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HMGCoA reductase inhibition partially mediates tumor necrosis factor α-induced apoptosis in human U-937 and HL-60 cells
BBRC Biochemical and Biophysical Research Communications 300 (2003) 397–402 www.elsevier.com/locate/ybbrc
HMGCoA reductase inhibition partially mediates tumor necrosis factor a-induced apoptosis in human U-937 and HL-60 cellsq Germ an Gallardo,a,* Felix L opez-Blanco,b Carlos M. Ruiz de Galarreta,a and Luisa F. Fanjula a
Departamento de Bioquımica, Biologıa Molecular y Fisiologıa, Edificio de Ciencias de la Salud, Universidad de Las Palmas de Gran Canaria, Las Palmas de Gran Canaria 35016, Spain b Departamento de Ciencias Clınicas, Secci on de Farmacologıa, 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 TNFa and 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. Ó 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 repress transcription of HMGCoA reductase gene while both non-sterol and sterol metabolites regulate its translation and degradation rates. Sterols and its metabolites 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 q Abbreviations: TNFa, tumor necrosis factor a; HMGCoA, 3hydroxy-3-methylglutaryl coenzyme A; C2-cer, N-acetylsphingosine. * Corresponding author. Fax: +34-928-451-441. E-mail address: [email protected] (G. Gallardo).
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. Mevalonate is the precursor for the synthesis of all isoprenoids [2] and prenyl groups required for posttranslational modification of several key proteins including 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 3kinase and the PKB/Akt survival pathway [8,9]. Tumor necrosis factor a (TNFa) produced by activated 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 initiated upon binding to the p55 receptor coupled to hydrolysis of sphingomyelin to yield ceramide [11] by two
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(02)02846-2
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different SMases [12]. Among others, ceramide serves as a 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 Biomol (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). ½14 CHMGCoA (57.5 mCi/mmol) and ½3 Hmevalonolactone (33 Ci/mmol) were obtained from Du Pont-New England Nuclear (Bad Homburg, Germany). Trans35 S-label (1175 Ci/mmol) was purchased from ICN Biomedicals (Irvine, CA, USA). Monoclonal pan-ras antibody 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), L -glutamine (2 mM), 1.12 g/l NaHCO3 , and antibiotics penicillin (100 U/ml) and streptomycin (100 lg=ml) at 37 °C in an atmosphere 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 15 min to deplete the cells of lovastatin [14]. Cells were lysed and thereafter a 50 lg aliquot of each sample was preincubated for 30 min at 37 °C in assay buffer (0.1 mM phosphate, pH 7.5, 5 mM DTT). Next, a reaction mixture containing ½14 CHMGCoA (100,000 dpm), ½3 Hmevalonolactone (4000 dpm), 2.5 mM NADPH, 30 mM glucose6-phosphate, and 3 U/ml glucose-6-phosphate dehydrogenase (final concentrations) was added [15]. The reaction was continued for 30 min at 37 °C and stopped by the addition of HCl (0.5 N final concentration). Aliquots of each sample were applied to a column (1 ml) of BioRex 5 [16]. ½14 CMevalonate and the internal standard ½3 Hmevalonate were eluted with 2 ml 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 75 min at 42 °C in 20 ll reaction 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 glyceraldehyde-3-phosphate dehydrogenase (G3PDH) sense and antisense primers (Clontech, Palo Alto, CA, USA). Reaction conditions were 1 min at 94 °C, 1.5 min at 58 °C, and 1.5 min 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 Polaroid 667 instant film (Sigma Chemical). Cellular apoptosis studies. To assess the existence of DNA laddering, low molecular weight DNA was isolated and subjected to DIG-11dUTP end labeling to allow chemiluminescence detection and thereafter 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-lamin 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 107 Þ that were preincubated for 2 h in methionine-free RPMI supplemented with 2% BSA, treated for 24 h, and pulsed with ½35 Smethionine (50 lCi=ml Trans35 SLabel) for the last 22 h. The cells were thereafter washed with PBS and sonicated in 20 mM Tris–HCl, pH 7.5, 250 mM sucrose, 1.2 mM EGTA, 50 mM b-mercaptoethanol, and 100 mM PMSF. Cytosolic fractions were