this way were stable and showed no sign of aggregation for at least a week. By contrast, solutions made up in buffer aggregated much faster, often being almost entirely precipitated after 24 hr. Aβ(1-42) was pretreated by dissolution in hexafluoroisopropanol and lyophilisation, before dissolving in 10 mM ammonium hydroxide, followed by adjustment of the pH to 7.2. These solutions started to aggregate and form fibrils immediately and were only usable for 2-3 days after solution. Solution of the commercial material directly into sodium hydroxide resulted in no NMR signal whatsoever, indicating significant aggregation using this method. Stock solutions of gangliosides or heparin were prepared at high concentration (approximately 100-200 mM), adjusted to pH 7.2 and added direct to the NMR tube. All NMR experiments were carried out on a Bruker DRX-500 equipped with a cryoprobe, and operated at 13 °C. 15 N HSQC experiments used gradient selection for water suppression and water flip-back pulses to minimise loss of magnetisation through exchange and relaxation processes. The 3D TOCSY-HSQC spectrum incorporated solvent suppression using gradients, with a 35 ms spin-lock at 8.3 kHz decoupler power. Processing of NMR data used FELIX (Accelrys Inc., San Diego, CA), and titration data were analysed using home-written scripts. Cross-peak intensities were measured within FELIX, transferred to a text file, and fitted to an exponential decay using a Marquardt nonlinear least-squares fitting based originally on a Numerical Recipes© algorithm. Binding constants were obtained by fitting to a standard equation using Excel (Microsoft).

RESULTS

The HSQC spectrum of Aβ(1-40) was assigned based on assignments described and kindly provided by Dr M. Zagorski [START_REF] Hou | Solution NMR studies of the Aβ(1-40) and Aβ(1-42) peptides establish that the Met35 oxidation state affects the mechanism of amyloid formation[END_REF]. Assignments were confirmed using a 3D TOCSY-HSQC experiment, and proved very similar to published assignments [START_REF] Hou | Solution NMR studies of the Aβ(1-40) and Aβ(1-42) peptides establish that the Met35 oxidation state affects the mechanism of amyloid formation[END_REF].

The HSQC spectrum is shown in Figure 2. Almost all backbone signals are resolved.

At 13 °C, all signals can be found except for the N-terminal residue and His6. His14 gives a weak signal, as expected because of its rapid amide exchange under these conditions [START_REF] Bai | Primary structure effects on peptide group hydrogen exchange[END_REF]. The reason for the absence of His6 in our HSQC spectra is less clear, but it was also not observable by Hou et al. [START_REF] Hou | Solution NMR studies of the Aβ(1-40) and Aβ(1-42) peptides establish that the Met35 oxidation state affects the mechanism of amyloid formation[END_REF]. On the basis of the chemical shifts, we concur with other authors [START_REF] Hou | Solution NMR studies of the Aβ(1-40) and Aβ(1-42) peptides establish that the Met35 oxidation state affects the mechanism of amyloid formation[END_REF]18] that the peptide is random coil in aqueous solution. The majority of HSQC peaks in Aβ were assigned in the same way, but the assignment is somewhat less complete due to its rapid aggregation, which limits the time available for 3D NMR experiments. The signals from Aβ(1-42) have very similar chemical shifts to those of Aβ(1-40) except at the C-terminus, implying no significant conformational differences between them.

GM1 forms micelles with a critical micelle concentration in the low µM range [19]. Thus, at concentrations >100 µM, as used here, it is essentially 100% micellar.

On titration of GM1 micelles into Aβ(1-40), chemical shift changes were seen in the NMR spectrum, as shown in Figure 3. The chemical shift changes are small, but are reproducible and specific, since many residues have essentially no change in shift.

Smaller chemical shift changes have been demonstrated to be biologically relevant on many previous occasions (eg [START_REF] Morrison | Solution NMR study of the interaction between NTF2 and nucleoporin FxFG repeats[END_REF][START_REF] Laguri | Solution structure and DNA binding of the effector domain from the global regulator PrrA (RegA) from Rhodobacter sphaeroides: insights into DNA binding specificity[END_REF]). Almost all the chemical shift changes were in the N-terminal half of the peptide, and were close to potentially positively charged residues: Glu3-Arg5 (close to Arg5 and His6) and Val12-Leu17 (close to His13, His14 and Lys16). However, the pK values of the three histidines are all approximately 6.5 [START_REF] Ma | Residue-specific pK a measurements of the β-peptide and mechanism of pH-induced amyloid formation[END_REF]. Hence at the pH of our measurements, 7.2, most of the histidines will be unprotonated. We therefore expect that the only residues significantly positively charged at this pH will be Arg5 and Lys16, together with Lys28. This makes it unlikely that the chemical shift changes are due only to coulombic interactions with the single negative charge in the GM1 headgroup, the sialic acid. In order to confirm this, a further titration was carried out at approximately physiological salt concentration (150 mM NaCl), which should markedly reduce purely coulombic interactions in water. The results (Figure 4) show reduced chemical shift changes, indicating a loss of affinity. Residues 14-17 still shift, but the changes seen in residues 3-13 are much reduced. This implies that although some of the binding and the chemical shift changes are primarily coulombic in origin, others (and in particular the changes seen in residues 15-17) are much less so. We note that other authors have concluded that binding to GM1 is not primarily coulombic, although again there are clearly coulombic interactions [START_REF] Choo-Smith | Acceleration of amyloid fibril formation by specific binding of Aβ-(1-40) peptide to ganglioside-containing membrane vesicles[END_REF][START_REF] Mclaurin | Structural transitions associated with the interaction of Alzheimer β-amyloid peptides with gangliosides[END_REF][START_REF] Bokvist | Two types of Alzheimer's β-amyloid (1-40) peptide membrane interactions: Aggregation preventing transmembrane anchoring Versus accelerated surface fibril formation[END_REF].

A further titration of Aβ(1-40) was carried out, using micelles of asialo-GM1 (Figure 1). The results are shown in Figure 5, in which the chemical shift ranges used in the plot are 75% of those used for Figure 3, and show that chemical shift changes with asialo-GM1 are similar to those seen for GM1 but reduced in magnitude for equivalent concentrations by approximately 25%. A reduction is expected in the magnitude of the shift change, because asialo-GM1 is known to bind less tightly than GM1 to Aβ. The extent of the difference has been reported differently. Choo-Smith et al. report no binding at all to asialo-GM1 [START_REF] Choo-Smith | Acceleration of amyloid fibril formation by specific binding of Aβ-(1-40) peptide to ganglioside-containing membrane vesicles[END_REF], while a factor of 2 has also been reported [START_REF] Ariga | Characterization of high-affinity binding between gangliosides and amyloid β-protein[END_REF]. Our data are in better agreement with the latter result. The overall similarity of the chemical shift changes for GM1 and asialo-GM1 implies that the binding interactions are similar, although with some differences in the region of His13-Gln15. This result therefore also implies that the interaction is not dominated by coulombic forces, since asialo-GM1 has no charge in the headgroup.

In a further experiment, Aβ(1-40) was titrated with heparin (Figure 6). The chemical shift changes again affect very similar residues to those affected by GM1, although the size and direction of the change is in several cases markedly different (eg Arg5, His13 and His14). Heparin is a polyanionic polysaccharide (consisting mainly of 2-deoxy-2-sulphamino-α-D-glucose 6-sulphate, α-L-iduronic acid 2-sulphate, 2acetamido-2-deoxy-α-D-glucose, β-D-glucuronic acid, and α-L-iduronic acid in random order), but otherwise has little structural similarity to GM1 micelles. Despite its completely different covalent structure, and presumably its three-dimensional structure, it causes similar chemical shift changes in Aβ to those caused by ganglioside micelles. Hence, many of the changes must reflect a general conformational propensity of the peptide on binding to a surface, especially to one carrying a negative charge (although the overall similarity of results from asialo-GM1 imply that a negative charge is not essential). However, the exact structure adopted when bound, indicated by the direction of the chemical shift changes for residues Phe4, Arg5, Val12 and His13 in particular, depends on the substrate. By contrast, Aβ(1-40) was also titrated with the pentasaccharide headgroup of GM1. No chemical shift changes could be observed at all (data not shown), implying a lack of interaction, and therefore the requirement for an extended surface for efficient binding. A similar observation has been made before [START_REF] Choo-Smith | The interaction between Alzheimer amyloid β(1-40) peptide and ganglioside G M1 -containing membranes[END_REF].

The chemical shift changes for Gln15, Lys16 and Leu17 on titration are in all cases similar, and show a reduction in chemical shift (ie, an upfield shift) for both 1 H and 15 N. These changes are those expected for a change from random coil to α-helix, and in the opposite direction for those expected on going from random coil to β-sheet [28,29]. The data therefore imply that this region of the peptide becomes more helical on binding, but that the interacting region N-terminal to this sequence has a more complicated and substrate-specific conformational change.

The affinity of Aβ(1-40) for GM1 micelles was estimated by fitting the chemical shift changes to a standard saturation curve [START_REF] Charlton | Polyphenol/peptide binding and precipitation[END_REF]. The numerical result requires an assumption as to the number of GM1 molecules in a micelle. Here we have assumed an aggregation number of 310 [START_REF] Cantu | Thermal hysteresis in ganglioside micelles investigated by differential scanning calorimetry and light-scattering[END_REF], which produces a dissociation constant for a micelle of approximately 5 µM, in approximate agreement with values produced by others for binding to vesicles containing GM1 [START_REF] Kakio | Interactions of amyloid β-protein with various gangliosides in raft-like membranes: Importance of GM1 ganglioside-bound form as an endogenous seed for Alzheimer amyloid[END_REF][START_REF] Kakio | Cholesterol-dependent formation of GM1 ganglioside-bound amyloid β-protein, an endogenous seed for Alzheimer amyloid[END_REF][START_REF] Choo-Smith | Acceleration of amyloid fibril formation by specific binding of Aβ-(1-40) peptide to ganglioside-containing membrane vesicles[END_REF]. This result implies that the fairly weak binding to GM1 does not act to concentrate Aβ directly (and therefore increase the local concentration of Aβ which might encourage fibrillisation); rather, it acts to fix Aβ into a fibrillogenic conformation. When the dissociation constant is calculated for GM1 monomers, it is much weaker at approximately 1 mM. The dissociation constant for heparin (per disaccharide repeat) is similar, implying that the binding to gangliosides is not particularly strong, in agreement with results from other groups [START_REF] Kakio | Formation of a membrane-active form of amyloid β-protein in raft-like model membranes[END_REF]. The calculations also imply that at 8 equivalents of GM1 (the maximum shown here), the shift changes are approximately 30% of maximal. Thus, 1 HN chemical shift changes on 100% binding to GM1 micelles are estimated to be approximately 0.06 ppm for residues 15-17, implying that the bound structure is not fully helical, which would produce shift changes of approximately 0.2 ppm [28].

