Ligands of Macrophage Scavenger Receptor Induce Cytokine … · include protein tyrosine kinases...

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Ligands of Macrophage Scavenger Receptor Induce Cytokine Expression

via Differential Modulation of Protein Kinase Signaling Pathways

Running title: MSR ligand-mediated MAPK in regulation of proIL-1/IL-1

Hsien-Yeh Hsu1, Show-Lan Chiu2, Meng-Hsuan Wen1, Kuo-Yen Chen1 and Kuo-Feng Hua1

Faculty of Medical Technology, Institute of Biotechnology in Medicine,National Yang-Ming University, 112, Taipei, TAIWAN1 and

National Laboratory of Foods and Drugs, National Health Administration, 115, Taipei, TAIWAN2

Address reprint requests and correspondence to:

Dr. Hsien-Yeh HsuFaculty of Medical TechnologyInstitute of Biotechnology in MedicineNational Yang-Ming University155 Li-Nong Street, Shih-PaiTaipei, TAIWAN

Tel. number: 011-886-2-2826-7252FAX number: 011-886-2-2826-4092Email: [email protected]

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on June 4, 2001 as Manuscript M011117200 by guest on A

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1The abbreviations used are: MSR, macrophage scavenger receptor-type A; LDL, low density

lipoprotein; OxLDL, oxidized LDL; IL-1, interleukin 1β; proIL-1, prointerleukin 1β; TNF, tumor

necrosis factor-α ; GA, geldanamycin; HB, herbimycin A; Hsp90, heat-shock protein 90; PTK,

protein tyrosine kinase; NRPTK, non-receptor protein tyrosine kinase; MAPK, mitogen activated

protein kinase; ERK, extracellular signal-regulated kinase; PAK, p21 activated kinase; JNK, c-JUN

NH2-terminal protein kinase; p38, p38 mitogen activated protein kinase; PLC-γ1, phospholipase C-

γ 1; PI 3-kinase, phosphatidylinositol-3-OH kinase; ICE (caspase 1), interleukin 1 converting

enzyme.

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ABSTRACT

Our previous works demonstrated that ligands of macrophage scavenger receptor (MSR)

induce protein kinases (PK) including protein tyrosine kinase (PTK) and upregulate uPA

expression (Hsu, H. Y., Hajjar, D. P., Khan, K. M., and Falcone, D. J. (1998) J. Biol. Chem. 273,

1240-1246). To continue to investigate MSR ligand-mediated signal transductions, we focus on

ligands, OxLDL and fucoidan induction of cytokines TNF and IL-1. In brief, in murine

macrophages J774A.1, OxLDL and fucoidan upregulate TNF production; additionally, fucoidan

but not OxLDL induces IL-1 secretion, prointerleukin 1 (proIL-1, precursor of IL-1) protein and

proIL-1 message. Simultaneously, fucoidan stimulates activity of interleukin 1 converting

enzyme. We further investigate molecular mechanism by which ligand binding-induced PK-

mediated mitogen activated protein kinase (MAPK) in regulation of expression of proIL-1 and IL-1.

Specifically, fucoidan stimulates activity of p21 activated kinase (PAK) and of MAPKs: ERK, JNK

and p38. Combined with PK inhibitors and genetic mutants of Rac1 and JNK in PK activity

assays, Western blotting analyses and IL-1 ELISA, the role of individual PK in regulation of proIL-

1/IL-1 was extensively dissected. Moreover, tyrosine phosphorylation of pp60Src as well as

association between pp60Src and Hsp90 play important roles in fucoidan-induced proIL-1

expression. We are the first to establish fucoidan-mediated two signaling pathways:

PTK(Src)/Rac1/PAK/JNK and PTK(Src)/Rac1/PAK/p38, but not PTK/PLC-γ1/PKC/MEK1/ERK

playing critical roles in proIL-1/IL-1 regulation. Our current results indicate and suggest a model

for MSR ligands differentially modulate specific PK signal transduction pathways, which regulate

atherogenesis-related inflammatory cytokines TNF and IL-1.


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Overexpression of macrophage scavenger receptors (MSR) on activated macrophages and

macrophage-derived foam cells in atherosclerotic lesions has been reported (2-5). Scavenger

receptor mediates the high affinity binding and internalization of modified lipoproteins including

OxLDL (6-8), implying an important role in lipid-laden foam cell formation during atherosclerosis

development and progression. In addition to OxLDL, a diverse group of polyanionic compounds

has been listed as ligands for MSR (9;10). The broad nature of ligand specificity in MSR may

explain receptor-multifaceted functions of macrophages such as adhesion (11;12), clearance of

pathologic substances (13;14), host defenses (9;15) and cytokine production (this paper and (9;16)).

However, the molecular mechanisms for MSR on macrophages possess the capacity of cytokine

induction remain not clear and need further investigation.

The activated macrophages not only transform into foam cells via uptaking OxLDL, but

also aberrantly produce a battery of inflammatory cytokines, which playing pivotal roles in the

process of atherogenesis. It has demonstrated that interleukin-1β (IL-1) and tumor necrosis

factor-α (TNF) (2;17-19) are the major inflammatory cytokines(2;18;19), altering the function of

macrophages in ways that appear to promote atherosclerosis (20). Recent studies show that

cytokines secreted from activated macrophages could be initiated or potentiated by one of MSR

ligands, OxLDL (21), thus dysregulated expression of MSR likely promoting the development of

localized inflammatory reactions. Indeed, the macrophage-derived inflammatory cytokines, IL-1

and TNF cause adjacent endothelial cells (EC) expression of procoagulant activity, changing EC

surface from antithrombotic to thrombotic.

On the other hand, fucoidan, the principal polysaccharide sulfate ester occurring in the

various species of Phaeophyceae (brown seaweed), though it is a non-lipoprotein recognized as an

MSR ligand and effectively competes with OxLDL in MSR ligand-binding studies (9;10). Up-

regulation of MSR expression in human THP-1 monocytic macrophages upon phorbol 12-myristate

13-acetate priming has been reported (16;22;23). Binding of fucoidan to MSR stimulates

urokinase-type plasminogen activator (uPA) secretion in human THP-1 macrophages (1), in

activated peritoneal murine macrophages, and in murine macrophage cell lines (J774A.1 and

RAW264.7) (24;25). The expression of uPA via MSR ligand-mediated signaling molecules

include protein tyrosine kinases (PTK), phospholipase C- γ1 (PLC-γ1), phosphatidylinositol-3-OH

kinase (PI 3-kinase) and protein kinase C, indicating a molecular model for regulation of uPA

expression by the specific ligand of MSR (1). Furthermore, herbimycin A inhibits fucoidan-

induced tyrosine phosphorylation of PLC-γ1 (1) suggesting that an unidentified PTK is involved in

fucoidan-induced signalings. In this paper, we demonstrate again that geldanamycin (GA) or


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herbimycin A (HB) inhibits fucoidan-induced various protein tyrosine phosphorylation and IL-1

expression. As it known that GA or HB inhibits the activation of certain tyrosine kinases;

however, their cellular target is heat-shock protein 90 (Hsp90) (26), which expression is induced by

cell stress. Obviously, the MSR-induced cytokines, IL-1 and TNF could lead to cellular stress

including inflammation. Both GA and HB, benzoquinone ansamycin antibiotics inhibit various

signal transduction proteins including non-receptor protein tyrosine kinase (NRPTK), pp60Src.

Pharmacologically, HB or GA binds in a specific manner to Hsp90 and inhibits pp60Src-Hsp90

heterocomplex formation (26-28). Although ligands of MSR such as fucoidan and polyinosinic

acid induce IL-1 production in human THP-1 macrophages (16), it lacks systematically to examine

MSR ligand-regulated signaling cascade for IL-1 production in macrophages. Hence, we propose

and investigate pp60Src and Hsp90, which may play certain roles in fucoidan-mediated effects such

as association between pp60Src and Hsp90 to form a complex in the regulation of fucoidan-induced

proIL-1 protein expression

In studies reported here, we observe both OxLDL and fucoidan up-regulate TNF

production; additionally, fucoidan but not OxLDL induces prointerleukin 1β (proIL-1) protein and

IL-1 secretion in murine macrophages J774A.1. We demonstrate for the first time that fucoidan

differentially stimulates signaling machinery including the NRPTK, pp60Src, p21-activated kinase

(PAK) and mitogen-activated protein kinases (i. e., ERK, JNK, and p38). Recent studies indicate

that IL-1 secretion is post-transcriptionally regulated via IL-1 converting enzyme (ICE, caspase 1)

(29-32). In here, we find fucoidan-mediated specific signalings regulate IL-1 secretion,

simultaneously, fucoidan induces ICE activity during alternation of proIL-1/IL-1 expression.