obtained by centrifugation at 100,000g for 30 min 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 activity in U-937 and HL-60 cells Because HMGCoA reductase basal activity is depressed by serum and to maximally induce the reductase activity, U937 and HL-60 cells were serum-deprived and treated overnight with lovastatin (20 and 10 lM, respectively), a potent competitive inhibitor of HMGCoA reductase [19] that at micromolar concentrations inhibits mevalonate synthesis and induces both expression and activity of HMGCoA reductase. Under these conditions of maximal induction, 30 ng/ml of TNFa inhibited in a time-dependent manner the HMGCoA reductase activity in U-937 cultures (Fig. 1), reaching an 80% inhibition after 6 h treatment. Likewise, addition of 20 lM cell permeable analogue C2-ceramide produced a similar inhibition in reductase activity (Fig. 1).One of a series of representative equivalent experiments performed on HL-60 cells is also presented in Fig. 1 (inset), whereas a 50% inhibition in HMGCoA reductase activity may be observed after 24 h treatment with 6 lM C2-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 transcription rate, we next measured HMGCoA reductase mRNA levels by RT-PCR. As expected (Fig. 2, left panel), overnight lovastatin treated and serum-deprived U937 cells had several fold higher levels of HMGCoA reductase mRNA than serum-cultured cells. In agreement with the activity experiments, treatment with 30 ng/ml TNFa decreased HMGCoA reductase mRNA levels
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Fig. 3. TNFa and ceramide inhibition of p21ras maturation in HL-60 cells. HL-60 (10 106 ) cells were treated with lovastatin ð10 lMÞ, TNFa (30 ng/ml), or C2-ceramide ð6 lMÞ, with or without mevalonate (20 mM) for 24 h and pulsed with [35 S]methionine. Cytosolic fractions were isolated and immunoprecipitated with an anti-pan-ras antibody and proteins were resolved by SDS–PAGE as described under ‘‘Materials and methods.’’ The lower panel shows quantization of the protein bands.
Fig. 1. TNFa and ceramide inhibit HMGCoA reductase activity in U937 and HL-60 cells. U937 cells ð5 106 Þ were serum-deprived and pretreated with 20 lM lovastatin overnight. TNFa (30 ng/ml circles) or C2-ceraminde (20 lM, 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 ð6 lMÞ. HMGCoA reductase was measured as described in ‘‘Materials and methods.’’ The values shown are means SD of triplicate determinations. One out of three representative experiments is shown.
nearly by 75% of the lovastatin control during the first hour and by 90% at 6 h. An 80% inhibition in reductase mRNA levels was also obtained after 6 h of treatment with C2-ceramide (20 lM). Equivalent experiments carried out with HL-60 cells produced similar results. As shown in Fig. 2 (right panel) treatment with C2-ceramide (6 lM) for 24 h inhibited HMGCoA reductase mRNA levels by about 65% of the lovastatin control. TNFa and ceramide inhibited p21ras maturation in HL-60 cells Isoprenylation of proteins is a process controlled by the availability of mevalonic acid. When prenylation is
inhibited, the association of Ras proteins to the plasma membrane is impaired, causing non-prenylated Ras to accumulate in the cytosolic fraction. Unmodified cytosolic Ras protein may then be visualized in SDS–PAGE as a band that migrates with an apparently higher molecular weight than the farnesylated Ras protein [20]. As shown in Fig. 3 and in agreement with previously described data, lovastatin, that was used as a positive control, produced an accumulation of immature Ras of 15-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%, respectively. Mevalonate partially reverted TNFa and ceramideinduced 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. 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 10 lM, respectively) overnight to maximally induce HMGCoA reductase. TNFa (30 ng/ml) or C2-ceramide ð20 lMÞ was added to U937 cells. C2-ceramide ð6 lMÞ 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.
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dering in response to lovastatin, TNFa, and C2-ceramide. Lamin proteolysis during apoptosis has been reported 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 proteolytic fragment was present in extracts from cells treated with lovastatin as well as with TNFa and ceramide. Mevalonate completely reverted lovastatin and partially counteracted TNFa and C2-ceramide effects.
