Báo cáo Y học: Neurotrophic signalling pathway triggered by prosaposin in PC12 cells occurs through lipid rafts doc

Preferential association of prosaposin and LRP-1 with lipid raft fractions To analyse the presence of prosaposin and the related receptor in lipid raft fractions of PC12 cells, we invest

in PC12 cells occurs through lipid rafts

Maurizio Sorice1,2, Sabrina Molinari1, Luisa Di Marzio3, Vincenzo Mattei1,2, Vincenzo Tasciotti1,2, Laura Ciarlo1, Masao Hiraiwa4, Tina Garofalo1,2and Roberta Misasi1

1 Dipartimento di Medicina Sperimentale, ‘Sapienza’ University, Rome, Italy

2 Laboratorio di Medicina Sperimentale e Patologia Ambientale, ‘Sapienza’ University, Rieti, Italy

3 Dipartimento di Scienze del Farmaco, Universita` G D’Annunzio, Chieti Scalo, Italy

4 Department of Neurosciences, University of California, San Diego, CA, USA

Prosaposin is a neurotrophic factor that has been

dem-onstrated to mediate trophic signalling events in

differ-ent cell types through its active region within the

saposin C domain [1,2] Prosaposin is also secreted into

various body fluids [3], and mRNA and protein levels

increase following peripheral nerve injury Exogenous

prosaposin promotes axonal sprouting in neural cells

and myelin lipid synthesis, and prolongs cell survival in

both Schwann cells and oligodendrocytes [4], suggesting

that secreted prosaposin may have neurotrophic and

neuroprotective roles Moreover, prosaposin treatment

induced pheochromocytoma cells (PC12) to enter the

S-phase of the cell cycle [5] and monocytic U937 cell

death prevention, reducing both necrosis and apoptosis

[6] This effect was achieved through rapid extracellular signal-regulated kinase (ERK) phosphorylation, sphin-gosine kinase (SphK) activation, with intracellular sphingosine 1-phosphate (S1P) production, and phos-phatidylinositol 3-kinase–Akt pathway involvement Thus, prosaposin appears to be a regulatory factor in the ceramide–S1P rheostat, which regulates cell fate, not only in cells of neurological origin [6]

Prosaposin triggers the signal cascade after binding

to a putative Go-coupled cell surface receptor [7] and⁄ or to the low-density lipoprotein (LDL) receptor-related protein (LRP-1) [8]

The presence of lipid-binding domains in prosaposin and saposins has been well demonstrated [9], and a

Keywords

lipid domains; PC12 cells; prosaposin; rafts;

sphingosine kinase

Correspondence

R Misasi, Department of Experimental

Medicine, ‘Sapienza’ University, Viale

Regina Elena 324, Rome 00161, Italy

Fax: +39 (6) 4454820

Tel: +39 (6) 49970663

E-mail: roberta.misasi@uniroma1.it

(Received 6 May 2008, revised 25 July

2008, accepted 5 August 2008)

doi:10.1111/j.1742-4658.2008.06630.x

Prosaposin is a neurotrophic factor that has been demonstrated to mediate trophic signalling events in different cell types; it distributes to surface membranes of neural cells and also exists as a secreted protein in different body fluids Prosaposin was demonstrated to form tightly bound complexes with a variety of gangliosides, and a functional role has been suggested for ganglioside–prosaposin complexes In this work, we provide evidence that exogenous prosaposin triggers a signal cascade after binding to its target molecules on lipid rafts of pheochromocytoma PC12 cell plasma mem-branes, as revealed by scanning confocal microscopy and linear sucrose gradient analysis In these cells, prosaposin is able to induce extracellular signal-regulated kinase phosphorylation, sphingosine kinase activation, and consequent cell death prevention, acting through lipid rafts These findings point to the role of lipid rafts in the prosaposin-triggered signalling path-way, thus supporting a role for this factor as a new component of the multimolecular signalling complex involved in the neurotrophic response

