- Email: [email protected]
Sirolimus upregulates aP2 expression in human monocytes and macrophages
Sirolimus Upregulates aP2 Expression in Human Monocytes and Macrophages Q.-Y. Liu and P. Nambi ABSTRACT Rapamycin (Sirolimus) is a potent immunosuppressive drug that reduces renal transplant rejection. Hyperlipidemia is a significant side effect of rapamycin treatment, and frequently leads to cardiovascular disease. Adipocyte fatty acid binding protein (aP2) is a member of the cytoplasmic fatty acid binding protein (FABP) family. aP2 has been shown to affect insulin sensitivity, lipid metabolism, lipolysis, and has recently been shown to play an important role in atherosclerosis. We found that aP2 messenger RNA (mRNA) was increased in human THP-1 cells after rapamycin treatment. Exposure of human differentiated THP-1 cells to rapamycin led to a time- and dose-dependent induction of aP2 mRNA expression. While aP2 expression was undetectable in undifferentiated THP-1 cells, aP2 was induced in these cells by rapamycin. These data suggest that rapamycininduced aP2 may play a role in increased triglyceride accumulation.
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APAMYCIN (Sirolimus) is a potent immunosuppressive drug that reduces renal transplant rejection. The immunosuppressive effects of rapamycin result from its inhibition of T-cell and B-cell activity. It blocks T-lymphocyte proliferation induced by most stimuli employing either calcium-dependent or calcium-independent pathways.1–3 Moreover, rapamycin inhibits interleukin-2– or interleukin-6 – dependent proliferation of purified normal human B lymphocytes stimulated by Staphylococcus aureus and soluble CD40 ligand in the mid-G1 phase of the cell cycle.4 Hyperlipidemia is a significant side effect of rapamycin treatment, which may lead to cardiovascular disease. It has been shown that during rapamycin treatment, total plasma cholesterol, triglycerides, and ApoC-III are increased by 50%, 95%, and 92%, respectively. These changes are dose-dependent and reversible.5 Adipocyte fatty acid binding protein (aP2) is a member of the cytoplasmic fatty acid binding protein (FABP) family, which are small proteins expressed in a tissue-specific manner and bind fatty acid ligands.6,7 The adipocyte FABP (aP2) that affects insulin sensitivity, lipid metabolism, and lipolysis has recently been implicated in the development of atherosclerosis.8,9 To understand the mechanism(s) of the hyperlipidemia caused by rapamycin, we have evaluated the expression of aP2 in THP1 monocyte/macrophage cells exposed to rapamycin by real-time polymerase chain reaction (TaqMan). Our data show that aP2 expression is up-
regulated by rapamycin in a dose- and time-dependent manner. METHODS Cell Culture All cell culture reagents were obtained from Gibco-BRL (Grand Island, NY, United States). Phorbol 12, 13-dibutyrate was obtained from Sigma. THP-1 cells obtained from ATCC were cultured in RPMI medium containing 10% fetal bovine serum (FBS). For gene expression analysis, THP1 cells were differentiated with 150 nmol/L Phorbol 12, 13-dibutyrate for 3 days followed by rapamycin treatment as indicated.
RNA Analysis Total RNA was isolated using QIAGEN RNA miniPrep kit (Qiagen, Valencia, Calif, United States). Quantitative RT-PCR assays were performed using an Applied Biosystems 7700 sequence detector. Briefly, each amplification mixture (50 uL) contained 50 ng total RNA, 400 nmol/L forward primer, 400 nmol/L reverse primer, 200 nmol/L dual-labeled fluorogenic probe (Applied Biosystems), 5.5 mmol/L MgCl2, 1 U RNase inhibitor, 1.25 U Gold Taq (Applied Biosystems), and 10 U Supscript II reverse transcriptase. The RT-PCR thermocycling parameters were 48°C for 30 From the Department of Cardiovascular and Metabolic Diseases Research, Wyeth Research, Collegeville, Pennsylvania, USA. Address reprint requests to Ponnal Nambi, PhD, Wyeth Research, RN-2275, 500 Arcola Road, Collegeville, PA 19426. E-mail: [email protected]
© 2004 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/04/$–see front matter doi:10.1016/j.transproceed.2004.10.086
Transplantation Proceedings, 36, 3229 –3231 (2004)
3229
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minutes, 95°C for 10 minutes, and 40 cycles at 95°C for 15 seconds and 60°C for 1 minute together with the samples and no-RT controls; a serially diluted RNA standard was analyzed in parallel. All samples were analyzed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in parallel in the same run using probe and primers from predeveloped assays for GAPDH (Applied Biosystems). All target gene expressions were normalized to the expression of GAPDH. Quantitative analysis was performed using the threshold procedure (Perkin-Elmer protocol), with relative amounts calculated from the standard curve.
Primers and Probes for RT-PCR The primers and probes used in these studies were as follows: human aP2 (Genbank accession no. BC003672) aP2-forward (5=-GGGCTTTGCCACCAGGA-3=) aP2-reverse (5=-TCATCACATGGGGATTCACACT-3=), aP2 TaqMan probe (FAM-TGGCTGGCATGGCCAAACCTAACA-TAMRA). Following initial optimization of the PCR protocols, the aP2 primer pairs successfully amplified single products from complementary DNA (cDNA), of the expected sequence (as confirmed by sequencing data). No products were detected when no-RT controls were analyzed (data not shown), demonstrating that the assay was specific for the transcribed aP2 product.
Statistical Analysis Statistical significance of differences between the 2 groups was determined using a Student t test; P ⬍ .05 was considered significant.
