Journal of Huazhong University of Science and Technology ~Vled Sci] ~ ~ ~ ~ ~ q~ ~ [ I~ q~ ( ~ ~ ~ ) ~[ ] 23 (2): 97-100, 2003 97

Induction of Very Low Density Lipoprotein Receptor (VLDLR) Transcription by VLDL Is Mediated by the Extracellular Signal-Regulated Kinase Signaling Pathway" WANG Yan (~ ~ ) , QU Shen (~ 4~), ZONG Yiqiang (~.. Y,.~), ZHANG Mingtao (~a~q,), WU Fan (~ The Department of Biochemistry ~- Molecular Biology Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030

}L)

Summary: To elucidate the intracellular signaling pathways for VLDL-induced VLDLR tran~fip- tion, Western blot analysis was u~d to examine phosphorylated ERK1/2 protein. It was found that that VLDL induced an increa~ in ERK1/2 activity in a protein kinase C (PKC)-dependent manner in murine RAW264.7 macrophages. By using different protein kinases inhibitors or activators it was observed that the effect of VLDL-induced VLDL receptor transcription, which is monitored by RT- PCR analysis of VLDL receptor mRNA, was not affected by the inhibitor of p38 kinase and cAMP analog, but completely abolished by pretreatment of the cells with PD 98059, an inhibitor of MEK and GF 109203X, an inhibitor of PKC. The~ results demonstrated that the PKC/ERK1/2 cascade is the essential signaling pathway by which VLDL activates VLDL receptor mRNA expre~ion. Key words: VLDL receptor; VLDL; signal transduction

Very low density lipoprotein receptor (VLDI.R) is structurally related to LDI.-R and belongs to the same family. While LDL-R binds apo-B and apo-E containing lipoproteins, VLDLR interacts preferen- tially with apo-E and triglyceride-rich lipoprotein [13. VI.DLR has been shown to contribute to the foam cell formation and play an important role in atheroge- nesis [~'3]. Our previous work suggested that VLDL increases VLDLR transcription, uptake of triglyc- eride-rich lipoprotein and enhances the formation of foam cells. But very little is known about the mecha- nism regulating the expression of VLDLR. In order to identify the signal pathway, which mediates VLDL-induced VLDL receptor transcription, we ex- amined the VI.DL-elicited signal transduction path- ways and specific kinases that are involved in VI.DL receptor transcription.

Two major signal pathways can be activated by VLDL: the PKC-dependent ERK1/2 pathway and p38 pathway [']. One in vitro study addressed the reg- ulation of the human VLDI. receptor promoter, indi- cating that the phosphorylated substrates of ERK1/ 2-NF-Y and C/EBP[3 might be important for tran- scriptional regulation of VI.DL receptor in hepatoma and BeWo ceils [s]. Moreover, 8-bromo-cAMP, an analog of cAMP, causes a suppression of VLDI. re- ceptor message in JEG-3 and BeWo cells [6~. There- fore, in the present studies, our investigation is fo- cused on the MAP kinase and PKA pathway.

1 MATERIALS AND METHODS

I. 1 Materials The RAW264. 7 cell line was obtained from

Shanghai Cell Bank. Tissue culture flasks, and mul- tiwell plates were purchased from Nunc (Roskilde,

WANG Yan, born in 1974, Ph.D. " This project was supported by a grant from National Natu- ral Sciences Foundation of China (Serial No. 39970307).

Denmark). Newborn calf serum and medium RP- MI1640 were from Hyclone, USA. PD 98059, Taq DNA Polymerase, M-rely , Oligo dT, dNTP were from Promega, USA. SB 203580 was from Cal- biochem. 8-bromo-eAMP and GF 109203X were from Sigma, USA. RNAeX Reageat was procured from Shanghai Huasun Biotechnology Inc. The primers for VI.DL receptor and glyceraldehydes- phosphate dehydrogenase (GAPDH) were synthe- sized by Shanghai Biotechnology Inc. Goat polyclonal antibody against phosphospecific ERK1/2 and an en- hanced chemiluminescence (ECI.) detection system was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-goat IgG-horseradish peroxidase antibody was from Sigma, USA. 1.2 Lipoprotein Preparation