During the course of the titration, reductions in peak intensity and increases in linewidth were seen in the HSQC spectrum. Such changes are expected due to the increased correlation time arising from binding to a large micelle, which reduces T 2 and therefore reduces intensity in multipulse experiments such as HSQC. For most protons, the increased linewidth and reduced intensity seen are consistent with a fastexchange limit equilibrium between the free and micelle-bound forms: fast exchange is expected from the the relatively weak binding affinity, which is in the low µM range. Thus, for the heparin titration, intensity changes were small and showed no clear variation along the sequence (except that the two C-terminal residues showed a lesser intensity reduction, implying little or no restriction in motional freedom arising from binding of the peptide to heparin). However, for the titrations with GM1, intensity changes were large and markedly non-uniform along the sequence (Figure 7). One would expect the changes to be larger than with heparin because of the much longer correlation time of the GM1 micelles. The non-uniformity is consistent either with greater motional restriction at some sites, or with exchange broadening. The latter explanation is less likely because the affinity is too weak to cause significant exchange broadening. As a way of handling the data, the intensities were fitted to an exponential curve (intensity against amount of GM1 added), the results of which are shown in Figure 8. For both GM1 and asialo-GM1, the most dramatic decrease in intensity is for residues 26-28, despite the fact that these residues show only very small chemical shift changes. There is also faster broadening at the C-terminal end, around Gly37. Loss of intensity can be caused by a large number of factors, all of which imply a change in conformation or environment. We therefore conclude that binding to GM1 micelles does affect the C-terminal part of the peptide, specifically around residues 27 and 37, even though no or only small chemical shift changes are seen here.

There are also marked decreases in intensity around His13 and Ser8 in the Nterminal end of the peptide on binding GM1, which are smaller or absent on titration with asialo-GM1. This implies a reduced interaction at these locations in asialo-GM1, an observation that is consistent with the chemical shift changes described above, which were smaller and different for asialo-GM1 for Ser8, Gly9, His13 and Gln15. Thus, both chemical shift changes and line-broadening imply an interaction with the sialic acid group in the region of residues 8-15.

HSQC experiments were also carried out on Aβ . This peptide differs from Aβ(1-40) only by the presence of Ile41 and Ala42. Immediately after separation of aggregates by treatment with hexafluoroisopropanol, followed by lyophilisation, sonication at high pH and adjustment to pH 7.2, HSQC spectra already indicated the presence of aggregated species, which increased in intensity with time (Figure 9). These signals were sharp and apparently in slow exchange with monomer, and only affected C-terminal residues (from G33 onwards). We therefore conclude that the peptide forms small well-defined aggregates, probably dimers because of their sharpness, in a time-dependent manner, centred on the C-terminus. The sharpness of the oligomer signals is inconsistent with their being as large as hexamers.

Titrations of Aβ(1-42) with GM1 showed chemical shift changes. The slow exchange between monomer and oligomer meant that we were able to monitor chemical shift changes for some of the C-terminal residues in both monomer and oligomer. We were therefore able to identify binding interactions for monomer and oligomer from the same solution. There were no significant chemical shift changes in either monomer or oligomer, for any of the residues showing signal splitting (ie, G33 onwards), implying that the C-terminal end of the peptide does not interact with GM1 micelles, either as monomers or oligomers. The lack of binding of the oligomer is of interest in the light of recent reports that the fibrillogenic form of Aβ is soluble oligomers [START_REF] Cleary | Natural oligomers of the amyloidprotein specifically disrupt cognitive function[END_REF]. By contrast, residues in the N-terminal end show shift changes comparable to those observed for Aβ(1-40), with the largest changes being for Y10, V12, K16 and L17. (We note that shift changes for H13 and Q15 were not measurable due to signal overlap and poor solubility.) We therefore conclude that Aβ(1-42) binds to GM1 micelles in a similar way to Aβ(1-40), and particularly in the region 10-17; and that it also undergoes a dimerisation or aggregation at the C-terminal end, which is independent of any binding to GM1.

DISCUSSION

There have been a large number of conformational studies of Aβ. Many of these are in unphysiological solvents or in SDS or lipid micelles, which are well known to have a tendency to push peptides towards helical structures. It is therefore not surprising to find that these studies tend to report helical conformations for Aβ. However, studies in water have suggested random coil states [START_REF] Hou | Solution NMR studies of the Aβ(1-40) and Aβ(1-42) peptides establish that the Met35 oxidation state affects the mechanism of amyloid formation[END_REF]18], a conclusion with which we agree.

In this study we demonstrate binding in the low µM range to GM1, asialo-GM1 and heparin, but no measurable binding to the isolated GM1 headgroup. Several experiments (both ours and those of others [START_REF] Mclaurin | Effect of amino-acid substitutions on Alzheimer's amyloid-β peptide-glycosaminoglycan interactions[END_REF]) have demonstrated that although there may be a coulombic element to the binding, the interactions are not limited to coulombic ones. Our results are therefore relevant to the physiological situation where the salt concentration is higher and coulombic interactions are weaker.

The chemical shift changes reported here demonstrate binding of Aβ to GM1 micelles, which is localised to the N-terminal half of the peptide. We confirm that binding to asialo-GM1 is weaker, specifically with differences around residues 12-15.

[This conclusion is radically different from one resulting from another NMR study [START_REF] Mandal | Alzheimer's disease: NMR studies of asialo (GM1) and trisialo (GT1b) ganglioside interactions with Aβ(1-40) peptide in a membrane mimic environment[END_REF]. However, that study was carried out using SDS micelles, which may be the reason for the different results.] Binding to heparin also involves the same residues, although the chemical shift changes (and therefore presumably the interactions and conformational changes on binding) are different. Our results therefore agree with the consensus of opinion, which is that high affinity binding is directed to the N-terminal end of the Aβ peptide [START_REF] Ariga | Characterization of high-affinity binding between gangliosides and amyloid β-protein[END_REF]. For binding to heparin, the region 12-17 has previously been identified [START_REF] Mclaurin | Effect of amino-acid substitutions on Alzheimer's amyloid-β peptide-glycosaminoglycan interactions[END_REF][START_REF] Brunden | pH-dependent binding of synthetic β-amyloid peptides to glycosaminoglycans[END_REF], and this region has also been demonstrated to be important for attachment to microglial cells and for neurotoxicity [START_REF] Giulian | The HHQK domain of β-amyloid provides a structural basis for the immunopathology of Alzheimer's disease[END_REF]. There is no binding to isolated pentasaccharide headgroups. The results therefore imply that Aβ binds to a range of surfaces in a similar way and with similar affinity, the binding being at the N-terminal end of the peptide.

Chemical shift changes (particularly of 15 N and 1 HN) are notoriously difficult to relate to specific conformational changes. However, there is wide agreement that helical regions have higher field 15 N and 1 HN shifts than random coil. The results reported here therefore imply that the short region from Gln15 to Leu17 becomes more helical on binding, to GM1, asialo-GM1 and heparin. It is perhaps significant that this is exactly the same region identified above as being important for neurotoxicity [START_REF] Giulian | The HHQK domain of β-amyloid provides a structural basis for the immunopathology of Alzheimer's disease[END_REF]. The region C-terminal to Leu17 has little or no observable conformational change. Intensity changes did however suggest some interaction in this part of the peptide, particularly around residues 27 and 37.

A comparison of chemical shift changes in Aβ on addition of GM1 micelles compared to asialo-GM1 or heparin implies that His13 interacts specifically with the sialic acid moiety. Because previous studies have identified the sialic acid as being important for the growth of amyloid fibres [10, [START_REF] Kakio | Interactions of amyloid β-protein with various gangliosides in raft-like membranes: Importance of GM1 ganglioside-bound form as an endogenous seed for Alzheimer amyloid[END_REF][START_REF] Matsuzaki | Interactions of amyloid β-peptide (1-40) with ganglioside-containing membranes[END_REF], the results imply that His13 binding may play an important role in fibrillogenesis. Indeed, His13 has previously been identified as crucial for rapid fibrillisation [START_REF] Kirkitadze | Identification and characterization of key kinetic intermediates in amyloid β-protein fibrillogenesis[END_REF]. It is relevant to note that rats, which differ in their Aβ from humans in only three positions (R5G, Y10F and H13R), do not form cerebral Aβ amyloid [START_REF] Selkoe | Biochemistry of altered brain proteins in Alzheimer's disease[END_REF].

Comparison of chemical shift changes in Aβ(1-40) and Aβ implies that binding to GM1 micelles is located at the N-terminal end, while the C-terminal end plays no part in binding, but does lead to oligomerisation in solution. We note that residues 1-28 alone are sufficient for formation of structured aggregates, but do not form fibrils [40]. The results therefore suggest that the N-terminal binding of the peptide to GM1 may provide the initial fibrillogenic seed, but that the C-terminus is required for propagation into fibrils. This raises the question of what this study implies about the formation of amyloid fibres in vivo. The study is consistent with the seeding/nucleation amyloid cascade model of Alzheimer's. By contrast, it implies that soluble Aβ aggregates, currently a very popular topic of study [START_REF] Cleary | Natural oligomers of the amyloidprotein specifically disrupt cognitive function[END_REF], may be off-pathway intermediates with no direct involvement in plaque formation. There is however a recent report [START_REF] Kokubo | Oligomeric proteins ultrastructurally localize to cell processes, especially to axon terminals with higher density, but not to lipid rafts in Tg2576 mouse brain[END_REF] that Aβ polymers are found associated with lipid rafts in mouse brain, but that oligomers are found particularly at axon terminals. It is therefore possible that both modes of polymerisation operate at the same time, in different regions of the brain. Multiple assembly pathways would be no surprise [START_REF] Goldsbury | Multiple assembly pathways underlie amyloid-β fibril polymorphisms[END_REF].

