HMGCoA reductase inhibition partially mediates tumor necrosisfactor a-induced apoptosis in human U-937 and HL-60 cellsq

Germ�aan Gallardo,a,* F�eelix L�oopez-Blanco,b Carlos M. Ruiz de Galarreta,a

and Luisa F. Fanjula

a Departamento de Bioqu�ıımica, Biolog�ııa Molecular y Fisiolog�ııa, Edificio de Ciencias de la Salud, Universidad de Las Palmas de Gran Canaria,

Las Palmas de Gran Canaria 35016, Spainb Departamento de Ciencias Cl�ıınicas, Secci�oon de Farmacolog�ııa, Edificio de Ciencias de la Salud, Universidad de Las Palmas de Gran Canaria,

Las Palmas de Gran Canaria 35016, Spain

Received 14 November 2002

Abstract

We have examined the effects of tumor necrosis factor a (TNFa) and its second messenger, ceramide, on HMGCoA reductase,

the rate-limiting enzyme in the mevalonate pathway. Treatment of human U-937 and HL-60 cells with TNFa or C2-ceramide

inhibited both expression and activity of HMGCoA reductase in a time-dependent manner. Maturation of p21ras was also inhibited

in a mevalonate-dependent fashion. The addition of mevalonate to both U-937 and HL-60 cells could also partially prevent TNFaand ceramide-induced apoptosis. These results support the hypothesis that the inhibition of HMGCoA reductase expression and the

subsequent decrease in prenylation of proteins such as p21ras are part of the mechanism by which TNFa induces apoptosis in these

cells.

� 2002 Elsevier Science (USA). All rights reserved.

Keywords: Tumor necrosis factor a; Ceramide; HMGCoA reductase; Apoptosis; Isoprenylation; Ras proteins

The rate-limiting enzyme in the mevalonate pathway

is 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA)

reductase (EC 1.1.1.34) that catalyzes the synthesis of

mevalonic acid from HMGCoA [1]. The enzyme is

regulated at multiple [2], transcriptional, translational,

and post-translational levels by signals that involve

sterol and non-sterol metabolite feedback. Sterols re-press transcription of HMGCoA reductase gene while

both non-sterol and sterol metabolites regulate its

translation and degradation rates. Sterols and its me-

tabolites may also induce changes in the enzyme activity

by reversible phosphorylation. In addition, a number of

mitogenic and non-mitogenic signals including phorbol

esters [3], insulin [4], interleukin-1b [5], platelet-derived

growth factor [6], and epidermal growth factor [4] have

been reported to modify HMGCoA reductase activity at

several levels.

Compelling evidences support the critical role of

mevalonic acid availability in basic cellular processes

including growth, differentiation, and apoptosis. Me-

valonate is the precursor for the synthesis of all isopre-

noids [2] and prenyl groups required for post-translational modification of several key proteins in-

cluding the small GTP-binding protein superfamily,

nuclear lamins, and heterotrimeric G-proteins [7].

Isoprenylation is a mandatory step for the association of

p21ras to membranes that allows the activation of PI 3-

kinase and the PKB/Akt survival pathway [8,9].

Tumor necrosis factor a (TNFa) produced by acti-

vated macrophages is a cytokine that influences growth,differentiation, and apoptosis processes in most cell

types [10] and plays important roles in virus inactivation

responses and inflammation. The TNFa effects are ini-

tiated upon binding to the p55 receptor coupled to hy-

drolysis of sphingomyelin to yield ceramide [11] by two

Biochemical and Biophysical Research Communications 300 (2003) 397–402

www.elsevier.com/locate/ybbrc

BBRC

qAbbreviations: TNFa, tumor necrosis factor a; HMGCoA, 3-

hydroxy-3-methylglutaryl coenzyme A; C2-cer, N-acetylsphingosine.* Corresponding author. Fax: +34-928-451-441.

E-mail address: ggallardo@pas.ulpgc.es (G. Gallardo).

0006-291X/02/$ - see front matter � 2002 Elsevier Science (USA). All rights reserved.

doi:10.1016/S0006-291X(02)02846-2

different SMases [12]. Among others, ceramide serves asa second messenger for TNFa apoptotic effects [13].