Blood was obtained from normolipidemic sub- jects among the medical staff at Tongji Hospital (Wuhan, China). Plasma was separated by centrifu- gation (600 g) at 4 ~ Human VI.DL (d ~ 1. 006 g/ml) was isolated from the plasma by ultracen- trifugation as previously described [7]. VLDL particles were sterilized through a 0. 45 mm filter, stored in sterile tubes at 4 'C, and used within 2 weeks. Total protein content in VI.DL was measured by the Lowry's method ~8] , with bovine serum albumin used as a standard. 1.3 Cell Culture and Stimulation

RAW264. 7 cells were cultured in RPMI 1640 supplemented with 10 0~ ( v / v ) heat-inactivated newborn calf serum, 2 mmol/L glutamine, and an- tibiotics at 37 *C and 5 % COz in a humidified incu- bator. For experiments, the cells were plated at a density of 5 • 10 s cells in 6-well plates and used after 24-h preincubation in serum-free medium. Cells were pretreated with different inhibitors or activators. And then they were incubated with VLDL. 1.4 Western Blot

Cells were washed twice with cold PBS and then lysed in protein extraction buffer (25 mmol/L Tris

98 Journal of Huazhong University of Science and Technology [-Med Sci] 23 (2): 97-100, 2003

C1, p H = 7 . 4 , 50 mmol/L NaC1, 0.5 ~ sodium de- oxycholate, 2 % Nonidet P-40, 0. 2 ~ SDS, 1 mmol/L EDTA, 0. 2 mmol/L sodium vanadate, 1 mmol/L phenylmethylsulfonyl fluoride, 10 lug/ml aprotinin, and 10 /~g/ml leupeptin). The extracts were incubated on ice for 15 rain and centrifuged at 13 000 rpm at 4 ~ to remove insoluble materials. Protein concentrations were determined by the Lowry's method according to instructions, with bovine serum albumin used as a standard. Equal amounts of protein (80 /tg/lane) were subjected to SDS-polyacrylamide gel electrophoresis. Stacking gels contained 4 % acrylamide/bisacrylamide (29 �9 1) and separating gels contained 12 % acrylamide/ bisacrylamide. Electrophoresis was carried out at 200 V in running buffer (25 mmol/L Tris, 250 mmol/L glycine, and 0. 1% SDS). The samples were elec- trophoretically transferred to nitrocellulose mem- branes using transfer buffer (48 mmol/L Tris, pH= 8. 3, 39 mmol/L glycine, and 20 % methanol) at 350 mA for 2 h at 4 ~ After transfer, nitrocellu- lose membranes were equilibrated in TBST (25 mmol/L Tris, p H = 8. 0, 125 mmol/L NaC1, and 0 . 1 % Tween 20) for 10 rain at room temperature and blocked with TBST containing 4 ~ dry skim milk (Nestle Foods, USA) overnight. The nitrocel- lulose membranes were incubated for 4 h at room temperature with the primary antibodies that were diluted in TBST containing 4 ~ milk (diluted 1 " 200). After washing with TBST, horseradish perox- idase anti-goat-conjugated secondary antibody diluted in TBST containing 4 ~ milk (1 " 10 000) was ap- plied for 1 h. The blots were washed once with TB- ST for 10 rain and then twice with TBST for 10 min. Bound horseradish peroxidase was detected us- ing ECL reagents by following the manufacturer's in- structions. 1 .5 RT-PCR Analysis 1.5. 1 RT Total RNA was isolated from RAW264. 7 cells by using RNAeX reagent by follow- ing manufacturer's instructions. 4 t~g RNA was mixed with 20 U mlv, 3 ttl of 10 mmol/I, dNTP, 1 t~l of oligo ( d T ) and 5 t~l of 5 • buffer, the reaction was performed at 42 'C, 1 h, at 95 'C, 5 min. cD- NA was stored at --20 C for later use. 1.5. 2 PCR cDNA (2 tA) was mixed with 2.5 tA of 10• 0. 5 t~l of 10 mmol/L dNTP, 2.5 ttl of 25 mmol/L MgC12, 1 ~1 of 10 pmol/t~l 5'primer and 3'primer, and 0. 5 U Taq DNA Polymerase, then water was added to the 25 t~l for reaction. The condition for VLDLR was as follows: for step 1, 95 C , 3 min, for step 2, 95 "C, 30 s; at 62 C , 30 s; 72 ~ 45 s, for 32 cycles; for step 3, 72 ~C, 10 min. The condition for GAPDH was as follows: for step 1, 95 ~ 3 min, for step 2, 95 ~ 30 s; 62 C , 30 s;72 C , 45 s, for 28 cycles; for step 3, 72 ~C, 10 rain. The products from the PCR were exam- ined by 1.5 ~ agarose gel electrophoresis with ethid- ium bromide (EB), and normalized by comparison to RT-PCR of mRNA of GAPDH, a constitutively ex-

pressed gene. Each EB-stained band was quantified using GIS gel analysis software.