The study also implies that the fibrillogenic seed nucleus involves an interaction of His13 with the sialic acid moiety of GM1. Aβ can bind to other nonfibrillogenic surfaces (heparin, for example), but without inducing the same structural change in Aβ. This implies that binding of Aβ in other conformations may merely lead to buildup of Aβ without the formation of fibrils -in other words, to diffuse plaques. Indeed, this could provide an explanation of why Aβ(1-42), which is less soluble than Aβ(1-40) and speeds up fibrillogenesis [START_REF] Kirkitadze | Identification and characterization of key kinetic intermediates in amyloid β-protein fibrillogenesis[END_REF], surprisingly tends to be found in diffuse rather than neuritic plaques [START_REF] Esiri | The neuropathology of Alzheimer's disease[END_REF]: we suggest that it binds and aggregates so rapidly that it does not have time to rearrange into the correct conformation to form the required seed. This suggestion is consistent with our observation that oligomerisation of Aβ(1-42) is rapid and independent of GM1 binding, and occurs in a different region of the peptide. It could also provide an explanation of the observation that the amount of soluble Aβ oligomer correlates with synaptic loss better than does the amount of insoluble Aβ [START_REF] Walsh | Amyloid-β oligomers: their production, toxicity and therapeutic inhibition[END_REF]: it may be that deposition of Aβ in the correct conformation requires multiple equilibration between soluble and insoluble forms. The formation of fibrils is thus seen to be a fine balance between over-rapid deposition (leading to diffuse plaques) and inadequate deposition (leading to small and non-aggressive plaques). In partial support of this argument, we note that treatment of transgenic mice (which develop Alzheimer's-like symptoms) with anti-Aβ antibody not only leads to a reduction in plaques (implying reversible binding of Aβ to plaques in vivo), but also gives a reduction in neuritic damage, implying that the neuritic damage is a consequence of the plaques [START_REF] Brendza | Anti-Aβ antibody treatment promotes the rapid recovery of amyloid-associated neuritic dystrophy in PDAPP transgenic mice[END_REF]. Only the N-terminal residues are shown, because there were effectively no changes in the C-terminal residues. Titrations are 0, 1, 2, 4 and 6.6 equivalents of GM1. Other conditions are as for Figure 3. Other conditions are as for Figure 3, except that the chemical shift ranges are ± 0.05 ppm for 1 H and ± 0.3 ppm for 15 N. The bars show the decay constant from the exponential fits of the experimental data (Figure 7), with the estimated fitting error. The numerical values of the decay constants therefore have no direct meaning, but serve as a measure of the extent of intensity loss during the titration. Data for His14 are not shown in B, because the intensity of His14 is too weak to allow fitting of the data. is also not affected by the addition of GM1, but there is an independent and timedependent loss of the signal from monomer (marked m) and increase of a signal from an oligomeric species (marked o). All signals are starting to decrease in the final spectrum because of precipitation of aggregates.

Figure legends

Figure 1

 1 Figure1The structure of gangliosides GM1 and asialo-GM1.

Figure 2 HSQC

 2 Figure 2 HSQC spectrum of 15 N-labelled Aβ(1-40) in water, pH 7.2, 13 °C. The small unlabelled peak at ( 1 H = 7.2, 15 N = 119 ppm) is probably an arginine sidechain NH.

Figure 3

 3 Figure 3 Chemical shift changes in Aβ(1-40) on addition of GM1 to 200 µM peptide in water.Each box shows results for a different residue, with 1 H shifts horizontal (increasing left to right, total range ± 0.02 ppm) and15 N shifts vertical (increasing bottom to top, total range ± 0.1 ppm). This representation therefore resembles the change seen in an HSQC spectrum, except that the directions of the axes are reversed. The start of the titration is indicated with a filled circle, and subsequent titrations are 1, 2, 4 and 8 equivalents of GM1. The size of the circles approximates the experimental uncertainty. No data are shown for His6 because it could not be observed.

Figure 4

 4 Figure 4 Chemical shift changes in Aβ(1-40) on addition of GM1 to 200 µM peptide in 150 mM NaCl.

Figure 5

 5 Figure 5 Chemical shift changes in Aβ(1-40) on addition of asialo-GM1 to 200 µM peptide in water.Other conditions are as for Figure3, except that the chemical shift ranges in each box are ± 0.015 ppm for 1 H and ± 0.075 ppm for15 N.

Figure 6

 6 Figure 6 Chemical shift changes in Aβ(1-40) on addition of heparin to 200 µM peptide in water.

Figure 7

 7 Figure 7 Intensity changes of HSQC peaks on addition of GM1 micelles to Aβ(1-40).The horizontal scale shows the molar ratio of GM1 to Aβ, with for each residue a range of 0 to 40 molar equivalents: the data are shown for 0, 1, 2, 4, 8, 12, 16, 20, 24, 28, 32, and 36 equivalents. The vertical scale shows the peak intensity relative to that in pure peptide, on a range from 0 to 100%. The intensity changes are fitted to an exponential decay.

Figure 8

 8 Figure 8 Loss of intensity during the titrations of (A) GM1 and (B) asialo-GM1into Aβ(1-40).

Figure 9

 9 Figure 9 Titration of GM1 into Aβ(1-42). HSQC spectra of Aβ(1-42) alone (left), with 8 mole equivalents of GM1 (centre) and 16 mole equivalents (right). The spectra were acquired sequentially, about 6 hours apart. The signals for glycines 9 and 29 are barely affected. The signal for glycine 38

Biochemical Journal Immediate Publication. Published on 21 Apr 2006 as manuscript BJ20060293 Copyright 2006 Biochemical Society

Acknowledgments

We thank the Biotechnology and Biological Sciences Research Council (UK) for a studentship (NTB) and for the award of a Japan Partnership Award to MPW. We thank the Wellcome Trust and BBSRC for grants for spectrometer and cryoprobe purchase.


Sonja Balthasar

Johanna Samulin

Hanna Ahlgren

Nina Bergelin

Mathias Lundqvist

Emil C Toescu

Margaret C Eggo

Kid Törnquist

Sphingosine 1-phosphate receptor expression profile and regulation of migration in human thyroid cancer cells

Keywords: sphingosine 1-phosphate, thyroid, tumour, calcium, motility, proliferation Biochemical

come

INTRODUCTION

Sphingosine 1-phosphate (S1P) is a bioactive lipid that regulates central cellular processes, such as cell growth, survival, differentiation and motility [START_REF] Radeff-Huang | G protein mediated signalling pathways in lysophospholipid induced cell proliferation and survival[END_REF][START_REF] Chalfant | Sphingosine 1-phosphate and ceramide 1phosphate: expanding roles in cell signalling[END_REF][START_REF] Ishii | Lysophospholipid receptors: signalling and biology[END_REF][START_REF] Takuwa | Subtype-specific differential regulation of Rho family G proteins and cell migration by the Edg family sphingosine-1-phosphate receptors[END_REF]. S1P is present in plasma and serum at high nanomolar concentrations [START_REF] Yatomi | Sphingosine 1-phosphate, a bioactive sphingolipid abundantly stored in platelets, is a normal constituent of human plasma and serum[END_REF][START_REF] Murata | Interaction of sphingosine 1-phosphate with plasma components, including lipoproteins, regulates the lipid receptor-mediated actions[END_REF]. Platelets store S1P and release it into the blood [START_REF] Yatomi | Sphingosine 1-phosphate, a bioactive sphingolipid abundantly stored in platelets, is a normal constituent of human plasma and serum[END_REF][START_REF] Yatomi | Sphingosine 1phosphate: synthesis and release[END_REF] where its concentration is tightly controlled by ectoenzyme activity and distribution among plasma proteins [START_REF] Murata | Interaction of sphingosine 1-phosphate with plasma components, including lipoproteins, regulates the lipid receptor-mediated actions[END_REF]8]. Extracellular S1P may also be produced locally by other cell types, e.g. glioma cells [START_REF] Edsall | Sphingosine kinase expression regulates apoptosis and caspase activation in PC12 cells[END_REF], mouse melanoma cells, and fibroblasts [START_REF] Yamamura | Sphingosine 1-phosphate regulates melanoma cell motility through a receptorcoupled extracellular action and in a pertussis toxin-insensitive manner[END_REF]. The effects of extracellular S1P are mediated by G-protein coupled receptors, termed S1P 1-5 [START_REF] Chun | International Union of Pharmacology. XXXIV. Lysophospholipid receptor nomenclature[END_REF], which regulate multiple intracellular pathways [START_REF] Ishii | Lysophospholipid receptors: signalling and biology[END_REF][START_REF] Taha | Sphingosine-1-phosphate receptors: receptor specificity versus functional redundancy[END_REF]13]. Some of the effects of S1P are mediated by intracellular S1P acting on unknown targets [START_REF] Spiegel | Exogenous and intracellularly generated sphingosine 1-phosphate can regulate cellular processes by divergent pathways[END_REF]. S1P may either inhibit or stimulate cellular motility, depending on cell type and S1P concentration [START_REF] Takuwa | Subtype-specific differential regulation of Rho family G proteins and cell migration by the Edg family sphingosine-1-phosphate receptors[END_REF][START_REF] Taha | Sphingosine-1-phosphate receptors: receptor specificity versus functional redundancy[END_REF]. Differences in migratory responses correlate well with differences in S1P receptor expression, both in vitro and in vivo [START_REF] Takuwa | Subtype-specific differential regulation of Rho family G proteins and cell migration by the Edg family sphingosine-1-phosphate receptors[END_REF][START_REF] Malchinkhuu | Role of p38 mitogen-activated kinase and c-Jun terminal kinase in migration response to lysophosphatidic acid and sphingosine-1-phosphate in glioma cells[END_REF][START_REF] Yamaguchi | Sphingosine-1-phosphate receptor subtype-specific positive and negative regulation of Rac and haematogenous metastasis of melanoma cells[END_REF][START_REF] Dolezalova | Biochemical regulation of breast cancer cell expression of S1P 2 (Edg-5) and S1P 3 (Edg-3) G protein-coupled receptors for sphingosine 1-phosphate[END_REF][START_REF] Czeloth | Sphingosine-1-phosphate mediates migration of mature dendritic cells[END_REF]. S1P receptors regulate migration by modulating the activity of the Rho family of GTPases [START_REF] Takuwa | Subtype-specific differential regulation of Rho family G proteins and cell migration by the Edg family sphingosine-1-phosphate receptors[END_REF][START_REF] Taha | Sphingosine-1-phosphate receptors: receptor specificity versus functional redundancy[END_REF]. S1P 1and S1P 3 -induced migration involves G i -mediated activation of phosphatidylinositol 3kinase (PI3K), Akt, and Rac [START_REF] Taha | Sphingosine-1-phosphate receptors: receptor specificity versus functional redundancy[END_REF]. S1P 2 signals via G 12/13 to activate Rho and to inhibit Rac-dependent signalling, which results in an inhibitory effect of S1P on migration [START_REF] Taha | Sphingosine-1-phosphate receptors: receptor specificity versus functional redundancy[END_REF][START_REF] Yamaguchi | Sphingosine-1-phosphate receptor subtype-specific positive and negative regulation of Rac and haematogenous metastasis of melanoma cells[END_REF][START_REF] Lepley | The G protein-coupled receptor S1P 2 regulates Rho/Rho kinase pathway to inhibit tumour cell migration[END_REF]. S1P 4 is mainly expressed in cells of the immune system [13], and stimulates migration of transfected CHO cells by activating cdc42 in a pertussis toxin-sensitive manner [START_REF] Kohno | Sphingosine 1phosphate promotes cell migration through the activation of Cdc42 in Edg-6/S1P4expressing cells[END_REF]. The effects of S1P 5 on migration are to our knowledge not clarified. S1P is implicated in several of the key cellular events in cancer growth and metastasis [START_REF] Ogretmen | Biologically active sphingolipids in cancer pathogenesis and treatment[END_REF]. On the other hand, S1P has been shown to inhibit metastasis of mouse melanoma cells in vivo [START_REF] Yamaguchi | Sphingosine-1-phosphate receptor subtype-specific positive and negative regulation of Rac and haematogenous metastasis of melanoma cells[END_REF]. S1P modulates central intracellular cascades, as well as regulates proliferation of rat thyroid cells, which express S1P 2 , S1P 3 and S1P 5 [22][START_REF] Törnquist | Sphingosine 1-phosphate stimulates Na+/H+ exchange in thyroid FRTL-5 cells[END_REF][START_REF] Okajima | Sphingosine-1-phosphate stimulates hydrogen peroxide generation through activation of phospholipase C-Ca2+ system in FRTL-5 thyroid cells: possible involvement of guanosine triphosphate-binding proteins in the lipid signalling[END_REF][START_REF] Björklund | Effects of sphingosine 1-phosphate on calcium signalling, proliferation and S1P 2 receptor expression in PC Cl 3 rat thyroid cells[END_REF].