Moreover, combining with pharmacological inhibitors and genetic mutants in protein kinase assays,

we investigate the molecular mechanism for MSR ligand-mediated signal transduction pathways in

the regulation of proIL-1/IL-1 expression. Specifically, we further dissect and confirm the role of

several key-signaling molecules in induction of proIL-1, and establish two signaling cascades, i.e.,

pathway of PTK(Src)/Rac1/PAK/JNK and pathway of PTK(Src)/Rac1/PAK/p38 in regulation of

proIL-1/IL-1 expression.


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EXPERIMENTAL PROCEDURES

Cell cultures--- Murine macrophage J774A.1 cells (J774A.1 cells) were obtained from ATCC

(Rockville, MD), propagated in RPMI 1640 medium supplemented with 10% heated-inactivated

fetal bovine serum and 2 mM L-glutamine (Life Technologies, Inc., MD) and cultured in 37°C, 5%

CO2 incubator. Using Histopaque -1077 method, human blood monocyte-derived macrophages

were isolated from blood of healthy persons obtained from Taiwan Blood Center (Taipei, Taiwan).

Materials---Histopaque -1077, fucoidan, sodium orthovanadate, phenylmethylsulfonyl

fluoride (PMSF), bovine serum albumin (fraction V), geldanamycin and curcumin were purchased

from Sigma Co. (St. Louis, MO). LipofectAMINE PLUS reagent were purchased from Life

Technologies, Inc. (Gaithersburg, MD). Immobilon PVDF membrane was purchased from

Millipore Inc. (Bedford, MA). DuPont non-radioactive Western Blot Chemiluminescence

Reagent, Renaissance , γ-32P-ATP (10 Ci/mmol) were purchased from DuPont NEN Research

Product Co. (Boston, MA). REZOl C&T was from PROtech Technology Co. (Taipei, Taiwan).

GeneAmp RNA PCR kit for RT-PCR amplification was purchased from Perkin Elmer Inc.

(Branchburg, New Jersey). Growth factors and antibodies: Anti-IL-1β, 3ZD monoclonal

antibody (a gift from NIH, Bethesda, MD, USA to Dr. H.-Y. Hsu), anti-PAK, rabbit polyclonal IgG,

anti-rabbit IgG-HRP, anti-mouse IgG-HRP and protein A/G plus-agarose were obtained from Santa

Cruz Biotechnology (Santa Cruz, CA); anti-phosphotyrosine, clone 4G10 (mouse monoclonal

IgG2bκ) was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY), monoclonal anti-

Hsp90 and monoclonal anti-Src (Oncogene Inc. product) were obtained from Calbiochem-

Novabiochem Corp. (La Jolla, CA). The CaspACE Assay System, Fluorometric was purchased

from Promega Co., WI. Kinase assay kits: p44/42 MAP Kinase Assay Kit, SAPK/JNK Assay Kit

and p38 MAP Kinase Assay Kit were purchased from Cell Signaling Technology (Beverly, MA).

Protein kinase inhibitors: PD98059 was from Cell Signaling Technology, PP1 was from Biomol

(Plymouth meeting, PA), calphostin C, and herbimycin A were from Calbiochem-Novabiochem

Corp., wortmannin, LY294002 and SB203580 were from Sigma Co. Oligonucleotides: primers

for TNF, proIL-1/IL-1, glyceraldehyde phosphate dehydrogenase (GAPDH) were synthesized from

local MD Bio. Inc. (Taipei, Taiwan). The dominant negative JNK construct (DN-JNK) was a gift

from Dr. Michael Karin (UCSD, San Diego, CA, USA) (33). The dominant negative Rac1

construct (DN-Rac1) and constitutive activated-Rac1 construct (CA-Rac1) were a gift from Dr. S.

Bagrodia (Cornell University, Ithaca, NY, USA) (34).


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Isolation of LDL for Preparation of OxLDL, RNA Isolation, RT and PCR Amplification for

detecting the expression of TNF or proIL-1/IL-1, Protein Assay (determined by Bio-Rad protein

assay), Western Blotting Analysis, and Enzyme-Linked Immunosorbent Assay for measurement of

TNF and IL-1---All detail methods and procedures were followed the previous methods (22;35).

Analysis of Interleukin 1 Converting Enzyme (ICE, caspase 1) Activity---To analyze the ICE

activity in J774A.1 cells basically was followed the protocols from The CaspACE Assay System,

Fluorometric, Promega Co. Briefly, the cells were incubated with fucoidan (25 µg/ml) or OxLDL

(5 µg/ml). At indicated time, the cells were washed twice with ice-cold PBS (without Ca+2,

Mg+2), harvested in 300 µl cell suspension buffer (25 mM pH 7.5 HEPES, 5 mM MgCl2, 5 mM

EDTA, 5 mM DTT, 2 mM PMSF, 10 µg/ml pepstatin A, 10 µg/ml leupeptin, from Promega Co.).

The suspended cells were stored in 1.5 ml centrifugation tubes, frozen and thawed three times then

centrifuged 12,000 xg at 4℃ for 15 min, discarded the pellets. The protein concentration of

supernatant in cell extract was determined, and the rest supernatant was saved at –80 ℃ freezer

for future use. For assaying ICE activity, 32 µl ICE-like enzyme assay buffer, 2 µl DMSO and 10

µl 100 mM DTT were added to 75 µg of protein of cell extract from tested samples at various times,

and then adjusted with water to 98 µl. For each tested sample, a negative control (the same as

above tested sample with additional 2 µl 2.5 mM ICE inhibitor (Ac-YVAD-CHO)) and a blank

control (32 µl ICE-like enzyme assay buffer, 10 µl 100 mM DTT, 2 µl DMSO and 54 µl water).

All the tested samples were incubated at 30 ℃ for 30 min, after incubation, 2 µl 2.5 mM ICE

substrate (Ac-YVAD-AMC) was added to each sample, additional 60 min incubation at 30 ℃,

then the intensity of fluorescence of tested sample was measured at excitation wavelength 360 nm,

emission wavelength 460 nm. The calculation of the relative fluorescence units (△FU) and the

caspase activity for each sample, as well as construction of standard curve and AMC calibration

curves were as described in the Technical Bulletin from Promega Co.

Assay Activity of Extracellular Signal-Regulated Kinase (ERK), of c-Jun NH2-terminal Kinase

(JNK), of p38 Mitogen Activated Protein Kinase (p38) and of p21 Activated Protein Kinase (PAK)

in Fucoidan-treated J774A.1 Cells with or without PK Inhibitors---Methods for assay activity of

these PKs including cell lysate preparation, immunoprecipitation of PK, in vitro PK reaction,

analysis of PK activity and quantification of PK activity were as described previously (35).

Transfection of JNK Dominant Negative Construct (DN-JNK), Rac1 Dominant Negative

Construct (DN-Rac1) and Constitutive Activated-Rac1 Construct (CA-Rac1) into J774A.1 Cells


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and Assay of JNK and p38 Activity as well as Assay of ProIL-1 Protein Expression in the

Transfected Cells upon Fucoidan Stimulation---Methods for transfection and MAPK activity assays

were as described (35). Briefly, the conditions for growing to be transfected J774A.1 cells were

identical to those of regular J774A.1 cells, except cells were sub-passaged the day before

transfection, and replaced with fresh medium. The transient transfection of DN-JNK, DN-Rac1,

CA-Rac1 or control (Control or CTR, i. e., the empty expression vector) at 10 µg DNA per 100 mm

plate into cells were conducted by using the LipofectAMINE PLUS ® reagent (Life Technologies,

Inc., USA) method as previous methods (35). The efficiency of both transfection and expression

were monitored by the HA-tag expression. DN-Rac1 transfected cells were stimulated with

fucoidan for 120 min and 240 min, respectively, then to assay the activity of JNK and p38 as

described above. DN-JNK transfected cells and DN-Rac1 transfected cells were harvested after

24 h, 48 h and 72 h (each sample treated with fucoidan for 8 h), as well as CA-Rac1 stably

transfected cells were harvested after fucoidan treatment for 4 h, 12 h or 24 h, respectively.

ProIL-1 protein expression in cells was detected by Western blotting analysis.