Abbreviations

CTxB, cholera toxin B subunit; D-PDMP, D -threo-1-phenyl-2-decanoylamin-3-morpholino-propanol; ERK, extracellular signal-regulated kinase; FITC, fluorescein isothiocyanate; HRP, horseradish peroxidase; HS, horse serum; LDL, low-density lipoprotein; LRP-1, low-density lipoprotein receptor-related protein; MbCD, methyl-b-cyclodextrin; PI, prodium iodide; p-Ser, phosphoserine; PT, pertussis toxin; S1P, sphingosine 1-phosphate; SphK, sphingosine kinase.

role has been suggested for ganglioside–prosaposin

complexes as structural components of membrane

sig-nalling lipid domains [10] In the last few years, lipid

rafts have been characterized in different cell types as

small and highly dynamic structures, envisaged as

lat-eral assemblies of specific lipids and proteins in cellular

membranes, proposed to function in processes such as

membrane transport, signal transduction, and cell

adhesion [11,12] They are enriched in certain lipids

(sphingolipids, including gangliosides, sphingomyelin

and cholesterol) that show the property of being

insol-uble in common detergents, such as Triton X-100 A

variety of specific proteins implicated in signal

trans-duction from plasma membrane to cytoplasm has also

been detected within these domains; some proteins are

costitutively enriched within lipid rafts and act through

them, and some others are recruited upon specific cell

stimulation [12,13]

In this work, we provide evidence that exogenous

prosaposin is able to induce ERK phosphorylation

and SphK activation, acting through lipid rafts,

proba-bly binding different target molecules on the cell

plasma membrane

These findings indicate that the proliferative and

antiapoptotic functions of prosaposin are dependent

on its association with lipid rafts

Results

Prosaposin association with lipid rafts in

PC12 cells

In order to reveal the possible association of

prosapo-sin with lipid rafts, we performed immunofluorescence

labelling, followed by scanning confocal microscopy

analysis Cells were labelled with antibody against

prosaposin (anti-769) and then with cholera toxin B

subunit (CTxB), which stains ganglioside GM1 as a

specific raft marker [14]

The analysis of prosaposin staining revealed an

uneven distribution over the cell surface (Fig 1), which

was also the case for GM1 fluorescence However, the

ganglioside distribution on the cell surface appeared to

be highly heterogeneous The merged image of the two

stainings clearly revealed yellow areas, which

corre-sponded to colocalization areas between prosaposin

and GM1 (Fig 1A) After triggering with prosaposin,

colocalization areas appeared as clusters, indicating

raft aggregation in patches upon cell stimulation

As it has been reported that prosaposin triggers a

signal cascade after binding to a putative Ga0-coupled

cell surface receptor [7], we performed a preliminary

analysis of the distribution of Ga0 heterotrimeric

protein, which might reflect a putative prosaposin receptor, and its association with GM1 In Fig 1B, the merged image of the staining clearly shows the pres-ence of colocalization areas, indicating, as expected, that Ga0 proteins are associated with lipid rafts After triggering with prosaposin, the labelled molecules, Ga0 protein and GM1, do not seem to modify their distri-bution, maintaining an evident colocalization pattern

in the merged picture

As prosaposin has been demonstrated to be a ligand for LRP-1 [8], we also decided to evaluate the possible association of this receptor with lipid rafts Scanning confocal microscopy showed a green fluorescence, cor-responding to LRP-1, unevenly distributed over the cell membrane, similar to the appearance of the red fluorescence corresponding to the raft marker GM1 [14] The merged image showed two distinct distribu-tion patterns, which revealed the absence of colocaliza-tion areas (Fig 1C, upper) When the cells were preincubated with prosaposin, a number of yellow-stained colocalization areas became evident (Fig 1C, lower), indicating recruitment of LRP-1 molecules to lipid rafts

The morphometric analyses aimed at evaluation of the percentages of cells showing prosaposin–GM1,

Ga0–GM1 and LRP-1–GM1 colocalization (i.e dis-playing yellow staining) are reported in Fig 1D