However, a possible role for S1P in human thyroid cells has not previously been investigated, nor has the importance of S1P in thyroid tumour growth and metastasis been addressed.

We have determined the relative expression level of S1P receptors in one human papillary (NPA), two human follicular (ML-1, WRO) and two human anaplastic (FRO, ARO) thyroid cancer cell lines, as well as in primary cultures of normal human thyroid cells. Furthermore, we have investigated the cellular responses to S1P stimulation (proliferation, adhesion, migration, and calcium signalling) in all of these cell types. The S1P receptor expression pattern differs significantly between the cell types investigated, which is reflected in the responses to S1P stimulation. Most notably, the follicular thyroid tumour cell line ML-1, which was originally isolated from an aggressive tumour [26],

showed high expression of the migration-inducing S1P receptors S1P 1 and S1P 3 . In line with this, nanomolar concentrations of S1P induced migration of ML-1 cells, whereas migration was inhibited by S1P in the other cell lines. Furthermore, the S1P 1/3 antagonist VPC 23019 inhibited migration towards S1P in ML-1 cells. In primary cultures of thyroid cells, S1P evoked a modest calcium response, but did not affect migration or proliferation. Our results implicate that aberrant S1P receptor expression may contribute to the aggressive phenotype of some thyroid tumours.

MATERIALS AND METHODS

Materials

Dulbecco's modified Eagle's medium (DMEM), bovine TSH, porcine insulin, fatty acid-free bovine serum albumin (BSA) and pertussis toxin were purchased from Sigma (St. Louis, MO). Coon's modified Ham's F-12 medium, foetal calf serum, penicillin/streptomycin, trypsin and L-glutamine were from Gibco (Grand Island, NY).

Type 2 collagenase was from Worthington Biochemicals (Lakewood, NJ). Non-essential amino acids and RPMI were obtained from Cambrex Bio Science (Verviers, Belgium). Cell culture ML-1 thyroid follicular cancer cells, kindly provided by Dr Johann Schönberger (University of Regensburg, Germany), were cultured essentially as previously described [26] in DMEM medium supplemented with 2 mM L-glutamine, 10% foetal calf serum (FCS), and 100 U/ml penicillin-streptomycin. NPA papillary and WRO follicular thyroid cancer cells were a kind gift from Dr Sylvia Asa (University Health Network and Toronto Medical Laboratories, Canada). FRO and ARO anaplastic thyroid cancer cell lines were generously provided by Dr James Fagin (University of Cincinnati, Ohio, USA). ARO, FRO, WRO and NPA cells were cultivated in RPMI medium supplemented with 0,1 mM non-essential amino acids, 2 mM L-glutamine, 10% foetal calf serum and 50 U/ml penicillin-streptomycin. Human thyroid follicular cells were prepared from surgical specimens as previously described [START_REF] Eggo | Functional human thyroid cells and their insulin-like growth factor-binding proteins: regulation by thyrotropin, cyclic 3',5' adenosine monophosphate, and growth factors[END_REF]. In brief, thyroid tissue (normal and multinodular goitre) was digested using 0.2% collagenase. Follicles were plated in Coon's F12 modification supplemented with 0.3 U/l TSH, 1 mg/l insulin, 10 5 U/l penicillin, 100 mg/l streptomycin and 1% FCS. After 72 h serum was omitted and the medium was changed every 3 days. All cell cultures were maintained in 37°C and 5% CO 2 in a humified cell culture chamber.

Relative quantitative RT-PCR

RNA was isolated with the Aurum total RNA isolation kit according to the manufacturer's instructions. RNA was quantitated with RiboGreen reagent and RNA quality and integrity was checked by absorbance spectrometry and agarose gel electrophoresis. Reverse transcription reactions were performed with SuperScript III reverse transcriptase and oligo(dT) primers from 0.5 g total RNA as described by the manufacturer.

Quantitative RT-PCR was performed on a LightCycler instrument (Roche) with LightCycler DNA Master Kit reagents, and the following cycling conditions: preincubation 95°C 10 min, amplification 45-50 cycles (95°C 15 sec, annealing T°C 5 sec, 72°C 9 sec). The primers used (0.5 M), and the primer-specific amplification conditions are listed in table 1. Relative mRNA levels were calculated with the LightCycler software. Control analyses, in which reverse transcriptase was omitted from the cDNA synthesis reaction, were performed for each of the RNA preparations to ensure that the preparations were not contaminated with genomic DNA. A melting curve analysis was performed for each run to ensure correct amplification. The data was related to the amount of total RNA [START_REF] Bustin | Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays[END_REF]. For comparison, the data was also analyzed with porphobilinogen deaminase (PBGD) as a reference gene.

Measurement of intracellular free calcium concentrations [Ca 2+ ] i

The cell lines were harvested with calcium-free Hank's buffered saline solution (Ca 2+ -free HBSS; NaCl, 118 mM; KCl, 4.6 mM; glucose, 10 mM; Hepes, 20 mM; pH 7.2) containing 0.02% EDTA and 0.1% trypsin. After washing the cells three times with HBSS (containing 1 mM CaCl 2 ) by pelleting, the cells were incubated with 1 M Fura 2-AM for 30 min at 37°C. After washing twice, cells were kept in darkness for a minimum of 10 min and washed again prior to the experiment. The cell suspension was added to a quartz cuvette, kept at 37°C, and stirred throughout the experiment. A 125 M stock solution of S1P was made in HBSS containing 4 mg/ml fatty acid-free BSA. The cells were stimulated with the indicated concentrations of S1P and Fura 2 emission was measured with a Hitachi 2000 fluorescence spectrophotometer. The excitation wavelengths were 340 and 380 nm, and emission was measured at 510 nm. The signal was calibrated by addition of 0.05% Triton X-10 to lyse the cells and obtain maximal fluorescence. Tris-base was used to elevate pH above 8.3 and extracellular Ca 2+ was chelated with 10 mM EGTA to obtain minimal fluorescence. Intracellular free calcium concentrations ([Ca 2+ ] i ) was calculated as described in [29], using a computer program designed for the fluorimeter and a K d -value of 224 nM for Fura 2.

The primary thyroid cells, cultured on poly-(D)-lysine-coated coverslips, were washed and incubated with 2 M Fura 2-AM as described above, and Ca 2+ signals were measured as described before [START_REF] Xiong | Changes in mitochondrial status associated with altered Ca 2+ homeostasis in aged cerebellar granule neurons in brain slices[END_REF]. Cells were placed on the stage of an upright Copyright 2006 Biochemical Society microscope (Olympus B W5 01), and visuali zed through a 60x water-immersion obj ective.

Images were captured using an intensified GenIV camera (U niversal Imaging, Marlow, U K), and analysed with MetaFluor software (U niversal Imaging Corporation, West Chester, PA). After measuring a baseline for 60 seconds, the cells were perfused with S1P for 60 seconds after which the perfusion was turned off for 30 seconds. Then S1P was washed away with HB SS, and the measurement was continued for five more minutes.

Measurement of intracellular inositol phosphates

Inositol phosphates were measured essentially as described by Oldham [START_REF] Oldham | Polyphosphoinositide turnover. In Receptor-effector coupling. A practical approach[END_REF]. Cells were plated onto 100 mm cell culture plates at a density of 750 000 cells/plate, and cultured as described above for four days. [ 3 H]-myo-inositol (1 Ci/ml) was added, and the cells were allowed to grow for an additional 48 hours. Cells were detached with trypsin solution, and washed with HBSS, after which they were incubated with 100 nM sphingosine 1-phosphate for 30 sec at 37°C. The reaction was terminated by adding an equal volume of ice-cold 10% perchloric acid, and then centrifuged for 5 min at +4°C. The supernatant was transferred to fresh tubes, and neutralized with freon:tri-noctylamine 1:1. The samples were centrifuged briefly, and the upper phase was passed through Amprep™ columns that had been activated with 1M KOH. After washing the columns with distilled water to remove neutral compounds, inositol phosphates were eluted with 5 ml 0.17 M KOH. 1 ml of the effluent was mixed with 4 ml scintillation fluid, and radioactivity was measured using a Wallac 1410 scintillation counter.