Statistical Analysis---Statistical differences between the experimental groups were examined

by analysis of variance, and statistical significance was determined at p < 0.05. The experiments

were conducted three times or as indicated, all data are expressed as mean + S.E. by guest on April 10, 2019

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RESULTS

Ligands of macrophage scavenger receptor (MSR), lipoprotein (i. e., OxLDL) and

nonlipoprotein (i. e., fucoidan) upregulate inflammatory cytokine, TNF expression in J774A.1 cells

The effect of OxLDL and fucoidan on expression of TNF protein expression by J774A.1

cells was initially determined. Cells were grown at various times in media with native LDL (5

µg/ml), OxLDL (5 µg/ml), fucoidan (25 µg/ml) or without any supplement (i. e., as control), the

released TNF in conditioned media was measured by enzyme-linked immunosorbent assay

(ELISA). Specifically, between 4h and 12h, OxLDL-treated cells produced about 600 pg/ml of

TNF, or about 100% more TNF than those of LDL-treated or of control cells (the background of

TNF is about 300 pg/ml); after 24h, the released TNF in OxLDL-treated cells returned to basal

level (Fig. 1 A). In contrast, cells treated with fucoidan for 4 h began to increase TNF releasing,

and enormous TNF produced by stimulated-cells to about 15,000 pg/ml and 30,000 pg/ml at 8 h

and 24 h, respectively (Fig. 1 B). We next determined whether TNF secretion in conditioned

medium was reflected by increasing TNF message stimulated by OxLDL and fucoidan in J774A.1

cells. Using RT-PCR method, we detected the alternation of TNF message between 2 h and 24 h,

and found that no significant increase of TNF mRNA was observed in either fucoidan-treated cells

(Fig. 1C) or OxLDL-treated cells (data not shown).

Fucoidan upregulates inflammatory cytokine, IL-1, prointerleukin-1β (proIL-1, IL-1

precursor) and proIL-1 message as well as stimulates activity of interleukin 1 converting enzyme

(ICE or caspase 1) in J774A.1 cells

To detect the effect of MSR ligands on another important inflammatory cytokine, IL-1

protein secretion, we used ELISA to quantitate mature IL-1 secretion in the conditioned medium of

J774A.1 cells. As shown in Fig. 2 A, approximate 10 pg/ml of IL-1 protein was detected in

conditioned medium in 8-h fucoidan-treated cells as compared with that of control cells or of native

LDL-incubated cells (both below detectable level). The IL-1 concentration increased as

prolonged fucoidan incubation time, at 48 h, the accumulated concentration of IL-1 in fucoidan-

treated cells was up to 50 pg/ml. In contrast, OxLDL barely stimulated IL-1 secretion in the cells

for all testing periods (Fig. 2A). Results indicate that in J774A.1 cells, ligation of different MSR

ligands leads to differentially induce IL-1 protein secretion.

To further investigate the molecular mechanism by which the secretion of mature IL-1

(MW 17-18 kD), initially, fucoidan-induced IL-1 precursor, proIL-1 (MW 34 kD) was detected by


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Western blotting analysis. As shown in Fig. 2 B, that fucoidan-induced proIL-1 protein was

detected between 2 h and 4 h, peaked at 8 h. After 12 h, proIL-1 protein decreased, and it

gradually returned to basal level around 24 h. There was no detectable proIL-1 protein in

OxLDL-treated cells for all periods (data not shown), and consistent with no IL-1 secretion in

OxLDL-treated cells. Moreover, using RT-PCR methods, we demonstrated that 2-h fucoidan

incubation, but not OxLDL induced proIL-1 mRNA expression as compared with native LDL-

treated cells or with control cells. Fucoidan-induced proIL-1 message peaked between 6 h and 8 h

(Fig. 2 C); at 12 h, it remained higher than that of control cells. After 24 h, induced proIL-1

message returned to basal level. The similar upregulation of proIL-1 mRNA detected by Northern

analyses is compatible to the message detection by RT-PCR methods (data not shown).

Post-transcriptional regulation and processing of proIL-1 protein into mature IL-1

secretion via ICE has been reported (31;32). Since incubation of cells with fucoidan induced

proIL-1 protein and simultaneously led to IL-1 secretion in a time fashion, we examined whether

fucoidan stimulates ICE activity during IL-1 secretion. As shown in Fig. 2 D, ICE activity

increased to 2 folds as compared to control cells after 6-h fucoidan stimulation; in contrast, there is

no detectable ICE activity in OxLDL-treated cells. Hence, we focus on fucoidan-mediated signal

transductions including protein kinase in the regulation of proIL-1/IL-1 in J774A.1 cells.

Fucoidan activates extracellular signal-regulated kinase (ERK) in J774A.1 cells---Our

previous results demonstrated that fucoidan transduces protein kinase signaling pathways in the

upregulation of uPA (1). To examine fucoidan-mediated signal transduction pathways in the

regulation of IL-1 gene expression, first we tested whether fucoidan stimulates mitogen activated

protein kinase (MAPK). Incubation of J774A.1 cells with fucoidan led to a modest

phosphorylation of Elk-1, a transcriptional factor, evidencing the activation of ERK induced by

fucoidan (Fig. 3 A). Experiments for time course of fucoidan-induced ERK activity were further

conducted. As detected by Western blot analysis with anti-phospho-Elk-1 antibody, which

recognizes the activated, serine 383-phosphorylated form of Elk-1 by activated ERK (36);

relatively there barely exists ERK activity in untreated cells. Upon fucoidan stimulation, activity

of ERK was detected around 30 min, and it reached to the maximal level about 2-fold at 120 min;

after 240 min, ERK activity gradually returned to basal level (Figs. 3 A and 3 B).

Role of PKC and MEK1 in fucoidan-induced proIL-1 protein expression---Fucoidan

induces activity of protein tyrosine kinase (PTK), PKC and ERK, simultaneously it stimulates

proIL-1 protein. We examined whether the PKC/MEK1/ERK pathway is one of fucoidan-induced

PTK downstream signaling cascades in the regulation of proIL-1. Initially, experiments were


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conducted for dose responses of calphostin C (PKC inhibitor, 0.25, 0.50, 1.0 and 5.0μM) and of

PD98059 (MEK1 inhibitor, 10, 25, 50 and 100μM) in inhibiting ERK activity. J774A.1 cells

were exposed to the indicated concentrations of inhibitors, followed by incubation with fucoidan,

the appropriated concentrations of calphostin C and PD98059 inhibiting ERK activity were

determined as 1.0μM and 50μM, respectively (data not shown). Results of Western blotting

analysis show that at indicated concentration, neither calphostin C (Fig. 4 A) nor PD98059 (Fig. 4

B) blocks fucoidan-induced proIL-1 protein, though calphostin C and PD98059 effectively inhibit

fucoidan-stimulated ERK activity, suggesting the PKC/MEK1/ERK pathway less involves in

fucoidan-induced proIL-1 protein expression.

Fucoidan stimulates c-JUN NH2-terminal protein kinase (JNK) activity and role of JNK in

fucoidan-induced proIL-1 protein---The inflammatory response of J774A.1 cells to fucoidan

resulting in induction of IL-1 expression prompted us one possibility for fucoidan activation of

stress-related JNK pathway. We examined whether fucoidan activates JNK. Cells incubated

with fucoidan leads to JNK activation (Fig. 5 A) as determined by Western blot analysis via anti-

phospho-c-Jun, an antibody that recognizes the activated, serine 63-phosphorylated form of c-Jun

(37;38). As in a time course fashion, JNK activity gradually increased around 60 min, and the

maximal activity of JNK was at 120 min about 13-fold of untreated cells (Figs. 5 A and 5 B).

After 240 min, induced JNK activity gradually returned to basal level. To investigate the role of

fucoidan-induced JNK activity, initially, we chose and examined curcumin (a JNK inhibitor) in

regulation of proIL-1 protein expression. As shown in Fig. 6 A, increasing curcumin above 0.1

µM, it gradually reduces fucoidan-induced proIL-1 protein, and completely inhibits proIL-1 protein

at concentration above 1 µM. Alternatively, experiments of transient transfection of dominant

negative JNK (DN-JNK) into J774A.1 cells were conducted, we directly studied the role of induced

JNK in the regulation of proIL-1. Results of Western blotting analyses indicate that upon

fucoidan stimulation, it is about 10-fold less proIL-1 protein in DN-JNK transfected cells than that

of control cells (Fig. 6 B); in addition, no proIL-1 protein can be detected in the 48 h and 72 h DN-

JNK transfected cells. Consistently, there is no detectable JNK activity in the fucoidan-treated

DN-JNK cells (data not shown).