Preferential association of prosaposin and LRP-1 with lipid raft fractions

To analyse the presence of prosaposin and the related receptor in lipid raft fractions of PC12 cells, we investi-gated the distribution of these proteins in fractions obtained by a 5–40% linear sucrose gradient, in either the absence or the presence of triggering with prosa-posin for 5 min (Fig 2) or 10 min (data not shown) Western blot results revealed that, in untreated cells, prosaposin was enriched mainly in fractions 5 and 6 After treatment, an increase of prosaposin content in these fractions was observed (Fig 2A) A distribution

in the same fractions was observed for Ga0 protein in untreated as well as in prosaposin-treated cells (Fig 2B) When PC12 cell fractions were analysed for LRP-1 distribution (Fig 2C), a band of 85 kDa, rec-ognized by a monoclonal antibody against LRP-1, was present in Triton X-100-soluble fractions (10 and 11); but, when cells were incubated with prosaposin, LRP-1 clearly switched to Triton X-100-insoluble fractions (4–6) As a control, we also analysed the distribution

of calnexin in both treated and untreated cells As expected, it was found in the fractions corresponding

to heavy membranes (Fig 2D) In contrast, GM1 was

present in the raft fractions, independently of

pros-aposin treatment, as revealed by TLC immunostaining,

using a monoclonal antibody against GM1 (Fig 2E)

Prosaposin induces ERK and SphK activation

through lipid rafts

We previously demonstrated that prosaposin induced

ERK phosphorylation and SphK activation in PC12

cells [5] To investigate the contribution of lipid rafts

to the prosaposin effect, we analysed ERK and SphK

activation following prosaposin stimulation, in either

the presence or the absence of pretreatment with

methyl-b-cyclodextrin (MbCD) or filipin III, as these

treatments induce cholesterol efflux from the plasma

membrane and, consequently, lipid raft disruption [15],

or the ceramide analogue

d-threo-1-phenyl-2-decanoyl-amin-3-morpholino-propanol (D-PDMP), which

inhib-its glucosylceramide synthase and thus leads to extensive depletion of endogenous glycosphingolipids [16] In Fig 3A, a western blot analysis shows that pretreatment with MbCD or filipin III, as well as D-PDMP, partially prevented the prosaposin-induced ERK activation In parallel experiments, ERK phos-phorylation by prosaposin was partially prevented by previous incubation of the cells with a natural ligand

of LRP-1, LDL, as well as with pertussis toxin (PT), which catalyses ADP-ribosylation of several G-proteins [17] (Fig 3B), indicating that LRP-1 receptor, as well

as the Ga0-coupled prosaposin receptor, are involved

in ERK phosphorylation As a loading control for immunoblotting experiments, an antibody against b-actin was used

Moreover, basal SphK activity was improved by prosaposin, and this effect was strongly inhibited (about 65%) by pretreatment with MbCD (Fig 4A)

Merge GM1

Prosaposin

Merge GM1

G α0

Prosaposin/GM1

Contr ol

+pr osaposin Contr

ol

+pr osaposin Contr

ol

+pr osaposin

70 60 50

40 30 20 10 0

Fig 1 Scanning confocal microscopic analysis of prosaposin, G a0 or LRP-1 association with the raft marker GM1 on the PC12 cell surface Cells were analysed in the absence or in the presence of prosaposin incubation (10 n M for 5 min at 37 C), and then labelled with antibody against prosaposin (anti-769), antibody against Ga0 or antibody against LRP-1, followed by FITC-conjugated anti-rabbit or anti-mouse IgG Then, cells were stained with Texas-red-conjugated CTxB (as GM1 staining) One representative cell is shown (A) Prosaposin–GM1 associa-tion in untreated (control) and treated (+ prosaposin) cells (B) Ga0–GM1 association in untreated (control) and treated (+ prosaposin) cells (C) LRP-1–GM1 association in untreated (control) and treated (+ prosaposin) cells (D) Morphometric analysis of data obtained by confocal microscopy Two hundred cells were counted for each sample Histograms indicate the percentage of cells with prosaposin–GM1, G a0 –GM1 and LRP-1–GM1 colocalization.