2 RESULTS

2. 1 Effect of VLDL on E R K 1 / 2 Activity To determine whether VLDL stimulates MAP

kinase, RAW264. 7 cells were treated with increas- ing concentration of VLDL (25 /tg/ml, 50 ~g/m[, 100/zg/ml) for 5 rain and with VLDL ( 100 ~g/ml) for various time (5--60 rain). Total cell protein was extracted and subject to Western Blot analysis for the phosphorylation of MAP kinase isoforms ERK1/2 in RAW264.7 cells. Figure 1 shows that VLDL acti- vates ERK1/2 in a dose-responsive fashion at concen- trations ranging from 25--100 ttg/ml (lane 1--4). VLDL induced a time-dependent activation of ERK1/2 with an obvious response noted after 5 min and lasting for at least 60 rain (lane 4--8).

1 2 3 4 5 6 7 8

p-ERKI p-ERK2

0 25 50 100 100 100 100 100VLDL(gg/ml) 5 5 5 5 10 15 30 60 Time(rain)

Fig. 1 VLDL activates ERK1/2 in RAW264.7 cells

2. 2 Effect of PKC on VLDL Induced- E R K 1 / 2 Activity

Protein kinase C was shown to be activated by VLDL in several cell lines. To determine whether PKC is involved in VLDL-induced MAPK kinase ac- tivation in RAW264.7 cells, the cells were pretreated with different concentrations (1/~mol/L, 5/~mol/L, 10 ptmol/L) of GF 109203X, a protein kinase C in- hibitor, for 1 h, and then incubated with VLDL (100 t~g/ml) for 10 rain. The data in fig. 2 show that, as expected, the inhibitor substantially blocks VLDL-induced MAP kinase activation, suggesting that VLDL stimulates MAP kinase in a protein ki- nase C-dependent manner.

+ + + + VLDL( 100 ~ g/ml) 10 5 1 + GF109203X( ~ tool/L)

ERKI ERK2

Fig. 2 Protein kinase C mediates VLDL-induced ERKI/2 activity

2 . 3 Roles of PKC, MAPK, PKA Pathway in VLDL-Induced VLDL Receptor mRNA Expression

To determine which kinase pathway is involved in VLDL-induced VLDL receptor message expres-

WANG Yan et al. VLDLR Transcription by VLDL 99

sion, the RAW264. 7 cells were pretreated with MEK1 inhibitor PD98059, p38 kinase inhibitor SB203580, PKC inhibitor GF 109203X or cAMP analogue 8-bromo-cAMP, respectively for 1 h, fol- lowed by 24-h VLDL incubation. Total RNA was isolated and the level of VLDI. receptor mRNA was analyzed by RT-PCR. The result showed that PD98059 and GF 109203X markedly reduced VI.DL receptor message. Fig. 3a is representative of four separate RT-PCR. Fig. 3b is the normalized VLDL mRNA level and shows that PD98059-treated cells contained only about 50 ~ of VLDL receptor mes- sage of VLDL-induced cells. GF 109203X-treated cells contained only about 55 ~ of VLDL receptor message of VLDI.-induced cells. While SB203580 at effective concentrations (10 /~mol/L--30 /~mol/I.) and 8-bromo-cAMP at effective concentrations (0. 2 retool/L--1.2 mmol/L ) had no or little effect (only high concentration groups are shown). On the basis of these data, we are led to conclude that the MEK/ ERK1/2 cascade involved PKC is the major signaling pathway for VLDL-induced VLDI. receptor tran- scription.

bp 1 2 3 4 5 6 7

VLDL (320bp)

GAPDH

(520bp)