Fucoidan stimulates p38 mitogen activated protein kinase (p38) activity and role of p38

in fucoidan-induced proIL-1 protein---To explore fucoidan-mediated additional signal transduction

pathways, we further examined whether fucoidan induces p38 activity, another important stress-

related MAPK member. Upon fucoidan stimulation, p38 activity gradually increased as detected

by Western blotting analysis with anti-phospho-ATF-2, an antibody that specifically recognizes the


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activated, threonine Thr71-phosphorylated form of ATF-2 (39) (Fig. 7 A). Result of time course

study of fucoidan-induced p38 activity indicated that at 60 min, p38 activity increased to 6-fold

than that of control cells. At 240 min, p38 activity of treated cells reached to about 10-fold as

compared with control cells; after 480 min, it returned to basal level (Figs. 7 A and 7 B).

To dissect the effect of fucoidan-induced p38 activity in ATF-2 phosphorylation, we

designed a series of experiments. Using a specific p38 inhibitor, SB203580 (SB), we initially

conducted the inhibitory dose response study of SB in fucoidan induction of p38 activity.

J774A.1 cells were exposed to various concentrations of SB (0.1, 1.0, and 10μM), followed by

incubation with fucoidan for various times. In the time course study, SB effectively inhibits

fucoidan-induced p38 activity at concentration above 1μM as compared with control untreated

cells, though 0.1μM of SB slightly reduces p38 activity (Fig. 8 A). Moreover, we further

examined the potential role of fucoidan-induced p38 activation and ATF-2 phosphorylation in

regulation of proIL-1 protein. J774A.1 cells pre-treated with or without SB, followed by

incubation of fucoidan, Western blotting analyses were conducted to study SB inhibitory dose

response (concentration: 0.1, 1.0, and 10μM) in proIL-1 protein expression. SB completely

inhibits fucoidan-induced proIL-1 protein at concentration above 1 µM as compared with control

cells (Fig. 8 B, samples 6 and 8 vs. sample 2); but no inhibition below 0.1 µM (Fig. 8 B, sample 4).

Fucoidan stimulates p21 activated protein kinase (PAK) activity---To further investigate

the possible upstream signaling networks involving in regulation of JNK or p38 activity, one of the

relevant signaling molecules, PAK was chosen. PAK activity in the time course study was

examined by using an immunoprecipitated radioactive protein kinase activity assay, with

phosphorylation of substrate, myelin basic protein (MBP) (40). As shown in Figs. 9 A and 9 B,

the maximal fucoidan-stimulated PAK activity in phosphorylation of MBP occurred at 30 min,

after 60 min the stimulated PAK activity returned to basal level.

Effect of Rac1 transfection on fucoidan-stimulated JNK and p38 activity as well as effect

of Rac1 transfection on fucoidan-induced proIL-1 protein expression---Activation of Rac1 GTPase

stimulates PAK activity in some cells (34). To dissect the relationship of fucoidan-induced signal

transduction among Rac1 and JNK and p38, experiments for transfection of dominant negative

Rac1 (DN-Rac1) construct were conducted, and examined JNK and p38 activity, respectively.

Upon fucoidan stimulation, there was a 10-fold lower JNK activity (Fig. 10 A, sample 2 vs. sample

1) and a 5-fold lower p38 activity (Fig. 10 B, sample 2 vs. sample 1) in DN-Rac1 transfected cells

than those of empty expression vector transfected control cells (sample 1 in Figs. 10 A and 10 B).


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Obviously, without fucoidan stimulation, no JNK and p38 activity is detected in cells transfected

with empty expression vector (sample 3 in Figs. 10 A and 10 B). To further investigate the role of

Rac1 in proIL-1 protein expression, experiments for effect of DN-Rac1 and constitutive activated-

Rac1 construct (CA-Rac1) on fucoidan- induced proIL-1 protein were conducted. Moreover, the

effects of DN-Rac1 and constitutive activated-Rac1 construct (CA-Rac1) on fucoidan-induced

proIL-1 protein were further investigated. As shown in Fig. 10 C, upon 8-h fucoidan stimulation,

proIL-1 was much less expressed in DN-Rac1 transfected cells (i. e., the 24-, 48- and 72-h post-

DN-Rac1 transfected cells) than that of empty expression vector transfected control cells. In

contrast, upon 12-h and 24-h fucoidan stimulation, there were more proIL-1 expression (a

superinduction) in CA-Rac1 transfected cells than that in control cells (Fig. 10 D). All of these

results indicate that in fucoidan-induced signal transduction networks, Rac1 mediates JNK and p38

activity and further regulates proIL-1 protein expression.

Effect of inhibitor of PI 3-kinase, wortmannin on fucoidan-induced JNK and p38 activity

as well as on fucoidan regulation of proIL-1 protein and IL-1 secretion---As demonstrated above,

PKC/MEK1/ERK pathway plays less role in fucoidan regulation of proIL-1 expression. To

explore other fucoidan-mediated signal transduction pathways involving in proIL-1 expression, we

used wortmannin, an inhibitor of PI 3-kinase to examine the potential role of PI 3-kinase in

fucoidan activation of JNK or p38. J774A.1 cells pre-incubated with or without wortmannin for

60 min, followed by treatment of fucoidan for additional 120 min or 240 min, the activity of JNK

and p38 were examined. In the absence of wortmannin, fucoidan induces activity of JNK and p38

approximate 13-fold (Fig. 11 A, sample 2 vs. sample 1) and 10-fold (Fig. 11 B, sample 2 vs. sample

1), respectively as compared with control untreated cells (sample 1). Interestingly, cells pre-

incubated with wortmannin, followed by treatment of fucoidan, JNK and p38 activity increase to

approximate 16-fold (Fig. 11 A, sample 4 vs. sample 1) and 23-fold (Fig. 11 B, sample 4 vs. sample

1), respectively as compared with control untreated cells.

Moreover, the roles of PI 3-kinase in fucoidan regulation of proIL-1 protein and IL-1

secretion were examined. Pre-incubation of cells with wortmannin for 60 min, followed by

exposure of cells to fucoidan for additional 8 h, proIL-1 protein in cell lysates was detected by

Western blotting analysis. Results showed that pre-incubation of wortmannin in fucoidan-treated

cells significantly increases proIL-1 production (Fig. 12 A, sample 4) as compared to fucoidan-

treated cells or wortmannin-treated cells (Fig. 12 A, sample 2 or sample 3) or to control untreated

cells (Fig. 12 A, sample 1). In addition, the effect of wortmannin on fucoidan-induced mature IL-

1 secretion detected by ELISA was examined. There was a 5-fold higher of IL-1 secretion (20


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pg/ml) detected in cells pre-treated with wortmannin prior to incubation of fucoidan (Fig. 12 B,

sample 4) as compared to cells (4 pg/ml) only incubated with fucoidan (Fig. 12 B, sample 2); as

expected, no IL-1 was in control cells (Fig. 12 B, sample 1). Without fucoidan, wortmannin

slightly increased IL-1 secretion in cells (Fig. 12 B, sample 3 vs. sample 1).

Fucoidan stimulates tyrosine phosphorylation of non-receptor protein tyrosine kinase

(NRPTK), pp60Src and induces the association (complex formation) between pp60Src and heat-

shock protein 90 (Hsp90) in J774A.1 cells---Although there is no functional PTK motif found in

cytoplasmic domain of MSR (9;41-43), ligation of fucoidan to MSR induces tyrosine

phosphorylation of various proteins in human THP-1 macrophages (1) and in J774A.1 cells,

implying MSR as a PTK-linked receptor. We hypothesize that NRPTK such as pp60Src involves

MSR ligand-mediated signal transductions. Pre-incubation of J774A.1 cells with specific

inhibitors of pp60Src, e. g., PP1 or PP2 followed by exposure to fucoidan. First, cell lysates were

immunoprecipitated (IP) with monoclonal anti-pp60Src IgG, and immune-complexes were

recovered via incubation with protein A/G plus-agarose. Immunoprecipitates were separated by

SDS-PAGE, then immunoblotted (IB) with monoclonal anti-phosphotyrosine IgG. As seen in Fig.

13 A, the 60 kD phosphotyrosyl protein was immunoreactive with anti-pp60Src IgG in fucoidan-

treated cells (sample 2), and less reaction in PP2-preincubated cells (sample 4). In contrast, no

difference of tyrosine phosphotyrosine between PP1-preincubated cells and control untreated cells

(samples 3 and 1). These data indicate that ligation of fucoidan to MSR in J774A.1 cells leads to

tyrosine phosphorylation of pp60Src, but PP1 and PP2 inhibit the tyrosine phosphorylation.