Measurement of [ 3 H]-thymidine incorporation and proliferation

The thyroid cancer cell lines were grown in medium containing 10% dextrantreated, charcoal-stripped FCS ( stripped FCS) for two days. Primary cultures of thyroid cells were grown in serum-free medium with or without TSH ( 0.3 U /l) and/or insulin ( 1 mg/l). The cells were stimulated with S1P for 24 h or 48 h, with 0.4-1 Ci/ml [ 3 H]thymidine added for the last 4 hours of the incubation. 10% FCS was used as a positive control for the primary thyroid cultures. At the end of the experiment the cells were washed three times with cold PB S, and the assays were terminated by addition of ice-cold 5% trichloroacetic acid ( TCA) . After 10 min on ice TCA was aspirated, the TCA-insoluble precipitate was dissolved in 0.1 N NaOH, and the radioactivity was measured. The results were confirmed with the CellTiter proliferation assay, as described by the manufacturer, and by cell counting.

Cell adhesion

Cell adhesion experiments were performed as described previously [START_REF] Wang | Involvement of focal adhesion kinase in inhibition of motility of human breast cancer cells by sphingosine 1-phospate[END_REF][START_REF] Paik | Sphingosine 1-phosphate-induced endothelial cell migration requires the expression of EDG-1 and EDG-3 receptors and Rho-dependent activation of v 3-and 1-containing integrins[END_REF][START_REF] Hong | Sphingosine-1-phosphate modulates growth and adhesion of ovarian cancer cells[END_REF] with modifications. The cells were washed with PB S, and changed into serum-free medium containing 0.1% fatty-acid free B SA ( SFM) the day before the experiment. Then the cells were preincubated with S1P for 1 or 24 h. 96-well plates were coated with 5 g/cm 2 collagen IV, collagen I or fibronectin for 1 h, after which the wells were rinsed, and blocked with 3% FAF-B SA for 30 min. Cells in SFM, with or without S1P, were added to each well and placed in 37°C in the cell culture chamber. The cells were allowed to attach for 1 h. The attached cells were fixed with 70% ethanol for 20 min, and stained with 1 mg/ml crystal violet in 20% methanol for 10 min. After washing with water the

Akt phosphorylation assay

The ML-1 cells (10 000 cells/ well) were plated on 9 6-well plates and allowed to grow for 24 hours, after which the medium was changed to SFM.

RESULTS

S1P receptor expression in human thyroid cancer cell lines and primary cultures

To find out which S1P receptors are expressed in thyroid cancer cell lines and thyroid primary cultures, we performed relative q uantification of mRNA for each receptor with RT-PCR. All cell types studied expressed several S1P receptors (Fig. 1).

The results are presented relative to total RNA [START_REF] Bustin | Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays[END_REF], with the detection limit for each gene defined as 1. For comparison, data was also normalized to the expression of the reference gene PB GD , which gave similar results (not shown). S1P 2 and S1P 5 mRNA were expressed in all cells, and S1P 2 was the dominating receptor in FRO, ARO and WRO cells (Fig. 1A). FRO cells expressed S1P 2 at clearly higher levels than the other cell lines.

Primary cultures and ML-1 cells expressed large amounts of S1P 3 compared to the other cell lines (Fig. 1A). In addition, ML-1 cells showed a comparatively high expression of S1P 1 and S1P 5 . Thus, the expression pattern in ML-1 cells was strikingly different compared to the other cell lines. S1P 4 expression was absent or minimal in all cell types (Fig. 1B). Based on these results, and preliminary calcium measurements, proliferation and migration experiments, we chose the ML-1 and FRO cell lines for further studies.

Effects of S1P on intracellular calcium concentration

Extracellular S1P evokes release of intracellular calcium, and a subsequent calcium inflow in many cell types [START_REF] Meyer Zu Heringdorf | Lysophospholipid receptor-dependent andindependent calcium signalling[END_REF]. We show that high concentrations of S1P evoked a small increase in [Ca 2+ ] i in primary thyrocytes (EC 50 =654 nM; Fig. 2A). ML-1 cells responded in a similar manner (EC 50 =986 nM; Fig. 2B), whereas FRO cells were one order of magnitude more sensitive to S1P stimulation (EC 50 =51 nM; Fig. 2C). Moreover, the maximal increase in [Ca 2+ ] i was substantially larger in FRO cells, compared to ML-1 cells and primary cultures. The calcium response in ML-1 cells was not sensitive to pertussis toxin-treatment (data not shown), but 100 nM S1P stimulated inositol phosphate production (mean±SEM 158±23% of control levels; p<0.01). The S1P 1 -agonist SEW-2871 did not evoke a calcium response in ML-1 cells. Furthermore, preincubation of the cells with the S1P 1/3 -specific antagonist VPC 23019 was without an effect on the S1Pevoked calcium response (data not shown). Copyright 2006 Biochemical Society

E ffects of S1P on proliferation

Since S1P regulates the proliferation of many cell types, including cancer cells and rat thyroid cells, we next investigated whether S1P has an effect on the proliferation of normal or malignant thyroid cells. S1P had no effect on [ 3 H]thymidine incorporation in thyroid primary cultures either in the presence or absence of insulin (1 mg/ l) or TSH (0.3 U / l) (Fig. 3A). However, the primary thyrocytes proliferated in response to 10% foetal calf serum, which was used as a control (Fig. 3A).

S1P inhibited [ 3 H]thymidine incorporation in ML-1 cells (Fig. 3B ). The inhibitory effect on proliferation was verified with cell counting (Fig. 3B ). When FRO cells were stimulated with S1P, they reverted to a rounded morphology and were lost during the washing steps of the thymidine incorporation experiment. Therefore, the effect of S1P on FRO-cell proliferation was assessed with the CellTiter assay and cell counting, revealing a modest decrease in FRO viability (Fig. 3B ).

E ffects of S1P on adhesion and migration

S1P may either stimulate or inhibit cellular adhesion and motility, depending on cell type and S1P concentration. We therefore tested the effect of S1P on the adhesion of thyroid cells. A 24-h preincubation with 100 nM or 3 M S1P did not significantly affect cellular adhesion to fibronectin or collagen IV in ML-1 or FRO cells (Fig. 4A). The adhesion properties of ML-1 cells were further tested in experiments using a short time of S1P incubation (1h) and in some experiments, a collagen I matrix. No significant effects of S1P on ML-1 cell adhesion were found in these experiments (data not shown). In contrast, 3 M S1P inhibited FRO cell adhesion to collagen I (Fig. 4B ).

We next tested if S1P affects the migration of thyroid cells. The migration experiments were performed across a collagen IV -coated Transwell-membrane, except for FRO cells, which failed to migrate on Matrigel, collagen IV or fibronectin matrices.

Therefore, collagen I was used as a matrix in experiments with FRO cells. The cells showed no or a very modest basal migration in the absence of a chemoattractant. S1P was without an effect on basal or serum-induced migration of primary cultures (not shown).

In contrast, S1P inhibited the migration of all the cell lines that expressed predominantly S1P 2 (ARO, WRO and FRO; Fig. 5 ). The effect was strongest in FRO cells, where as little as 10 nM S1P completely blocked migration towards FCS (Fig. 5 B ).

In contrast to the other cell lines, nanomolar concentrations of S1P stimulated ML-1 cell migration (Fig. 6). The concentration-response curve was bell shaped, and maximal migration was achieved with 100 nM S1P (Fig. 6A,B ). The migratory effects of 100 nM S1P and 10% FCS were additive (Fig. 6A,D ). In contrast, 3 M S1P inhibited migration towards FCS (Fig. 6A,D ). This c ould not be explained by the S1P-mediated inhibition of proliferation, since incubation times longer than 8 hours were needed to reveal the effect of S1P on cell number (data not shown).

S1P-induced ML-1 cell migration is mediated by S1P 1/ 3 and G i -PI3K

Previous results have shown that migration evoked through S1P 1 and S1P 3 is mediated by G i proteins [START_REF] Taha | Sphingosine-1-phosphate receptors: receptor specificity versus functional redundancy[END_REF]. Therefore we tested the effect of Ptx pre-treatment (100 ng/ ml overnight) on S1P-induced migration. In Ptx-treated cells, the S1P-induced migration was totally abolished (Fig. 6A,C). Furthermore, Ptx reversed the effect of 100 nM S1P, revealing a strong inhibition on FCS-induced migration (Fig. 6A,D ). Ptx was without an effect on the inhibition of migration by 3 M S1P (Fig. 6D ).

To investigate which of the S1P receptors are involved in S1P-induced ML-1 migration, we used the S1P 1 agonist SE W-28 7 1 [START_REF] Sanna | Sphingosine 1-phosphate (S1P) receptor subtypes S1P1 and S1P3, respectively, regulate lymphocyte recirculation and heart rate[END_REF]37 ] and the S1P 1/ 3 -antagonist V PC 23019 [START_REF] Davis | Sphingosine 1-phosphate analogs as receptor antagonists[END_REF]. 1 M SE W-28 7 1 did not evoke migration, whereas a 1h pre-treatment with 1 M V PC 23019 potently inhibited S1P-evoked migration (Fig. 7 A). In the presence of FCS, the S1P-induced migration was reduced but not abolished in V PC 23019 -treated cells (Fig. 7 B ). On the other hand, 3 M S1P had no inhibitory effect on migration in V PC 23019 -treated cells (Fig. 7 B ). Thus, our results suggest that the effects of S1P on ML-1 cell migration are mediated by S1P 1 and/ or S1P 3 , but that activation of S1P 1 alone is not sufficient to induce migration.