Using U-937 and HL-60 cells, two promyelocytic

leukemia cell lines, we sought to investigate if changes in

HMGCoA reductase expression and/or activity and

p21ras processing may occur in response to TNFa and

ceramide, as a part of the mechanism underlying the

induction of apoptosis by this cytokine in both cell lines.

Materials and methods

Materials. N-Acetylsphingosine (C2-cer) was obtained from Bio-

mol (Plymouth Meeting, PA, USA) and tumor necrosis factor a(TNFa) was from Boehringer–Mannheim (Mannheim, Germany).

Lovastatin was a gift of Merck Sharp and Dohme Laboratories

(USA). ½14C�HMGCoA (57.5mCi/mmol) and ½3H�mevalonolactone

(33Ci/mmol) were obtained from Du Pont-New England Nuclear (Bad

Homburg, Germany). Trans35S-label (1175Ci/mmol) was purchased

from ICN Biomedicals (Irvine, CA, USA). Monoclonal pan-ras anti-

body was obtained from Oncogene Science (Cambridge, MA, USA)

and anti-lamin B antibody was from Santa Cruz Biotechnology (Santa

Cruz, CA, USA). Mevalonolactone, Hoechst no. 33258, and all other

reagents were obtained from Sigma Chemical (St. Louis, MO, USA).

Cell culture. Stock cultures of U-937 and HL-60 cell lines were

routinely grown in RPMI 1640 medium (Gibco, Grand Island, NY,

USA) supplemented with 10% v/v FBS (Biowhittaker, Walkersville,

USA), LL-glutamine (2mM), 1.12 g/l NaHCO3, and antibiotics peni-

cillin (100U/ml) and streptomycin (100lg=ml) at 37 �C in an atmo-

sphere of 5% CO2.

Assay of HMGCoA reductase activity. The assay was performed in

serum-deprived cells pretreated overnight with lovastatin. At the end

of the experiments cells were washed and incubated with medium for

15min to deplete the cells of lovastatin [14]. Cells were lysed and

thereafter a 50lg aliquot of each sample was preincubated for 30min

at 37 �C in assay buffer (0.1mM phosphate, pH 7.5, 5mMDTT). Next,

a reaction mixture containing ½14C�HMGCoA (100,000 dpm),

½3H�mevalonolactone (4000 dpm), 2.5mM NADPH, 30mM glucose-

6-phosphate, and 3U/ml glucose-6-phosphate dehydrogenase (final

concentrations) was added [15]. The reaction was continued for 30min

at 37 �C and stopped by the addition of HCl (0.5N final concentra-

tion). Aliquots of each sample were applied to a column (1ml) of Bio-

Rex 5 [16]. ½14C�Mevalonate and the internal standard ½3H�mevalonate

were eluted with 2ml water and radioactivity was determined in a

Wallac liquid scintillation counter.

Reverse transcriptase-polymerase chain reaction. One lg of total

RNA from each sample was incubated for 75min at 42 �C in 20llreaction mixture for the synthesis of cDNA. Aliquots of cDNA were

used as template for PCR with: human HMGCoA reductase sense

primer (50-TACCATGTCAGGGGTACGTC-30) and antisense primer

(50-CAAGCCTAGAGACATAATCATC-30) or a commercial glycer-

aldehyde-3-phosphate dehydrogenase (G3PDH) sense and antisense

primers (Clontech, Palo Alto, CA, USA). Reaction conditions were

1min at 94 �C, 1.5min at 58 �C, and 1.5min at 72 �C for 22 cycles

(HMGCoA reductase) or 18 cycles (G3PDH). The amplified products

were resolved by 1.8% agarose gel electrophoresis and transferred to

positively charged nylon membranes. Chemiluminescent detection was

performed with a commercially available DIG luminescent detection

kit (Boehringer–Mannheim) and the membranes were exposed to Po-

laroid 667 instant film (Sigma Chemical).

Cellular apoptosis studies. To assess the existence of DNA ladder-

ing, low molecular weight DNA was isolated and subjected to DIG-11-

dUTP end labeling to allow chemiluminescence detection and there-

after DNA samples were resolved by 1.8% agarose gel electrophoresis

as described [17].

Morphological alterations of apoptotic nuclei were visualized by

staining with DNA-binding fluorochrome bisbenzimide (Hoechst no.