Herbimycin A (HB) inhibits fucoidan-stimulated protein tyrosine phosphorylation (1),

suggesting unidentified PTK involve fucoidan-mediated signalings such as stress-related JNK and

p38 activity. As it known that one of HB cellular targets is Hsp90 (26), which expresses under

certain stress. Geldanamycin (GA) and HB inhibit signal transduction proteins including NRPTK,

pp60Src. Pharmacologically, GA and HB binds in a specific manner to Hsp90 and inhibits

pp60Src-Hsp90 heterocomplex formation (26-28). Here J774A.1 cells pre-incubated with GA and

HB as well as PP1 and PP2 followed by exposure to fucoidan, we further evaluated the association

(interaction) of pp60Src and Hsp90 by performing immune-precipitation with anti-Hsp90 IgG and

probing the resultant blots with anti-pp60Src IgG (Fig. 13 B). Whereas pp60Src co-precipitated

with Hsp90 in fucoidan-treated cells (sample 2), interaction between pp60Src and Hsp90 was

greatly decreased in cells pre-incubated with HB (sample 3) or GA (sample 4). Interestingly,

there was a similar complex formation between pp60Src and Hsp90 in the cells pre-treated with

PP1 and PP2 (samples 5 and 6), indicating inhibition of Src kinase activity does not interfere the


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specific association. Second, in the converse experiments, cell lysates were IP with monoclonal

anti-pp60Src IgG and IB with monoclonal anti- Hsp90 IgG. Similar results were observed as Fig.

13 B, the 90 kD protein was immunoreactive with anti-Hsp90 IgG in fucoidan-treated cells; but

there was no association in GA- or HB-pre-incubated cells (data not shown).

Roles of tyrosine phosphorylation of pp60Src as well as of association between pp60Src

and Hsp90 in the regulation of fucoidan-induced proIL-1 protein expression---We further

investigate the biological significance of fucoidan- stimulated tyrosine phosphorylation of pp60Src

and association of pp60Src with Hsp90 in regulation of proIL-1 protein. Results of Western

blotting of cell lysates with anti-IL-1 antiserum show fucoidan-induced proIL-1 protein expression

can be completely blocked in cells pretreated with inhibitors for pp60Src-Hsp90 complex (i. e., GA

or HB) (Fig. 14, samples 3 and 4 vs. sample 2) in a does response study (data not shown). In

contrast, fucoidan-induced proIL-1 protein can be partially reduced by inhibitors of pp60Src (PP1

or PP2) (Fig. 14, samples 5 and 6 vs. sample 2); relatively, PP1 exerts more inhibitory effect than

that of PP2 (Fig. 14, sample 5 vs. sample 6). Similarly, there was no detectable IL-1 secretion in

cells pretreated with these inhibitors (data not shown).


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DISCUSSION

In atherosclerotic lesions, activated macrophages via overexpressed scavenger receptor

aberrantly uptaking OxLDL, are also the main source for secretion of inflammatory cytokines, TNF

(2;17-19) and IL-1 (17;19). Yet it is unclear whether there is a relationship between receptor

ligand binding and stimulation of cytokine expression. Fucoidan, a polyanionic polysaccharide

has been used as an effective competitor for OxLDL or modified LDL in receptor binding study

(9;10). Moreover, fucoidan stimulates production of proteases (1;9;24) and cytokines in

macrophages (this paper and 9;16); however, there is no molecular mechanism to date for fucoidan-

mediated reactions. Here using ELISA, we demonstrate that during 24 h fucoidan induces about

50-fold TNF production more than OxLDL dose in J774A.1 cells. Based on time course of TNF

production (Fig. 1 A vs. 1B), there exist different patterns and mechanisms for TNF induction by

the two ligands, though TNF pre-exists in untreated cells. In addition, only the relatively lower

concentration of OxLDL (5 µg/ml) would induce TNF, there is no induction of TNF at a higher

concentration of OxLDL (50 µg/ml) (data not shown) which comparable to the previous

demonstration (21). Using RT-PCR for TNF mRNA, there is no apparent difference of TNF

message among cells treated with fucoidan and OxLDL and untreated cells. The fact that increase

of TNF production, but no alteration of TNF message under fucoidan stimulation, indicates that

fucoidan regulation of TNF expression is likely at post-transcriptional level.

Neither IL-1 nor proIL-1 pre-exists in quiescent J774A.1 cells. Upon fucoidan

stimulation, IL-1 can be detected by ELISA after 6-8 h; IL-1 secretion is consistent with the

sequential times for synthesis of proIL-1 mRNA and proIL-1 protein under stimulation for 2 h and

4 h, respectively. The ICE activity of fucoidan-treated cells peaks around 6 h and simultaneously

IL-1 secretion increases, which reflects that active ICE hydrolyzes proIL-1 into IL-1 as in various

cells (31;32); though the reason for continuous increasing IL-1 needs further study. Our results

indicate that there are complicated mechanisms, likely at transcriptional, post-transcriptional and

post-translational levels for fucoidan induction of proIL-1 protein and IL-1 secretion in

macrophages. Surprisingly, there is no detectable expression of proIL-1/IL-1 in the cell incubated

with OxLDL. One of explanations for the results is that binding of different ligands to the

conserved lysine clusters of collagen-like domain in MSR (43-45) may trigger on different signal

transductions (our unpublished data and (44;45)). This is the first time we demonstrate that

different MSR ligands transduce diverse signalings and further lead to different biochemical

reactions such as cytokine production.


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Ligation of MSR induces signal transduction pathways in macrophages (1;16;46;47), for

example, binding of fucoidan to MSR in human monocytic macrophage THP-1 cells induced PKC

signaling-mediated IL-1 secretion (16) and also stimulated protein tyrosine phosphorylation

including PLC-γ1, PI 3-kinase and PKC activity leading to upregulated uPA expression (1). To

further focus on molecular mechanism by which fucoidan-mediated signaling cascades including

MAPK in regulation of proIL-1/IL-1 expression, we utilized specific pharmacological antagonists

such as calphostin C, PD98059, wortmannin, curcumin and SB203580, which inhibit the

phosphorylation of PKC (1;48), MEK1 (49), PI 3-kinase (50), JNK (51;52) and p38 (53),

respectively. Since PKC and MEK1 involve in the induction of ERK activity (54) and ligation of

fucoidan induces ERK activity, we examined the roles of PKC and MEK1 in fucoidan regulation of

proIL-1/IL-1. Initially, both calphostin C and PD98059 inhibit fucoidan-stimulated ERK activity,

but fail to block fucoidan-induced proIL-1 protein and IL-1 secretion. Results indicate fucoidan-

induced PKC/MEK1/ERK pathway does not involve in regulation of proIL-1/IL-1 expression,

though we could not rule out the possibility of cross-talking between MEKK members and ERK.

Similarly, using pharmacological inhibitors and genetic dominant mutants, we established

fucoidan-mediated signaling relationship among JNK and p38 activity and regulation of proIL-1

protein expression. In preliminary studies, we determined whether OxLDL or fucoidan induce

cytokines in fully differentiated human macrophages. Peripheral blood monocytes were allowed

to differentiate in culture at indicated times, conditioned medium and cell lysates were collected

and prepared. Exposure of monocyte-derived macrophages to OxLDL or fucoidan increased TNF

secretion as detected by ELISAs; but only fucoidan induced proIL-1/IL-1 expression. Moreover,

fucoidan activated ERK, JNK and p38 in human macrophages examined by protein kinase assays,

which similar to those in J774A.1 cells.

Upstream signaling molecules, Rac1 and Cdc42 activating JNK activity have been

reported (55;56), and likely p38 activity is activated in a similar way (57). PAK family of protein

kinase has been recognized as one of main targets to interact with the GTPases of Rac1/Cdc42, and

generating downstream signaling networks (58). We demonstrate that fucoidan quickly stimulates

PAK activity, followed by increasing activity of JNK and p38, as well as induction of proIL-1

protein. The current results of fucoidan-induced PAK, JNK and p38 activity are comparable with

recent demonstration of Rac1 activation of PAK-mediated signalings (59), leading to induction of

activity of JNK (34) and p38 (57), respectively. Upon fucoidan stimulation, DN-Rac1-transfected

cells express lower activity of JNK and p38 (Figs. 10 A and 4 B) as well as less proIL-1 protein

production (Fig. 10 C) than those of empty expression vector-transfected control cells do.