In order to confirm these findings, using a highly

specific antibody against SphK1, untreated and

prosa-posin-stimulated cells were immunoprecipitated with

an antibody against SphK1 and analysed by western

blot with an antibody against phosphoserine (p-Ser)

(Fig 4B) This revealed that pretreatment with MbCD

or filipin III almost completely abolished the prosapo-sin-induced SphK1 activation, indicating that the pro-saposin-triggered signalling pathway occurs through lipid rafts Moreover, the results also suggested that the ERK pathway may act upstream of SphK activa-tion, as the MEK inhibitor PD98059 prevented pro-saposin-induced SphK1 activation

Protective effect of prosaposin against cell apoptosis occurs through lipid rafts

To verify whether the protective effect of prosaposin against cell apoptosis involved lipid rafts, cells were incubated with staurosporine, in either the presence or the absence of prosaposin, with or without pretreat-ment with 5 mm MbCD, and stained with Hoe-chst 33258 (Fig 5A) The nuclei of control (a) as well

as prosaposin-stimulated (c) PC12 cells were stained uniformly with this dye, whereas treatment with MbCD alone showed only a negligible effect on cell apoptosis (e) As expected, treatment of cells with staurosporine caused nuclear condensation and fragmentation (b), whereas in cells treated with prosaposin plus stauro-sporine, a decrease in the number of apoptotic cells was observed (d) When cells were pretreated with MbCD and incubated with prosaposin and stauro-sporine (f), the protective effect of prosaposin against apoptosis was partially inhibited, indicating that the signalling pathways involved in cell death prevention triggered by prosaposin occur through lipid rafts

In addition, DNA fragmentation was quantified as a hypodiploid peak by cytofluorimetric analysis (Fig 5B) As reported previously [5], a much larger hypodiploid peak was observed after 24 h of stauro-sporine incubation (37.4 ± 4%), as compared to untreated cells (4.9 ± 0.8%); preincubation of cells with prosaposin provided protection against stauro-sporine-induced apoptosis, as indicated by the decrease

in hypodiploid cell number (14.8 ± 2%) This effect was significantly inhibited (P < 0.001) when cells were pretreated with MbCD and then incubated with prosaposin and staurosporine (35.3 ± 2.8%)

Moreover, we evaluated the early stages of programmed cell death by analysing fluorescein isothio-cyanate (FITC)-conjugated annexin V⁄ propidium iodide (PI) staining of treated and untreated cells by flow cytometry (Fig 5C) Once again, it was confirmed that preincubation with MbCD partially prevented the protective effect of prosaposin against apoptosis It is

of note that the analysis of MbCD-treated cells revealed annexin V binding to less than 20% of cells Thus, pretreatment with MbCD, under our experi-mental conditions, has to be considered as a ‘mild’

A

B

C

D

E

Fig 2 Subcellular distribution of prosaposin, G a0 and LRP-1 in

PC12 sucrose gradient membrane fractions Cells, untreated or

treated with 10 n M prosaposin, were lysed in lysis buffer, and the

supernatant (postnuclear fraction) was subjected to sucrose density

gradient separation After centrifugation, the gradient was

fraction-ated, and each fraction was analysed by western blotting with

anti-body against prosaposin (A), Ga0 (B), LRP-1 (C) or calnexin (D).

Alternatively, gangliosides were extracted from each fraction and

separated by HPTLC, using silica gel 60 HPTLC plates and

chloro-form ⁄ methanol ⁄ 0.25% aqueous KCl (5 : 4 : 1 v ⁄ v ⁄ v) as eluent

sys-tem The plates were immunostained with monoclonal antibody

against GM1 (GMB16) (E).

treatment, without severe changes in membrane

orga-nization or signs of distress in the cells, according to

Ottico et al [18]