Discussion
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 ð20 lMÞ, TNFa (30 ng/ml), and C2-ceramide ð20 lMÞ with or without mevalonate (20 mM), and (A) apoptotic nuclei were evaluated by fluorescence microscopy or (B) DNA fragmentation 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 ð10 lMÞ, TNFa (30 ng/ml), and C2-ceramide ð6 lMÞ with or without mavalonate (20 mM) for 24 h.
of these cells. Fig. 4, upper panel, shows that treatment of U937 cells for 6 h with lovastatin, TNFa or C2ceramide increases the number of cells that exhibit apoptosis related morphological changes in their nuclei visualized by bisbenzimide staining. When mevalonate levels 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-
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 sphingomyelin hydrolysis to ceramide [30,31], the intracellular messenger that mediates several cellular responses to TNFa including apoptosis [13]. This seems to be the case for the TNFa effects on HMGCoA reductase in U937 and HL-60 cells because treatment with C2-ceramide, a cell-permeant ceramide analogue, results in a similar inhibition of the mRNA and enzyme activity levels. In sharp contrast with the above-described results, both TNFa and ceramide induced the expression and increased the activity of HMGCoA reductase in human 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 transcription [33]. Moreover, TNFa stimulates DNA synthesis in hepatocytes [34] and is implicated in hepatic regeneration processes [35,36], while in U-937 and HL60 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 activity 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
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cycle would be the result of a balance between the effects of 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 membrane association and/or contributing to specific protein–protein interactions (reviewed in [39]). Isoprenylation of p21ras results in the protein association to membranes that in turn allows binding of PI 3-kinase and activation of the PKB/Akt survival pathway [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, another HMGCoA reductase inhibitor, inhibits both isoprenylation 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, TNFa inhibits the expression and activity of HMGCoA reductase. We have also shown that the predictable decrease in mevalonate availability after HMGCoA reductase inhibition causes accumulation of p21ras immature forms in the cytosol and most likely apoptosis. It is open to future studies if PI 3-kinase and/or PKB activation 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 which the cytokine and its messenger induce apoptosis in U937 and HL-60 cells.
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Acknowledgment This work was supported by DGICYT: PM95-0131.
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HMGCoA reductase inhibition partially mediates tumor necrosis factor a-induced apoptosis in human U-937 and HL-60 cellsq Germ an Gallardo,a,* Felix L opez-Blanco,b Carlos M. Ruiz de Galarreta,a and Luisa F. Fanjula a
Departamento de Bioquımica, Biologıa Molecular y Fisiologıa, Edificio de Ciencias de la Salud, Universidad de Las Palmas de Gran Canaria, Las Palmas de Gran Canaria 35016, Spain b Departamento de Ciencias Clınicas, Secci on de Farmacologıa, 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 TNFa and 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. Ó 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 repress transcription of HMGCoA reductase gene while both non-sterol and sterol metabolites regulate its translation and degradation rates. Sterols and its metabolites 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 q Abbreviations: TNFa, tumor necrosis factor a; HMGCoA, 3hydroxy-3-methylglutaryl coenzyme A; C2-cer, N-acetylsphingosine. * Corresponding author. Fax: +34-928-451-441. E-mail address: [email protected] (G. Gallardo).