Signalling through PI3K-Akt has been shown to mediate S1P-induced migration [START_REF] Takuwa | Subtype-specific differential regulation of Rho family G proteins and cell migration by the Edg family sphingosine-1-phosphate receptors[END_REF][START_REF] Taha | Sphingosine-1-phosphate receptors: receptor specificity versus functional redundancy[END_REF]. In addition, S1P-evoked Akt activation is p38 MAPK-dependent in some model systems [START_REF] Baudhuin | Akt activation induced by lysophosphatidic acid and sphingosine-1-phosphate requires both mitogen-activated protein kinase kinase and p38 mitogen activated protein kinase and is cell-line specific[END_REF]. Treatment of ML-1 cells with the potent PI3K-inhibitor Wortmannin (100 ng/ ml, 1h) inhibited S1P-induced migration, but did not affect S1P-induced inhibition of migration (Fig. 7 C,D ). However, inhibiting p38 MAPK with SB 2305 8 0 (5 M, 1h) did not change the effects of S1P on migration (results not shown).

Next, we studied the S1P-induced phosphorylation of Akt in ML-1 cells with phosphorylation site-specific antibodies. 100 nM S1P, but not 3 M S1P, increased Akt phosphorylation on Ser-47 3 (Fig. 8 ). Consistently with its effects on migration, the S1P 1/ 3 antagonist V PC 23019 inhibited S1P-induced Akt phosphorylation (Fig. 8 A). However, although 1 M SE W-28 7 1 failed to stimulate ML-1 cell migration, it induced Akt Ser-47 3 phosphorylation more potently than S1P itself (Fig. 8 A). In contrast, we were not able to detect any phosphorylation of Akt Thr-308 with S1P or SE W-28 7 1, or in cells cultured in the presence of 10% FCS (results not shown).

S1P was unable to increase Ser-47 3 phosphorylation of Akt in Ptx (100 ng/ ml)or Wortmannin (100 ng/ ml)-pre-treated ML-1 cells (Fig. 8 B ). These two inhibitors reduced the basal Ser-47 3 phosphorylation of Akt as well. Thus, our results strongly suggest that, in ML-1 cells, the Gi-PI3K pathway mediates both S1P-induced migration and S1P-induced Akt phosphorylation, but that Akt phosphorylation on Ser-47 3 alone is not sufficient for the migratory response. The stimulatory effect of S1P on ML-1 cells is due to both chemotaxis and chemokinesis

To test whether S1P-induced ML-1 cell migration is chemotactic or chemokinetic, experiments where 100 nM S1P was added to both the upper and the lower well of the migration chamber (chemokinesis) were compared to experiments where S1P was present only in the lower well (chemotaxis). S1P stimulated migration in both experimental settings, but migration was less prominent in the absence of a concentration gradient (Fig. 10A). When the experiment was repeated in the presence of 10% FCS in the lower well, 100 nM S1P was not able to significantly stimulate migration when added to both compartments of the Transwell chamber (Fig 10B ). On the other hand, the inhibition of FCS-induced migration by 3 M S1P was similar in both experimental settings. These data indicate that S1P has both chemotactic and chemokinetic stimulatory effects on ML-1 cell migration, but that the inhibition of migration is independent of a concentration gradient.

DISCUSSION

In the present investigation we have addressed the role of S1P in thyroid cell proliferation, adhesion, migration, and calcium signalling. We show that human thyroid cells, as well as human thyroid cancer cell lines, express several S1P receptors at the mRNA level. In primary cultures of human thyroid cells, S1P evokes intracellular calcium signalling but neither stimulates proliferation nor affects motility. This is an important finding, indicating that under normal physiological conditions, S1P is not a maj or factor regulating growth and migrati on of thyroid cells. However, our results implicate that S1P may be of importance in thyroid cancer biology. We show that S1P can regulate the proliferation and adhesion of some types of thyroid cancer cells. Most importantly, S1P potently modulates the migration of thyroid cancer cells.

S1P receptor activation regulates proliferation in many cell types [START_REF] Radeff-Huang | G protein mediated signalling pathways in lysophospholipid induced cell proliferation and survival[END_REF]. Since S1P stimulates D NA synthesis in FRTL-5 rat thyroid cells [22], but inhibits D NA synthesis in PC Cl 3 rat thyroid cells [START_REF] Björklund | Effects of sphingosine 1-phosphate on calcium signalling, proliferation and S1P 2 receptor expression in PC Cl 3 rat thyroid cells[END_REF], it was of interest to test the effect of S1P on human thyroid cell proliferation. However, our results show that S1P does not induce proliferation of human thyroid cells in primary culture. In contrast, S1P reduces cell number in some thyroid cancer cell lines, but we did not detect apoptosis (B althasar and Tö rnq uist, unpublished results). Furthermore, S1P modulates cellular adhesion to extracellular matrix proteins in some cell types [START_REF] Hong | Sphingosine-1-phosphate modulates growth and adhesion of ovarian cancer cells[END_REF][START_REF] Van Brocklyn | Sphingosine-1-phosphate stimulates motility and invasiveness of human glioblastoma multiforme cells[END_REF]. We did not observe any significant effects of S1P on the adhesion to fibronectin, collagen I or collagen IV in ML-1 cells. In contrast, the adhesion to collagen I was attenuated in FRO cells preincubated with S1P.

There are many implications of S1P-receptor-mediated regulation of tumour cell invasiveness. S1P affects the migration of many tumour cell lines [10,15 ,16,40,41]. It is well established that S1P 1 and S1P 3 mediate stimulation of migration by coupling to G i , whereas S1P 2 mediates inhibition of migration via G 12/ 13 [START_REF] Takuwa | Subtype-specific differential regulation of Rho family G proteins and cell migration by the Edg family sphingosine-1-phosphate receptors[END_REF][START_REF] Taha | Sphingosine-1-phosphate receptors: receptor specificity versus functional redundancy[END_REF]. The S1P receptor expression pattern, and conseq uently the migratory response to S1P, may change upon cellular transformation [START_REF] Malchinkhuu | Role of p38 mitogen-activated kinase and c-Jun terminal kinase in migration response to lysophosphatidic acid and sphingosine-1-phosphate in glioma cells[END_REF]. Furt hermore, overexpression of S1P 1 enhances in vivo metastasis of B 16F10 mouse melanoma cells, whereas overexpression of S1P 2 inhibits it [START_REF] Yamaguchi | Sphingosine-1-phosphate receptor subtype-specific positive and negative regulation of Rac and haematogenous metastasis of melanoma cells[END_REF]. In our hands, S1P does not affect the migration of normal human thyroid cells, although the cells express mRNA for several S1P receptors. However, at physiological concentrations S1P may either inhibit or activate migration of thyroid tumour cell lines.

In three out of the five cell lines studied (i.e. WRO, FRO and ARO cells), the dominating S1P receptor expressed was S1P 2 . In line with this, nanomolar concentrations of S1P attenuated migration in all of these cell lines. The FRO cells were the most sensitive to S1P, and as little as 10 nM S1P completely inhibited serum-induced migration.

Consistently, the FRO cells expressed the S1P 2 receptor abundantly, compared to the other cell lines.

The migratory response of ML-1 cells was strikingly different from the other cell lines: low nanomolar concentrations of S1P stimulated ML-1 cell migration, whereas micromolar concentrations inhibited it. The ML-1 cell line was originally derived from a follicular thyroid carcinoma that was locally invasive, and had metastasized to the lymph nodes [26]. Given that the active concentration of S1P in human plasma is in the low nanomolar range [START_REF] Murata | Interaction of sphingosine 1-phosphate with plasma components, including lipoproteins, regulates the lipid receptor-mediated actions[END_REF], this suggests that S1P may have contributed to the aggressive and metastasing behaviour of the original tumour. ML-1 cells strongly expressed S1P receptors known to stimulate migration (S1P 1 and S1P 3 ), as well as the inhibitory receptor S1P 2 .

According to previous investigations, S1P-evoked migration is mediated by G idependent activation of PI3K and Akt kinase, resulting in the activation of Rac [START_REF] Takuwa | Subtype-specific differential regulation of Rho family G proteins and cell migration by the Edg family sphingosine-1-phosphate receptors[END_REF][START_REF] Taha | Sphingosine-1-phosphate receptors: receptor specificity versus functional redundancy[END_REF]. This seems to be the case also in ML-1 cells, based on the following observations.

Inhibition of G i with Ptx completely abolished S1P-induced migration. Inhibition of PI3K

with Wortmannin attenuated the migratory effect of S1P. Furthermore, S1P phosphorylated Akt kinase on Ser-473, through a Ptx-and Wortmannin-sensitive mechanism. Akt activity is regulated by phosphorylation on Ser-473 and Thr-308, but the mechanism of these phosphorylation events is poorly understood and controversial [START_REF] Hresko | mTOR-RICTOR is the Ser-473 kinase for Akt/protein kinase B in 3T3-L1 adipocytes[END_REF].

In ML-1 cells, Thr-308 phosphorylation seems not to be needed for migration, since neither FCS nor S1P were able to induce it. The mechanism by which S1P

phosphorylates Akt on Ser-473 in ML-1 cells remains to be investigated. Akt activation by S1P is p38 MAPK-dependent in many cell lines, including ovarian cancer cells [START_REF] Baudhuin | Akt activation induced by lysophosphatidic acid and sphingosine-1-phosphate requires both mitogen-activated protein kinase kinase and p38 mitogen activated protein kinase and is cell-line specific[END_REF],

and p38 MAPK activity is involved in S1P-induced migration of endothelial and smooth muscle cells [START_REF] Fegley | Sphingosine-1phosphate stimulates smooth muscle cell migration through G i-and PI3-kinasedependent P38 MAPK activation[END_REF][START_REF] Liu | Differential regulation of sphingosine-1-phosphate-and VEGF-induced endothelial cell chemotaxis. Involvement of G i 2 -linked Rho kinase activity[END_REF][START_REF] Kimura | Sphingosine 1phosphate stimulates proliferation and migration of human endothelial cells possibly through the lipid receptors, Edg-1 and Edg-3[END_REF]. However, inhibition of p38 MAPK with SB 230580 did not affect S1P-induced ML-1 cell migration. This is similar to the situation in S1P 1 -or S1P 3transfected CHO cells where PI3K, but not p38 MAPK activity, is needed for S1Pinduced migration [START_REF] Okamoto | Inhibitory regulation of Rac activation, membrane ruffling, and cell migration by the G protein-coupled sphingosine-1-phosphate receptor EDG5 but not EDG1 or EDG3[END_REF].

We used the S1P 1 -specific agonist SEW-2871 to mimic signalling through S1P 1 .