Z

V : VLDL

l ' 0 ~ ~ l l l ~ ! j

~ 0 . 8 ~o. 6

0.4 ~0.2 ,,,. 0

Control V V+pd V+gf V+sb V+8-br

Fig. The effects of PD98059, GF109203X, SB203580, 8-Br-cAMP on VLDL induc- tion of VLDLR mRNA expression Upper: RT-PCR results Lane: 1. DNA Marker; 2. Control cells 3. VLDL (100 ~tg/ml); 4. VLDL + PD98059 ( 10 t~M ); 5. VLDL + GF109203X (1 t~M ); 6. VLDL -k SB203580 (30 pM); 7. VLDL -k 8-Br- cAMP (1.2 retool/L) Lower: The normalized VLDLR mRNA levels

3 DISCUSSION

The VLDL receptor is thought to be involved in the pathogenesis of atherosclerosis. VLDL receptor is expressed in smooth muscle cells and macrophages of atherosclerotic lesions and VLDL receptor mRNA level are very high in atherosclerotie lesions cz'3] Moreover, the VLDL receptor, unlike LDL recep- tor, is not down-regulated by excess LDL or VLDL and apoE-containing lipoproteins are accumu- lated without any restriction CgJ. Our previous study showed that VLDLR mRNA is upregulated by VLDIJVI.DL and this regulation is important in foam cell formation. In present study, we investigat- ed the signal mechanism involved in VLDL-induced VLDL receptor transcription.

It has been reported that in hepatoma and BeWo cells, two cis-elements of the VLDLR promoter--an inverted CCAAT box and site I')--binds transcrip- tional factor NF-Y and C/EBPI3, which may be im- portant for transcriptional regulation of VI.DL recep- tor E~3. Since NF-Y and C/EBP/3 are the phosphory- lated substrates of ERK1/2 E12"laJ and studies about the smooth muscle cell (SMC) proliferation showed that VLDL and VLDL hydrolysis-derived fatty acids activated MAP kinases ~4~. Our studies, for the first time, showed that VLDL activated RAW264.7 cells ERK kinase in a protein kinase C-dependent manner. Furthermore, we are interested in the role of MAPK signal pathway in VLDI.-induced VLDL receptor transcription. Using inhibitors of different signal pathways, we demonstrated that PD 98059 (in- hibitor of MEK) and GF 109203X (inhibitor of PKC) effectively reduced VLDL activation of VLDL receptor mRNA, suggesting that VLDL induced- VLDL transcription is mediated by VLDI. stimulat- ing P K C / E R K 1 / 2 signal pathway, which may acti- vate related transcriptional factors and increase VLDLR transcription.

The cAMP-dependent protein kinase ( P K A ) has been reported to downregulate the trophoblast VI.DL receptor. Treatment of JEG-3 and BeWo cells with 8-bromo-cAMP caused a substantial suppression of VI.DI. receptor message ~6~. Moreover, increasing cAMP concentration may increase, decrease, or have no effect on ERK activity depending on cell type E12'~33, indicating that VLDL activates p38 activi- ty in SMC cells. We also examined the role of PKA and p38 in VLDL regulation of VLDL receptor mR- NA expression, we found that 8-bromo-cAMP and SB203580 had no effect on VLDL receptor mRNA expression.

MAP kinase-mediated signal transduction path- ways is the main signal transduction system con- tributing to cell growth and differentiation. Accumu- lating evidence indicated that ERK1/2 might play an important role in atherogenesis and there are several reasons for this: ERK protein levels in protein ex- tracted from atherosclerotic lesions were 2- to 3- folds

100 Journal of Huazhong University of Science and Technology [Med Sci] 23 (2)~ 97-100, 2003

higher, than the vessels of chow-fed rabbits, and their activities were elevated 3- to 5-folds over those of the normal vessels r:4~. Proliferation of vascular SMCs is a hallmark in the pathogenesis of atherosclerosis, VLDL, ~ V L D L , LDL and oxidized LDL are mito- genie to SMCs and have been demonstrated to induce SMCs proliferation via ERK signal pathways. The inhibitor of E R K 1 / 2 , PD98059 inhibits proliferation of SMCs C~5' ~6~. Moreover, ERK signal transduetion pathway is involved in expression of lipoprotein re- ceptors, such as LDL receptor and MSR ~17' as3. Our study provided, for the first time, the evidence that VLDL stimulates ERK activity and upregulates the mRNA expression of VLDL receptor.