Moreover, fucoidan induces higher JNK and p38 activity (unpublished data) and more proIL-1


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protein production (Fig. 10 D) in CA-Rac1-stably transfected J774A.1 cells than those of control

cells. Together, results of using Rac1 various genetic mutants evidence and support that levels of

Rac1 and PAK are at the upstream of JNK and p38. In addition, we demonstrate that the

important roles in fucoidan-mediated signaling pathways of PTK/Rac1/PAK/JNK and of

PTK/Rac1/PAK/p38 in regulation of proIL-1 expression. Furthermore, inhibitors of PI 3-kinase,

wortmannin and LY294002 (data not shown) up-regulate JNK and p38 activity as well as increase

proIL-1 protein and superinduce IL-1 secretion suggest under normal conditions, endogenous PI 3-

kinase and/or PI 3-kinase-related downstream signalings inhibit fucoidan-stimulated JNK and p38

activity, and further suppress fucoidan- induced proIL-1/IL-1 expression.

Our results demonstrate that ligation of fucoidan to MSR induces multiple protein

kinases activity, mitogen-like signalings and stimulation of proIL-1/IL-1 expression in

macrophages, which comparable with cells treated with certain cytokines (35;60-63). There is no

functional tyrosine kinase motif in MSR cytoplasmic domain (9;41-43); however, engagement of

MSR with fucoidan leads to quickly trigger herbimycin A-blockable protein tyrosine

phosphorylation (1). Here, using specific inhibitors, we further demonstrate for the first time

fucoidan-mediated signalings are via MSR likely associated with cytosolic tyrosine kinase, e. g.,

pp60Src in concert with Hsp90; or via other receptor(s), but which, if any, of these mediate signal

transductions and regulate proIL-1 protein and IL-1 secretion. Although the physiologic ligands

for MSR are currently unknown, comparing polysaccharide structure of fucoidan with the relevant

structure of another MSR ligand, lipopolysaccharide (LPS) (9;14), fucoidan might exert LPS-like

signalings such as activation of MAPKs and induction of IL-1 as LPS (our unpublished data and

(64)). Phosphatidylserine (PS), one of effective competitors for MSR binding (9), which

translocates from the inner face of the cell plasma membrane to the cell surface after initiating

apoptosis. The role for MSR in the phagocytosis and clearance of apoptotic thymocytes has been

reported (65), it is likely that PS on dying cells become an endogenous ligand of MSR upon

apoptosis. Besides, another MSR ligand is advanced glycation end products of protein related to

age-enhanced disease processes including atherosclerosis, which are endocytic uptaken by

scavenger receptors on macrophages (66). Considering the physiological relevance of our

observations in fucoidan-mediated signalings and the potential endogenous ligand(s) as fucoidan,

we speculate that similar transducing signals might arise upon the ligation of MSR and the

sequential biological impacts need further investigation.

In summary, continuing to exposure MSR acting as a functional signaling receptor, we are

the first to examine MSR ligands differentially induce several transducing signal cascades, and


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further systematically to dissect the molecular mechanisms by which fucoidan-mediated signalings

in regulation of IL-1. Specifically, we establish signal transduction pathways of PTK (Src)→

Rac1 → PAK → → JNK and of PTK (Src)→ Rac1 → PAK → → p38 in the regulation of proIL-1

protein and IL-1 secretion (Fig. 15). In contrast, although fucoidan induces activity of PKC,

MEK1 and ERK, there is less role for the pathway of PTK → PLC-γ1 → PKC → MEK1 → ERK

in proIL-1/IL-1 regulation (Fig. 15). On the other hand, fucoidan-induced ICE activity results in

the alternation relationship between degradation of proIL-1 protein and increase of mature IL-1

secretion with time, suggesting fucoidan probably mediate transcriptional, post-transcriptional and

post-translational regulation of IL-1 expression. Moreover, considering the important role of IL-1

in stimulating inflammatory abnormality, the model for ligation of MSR-mediated signal

transduction pathways in regulation of proIL-1/IL-1 expression providing certain biological

significance in the development of vascular-related diseases.

Acknowledgments--- This work was supported by research grants of NSC 89-2320-B-010-083 (H.-

Y. Hsu) from the National Science Council, Taiwan, of NHRI-GT-EX89S937L (H.-Y. Hsu) from

National Health Research Institutes, Taiwan, and of Program for Promoting Academic Excellence

of Universities, 89-B-FA22-2-4 (H.-Y. Hsu) from the Ministry of Education, Taiwan. H.-Y. Hsu

is awarded by Medical Research and Advancement Foundation in Memory of Dr. Chi-Shuen Tsou.

We gratefully acknowledge that OxLDL supplied from Dr. Ming-Shi Shiao (Veteran General

Hospital, Taipei, Taiwan), DN-JNK construct from Dr. M. Karin (UCSD, San Diego, CA, USA),

DN-Rac1 and CA-Rac1 constructs from Dr. S. Bagrodia (Cornell University, Ithaca, NY, USA),

and Anti-IL-1β, 3ZD monoclonal antibody, a gift from NIH (Bethesda, MD, USA).


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FIGURE LEGENDS

Fig. 1. Effect of MSR ligands, OxLDL and fucoidan on TNF production and TNF mRNA in

murine macrophage J774A.1 cells. A. Effect of OxLDL on TNF production in cells.

Cumulative TNF is presented as concentration of TNF in cell conditioned medium treated with

OxLDL (5 µg/ml) collected at the indicated time within 24 h, and measured using an ELISA as

described in EXPERIMENTAL PROCEDURES. The data are reported as a representative of

three experiments (n = 3). B. Effect of fucoidan on TNF production in cells. The similar

experiments and measurement for TNF production in fucoidan (25 µg/ml)-treated cells as above A.,

one of three experiments is presented (n = 3). C. RT-PCR analysis of expression of TNF mRNA

in cells. Total RNA was isolated from cells grown in serum-free media in the presence of OxLDL

(5 µg/ml) or fucoidan (25 µg/ml) for various times as indicated. For fucoidan treatment, ethidium

bromide-stained agarose gel with amplified TNF cDNA at 692 bp and normalized by comparison to

RT-PCR of mRNA of glyceraldehyde phosphate dehydrogenase (GAPDH) at 450 bp, a

constitutively expressed gene are indicated with arrows for TNF and GAPDH, respectively. The

detailed method is described in EXPERIMENTAL PROCEDURES. One of three experiments is

presented (n = 3). Similar results of RT-PCR for TNF mRNA were found in OxLDL-treated cells

(data not shown).


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Fig. 2. Effect of fucoidan and OxLDL on expression of IL-1, proIL-1 protein and proIL-1

mRNA as well as on stimulation of interleukin 1 converting enzyme (ICE or caspase 1)

activity in J774A.1 cells. A. Effect of fucoidan and OxLDL on IL-1 secretion in cells. Cells

were treated with fucoidan (25 µg/ml) or OxLDL (5 µg/ml), conditioned media were harvested at

the time indicated within 48 h. Concentrated media were assayed for IL-1 concentration using IL-

1 specific ELISA, one of four experiments is presented (n = 4). B. Western blotting analysis of

proIL-1 protein expression in fucoidan-treated cells. Cells were treated with fucoidan or OxLDL

for various times as indicated, whole cell lysates were analyzed by Western blot with anti-IL-1

antiserum, as described in EXPERIMENTAL PROCEDURES. The proIL-1 (34 kD), and α -

tubulin (as an internal control) are indicated as arrows on the right side, one of four experiments is

presented (n = 4), no proIL-1 protein was found in OxLDL-treated cells (data not shown). C. RT-

PCR analysis of proIL-1 mRNA expression in cells. Total RNA was isolated from cells treated

with fucoidan or OxLDL within 24 h. Ethidium bromide-stained agarose gel with amplified

proIL-1 mRNA at 563 bp and normalized by comparison to RT-PCR of GAPDH mRNA are

indicated with arrows for proIL-1 and GAPDH. One of four experiments is presented, no proIL-1

mRNA was found in OxLDL-treated cells (data not shown). D. Time-dependent activation of ICE

activity by OxLDL and fucoidan in J774A.1 cells. Cells were treated with OxLDL and fucoidan

for indicated time. Cell extracts (75 µg of protein) were incubated in the presence of the

fluorescent caspase 1 substrate Ac-YVAD-CHO (50 µM) for 1 h at 30 ℃. Caspase 1 activity was

measured fluormetrically after substrate cleavage with excitation at 360 nm and emission at 460 nm.