SEW-2871 has been reported to induce migration in S1P 1 -transfected CHO cells [START_REF] Sanna | Sphingosine 1-phosphate (S1P) receptor subtypes S1P1 and S1P3, respectively, regulate lymphocyte recirculation and heart rate[END_REF].

However, SEW-2871 did not stimulate migration of ML-1 cells, although it potently phosphorylated Akt. Antagonizing S1P 1 and S1P 3 with VPC 23019 inhibited S1Pinduced migration substantially. These data suggest that S1P 1 and/or S1P 3 are involved in S1P-induced migration in ML-1 cells, but that S1P 1 alone is not able to induce migration.

On the other hand, the S1P-evoked inhibition of migration towards FCS was abolished by VPC 23019, suggesting that S1P 3 may mediate different effects on migration at different agonist concentrations. This might be possible by switching G protein coupling from G i to G 12/13 [START_REF] Sugimoto | Inhibitory and stimulatory regulation of Rac and cell motility by G12/13-Rho and Gi pathways integrated downstream of a single G protein-coupled sphingosine-1-phosphate receptor isoform[END_REF]. Indeed, inhibiting G i with pertussis toxin reversed the effect of 100 nM S1P, resulting in a strong inhibition of ML-1 cell migration. The same effect of Ptx-treatment has been reported in S1P 3 -transfected CHO cells [START_REF] Sugimoto | Inhibitory and stimulatory regulation of Rac and cell motility by G12/13-Rho and Gi pathways integrated downstream of a single G protein-coupled sphingosine-1-phosphate receptor isoform[END_REF]. Whether the inhibitory effect on migration is mediated by S1P 3 , S1P 2 , or both receptors in ML-1 cells remains to be tested.

S1P-receptors mediate inhibition of migration by G 12/13 -evoked stimulation of Rho and inactivation of Rac [START_REF] Taha | Sphingosine-1-phosphate receptors: receptor specificity versus functional redundancy[END_REF][START_REF] Yamaguchi | Sphingosine-1-phosphate receptor subtype-specific positive and negative regulation of Rac and haematogenous metastasis of melanoma cells[END_REF][START_REF] Lepley | The G protein-coupled receptor S1P 2 regulates Rho/Rho kinase pathway to inhibit tumour cell migration[END_REF][START_REF] Okamoto | Inhibitory regulation of Rac activation, membrane ruffling, and cell migration by the G protein-coupled sphingosine-1-phosphate receptor EDG5 but not EDG1 or EDG3[END_REF][START_REF] Sugimoto | Inhibitory and stimulatory regulation of Rac and cell motility by G12/13-Rho and Gi pathways integrated downstream of a single G protein-coupled sphingosine-1-phosphate receptor isoform[END_REF][START_REF] Arikawa | Ligand-dependent inhibition of B16 melanoma cell migration and invasion via endogenous S1P2 G protein-coupled receptor[END_REF]. However, Rho activity is also important for carcinoma cell motility in general [START_REF] Oxford | Ras superfamily monomeric G proteins in carcinoma cell motility[END_REF], as well as S1P-induced endothelial and smooth muscle cell migration [START_REF] Liu | Differential regulation of sphingosine-1-phosphate-and VEGF-induced endothelial cell chemotaxis. Involvement of G i 2 -linked Rho kinase activity[END_REF][START_REF] Galaria | Differential regulation of ERK1/2 and p38 MAPK by components of the Rho signalling pathway during sphingosine-1-phosphate-induced smooth muscle cell migration[END_REF]. The downstream effects of Rho are partly mediated by Rho kinases ( ROCKI/II) , whose effect on S1P-modulated migration is controversial. Inhibition of ROCK abolishes S1P-induced inhibition of migration in glioblastoma cells [START_REF] Lepley | The G protein-coupled receptor S1P 2 regulates Rho/Rho kinase pathway to inhibit tumour cell migration[END_REF], but has no effect in S1P 2 -transfected CHO cells or B 16 melanoma cells [START_REF] Sugimoto | Inhibitory and stimulatory regulation of Rac and cell motility by G12/13-Rho and Gi pathways integrated downstream of a single G protein-coupled sphingosine-1-phosphate receptor isoform[END_REF][START_REF] Arikawa | Ligand-dependent inhibition of B16 melanoma cell migration and invasion via endogenous S1P2 G protein-coupled receptor[END_REF]. On the other hand, inhibiting ROCK blocks S1P-induced migration in endothelial cells [START_REF] Liu | Differential regulation of sphingosine-1-phosphate-and VEGF-induced endothelial cell chemotaxis. Involvement of G i 2 -linked Rho kinase activity[END_REF]. In ML-1 cells, inhibition of Rho or the Rho kinases ROCKI/II further enhanced the stimulatory effect of 100 nM S1P. This suggests that S1P

activates Rho/ROCK-dependent inhibitory signalling pathways simultaneously with the stimulatory G i-PI3K pathway, and that the balance between these signals determines the final migratory response. The ROCKI/II inhibitor Y -27632 clearly had a stronger effect on S1P-induced migration than the Rho inhibitor C3 exotoxin. In the presence of C3 exotoxin the ML-1 cells had a rounded morphology, indicating that the cells responded to C3 exotoxin. It is, however, possible that the inhibition of Rho was incomplete.

Inhibition of ROCK increased the migration towards serum per se, and S1P could not further enhance the migratory response. A possible explanation for this could be that in the presence of the ROCK inhibitor and serum, maximal migration is already achieved, and a further migration in response to S1P is not possible. Surprisingly, inhibition of ROCKI/II did not reverse the inhibitory effect of 3 M S1P on ML-1 cell migration. This implicates that the inhibition of migration by high concentrations of S1P is mediated via a mechanism not dependent on ROCK.

S1P-induced activation of Rac requires calcium mobilization in endothelial cells [START_REF] Mehta | Sphingosine 1-phosphate-induced mobilization of intracellular Ca2+ mediates Rac activation and adherens junction assembly in endothelial cells[END_REF] and calcium is involved in the regulation of motility [START_REF] Huang | Identification of channels promoting calcium spikes and waves in HT1080 tumour cells: their apparent roles in cell motility and invasion[END_REF]. All known S1P receptors can mediate increases in [Ca 2+ ] i [START_REF] Ishii | Lysophospholipid receptors: signalling and biology[END_REF]. Furthermore, overexpression of S1P 1 mediates inhibition of calcium signals evoked by S1P or other substances [START_REF] Meyer Zu Heringdorf | Lysophospholipid receptor-dependent andindependent calcium signalling[END_REF]. In thyroid cells in primary culture, and in ML-1 cells, the S1P-evoked increase in [Ca 2+ ] i was similar in sensitivity and magnitude to that observed in rat thyroid cell lines [22,[START_REF] Björklund | Effects of sphingosine 1-phosphate on calcium signalling, proliferation and S1P 2 receptor expression in PC Cl 3 rat thyroid cells[END_REF]. FRO cells were much more sensitive, both regarding the concentration of the ligand, and the amplitude of the response. Similar to what has been observed in rat thyroid cells [START_REF] Okajima | Sphingosine-1-phosphate stimulates hydrogen peroxide generation through activation of phospholipase C-Ca2+ system in FRTL-5 thyroid cells: possible involvement of guanosine triphosphate-binding proteins in the lipid signalling[END_REF][START_REF] Björklund | Effects of sphingosine 1-phosphate on calcium signalling, proliferation and S1P 2 receptor expression in PC Cl 3 rat thyroid cells[END_REF],

S1P stimulated inositol phosphate production in both ML-1 cells and FRO cells [START_REF] Afrasiabi | Sphingosylphosphorylcholine enhances calcium entry in thyroid FRO cells by a mechanism dependent on protein kinase C[END_REF],

indicating that the PLC-IP 3 pathway is involved. Neither pertussis toxin nor VPC 23019 affected S1P-evoked calcium signalling in ML-1 cells. Moreover, SEW-2871 failed to evoke an increase in [Ca 2+ ] i , although it has been shown to evoke calcium responses in S1P 1 -transfected CHO cells [START_REF] Sanna | Sphingosine 1-phosphate (S1P) receptor subtypes S1P1 and S1P3, respectively, regulate lymphocyte recirculation and heart rate[END_REF]. These data, together with the high expression of S1P 2 in FRO cells, implicate that S1P 2 receptor expression is central in regulating calcium signalling in thyroid cancer cells. Whether or not the S1P-evoked calcium signal is involved in the regulation of ML-1 or FRO cell migration remains to be investigated.

Tumour growth and metastasis is dependent on adhesion, migration, invasion, angiogenesis, and proliferation. S1P has been shown in several investigations to affect all of these steps, and is therefore a candidate molecule for regulating metastatic behaviour.

Our investigation is the first to show that S1P may be of importance in the metastatic behaviour of aggressive thyroid tumours. As the five receptors mediating S1P responses have partly different signalling mechanisms, the receptor combination and level of expression is important for the effect of S1P. Our data indicate that S1P receptors, which mediate migratory responses may be up regulated in some thyroid tumours, and that S1P is important for the aggressive phenotype of these tumours. The co-expression of several S1P receptors differentially regulating migration in a tumour opens possibilities for therapeutic intervention. The primers for S1P 2-5 were designed with the EMBOSS ePrimer3. The primer sequences for S1P 1 [START_REF] Davaille | Antiproliferative properties of sphingosine 1-phosphate in human hepatic myofibroblasts[END_REF] and PBGD [START_REF] Westerman | Quantitative reverse transcriptionpolymerase chain reaction measurement of HASH1 (ASCL1), a marker for small cell lung carcinomas with neuroendocrine features[END_REF] are from the literature. s = sense strand, as = antisense strand.