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. Mevalonate is the precursor for the synthesis of all isoprenoids [2] and prenyl groups required for posttranslational modification of several key proteins including 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 3kinase and the PKB/Akt survival pathway [8,9]. Tumor necrosis factor a (TNFa) produced by activated 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 initiated upon binding to the p55 receptor coupled to hydrolysis of sphingomyelin to yield ceramide [11] by two
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(02)02846-2
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different SMases [12]. Among others, ceramide serves as a 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 Biomol (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). ½14 CHMGCoA (57.5 mCi/mmol) and ½3 Hmevalonolactone (33 Ci/mmol) were obtained from Du Pont-New England Nuclear (Bad Homburg, Germany). Trans35 S-label (1175 Ci/mmol) was purchased from ICN Biomedicals (Irvine, CA, USA). Monoclonal pan-ras antibody 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), L -glutamine (2 mM), 1.12 g/l NaHCO3 , and antibiotics penicillin (100 U/ml) and streptomycin (100 lg=ml) at 37 °C in an atmosphere 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 15 min to deplete the cells of lovastatin [14]. Cells were lysed and thereafter a 50 lg aliquot of each sample was preincubated for 30 min at 37 °C in assay buffer (0.1 mM phosphate, pH 7.5, 5 mM DTT). Next, a reaction mixture containing ½14 CHMGCoA (100,000 dpm), ½3 Hmevalonolactone (4000 dpm), 2.5 mM NADPH, 30 mM glucose6-phosphate, and 3 U/ml glucose-6-phosphate dehydrogenase (final concentrations) was added [15]. The reaction was continued for 30 min at 37 °C and stopped by the addition of HCl (0.5 N final concentration). Aliquots of each sample were applied to a column (1 ml) of BioRex 5 [16]. ½14 CMevalonate and the internal standard ½3 Hmevalonate were eluted with 2 ml 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 75 min at 42 °C in 20 ll reaction 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 glyceraldehyde-3-phosphate dehydrogenase (G3PDH) sense and antisense primers (Clontech, Palo Alto, CA, USA). Reaction conditions were 1 min at 94 °C, 1.5 min at 58 °C, and 1.5 min 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 Polaroid 667 instant film (Sigma Chemical). Cellular apoptosis studies. To assess the existence of DNA laddering, low molecular weight DNA was isolated and subjected to DIG-11dUTP end labeling to allow chemiluminescence detection and thereafter 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-lamin 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 107 Þ that were preincubated for 2 h in methionine-free RPMI supplemented with 2% BSA, treated for 24 h, and pulsed with ½35 Smethionine (50 lCi=ml Trans35 SLabel) for the last 22 h. The cells were thereafter washed with PBS and sonicated in 20 mM Tris–HCl, pH 7.5, 250 mM sucrose, 1.2 mM EGTA, 50 mM b-mercaptoethanol, and 100 mM PMSF. Cytosolic fractions were obtained by centrifugation at 100,000g for 30 min 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 activity in U-937 and HL-60 cells Because HMGCoA reductase basal activity is depressed by serum and to maximally induce the reductase activity, U937 and HL-60 cells were serum-deprived and treated overnight with lovastatin (20 and 10 lM, respectively), a potent competitive inhibitor of HMGCoA reductase [19] that at micromolar concentrations inhibits mevalonate synthesis and induces both expression and activity of HMGCoA reductase. Under these conditions of maximal induction, 30 ng/ml of TNFa inhibited in a time-dependent manner the HMGCoA reductase activity in U-937 cultures (Fig. 1), reaching an 80% inhibition after 6 h treatment. Likewise, addition of 20 lM cell permeable analogue C2-ceramide produced a similar inhibition in reductase activity (Fig. 1).One of a series of representative equivalent experiments performed on HL-60 cells is also presented in Fig. 1 (inset), whereas a 50% inhibition in HMGCoA reductase activity may be observed after 24 h treatment with 6 lM C2-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 transcription rate, we next measured HMGCoA reductase mRNA levels by RT-PCR. As expected (Fig. 2, left panel), overnight lovastatin treated and serum-deprived U937 cells had several fold higher levels of HMGCoA reductase mRNA than serum-cultured cells. In agreement with the activity experiments, treatment with 30 ng/ml TNFa decreased HMGCoA reductase mRNA levels
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Fig. 3. TNFa and ceramide inhibition of p21ras maturation in HL-60 cells. HL-60 (10 106 ) cells were treated with lovastatin ð10 lMÞ, TNFa (30 ng/ml), or C2-ceramide ð6 lMÞ, with or without mevalonate (20 mM) for 24 h and pulsed with [35 S]methionine. Cytosolic fractions were isolated and immunoprecipitated with an anti-pan-ras antibody and proteins were resolved by SDS–PAGE as described under ‘‘Materials and methods.’’ The lower panel shows quantization of the protein bands.