33258) as described [18].

Lamin B and its cleaved fragment were detected using an anti-la-

min B antibody at a 1:5000 dilution and a secondary antibody at a

same dilution to allow visualization by an ECL detection system

(Amershan Pharmacia Biotech).

Analysis of post-translational processing of p21ras. It was performed

with extracts obtained from HL-60 cells ð1� 107Þ that were preincu-

bated for 2 h in methionine-free RPMI supplemented with 2% BSA,

treated for 24 h, and pulsed with ½35S�methionine (50lCi=ml Trans35S-

Label) for the last 22 h. The cells were thereafter washed with PBS and

sonicated in 20mM Tris–HCl, pH 7.5, 250mM sucrose, 1.2mM

EGTA, 50mM b-mercaptoethanol, and 100mM PMSF. Cytosolic

fractions were obtained by centrifugation at 100,000g for 30min in a

Beckman TLA 100. Aliquots of each sample were immunoprecipitated

with pan-ras antibody and Ras proteins were resolved by 15% SDS–

PAGE, visualized in a screen storage system (Molecular Dynamics,

Sunnyvale, CA, USA), and quantified with the software provided by

the manufacturer.

Results

TNFa and ceramide inhibited HMGCoA reductase activ-ity in U-937 and HL-60 cells

Because HMGCoA reductase basal activity is de-

pressed by serum and to maximally induce the reductaseactivity, U937 and HL-60 cells were serum-deprived and

treated overnight with lovastatin (20 and 10lM, re-

spectively), a potent competitive inhibitor of HMGCoA

reductase [19] that at micromolar concentrations inhib-

its mevalonate synthesis and induces both expression

and activity of HMGCoA reductase. Under these con-

ditions of maximal induction, 30 ng/ml of TNFa inhib-

ited in a time-dependent manner the HMGCoAreductase activity in U-937 cultures (Fig. 1), reaching an

80% inhibition after 6 h treatment. Likewise, addition of

20lM cell permeable analogue C2-ceramide produced a

similar inhibition in reductase activity (Fig. 1).One of a

series of representative equivalent experiments per-

formed on HL-60 cells is also presented in Fig. 1 (inset),

whereas a 50% inhibition in HMGCoA reductase ac-

tivity may be observed after 24 h treatment with 6lMC2-ceramide.

TNFa and ceramide inhibited HMGCoA reductase

mRNA levels in U-937 and HL-60 cells

Since changes in HMGCoA reductase activity might

be attributed to a decrease in the reductase gene tran-

scription rate, we next measured HMGCoA reductase

mRNA levels by RT-PCR. As expected (Fig. 2, left pa-nel), overnight lovastatin treated and serum-deprived U-

937 cells had several fold higher levels of HMGCoA re-

ductase mRNA than serum-cultured cells. In agreement

with the activity experiments, treatment with 30 ng/ml

TNFa decreased HMGCoA reductase mRNA levels

398 G. Gallardo et al. / Biochemical and Biophysical Research Communications 300 (2003) 397–402

nearly by 75% of the lovastatin control during the firsthour and by 90% at 6 h. An 80% inhibition in reductase

mRNA levels was also obtained after 6 h of treatment

with C2-ceramide (20lM). Equivalent experiments car-

ried out with HL-60 cells produced similar results. As

shown in Fig. 2 (right panel) treatment with C2-ceramide

(6lM) for 24 h inhibited HMGCoA reductase mRNA

levels by about 65% of the lovastatin control.

TNFa and ceramide inhibited p21ras maturation in HL-60cells

Isoprenylation of proteins is a process controlled by

the availability of mevalonic acid. When prenylation is

inhibited, the association of Ras proteins to the plasmamembrane is impaired, causing non-prenylated Ras to

accumulate in the cytosolic fraction. Unmodified cyto-

solic Ras protein may then be visualized in SDS–PAGE

as a band that migrates with an apparently higher mo-

lecular weight than the farnesylated Ras protein [20]. As

shown in Fig. 3 and in agreement with previously de-

scribed data, lovastatin, that was used as a positive

control, produced an accumulation of immature Ras of15-fold over the levels of control untreated cells, while

TNFa or C2-ceramide decrease in HMGCoA reductase

activity resulted in inhibition of p21ras processing,

causing immature Ras to accumulate 10- or 6-fold. The

addition of mevalonate reverted the effects of lovastatin,

TNFa, and C2-ceramide by 94%, 68%, and 78%, re-

spectively.