Discussion

In this work, we provide evidence that exogenous

pro-saposin binds its target molecule on lipid rafts of the

cell plasma membrane and induces ERK

phosphoryla-tion and SphK activaphosphoryla-tion through these microdomains

These findings indicate that the trophic and

antiapop-totic signalling pathways triggered by prosaposin occur

through lipid rafts

The presence of prosaposin within lipid rafts is

sup-ported by two independent experimental approaches,

i.e scanning confocal microscopy and sucrose gradient

in the presence of Triton X-100, taking advantage of

the insolubility of these microdomains in the detergent

Our findings revealed significant colocalization areas

between prosaposin and ganglioside GM1, which,

although it represents a minor ganglioside component

in these cells [19], is a well-known marker of lipid rafts

[14] This prosaposin distribution pattern was

confirmed by western blot analysis of sucrose gradient

Triton X-100 fractions, which revealed that prosaposin

was enriched in Triton X-100-insoluble fractions After

treatment, an increase in prosaposin content in raft

fractions was observed These experiments indicate

that prosaposin is associated with lipid rafts, where it

may bind different molecules, including a Ga0-coupled

receptor and LRP-1 As already reported [20],

hetero-trimeric G-proteins have also been detected in lipid

rafts; thus, the analysis of Ga0-protein distribution on PC12 cells, as expected, clearly showed the presence of colocalization areas with raft marker molecules, indi-cating stable Ga0-protein localization within lipid rafts The same result was confirmed by western blot analy-sis of sucrose gradient Triton X-100 fractions, conanaly-sis- consis-tent with the view that the putative prosaposin receptor is Ga0-coupled [7] Interestingly, our findings show recruitment of LRP-1 molecules to lipid rafts only after prosaposin triggering This finding is in agreement with the observation that LRP-1 associates transiently with lipid rafts and that its distribution into membrane microdomains is cell type specific [21] These multifunctional lipoprotein receptors are estab-lished cargo transporters, but their expression at the cell surface and agonistic binding of diverse biological ligands are now thought to potentially evoke signalling pathways involved in cell fate determination [22] In addition, our data suggest that the LRP-1 receptor, as well as the Ga0-coupled receptor, may play a role in the prosaposin-triggered signal pathway leading to ERK phosphorylation Indeed, in other cell systems, activation of the MEKK–JNK–cJun signalling cascade and of the Mek1–ERK1⁄ 2 pathway by LRP-1 have been reported [23]

However, the main finding of this study is the demonstration that prosaposin induces ERK phos-phorylation and SphK activation through lipid rafts

In previous studies, we demonstrated that prosaposin treatment induces PC12 entry in the S-phase of the cell cycle and prevents apoptosis by activation of ERKs and SphK in PC12 cells [5], as well as in

A

B

Fig 3 Effect of MbCD, filipin III and

D-PDMP on ERK phosphorylation induced

by prosaposin (A) PC12 cells, untreated or

treated with 10 n M prosaposin, were lysed

in lysis buffer and analysed by western blot

with monoclonal antibody against

phospho-p44⁄ p42 A representative example of three

experiments is shown (B) PC12 cells,

prein-cubated with LDL (natural ligand of LRP-1)

(10 lgÆmL)1per 106cells for 30 min at

4 C) or with PT (inhibitor of G a0 -coupled

receptor function) (100 ngÆmL)1for 30 min

at 37 C), were treated with 10 n M

prosa-posin, as above, lysed in lysis buffer, and

analysed by western blot with monoclonal

antibody against phospho-p44 ⁄ p42 A

repre-sentative example of three experiments is

shown Approximately equal protein loading

of the gel was verified using an antibody

against b-actin.