Fig. 1. TNFa and ceramide inhibit HMGCoA reductase activity in U937 and HL-60 cells. U937 cells ð5 106 Þ were serum-deprived and pretreated with 20 lM lovastatin overnight. TNFa (30 ng/ml circles) or C2-ceraminde (20 lM, 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 ð6 lMÞ. HMGCoA reductase was measured as described in ‘‘Materials and methods.’’ The values shown are means SD of triplicate determinations. One out of three representative experiments is shown.
nearly by 75% of the lovastatin control during the first hour and by 90% at 6 h. An 80% inhibition in reductase mRNA levels was also obtained after 6 h of treatment with C2-ceramide (20 lM). Equivalent experiments carried out with HL-60 cells produced similar results. As shown in Fig. 2 (right panel) treatment with C2-ceramide (6 lM) for 24 h inhibited HMGCoA reductase mRNA levels by about 65% of the lovastatin control. TNFa and ceramide inhibited p21ras maturation in HL-60 cells Isoprenylation of proteins is a process controlled by the availability of mevalonic acid. When prenylation is
inhibited, the association of Ras proteins to the plasma membrane is impaired, causing non-prenylated Ras to accumulate in the cytosolic fraction. Unmodified cytosolic Ras protein may then be visualized in SDS–PAGE as a band that migrates with an apparently higher molecular weight than the farnesylated Ras protein [20]. As shown in Fig. 3 and in agreement with previously described data, lovastatin, that was used as a positive control, produced an accumulation of immature Ras of 15-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%, respectively. Mevalonate partially reverted TNFa and ceramideinduced 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. 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 10 lM, respectively) overnight to maximally induce HMGCoA reductase. TNFa (30 ng/ml) or C2-ceramide ð20 lMÞ was added to U937 cells. C2-ceramide ð6 lMÞ 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.
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dering in response to lovastatin, TNFa, and C2-ceramide. Lamin proteolysis during apoptosis has been reported 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 proteolytic fragment was present in extracts from cells treated with lovastatin as well as with TNFa and ceramide. Mevalonate completely reverted lovastatin and partially counteracted TNFa and C2-ceramide effects.
Discussion
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 ð20 lMÞ, TNFa (30 ng/ml), and C2-ceramide ð20 lMÞ with or without mevalonate (20 mM), and (A) apoptotic nuclei were evaluated by fluorescence microscopy or (B) DNA fragmentation 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 ð10 lMÞ, TNFa (30 ng/ml), and C2-ceramide ð6 lMÞ with or without mavalonate (20 mM) for 24 h.
of these cells. Fig. 4, upper panel, shows that treatment of U937 cells for 6 h with lovastatin, TNFa or C2ceramide increases the number of cells that exhibit apoptosis related morphological changes in their nuclei visualized by bisbenzimide staining. When mevalonate levels 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-
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 sphingomyelin hydrolysis to ceramide [30,31], the intracellular messenger that mediates several cellular responses to TNFa including apoptosis [13]. This seems to be the case for the TNFa effects on HMGCoA reductase in U937 and HL-60 cells because treatment with C2-ceramide, a cell-permeant ceramide analogue, results in a similar inhibition of the mRNA and enzyme activity levels. In sharp contrast with the above-described results, both TNFa and ceramide induced the expression and increased the activity of HMGCoA reductase in human 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 transcription [33]. Moreover, TNFa stimulates DNA synthesis in hepatocytes [34] and is implicated in hepatic regeneration processes [35,36], while in U-937 and HL60 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 activity 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
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cycle would be the result of a balance between the effects of 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 membrane association and/or contributing to specific protein–protein interactions (reviewed in [39]). Isoprenylation of p21ras results in the protein association to membranes that in turn allows binding of PI 3-kinase and activation of the PKB/Akt survival pathway [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, another HMGCoA reductase inhibitor, inhibits both isoprenylation 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, TNFa inhibits the expression and activity of HMGCoA reductase. We have also shown that the predictable decrease in mevalonate availability after HMGCoA reductase inhibition causes accumulation of p21ras immature forms in the cytosol and most likely apoptosis. It is open to future studies if PI 3-kinase and/or PKB activation 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 which the cytokine and its messenger induce apoptosis in U937 and HL-60 cells.
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Acknowledgment This work was supported by DGICYT: PM95-0131.
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