The detailed method is described in the section of EXPERIMENTAL PROCEDURES or as in the

instruction manual of “The CaspACE Assay System, Fluorometric, Promega Co”. Experiments

were repeated three times, a representative result is shown in the figure.


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Fig. 3. Time course of fucoidan- induced p44/42 ERK activity in J774A.1 cells. A.

Analysis of p44/42 ERK activity in fucoidan-treated J774A.1 cells. The ERK-induced

phosphorylation of Elk-1 was measured by quantitative immunoblotting with phospho-Elk-1

(Ser383) antibody and Phototope®-HRP Western Detection System. Briefly, whole cell lysates

(200 µg protein) at various times were incubated with 20 µl of immobilized Phospho-p44/42 ERK

(Thr202 and Tyr204) monoclonal antibody (50% slurry) at 4 ℃ for 24 h. The

immunoprecipitated active (phosphorylated) ERK was centrifuged at 4 ℃ for 10 min, pellet

washed twice with 1X lysis buffer and 1X kinase buffer, respectively, then resuspended in 1X

kinase buffer. The kinase reactions were performed in the presence of 200 µM of cold ATP and 2

µg of Elk-1 fusion protein at 30 ℃ for 30 min. The Elk-1 phosphorylation was measured by

Western blotting of nonradioactive labeled samples using phospho-Elk-1 (Ser383) antibody plus

Phototope®-HRP Western Detection System via chemiluminescence. The detailed method is

described in the section of EXPERIMENTAL PROCEDURES or in NEB instruction manual. B.

Histograms represent quantification by PhosphorImager® of phospho-Elk-1 (Ser383) for fucoidan-

activated ERK activity in each sample with using ImageQuaNT® software from Molecular

Dynamics. All data of relative activity are expressed as comparison with untreated J774A.1 cells,

(i. e., t = 0, activity of control cells defined as 1). Similar experiments were repeated four times

and a representative result is shown in the figure.


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Fig. 4. Effect of PKC inhibitor (calphostin C) and of MEK 1 inhibitor (PD98059) on

fucoidan-regulated proIL-1 protein expression. A. Effect of calphostin C on fucoidan-induced

proIL-1 protein expression. First, dose response study for effect of inhibitor of PKC, calphostin C

(various concentrations: 0.1, 0.25, 0.50, and 1.0 µM) on fucoidan-induced proIL-1 protein was

studied, the effective concentration of calphostin C was determined as 1 µM (data not shown).

Followed by cells incubated with or without calphostin C (1 µM) for 1 h, cells were divided into

two groups, fucoidan-untreated (#1 and 3) and fucoidan-treated (#2 and 4). For fucoidan-treated

cells means cells incubated with fucoidan (25 µg/ml) for additional 8 h. After incubation, samples

were subjected to Western blot analysis of proIL-1 as described in Fig. 2 B. The indicated arrows

on the right side represent position of proIL-1 and α -tubulin, respectively. This experiment is a

representative of three similar experiments. B. Effect of inhibitor of MEK 1, PD98059 on

fucoidan-induced proIL-1 protein expression. As above A., “dose response study for effect of

calphostin C on fucoidan-induced proIL-1 protein”, similarly, the effective concentration of

PD98059 for MEK 1 was determined as 50 µM. Followed by cells incubated with or without

PD98059 (50 µM) for 1 h, cells were divided into two groups, fucoidan-untreated (#1 and 3) and

fucoidan-treated (#2 and 4). For fucoidan-treated cells means cells incubated with fucoidan (25

µg/ml) for additional 8 h. After treatments, the samples were subjected to Western blot analysis

as described in above A. This experiment is a representative of three similar experiments.


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Fig. 5. Time course of fucoidan-activated JNK activity. A. Analysis of JNK activity in

fucoidan-treated J774A.1 cells by quantitative Western blotting using Phospho-c-Jun (Ser63)

antibody and Phototope®-HRP Western Detection System. Briefly, whole cell lysates (250 µg

protein) were incubated overnight with 2 µg of c-Jun (1-89) fusion protein beads at 4 ℃ for 24 h.

The active complex of JNK/c-Jun fusion protein beads was centrifuged at 4 ℃ for 10 min, pellet

washed twice with 1X lysis buffer and 1X kinase buffer, respectively, then resuspended in 1X

kinase buffer. The kinase reactions were performed in the presence of 100 µM of cold ATP at 30

℃ for 30 min. The phosphorylation of c-Jun at Ser63 was measured by Western blotting of

nonradioactive labeled samples using phospho-c-Jun (Ser63) antibody plus Phototope®-HRP

Western Detection System via chemiluminescence. The detailed method is described in the

section of EXPERIMENTAL PROCEDURES or in NEB instruction manual. B. Histograms

represent quantification by PhosphorImager ® of phospho-c-Jun (Ser63) for fucoidan-activated

JNK activity in each sample with using ImageQuaNT® software from Molecular Dynamics. All

data of relative activity are expressed as comparison with untreated cells, (t = 0, activity of control

cells defined 1). Similar experiments were repeated four times and a representative result is

shown in the figure.

Fig. 6. Effect of curcumin and DN-JNK transfection on fucoidan-regulated proIL-1 protein

expression. A. Effect of curcumin on fucoidan-regulated proIL-1 protein. Cells were treated

with various concentrations (0.1, 1.0, 10 and 100 µM) of curcumin as described in

EXPERIMENTAL PROCEDURES. After 24-h curcumin treatment, cells were treated with

fucoidan for 8 h and Western blotting analyses of proIL-1 protein expression were conducted as

described in above Fig. 2 B. Similar results were obtained in three separate experiments. B.

Effect of DN-JNK transfection on fucoidan-regulated proIL-1 protein expression. Cells were

transient transfected with DN-JNK or with the empty expression vector as control (Control), and

waited for various times, i. e., 24 h, 48 h and 72 h as indicated and described in EXPERIMENTAL

PROCEDURES. After 24-h, 48-h or 72-h DN-JNK transfection, cells were treated with fucoidan

for additional 8 h; then Western blotting analyses of proIL-1 expression were conducted as

described in above Fig. 2 B. The similar data are representative of three separate experiments.


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Fig. 7. Time course of fucoidan-activated p38 activity. A. Analysis of p38 activity in

fucoidan-treated J774A.1 cells. The p38-induced phosphorylation of ATF-2 was measured by

quantitative immunoblotting with phospho-ATF-2 (Thr71) antibody and Phototope®-HRP Western

Detection System. Briefly, whole cell lysates (200 µg protein) at various times were incubated

with 20 µl of resuspended immobilized Phospho-p38 MAP kinase (Thr180/Tyr182) monoclonal

antibody at 4 ℃ for 24 h. The immunoprecipitated active (phosphorylated) p38 was centrifuged

at 4 ℃ for 10 min, pellet washed twice with 1X lysis buffer and 1X kinase buffer, respectively,

and resuspended in 1X kinase buffer. The kinase reactions were performed in the presence of 200

µM of cold ATP and 2 µg of ATF-2 fusion protein at 30 ℃ for 30 min. The phosphorylation of

ATF-2 at Thr71 was measured by Western blotting of nonradioactive labeled samples using

phospho-ATF-2 (Thr71) antibody plus Phototope®-HRP Western Detection System via

chemiluminescence. The detailed method is described in the section of EXPERIMENTAL

PROCEDURES or in NEB instruction manual. B. Histograms represent quantification by

PhosphorImager® of phospho-ATF-2 (Thr71) for fucoidan stimulates p38 activity in each sample

with using ImageQuaNT® software from Molecular Dynamics. All data of relative activity are

expressed as comparison with untreated cells, (t = 0, activity of control cells defined as 1).

Similar experiments were repeated four times and a representative result is shown in the figure.

Fig. 8. Effect of p38 inhibitor, SB203580 on fucoidan-induced p38 activity and on

fucoidan-induced proIL-1 protein expression. A. Effect of inhibitor of p38, SB203580 on

fucoidan-induced p38 activity. J774A.1 cells were pre-treated with various concentrations (0.1,

1.0 and 10 µM) of SB203580, followed by incubation with fucoidan (25 µg/ml) for additional

times as above Fig. 7. After indicated treatments, samples were subjected to analysis of p38

activity via phosphorylation of ATF-2, and the representation of histograms as described in Fig. 7.