different cell types [6] Here, we confirm and extend

these findings, demonstrating the involvement of lipid

rafts Indeed, pretreatment with MbCD or with

fili-pin III, which are able to induce cholesterol efflux

from the membrane and, consequently, raft

disrup-tion, almost completely prevented ERK and SphK

activation, with a prosaposin antiapoptotic effect At

the same time, according to Ottico et al [18], MbCD

pretreatment under our experimental conditions does

not induce severe changes in membrane organization

or signs of distress in the cells, although the lipid

domains of plasma membranes lost the ability to sort

specific signalling proteins The finding that

pretreat-ment of cells with D-PDMP also inhibited

prosapo-sin-induced ERK phosphorylation suggests two

different considerations: (a) this finding may

consti-tute functional confirmation of the well-known ability

of prosaposin to bind gangliosides [9]; and (b) as gangliosides are well-known components of lipid rafts [14], this result strongly supports the view that pro-saposin-triggered signal transduction occurs through lipid rafts Moreover, we provide evidence that pro-saposin also induces SphK activation through lipid rafts Most cells express both SphK1 and SphK2; our findings suggest that in our system prosaposin may act through SphK1, as detected by a highly spe-cific antibody The involvement of SphK-1 activation

in our system is in agreement with the notion that the activation of SphK-1 by external stimuli, includ-ing growth factors, results in accumulation of intra-cellular S1P, and, consequently, increased cell proliferation and suppression of apoptosis [24] Interestingly, our findings suggest that the ERK pathway is upstream of SphK activation, as the MEK

A

B

Fig 4 Effect of MbCD on prosaposin-induced SphK activation (A) Cells were preincubated with 5 m M MbCD and stimu-lated with 10 n M prosaposin After cell lysis, cytosolic fractions were prepared and SphK activity was measured in the supernatant by incubating 50 l M sphingosine-b-octylgluco-side and [ 32 P]ATP[cP] The modulation of SphK activity by MbCD is evaluated as pmo-les of N-caproyl-S1P Error bars represent standard deviation (B) Effect of MbCD and filipin III on SphK phosphorylation induced

by prosaposin Cells, untreated or treated with 10 n M prosaposin, were lysed in lysis buffer and immunoprecipitated with a rabbit polyclonal antibody against SphK1 (M-209)

or, as a control, with rabbit IgG with irrele-vant specificity The immunoprecipitates were analysed by western blot with mono-clonal antibody against p-Ser The antibody against p-Ser was stripped, and the mem-brane was then reprobed with goat poly-clonal antibody against SphK1 (M-13) A representative example of three experi-ments is shown.

inhibitor PD98059 was able to inhibit the

prosaposin-induced SphK1 activation However, we cannot

exclude the possibility that additional transduction

pathway(s) triggered by prosaposin may be responsible

for SphK activation

Taken together, these findings point to a role of

lipid rafts in the prosaposin-triggered signalling

path-way, thus supporting a role for this factor as a new

component of the multimolecular signalling complex

involved in the neurotrophic response

Further studies are in progress in order to purify and sequence the putative prosaposin receptor(s)

Experimental procedures Materials and cells

Milk prosaposin was prepared as previously reported [25]

An antibody against the active 22-mer prosaposin peptide (anti-769) [1] and a monoclonal antibody against

prosapo-a b

c d

e

A

Control

+prosaposin

+M βCD +MβCD + prosaposin + STS

+prosaposin + STS +STS

B

C

f

Fig 5 Effect of MbCD on the antiapoptotic activity of prosaposin (A) PC12 cells, incubated with or without 5 m M MbCD, in the presence

or absence of 10 n M prosaposin, were treated with 1 l M staurosporine (STS) and stained with Hoechst 33258 Nuclei of control cells were stained uniformly with Hoechst (a), as well as those of cells treated with prosaposin alone (c) Treatment of cells with staurosporine caused nuclear fragmentation and condensation (b) Pretreatment of cells with prosaposin prevented apoptosis (d) This effect was partially inhibited

by preincubation with MbCD (f) Treatment with MbCD alone did not show any effect on cell apoptosis (e) A representative example of three independent experiments (B) Flow cytometry analysis of hypodiploid cells Histograms represent the percentage of hypodiploid peaks,

as detected by PI staining Mean ± standard deviation of five independent experiments STS versus prosaposin + STS, P < 0.001; MbCD + prosaposin + STS versus prosaposin + STS, P < 0.001 (C) Flow cytometry analysis of early stages of cell apoptosis by annexin V ⁄ PI staining Histograms represent the percentage of annexin V-positive cells Mean ± standard deviation of five independent experiments STS versus prosaposin + STS, P < 0.001; MbCD + prosaposin + STS versus prosaposin + STS, P < 0.001.