Mevalonate partially reverted TNFa and ceramide-

induced apoptosis in U937 and HL60 cells

TNFa and ceramide induce apoptosis in U937 and

HL-60 cells [21], therefore we next sought to investigate

if the inhibition of HMGCoA reductase by the cytokine

and its messenger was related to the apoptotic response

Fig. 3. TNFa and ceramide inhibition of p21ras maturation in HL-60

cells. HL-60 (10� 106) cells were treated with lovastatin ð10lMÞ,TNFa (30 ng/ml), or C2-ceramide ð6lMÞ, with or without mevalonate

(20mM) for 24 h and pulsed with [35S]methionine. Cytosolic fractions

were isolated and immunoprecipitated with an anti-pan-ras antibody

and proteins were resolved by SDS–PAGE as described under ‘‘Ma-

terials and methods.’’ The lower panel shows quantization of the

protein bands.

Fig. 2. TNFa and ceramide decrease HMGCoA reductase mRNA levels in U937 and HL-60 cells. U937 cells (left panel) or HL-60 cells (right panel)

were serum-deprived and treated with lovastatin (20 and 10lM, respectively) overnight to maximally induce HMGCoA reductase. TNFa (30 ng/ml)

or C2-ceramide ð20lMÞ was added to U937 cells. C2-ceramide ð6lMÞ was added to HL-60 cells. The lower panel shows quantization of HMGCoA

reductase mRNA against G3PDH mRNA levels. One out of three representative experiments is shown.

Fig. 1. TNFa and ceramide inhibit HMGCoA reductase activity in

U937 and HL-60 cells. U937 cells ð5� 106Þ were serum-deprived and

pretreated with 20lM lovastatin overnight. TNFa (30 ng/ml circles) or

C2-ceraminde (20lM, squares) was added during 1, 3, or 6 h. In the

inset an equivalent experiment was performed with HL-60 cells treated

with C2-ceramide ð6lMÞ. HMGCoA reductase was measured as de-

scribed in ‘‘Materials and methods.’’ The values shown are

means� SD of triplicate determinations. One out of three represen-

tative experiments is shown.

G. Gallardo et al. / Biochemical and Biophysical Research Communications 300 (2003) 397–402 399

of these cells. Fig. 4, upper panel, shows that treatment

of U937 cells for 6 h with lovastatin, TNFa or C2-

ceramide increases the number of cells that exhibit ap-

optosis related morphological changes in their nuclei

visualized by bisbenzimide staining. When mevalonatelevels were restored, apoptosis was completely reverted

in lovastatin treated cells and partially prevented in the

cells treated with TNFa or C2-ceramide. In the middle

panel of the same figure, increased DNA fragmentation

can be observed in U937 cells in response to lovastatin,

TNFa, and ceramide. As in the previous experiment

mevalonate prevented apoptosis distinctive DNA lad-

dering in response to lovastatin, TNFa, and C2-cera-mide.

Lamin proteolysis during apoptosis has been re-

ported in various cell lines [22–24], including HL60 cells

[25–27] that primarily express lamin B. Fig. 4, lower

panel, shows a representative lamin B Western blot

performed with proteins obtained from HL60 cells

treated with lovastatin, TNFa, and C2-ceramide with or

without mevalonate for 24 h. The lamin B 45 kDa pro-teolytic fragment was present in extracts from cells

treated with lovastatin as well as with TNFa and cera-

mide. Mevalonate completely reverted lovastatin and

partially counteracted TNFa and C2-ceramide effects.