All data of p38 relative activity are expressed as comparison with untreated cells, (t = 0, activity of

control cells defined as 1). Experiments were repeated and a representative of three similar results

is shown in the figure. B. Effect of p38 inhibitor, SB203580 on fucoidan-induced proIL-1 protein.

Followed by cells pre-treated with SB203580 (0.1, 1.0 and 10 µM) or without for 60 min, cells

were divided into two groups, fucoidan-untreated (1, 3, 5 and 7) and fucoidan-treated (2, 4, 6 and 8).

For fucoidan-treated cells means cells incubated with fucoidan (25 µg/ml) for additional 8 h.

After treatments, samples were subjected to Western blot analysis of proIL-1 protein as described.

Both proIL-1 and α -tubulin are as indicated. Experiments were a representative of three similar

results is shown in the figure.


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Fig. 9. Time course of fucoidan-stimulated PAK activity in J774A.1 cells. A. Activity of

PAK in fucoidan-treated J774A.1 cells. Samples were examined in a radioactive immune-

complex kinase assay method, using myelin basic protein (MBP) as substrate with additional 1 µl

γ-32P-ATP (5 µCi, 330 µM), incubated at 30 ℃ for 15 min. Equal numbers of cells were

treated with fucoidan at each time point as indicated. The detailed method is described in the

section of EXPERIMENTAL PROCEDURES. B. Histograms representing fucoidan-stimulated

PAK activity are quantified by PhosphorImager® of γ-32p-ATP of each sample via using

ImageQuaNT® software from Molecular Dynamics. All data of relative activity are expressed as

comparison with untreated cells, (t = 0, activity of control cells is 1). Similar experiments were

repeated four times and a representative result is shown in the figure.

Fig. 10. Effect of Rac1 transfection on fucoidan-stimulated JNK and p38 activity as well as

effect of Rac1 transfection on fucoidan-induced proIL-1 protein expression in J774A.1 cells.

In the following A, B, and C, cells were transient transfected with dominant negative Rac1 (DN-

Rac1) or with the empty expression vector as “Control” described in EXPERIMENTAL

PROCEDURES. A. Effect of DN-Rac1 transfection on reduction of fucoidan-stimulated JNK

activity. After 24-h transfection, cells were treated with or without fucoidan for 120 min, and

JNK activity of the treated cells was analyzed as described above. B. Effect of DN-Rac1

transfection on reduction of fucoidan-stimulated p38 activity. After 24-h transfection, cells were

treated with or without fucoidan for 240 min, and p38 activity of the treated cells was analyzed as

described above. C. Effect of DN-Rac1 transfection on reduction of fucoidan- induced proIL-1

protein expression. After 24-h, 48-h and 72-h transfection, cells were treated with fucoidan for 8

h, whole cell lysates were analyzed by Western blot with anti-IL-1 antiserum, as described in

EXPERIMENTAL PROCEDURES. The proIL-1 (34 kD) and α -tubulin are indicated as arrows

on the right side. D. Transfection of constitutive activated-Rac1 (CA-Rac1) increases fucoidan-

induced proIL-1 protein expression. Two groups of cells: group 1, cells stably transfected with

CA-Rac1 construct and group 2, cells transfected by the empty expression vector (i. e., control)

were prepared and used for the experiments. Cells were treated with or without fucoidan for 4 h,

12 h and 24 h as indicated, whole cell lysates were analyzed by Western blot with anti-IL-1

antiserum, proIL-1 is as indicated. All experiments in A, B, C and D were repeated and a

representative of three similar results (n = 3) is shown in the figures.


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Fig. 11. Effect of PI 3-kinase inhibitor, wortmannin on fucoidan-induced JNK and p38

activity in J774A.1 cells. A. Effect of wortmannin on fucoidan-stimulated JNK activity. Cells

were pretreated with wortmannin (25 µM) for 1 h prior to stimulating with fucoidan (25 µg/ml) for

additional 120 min, followed by nonradioactive method for JNK kinase assay as described

previously. B. Effect of wortmannin on fucoidan-stimulated p38 activity. Cells were pretreated

with wortmannin (25 µM) for 1 h prior to stimulating with fucoidan (25 µg/ml) for additional 240

min, followed by nonradioactive method for p38 kinase assay as described previously. All data of

relative JNK and p38 activity are expressed as comparison with untreated cells, (t = 0, activity of

control cells is 1). Similar results were obtained in four separate experiments.

Fig. 12. Effect of PI 3-kinase inhibitor, wortmannin on fucoidan-induced proIL-1 expression

and IL-1 secretion in J774A.1 cells. A. Effect of wortmannin on fucoidan-induced proIL-1

expression. Followed by cells incubated with or without wortmannin (25 µM) for 1 h, the cells

were divided into two groups, fucoidan-untreated (samples 1 and 3) and fucoidan-treated (samples

2 and 4). For fucoidan-treated groups were cells incubated with fucoidan (25 µg/ml) for

additional 8 h. After treatments, the samples were subjected to Western blot analysis of proIL-1

as described above. This experiment is a representative of three similar experiments. B. Effect

of wortmannin on fucoidan-induced IL-1 secretion. Cells were treated as described in A., after

indicated treatments, concentrated conditioned media were collected at 8 h. IL-1 content in tested

samples were assayed by an IL-1-specific ELISA method as described above. The data are

reported as a representative of four separate experiments (n = 4).


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Fig. 13. Fucoidan stimulates tyrosine phosphorylation of pp60Src and induces the

association (complex formation) between pp60Src and Hsp90. A. Fucoidan stimulates tyrosine

phosphorylation of pp60Src. J774A.1 cells were pre-incubated with inhibitors of pp60Src, PP1 (1

μM) and PP2 (1 μM), respectively, followed by exposure to fucoidan. Various cell lysates

were IP with monoclonal anti-pp60Src IgG at room temperature for 4 h, and immune-complexes

were recovered via incubation with protein A/G plus-agarose at 4 ℃ for 24 h.

Immunoprecipitates were separated by SDS-PAGE, and IB with monoclonal anti-phosphotyrosine

IgG. Visualization of protein tyrosine phosphorylation on each immunoblot was performed with

Renaissance®, DuPont Western Blot Chemiluminescence Reagent, NEN Research Products as

described (1). Similar results were obtained in three separate experiments. B. Fucoidan induces

the association (complex formation) between pp60Src and Hsp90. Cells pre-incubated with GA

(5 μM) and HB (1 μM), as well as inhibitors of Src: PP1 (1 μM) and PP2 (1 μM), followed

by exposure to fucoidan. Various cell lysates were IP with monoclonal anti-Hsp90 IgG at room

temperature for 4 h, and immune-complexes were recovered via incubation with protein A/G plus-

agarose at 4 ℃ for 24 h. Immunoprecipitates were separated by SDS-PAGE and IB with

monoclonal pp60Src IgG. Protein visualization on each immunoblot was performed with

Renaissance®, DuPont Western Blot Chemiluminescence Reagent as described. The positions of

pp60Src and the IgG heavy chain (IgG (H)) are indicated. Molecular weight markers are

indicated in kilodaltons (kD). Similar results were obtained in three separate experiments.

Fig. 14. Roles of tyrosine phosphorylation of pp60Src as well as of association between

pp60Src and Hsp90 in the regulation of fucoidan-induced proIL-1 protein expression.

Initially, dose response study (concentration of GA and HB from 1 to 15 µM) for effect of GA and

HB on fucoidan-induced proIL-1 protein was performed (data not shown) and the effective

concentration was determined as 1 µM of GA and 1 µM of HB. Pre-incubation of J774A.1 cells

with GA (5 µM) and HB (1 µM) as well as with inhibitors of Src, PP1 (1 µM) and PP2 (1 µM) for

30 min, followed by exposure of cells to fucoidan (25 µg/ml). ProIL-1 protein expression in

various cell lysates was analyzed by Western blot with anti-IL-1 antiserum, as in Fig. 2. The

experiment is a representative of three similar experiments (n = 3).

Fig. 15. The proposed fucoidan-mediated signal transduction pathways in the regulation of

proIL-1 protein expression and IL-1 secretion.


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Hsien-Yeh Hsu, Show-Lan Chiu, Meng-Hsuan Wen, Kuo-Yen Chen and Kuo-Feng Huamodulation of protein kinase signaling pathways

Ligands of macrophage scavenger receptor induce cytokine expression via differential

published online June 4, 2001J. Biol. Chem.

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Ligands of Macrophage Scavenger Receptor Induce Cytokine … · include protein tyrosine kinases...

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