sin [26] were employed A rabbit polyclonal antibody

against SphK1 (M-209) and a goat polyclonal antibody

against SphK1 (M-13) (Santa Cruz Biotechnology Inc.,

Santa Cruz, CA, USA) were employed for

immunoprecipi-tation and detection of SphK1 in western blots

The rat pheochromocytoma cell line PC12 was cultured

as previously described [5] Experiments investigating the

signalling pathway triggered by prosaposin were performed

with or without pretreatment with LDL (Sigma Chemical

Co., St Louis, MO, USA, or purified from human plasma

as described in [6]), as a natural ligand of LRP-1, or PT

(recombinant holotoxin; Calbiochem, San Diego, CA,

USA), which binds the Ga0-coupled molecules In

experi-ments aimed at investigating the signalling pathway

trig-gered by prosaposin through lipid rafts, cells were

pretreated with MbCD (5 mm for 30 min at 37C),

fili-pin III (10 lm for 30 min at 37C) or D-PDMP (30 lm for

5 days at 37C) (all purchased from Sigma) Optimal

con-centrations and incubation times were carefully checked on

the basis of morphological and viability tests In particular,

a Trypan blue exclusion test revealed incorporation into

less than 20% of cells pretreated with MbCD or filipin III

The effect of D-PDMP on glycosphingolipid depletion and

cholesterol distribution was checked as previously described

[27] (data not shown)

Immunofluorescence staining

PC12 cells were cultured onto coverslips and maintained in

DMEM, containing 2% horse serum, for 24 h before

pro-saposin treatment (10 nm for 5 min at 37C) and labelling

Cells were rinsed, and fixed in 4% formaldehyde in

NaCl⁄ Pifor 30 min at 4C After washes, cells were

incu-bated for 1 h with rabbit polyclonal antibody against Ga0

(anti-769) (Santa Cruz Biotechnology) or mouse

mono-clonal antibody against LRP⁄ a2MR (Immunological

Sciences), and this was followed by addition for 45 min of

Texas red-conjugated antibody against rabbit or mouse

IgG (Calbiochem), and addition of FITC-conjugated CTxB

(Molecular Probes, Invitrogen Corps, Carlsbad, CA, USA)

Cells were analysed, and images were collected as

previ-ously described [28]

Morphometric analyses in double labelling experiments

were carried out by evaluating at least 200 cells for each

sample, at the same magnification (·630)

Isolation and analysis of lipid raft fractions

Lipid raft fractions from PC12 cells, untreated or treated

with prosaposin (10 nm for 5 or 10 min at 37C), were

iso-lated as previously described [29], and the fractions were

sub-jected to western blot analysis Samples were normalized

for cell number Blots were probed with monoclonal

anti-body against prosaposin, monoclonal antianti-body against

LRP⁄ a2MR, antibody against Ga0, or polyclonal antibody

against calnexin (Sigma) overnight at 4C All the antibody dilutions for blotting and intermediate washes were performed with NaCl⁄ Tris, containing 0.05% Tween-20 Antibody binding was detected with ECL Tm peroxidase antibody against rabbit or mouse IgG (Amersham Bio-sciences, Amersham, UK), and immunoreactivity assessed

by chemiluminescence Alternatively, gangliosides were extracted from each fraction according to Svennerholm & Fredman [30] and separated by HPTLC, using silica gel 60 HPTLC plates (Merck, Darmstadt, Germany) Chromato-graphy was performed in chloroform⁄ methanol ⁄ 0.25% aqueous KCl (5 : 4 : 1 v⁄ v ⁄ v) The plates were immuno-stained for 1 h at room temperature with GMB16 monoclo-nal antibody against GM1 (Seikagaku Corp, Chuo-ku, Tokyo, Japan) and then with horseradish peroxidase (HRP)-conjugated anti-mouse IgM (Sigma) Immunoreactivity was assessed by chemiluminescence