Discussion

We herein present results that show for the first time

that TNFa downregulates both expression and activity

of HMGCoA reductase in U-937 and HL-60 cells. Both

cell lines are well-known model systems for the study of

the apoptotic effects of TNFa [28,29]. Also in U-937 and

HL-60 cells as in many others, TNFa induces sphingo-

myelin hydrolysis to ceramide [30,31], the intracellular

messenger that mediates several cellular responses toTNFa including apoptosis [13]. This seems to be the

case for the TNFa effects on HMGCoA reductase in U-

937 and HL-60 cells because treatment with C2-cera-

mide, a cell-permeant ceramide analogue, results in a

similar inhibition of the mRNA and enzyme activity

levels. In sharp contrast with the above-described re-

sults, both TNFa and ceramide induced the expression

and increased the activity of HMGCoA reductase inhuman hepatoma HepG2 cells (data not shown). This

discrepancy comes not as a surprise since in human

hepatocytes [32] ceramide induces maturation of

SREBP-1 (sterol regulatory element (SRE)-binding

protein-1), a transcription factor that activates both

LDL receptor and HMGCoA reductase gene tran-

scription [33]. Moreover, TNFa stimulates DNA syn-

thesis in hepatocytes [34] and is implicated in hepaticregeneration processes [35,36], while in U-937 and HL-

60 cell lines the cytokine acts as a potent inductor of

apoptosis [21].

We have also shown in this work that the inhibition

of HMGCoA reductase activity caused by TNFa and

ceramide may be correlated to a decrease in p21ras

maturation and to the induction of apoptosis because

both effects can be partially prevented by mevalonate.This is in agreement with previous reports showing that

in HL-60 cells inhibition of HMGCoA reductase acti-

vity with lovastatin results in apoptosis that may be

attributed to impairment in the p21ras processing [37].

The mechanisms underlying cellular apoptotic demise

are not completely understood [38]. In general, it can be

assumed that the cell fate at any given moment of its life

Fig. 4. Mevalonate effects on TNFa and ceramide-induced apoptosis

in U937 and HL-60 cells. Triplicates of 5� 105 U937 cells were treated

for 6 h with lovastatin ð20lMÞ, TNFa (30 ng/ml), and C2-ceramide

ð20lMÞ with or without mevalonate (20mM), and (A) apoptotic nu-

clei were evaluated by fluorescence microscopy or (B) DNA frag-

mentation was assessed as described under ‘‘Materials and methods.’’

(C) shows a representative lamin Western blot of proteins obtained

from HL-60 cells treated with lovastatin ð10lMÞ, TNFa (30 ng/ml),

and C2-ceramide ð6lMÞ with or without mavalonate (20mM) for 24 h.

400 G. Gallardo et al. / Biochemical and Biophysical Research Communications 300 (2003) 397–402

cycle would be the result of a balance between the effectsof multiple pro/anti-apoptotic and pro/anti-mitogenic

signals [38]. Prenylation of proteins plays a crucial role

in the maintenance of nuclear structures implicated in

pro-mitogenic pathways, because it ensures proper

function of the modified protein, facilitating its mem-

brane association and/or contributing to specific pro-

tein–protein interactions (reviewed in [39]).

Isoprenylation of p21ras results in the protein associ-ation to membranes that in turn allows binding of PI

3-kinase and activation of the PKB/Akt survival path-

way [8,9]. Thus a decrease in mevalonic acid availability

that would impair p21ras maturation [40] would also

disrupt PKB/Akt activation. Lovastatin that inhibits

HMGCoA reductase and thereby protein isoprenylation

also inhibits PI 3-kinase activity in PDGF stimulated

NIH-3T3 cells [41]. Likewise simvastatin, anotherHMGCoA reductase inhibitor, inhibits both isopreny-

lation of Ras protein and PI 3-kinase activity in L6

myoblast [42].

We have provided experimental evidences to support

the hypothesis that in U937 and HL-60 cells, TNFainhibits the expression and activity of HMGCoA re-

ductase. We have also shown that the predictable de-

crease in mevalonate availability after HMGCoAreductase inhibition causes accumulation of p21ras im-

mature forms in the cytosol and most likely apoptosis. It

is open to future studies if PI 3-kinase and/or PKB ac-

tivation are also prevented as a result of TNFa effects on

HMGCoA reductase activity and p21ras processing.

Would this be true, interruption of the PI 3-kinase/PKB/

Akt survival pathway as a result of p21ras incomplete

maturation might be a part of the mechanism by whichthe cytokine and its messenger induce apoptosis in U937

and HL-60 cells.

Acknowledgment

This work was supported by DGICYT: PM95-0131.

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