REFERENCES

1 Sadao T, Jinya S, Mitsuyuki K et al. Enhancement of the binding of triglyceride-rich lipoproteins to the very low density lipoprotein receptor by apolipoprotein E and lipoprotein lipase. J Biol Chem, 1995,270(26), 15747

2 Hiltunen T P, Luoma J S, Nikkari T eta/. Expression of LDL receptor, VLDL receptor, LDL receptor-related protein, and scavenger receptor in rabbit atherosclerotic lesions: marked induction of scavenger receptor and VLDL receptor expression during lesion development. Circulation, 1998,97(11) : 1079

3 Kosaka S, Takahashi S, Masamura K et al. Evidence of macrophage foam cell formation by very low-density lipoprotein receptor, interferon-gamma inhibition of very low-density [ipoprotein receptor expression and foam cell formation in macrophages. Circulation, 2001,103 (8), 1142

4 Ioanna G B, Heiner K, Hans Vet a/. Effects of authen- tic and VLDL hydrolysis-derived fatty acids on vascular smooth muscle cell growth. British Journal of Pharma- cology, 2001,132 (8) : 1725

5 Kreuter R, Sou,at A K, Wade D P. Transcription fac- tors CCAAT/enhancer-binding protein beta and nuclear factor-Y bind to discrete regulatory elements in the very low density lipoprotein receptor promoter. J Lipid Res, 1999,40(3):376

6 Wittmaack F M, Gafvel M E et al. Location and Regula- tion of the human very low density Iipoprotein/ apolipoprotein-e receptor: trophoblast expression predicts a role for the receptor in placental lipid transport. En- docrinology, 1995,136 (1) : 340

7 ~ - ~ - , ~ . ~ , ~ - ~ . ~ t ~ t g , ~ ' ~ ' l ~ t ~ ' g # .

~ ~P 4it, 1995,24(3).169 8 Lowry O H, Rosebrough N J, Farr A L a al. Protein

measurement with the Folin phenol reagent. J Biol Chem, 1951,193,265

9 Wada Y, Homma Y, Nakazato K et al. Effect of overex- pre.~sion of very low density lipoprotein receptor on cell growth. Heart Vessels, 2000,15(2),74

10 Marziali G, Perrotti E, Ilari R et al. The activity of the CCAAT-box binding factor NF-Y is modulated through the regulated expression of its A subunit during mono- cyte to macrophage differentiation, regulation of tissue- specific genes through a ubiquitous transcription factor. Blood, 1999,93(2) .519

11 Wood J L, Russo A F. Autoregulation of cell-specific MAP kinase control of the tryptophan hydroxylase pro- moter. J Biol Chem, 2001,276(24) :21262

12 Hafner S, Adler H S, Mischak H et al. Mechanism of inhibition of Raf-1 by protein kinase A. Mol Cell Biol, 1994,14..6696

13 Grewal S S, Horgan A M, York R D et al. Neuronal calcium activates a Rapl and B-Raf signaling pathway via the cyclic adenosine monophosphate-dependent pro- tein kinase. J Biol Chem, 2000,275,3722

14 Yanhua H, Hermann Dietrich, Bernhard Metzler et al.

Hyperexpression and activation of extracellular signal- regulated kinases (ERKI/2) in atheroscierotic lesions of cholesterol-fed rabbits. Arteriosc[er Thromb Vase Biol, 2000,20.18

15 Kusuhara M, Chair A, Cadet Aet al. Oxidized LDL stimulates mitogen-activated protein kina~s in smooth muscle cells and macrophages. Arterioscler Thromb Vase Biol, 1 9 9 7 , 1 7 ( 1 ) . 1 4 1

16 Zhao D, Letterman J, Schreiber B M. Beta-Migrating very low density lipoprotein (beta VLDL) activates smooth muscle cell mitogen-activated protein (MAP) ki- nase via G protein-coupled receptor-mediated transactiva- tion of the epidermal growth factor (EGF) receptor: ef- fect of MAP kinase activation on beta VLDL plus EGF- induced cell proliferation. J Biol Chem, 2001,276 (33) �9 30579

17 Cong L, Fredric B K, Thomas E et al. Induction of low- density lipoprotein receptor (LDLR) transcription by on- costatin M is mediated by the extracellular signal-regulat- ed kinase signaling pathway and the repeat 3 element of the LDLR promoter. J Biol Chem, 1999,274(10):6747

18 Hsien-Yeh H, Yuh-Ching T. Tumor necrosis factor-a- mediated protein kinases in regulation of scavenger recep- tor and foam cell formation on macrophage. J Biol Chem, 2000,275(52) ,41035

(Received Dec. 12, 2002)