Analysis of ERK and SphK activation

PC12 cells (6.0· 107

), washed in serum-free medium, were incubated with prosaposin (10 nm for 5 min at 37C) or,

as a positive control, with 4a-phorbol 12-myristate 13-ace-tate (Sigma) (50 ngÆmL)1for 2 min at 37C) (not shown),

in DMEM medium containing 2% horse serum In parallel experiments, cells were preincubated with MbCD (Sigma) (5 mm for 30 min at 37C), with filipin III (Sigma) (10 lm for 30 min at 37C), with D-PDMP (Sigma) (30 lm for

5 days at 37C), with LDL (10 lgÆmL)1 per 106 cells for

30 min at 4C) or with PT (100 ngÆmL)1, for 30 min at

37C) in the same medium, in either the presence or the absence of prosaposin Cells were washed twice with ice-cold NaCl⁄ Piand analysed for ERK and SphK, as previ-ously described [6] A loading control was performed using

a monoclonal antibody against b-actin (mouse IgG1, clone

(Sigma) Alternatively, cell-free lysates from unstimulated PC12 or PC12 cells stimulated with prosaposin, in the presence or absence of 50 lm MEK inhibitor PD98059, filipin III or MbCD, were immunoprecipitated with a rab-bit polyclonal antibody against SphK1 (M-209) (Santa Cruz) In brief, cells were lysed in lysis buffer, including

Na3VO4, phenylmethanesulfonyl fluoride and protease inhibitors To preclear nonspecific binding, cell-free lysates were mixed with protein G–Sepharose beads (Bio-Rad, Hercules, CA, USA) and stirred in a rotary shaker for 1 h

at 4C After centrifugation (1000 g for 6 min), the super-natant was immunoprecipitated with antibody against SphK1 The immunoprecipitates were subjected to 10% SDS⁄ PAGE The proteins were electrophoretically trans-ferred to a nitrocellulose membrane (Bio-Rad), and then, after blocking with NaCl⁄ Tris containing Tween-20 and 5% milk, probed with monoclonal antibody against p-Ser

respectively Bound antibodies were visualized with

HRP-conjugated anti-mouse IgG (Sigma Aldrich), and

immunoreactivity was assessed by the chemiluminescence

(Amersham) To confirm the positive band as SphK1, the

antibody against p-Ser was stripped from the nitrocellulose,

and the membrane was then reprobed with goat polyclonal

antibody against SphK1 (M-13) (Santa Cruz) as a loading

and reactivity control

Evaluation of cell death

Subconfluent PC12 cells, incubated in either the presence or

the absence of 10 nm prosaposin for 30 min, were treated

with 1 lm staurosporine (Sigma) for 24 h at 37C In

par-allel experiments, cells were preincubated with 5 mm

MbCD for 30 min at 37C

Apoptosis was measured by both morphological analysis

and flow cytometry Morphological analysis of the nuclei

was performed by staining the cells with Hoechst 33258

(Sigma), 5 lgÆmL)1, in 30% glycerol⁄ NaCl ⁄ Pi for 20 min

Cells were examined in an inverted fluorescence microscope

(320 nm UV excitation) Viable cells were identified by their

intact nuclei, and fragmented or condensed nuclei were

scored as apoptotic DNA fragmentation consistent with

apoptosis was quantified as a hypodiploid peak by PI

stain-ing and cytofluorimetric analysis, as previously described

[31] Alternatively, apoptosis was also quantified by flow

cytometry after double staining using FITC-conjugated

annexin V⁄ PI apoptosis detection kit (Eppendorf, Milan,

Italy), which allows discrimination between early apoptotic,

late apoptotic and necrotic cells In this case, in order to

detect the early stages of apoptosis, cells incubated in either

the presence or the absence of 10 nm prosaposin for 30 min

were treated with 1 lm staurosporine (Sigma) for 3 h at

37C Again, in parallel experiments, cells were preincubated

with 5 mm MbCD for 30 min at 37C

Acknowledgements

This work was supported by a grant to RM from

Uni-versity ‘Sapienza’ School of Medicine, Rome, Italy

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