Biochemical Journal Immediate Publication. Published on 6 Jun 2006 as manuscript BJ20060299

Copyright 2006 Biochemical Society

  Cell culture plastic ware as well as human type I and type IV collagen and fibronectin were purchased from Becton Dickinson Biosciences (Bedford, MA). [methyl-3 H]thymidine (82 Ci/mmol) was from Amersham Biosciences, Amersham, UK) and CellTiter 96® AQueous One Solution cell proliferation assay from Promega Corp (Madison, WI). Transwell™ inserts for migration assays and 96-well plates for adhesion experiments were from Corning Inc (Corning, NY). Sphingosine 1-phosphate and SEW-2871 were from Biomol (Plymouth, PA). VPC 23019 was obtained from Avanti Polar Lipids (Alabaster, AL), SB20350, Wortmannin and Y-27632 from Calbiochem (San Diego, CA), and C. botulinum C3 exotoxin from List Biological Laboratories Inc (Campbell, CA). The ProJect transfection reagent, and the BCA protein assay reagent kit were purchased from Pierce Biotechnology (Rockford, IL). The FACE™ AKT ELISA kit was from Active Motif (Carlsbad, CA). The Akt and phospho-Thr-308-specific Akt antibodies used in Western blotting, as well as the positive and negative protein controls for phosphorylated Akt, were purchased from Cell Signaling Technology (Danvers, MA). The HRP-conjugated goat anti-rabbit antibody, and the Aurum™ total RNA isolation kit were obtained from BioRad laboratories (Hercules, CA). Nitrocellulose membranes were from Schleicher & Schuell (Dassel, Germany). The RiboGreen® RNA quantification reagent, and Fura 2-AM were purchased from Molecular Probes (Eugene, OR). SuperScript III reverse transcriptase was from Invitrogen (Carlsbad, CA), and oligo(dT) primers from Promega Corp (Madison, WI). The LightCycler FastStart DNA Master SYBR Green kit was obtained from Roche Diagnostics (Mannheim, Germany). PCR primers were synthesized by TAGC Copenhagen (Denmark). All chemicals for RNA isolation and RT-PCR were of molecular biology grade, and all other chemicals of reagent grade.

Biochemical Journal Immediate Publication. Published on 6

 6 Jun 2006 as manuscript BJ20060299Copyright 2006 Biochemical Society wells were allowed to dry. The crystal violet was dissolved in 10% acetic acid, performed on 6.5 mm-diameter Transwell chambers with 8 M pore size. The membranes were coated with 5 g/ cm 2 collagen IV , and reconstituted with SFM for 1h at 37°C in the cell culture incubator prior to the experiment. FRO cells failed to migrate on a collagen IV matrix, and therefore collagen I (5 g/ cm 2 ) was used. The cell lines were changed into SFM the day before the experiment. In some experiments the cells were preincubated with one of the following inhibitors: 100 ng/ ml pertussis toxin (Ptx) overnight; 5 M SB 2035 8 0 for 1 hour; 10 M Y -27 632 for 1 hour; 100 ng/ ml Wortmannin for 1 hour; 1 M V PC 23019 for 1 hour; 100 ng/ ml C3 exotoxin in the presence of ProJ ect protein transfection reagent for 4 hours. 200 l cell suspension was added to the upper wells, and 8 00 l SFM with or without S1P and/ or stripped FCS to the lower wells. In e xperiments with inhibitors, the inhibitor was present in both chambers during the experiment. With ML-1 cells, additional experiments where S1P was added to both the upper and the lower wells were performed to test whether the S1P-induced migration was due to chemotaxis or chemokinesis. The cells were allowed to migrate in the incubator for 4-8 h towards S1P+ stripped FCS or 20 h towards S1P alone, after which the unmigrated cells from the top side of the membrane were wiped off with a cotton swab. The migrated cells were fixed in 2% paraformaldehyde (PFA) in PB S for 10 min, and stained with 0.1% crystal violet in 20% methanol for 5 min. The membranes were rinsed and allowed to dry. The cells were Biochemical Journal Immediate Publication. Published on 6 Jun 2006 as manuscript BJ20060299 Copyright 2006 Biochemical Society counted with 40x magnification in eight microscopic fields in a straight line bisecting the membrane.

  The role of the Rho-ROCK pathway in S1P-induced migration of ML-1 cells Previous investigations have indicated that the S1P 2 receptor suppresses S1Pinduced migration by signalling through Rho and ROCK[START_REF] Lepley | The G protein-coupled receptor S1P 2 regulates Rho/Rho kinase pathway to inhibit tumour cell migration[END_REF]. To investigate whether Rho and ROCK are involved in S1P-induced migration in ML-1 cells, the cells were treated with the Rho inhibitor C3 exotoxin (100 ng/ ml, 4h) or the ROCKI/ II inhibitor Y -27 632 (10 M, 1h). B oth inhibition of ROCKI/ II and Rho increased S1P-induced migration in the absence of serum, although the effect of C3 exotoxin was modest compared to that of Y -27 632 (Fig.9A,B ). Moreover, in Y -27 632-treated cells, 3 M S1P had a modest migratory effect (Fig.9 B).Likewise, C3 exotoxin increased S1P-induced migration towards FCS (Fig.9 C). In contrast, Y -27 632 did not abrogate the inhibitory effect of 3 M S1P on FCS-induced migration (Fig.9 D). Migration towards FCS was significantly increased in Y -27 632-treated cells , and could not be further enhanced by S1P (Fig.9 D). The results suggest that S1P and FCS regulate ML-1 cell migration by activating stimulatory and suppressor pathways simultaneously, and that the suppression of migration involves Rho and ROCK signalling. However, high concentrations of S1P may suppress migration by activating some other pathway.

Fig. 1 .

 1 Fig. 1. S1P receptor expression in thyroid cancer cell lines and primary cultures.

Fig. 2 .Fig. 3 . 3 U

 233 Fig. 2. S1P evokes intracellular calcium signals in thyroid cells. Calcium concentration response curves and representative traces of changes in [Ca 2+ ] i

Fig. 4 .

 4 Fig. 4. S1P modulates the adhesion of FRO cells, but not ML-1 cells.

Fig. 5 .

 5 Fig. 5. S1P inhibits migration of thyroid cancer cells.

Fig. 6 .

 6 Fig. 6. S1P regulates ML-1 cell migration.

Fig. 7 .

 7 Fig. 7. S1P-evoked ML-1 cell migration is inhibited by the S1P 1/3 antagonist VPC 23019

Fig. 8 . 1 M

 81 Fig. 8. S1P induces Akt phosphorylation on Ser-473 in ML-1 cells.

Fig. 9 .

 9 Fig. 9. S1P-evoked ML-1 cell migration is regulated by Rho and ROCK.

Fig. 10 .

 10 Fig. 10. S1P has both chemotactic and chemokinetic effects on ML-1 cell migration.

Biochemical Journal Immediate Publication. Published on 6 Jun 2006 as manuscript BJ20060299

  The following day the cells were stimulated with S1P for 10 min. In some experiments, the cells were treated

	and 15 l of commercial control extracts were separated by SDS-PAGE on 10%
	polyacrylamide gels. The proteins were transferred electrophoretically to nitrocellulose
	membranes. The membranes were blocked with 5% non-fat dry milk in Tris-buffered
	saline (NaCl 150 mM; Tris-base 20 mM; pH 7.5) with 0.1% Tween 20 for 1 h. The blots
	were then incubated with phospho-Thr-308-specific Akt antibody (1:1000) diluted in
	TBS containing 5% non-fat dry milk and 0.1% Tween 20 overnight at +4°C, washed, and
	incubated with peroxide-conjugated rabbit antibodies (1:3000) for 1h at room
	NaCl, 150 mM; EDTA, 7 mM; NP-40, 0.5%; PMSF, 0.2 mM; pH 7.7) with a cell
	scraper. Protein concentration was determined with a BCA protein assay reagent kit
	following the manufacturer's instructions. The cell extracts were mixed with 0.5 volumes
	of 3xSDS-buffer (glycerol, 30%; SDS, 3%; Tris-HCl, 0.1875 M, pH 6.8; bromophenol
	blue, 0.015%; -mercaptoethanol, 3%) and boiled for 2 min. 10-50 g of protein samples

with Ptx (100 ng/ ml, o/ n) or Wortmannin (100 ng/ ml for 1 h) before and during stimulation. The cells were fixed with 4% PFA in PB S 20 min. The FACE -Akt E LISA was performed with an antibody recognizing Ser-47 3-phosphorylated or total Akt according to the instructions provided by the manufacturer, and the absorbance was read at 45 0 nm.

Western blotting

ML-1 cells were cultured in SFM for one day and stimulated with 100 nM S1P for 10, 30 or 60 minutes. In some experiments, the cells where stimulated with 1 M SEW-2871 for 10 minutes. At the end of the stimulation, the cells were briefly rinsed with ice-cold HBSS and extracted in 200 l ice-cold lysis buffer (Tris-base, 10 mM; temperature. The proteins were incubated in ECL solution (luminol, 1.25 mM; pcoumaric acid, 0.68 mM; H 2 O 2 , 0.01%) for 1 min, and the membranes were exposed to Hyperfilm™ (Amersham Biosciences, Buckinghamshire, UK) for 1-5 min. The membranes were stripped with 0.1 M glycin (pH 2.5), rinsed with 1M Tris-HCl (pH 7.6), and reprobed with total Akt antibody (1:1000) as described above.

Biochemical Journal Immediate Publication. Published on 6 Jun 2006 as manuscript BJ20060299

Table 1 .

 1 PCR primers and cycling conditions for quantification of S1P-receptors in

		thyroid cancer cells.
	mRNA	primer sequences	Product length	[MgCl 2 ], mM	Annealing T°C	Product melting T°C
	S1P 1	s GGCTGGAACTGCATCAGTGCG as GAGCAGCGCCACATTCTCAGAGC	223	4	60	89-90
	S1P 2	s CCGAAACAGCAAGTTCCACT as CCAGGAGGCTGAAGACAGAG	197	2	61	90
	S1P 3	s AAGGCTCAGTGGTTCATCGT as GCTATTGTTGCTGCTGCTTG	201	2	61	92-93
	S1P 4	s CCTTCAGCCTGCTCTTCACT as AAGAGGATGTAGCGCTTGGA	223	4	64	94
	S1P 5	s AGGACTTCGCTTTTGCTCTG as TCTAGAATCCACGGGGTCTG	201	3	59	87
	PBGD	s TCCAAGCGGAGCCATGTCTG as AGAATCTTGTCCCCTGTGGTGGA	204	3	62	89

Biochemical Journal Immediate Publication. Published on 6 Jun 2006 as manuscript BJ20060299 Copyright 2006 Biochemical Society

Acknowledges

This work was financially supported by the K Albin Johansson foundation, Svenska Kulturfonden, the Magnus Ehrnrooth foundation, Stiftelsens för Åbo Akademi forskningsinstitut, the Liv och Hälsa Foundation, and the Centre of Excellence in Cell Stress (Åbo Akademi University).

Figure 1 A.