RESULTS
Treatment of differentiated THP-1 cells with increasing concentrations (2 nmol/L to 80 nmol/L) of rapamycin for 24 hours resulted in a dose-dependent increase in aP2 expression (Fig 1). The maximum increase (3-fold over basal) was observed with 10 nmol/L of rapamycin, after which there was a decrease in the response. When the cells were treated for 48 hours with the same concentrations of rapamycin, the response profile was similar to that observed with 24-hour treatment except the fold induction was much higher (16fold increase over basal of 10 nmol/L rapamycin) after 48-hour treatment (Fig 2). Treatment of undifferentiated THP-1 cells with increasing concentrations of rapamycin also resulted in a dosedependent increase in aP2 expression (Fig 3). Although the maximal response in differentiated THP-1 cells occurred on exposure to 10 nmol/L rapamycin, the maximum response in undifferentiated THP-1 cells required 80 nmol/L rapamycin.
Fig 1. THP-1 cells were incubated in RPMI medium supplemented with 10% FBS and were differentiated for 72 hours with phorbol ester. Various concentrations of rapamycin were added to the medium and were incubated for an additional 24 hours. Total RNA of the cells were isolated and analyzed using TaqMan as described under Materials and Methods. Data are expressed as the fold increase above vehicle treatment and are expressed as the mean ⫹ SEM of triplicate samples.
addition to adipocytes, aP2 is also expressed in macrophages, playing a critical role in the development of atherosclerosis. ApoE knockout mice that are devoid of aP2 were protected from the development of atherosclerosis.9 Therefore, the present investigation was undertaken to delineate the mechanism(s) involved in rapamycin-mediated triglyceride increase. As shown in these studies, exposure of differentiated THP-1 cells (human macrophage cell line) to increasing concentrations of rapamycin resulted in a timedependent and dose-dependent increase in aP2 expression. The maximal expression was observed with 10 nmol/L rapamycin, at higher concentrations, the increase in aP2 expression was smaller. Because aP2 is a marker of differentiation, it was not detectable in undifferentiated macrophages (monocytes). Treatment of undifferentiated cells with rapamycin also resulted in a dose-dependent increase in aP2 expression, although the dose-response curve was shifted to the right compared with that observed in differentiated cells. Thus, the present data indicate that a rapamycin-mediated increase in triglyceride accumulation
DISCUSSION
Although rapamycin has been shown to be an effective immunosuppressant, its side effects include increases in plasma cholesterol and triglycerides and the development of atherosclerosis. Fatty acid binding protein, especially aP2 (originally identified in adipocytes), has been shown to be involved in the differentiation of adipocytes, lipolysis, insulin resistance, and obesity.10,11 Recent data indicate that in
Fig 2. Differentiated THP-1 cells (as explained in the legend of Fig 1) were treated with increasing concentrations of rapamycin for 48 hours. aP2 mRNA levels were quantitated using TaqMan. Data are expressed as explained in Fig 1.
SIROLIMUS UP-REGULATES aP2
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to regulate aP2 expression in undifferentiated THP-1 cells. As shown in Fig 3, and in agreement with the previous studies, there was no detectable aP2 messenger RNA (mRNA) expression in undifferenteated THP-1 cells. aP2 expression was induced in undifferentiated THP-1 cells after treatment with rapamycin for 48 hours (Fig 3). Thus, the data presented herein suggest that rapamycin induces aP2 accumulation, which may be responsible for the triglyceride accumulation. REFERENCES Fig 3. Undifferentiated THP-1 cells were incubated in RPMI medium containing 10% FBS plus rapamycin (2 nmol/L– 80 nmol/L) for 48 hours. The aP2 mRNA expression levels were analyzed using TaqMan. Because there was no aP2 expression in the vehicle-treated undifferentiated THP-1 cells, the data are represented as percent maximum. In this experiment, 80 nmol/L treatment gave the maximum response and this was kept as 100%.
might be mediated through the up-regulation of aP2 expression. It has been shown that aP2 was not expressed in resting monocytes and a significant increase in its expression was observed following treatment of cells with Phorbol 12, 13-dibutyrate. We then addressed the ability of rapamycin
1. Sehgal SN, Molnar-Kimber K, Ocain TO, et al: Med Res Rev 14:1, 1994 2. Wood MA, Bierer BE: Rapamycin. Persp Drug Disc Des 2:163, 1994 3. Sehgal SN, Camardo JS, Scarola JA, et al: Hypertension 4:482, 1995 4. Aagaard-Tillery KM, Jelinek D: Cell Immunol 156:493, 1994 5. Morrisett JD, Abdel-Fattah G, Hoogeveen R, et al: J Lipid Res 43:1170, 2002 6. Glatz JF, and van der Vusse GJ: Prog Lipid Res 35:243, 1996 7. Cae NR, Bernlohr DA: Biochim Biophys Acta 1391:287, 1998 8. Biird HB, Fazio S, Linton MF: Curr Opin Lipidol 13:141, 2002 9. Kakowski L, Boord JB, Maeda K, et al: Nat Med 7:699, 2001 10. Hotamisligil GS, Johnson RS, Distel RJ, et al: Science 274:1377, 1996 11. Uysal KT, Scheja L, Wiesbrock SM, et al: Endocrinology 141:3388, 2000
R
APAMYCIN (Sirolimus) is a potent immunosuppressive drug that reduces renal transplant rejection. The immunosuppressive effects of rapamycin result from its inhibition of T-cell and B-cell activity. It blocks T-lymphocyte proliferation induced by most stimuli employing either calcium-dependent or calcium-independent pathways.1–3 Moreover, rapamycin inhibits interleukin-2– or interleukin-6 – dependent proliferation of purified normal human B lymphocytes stimulated by Staphylococcus aureus and soluble CD40 ligand in the mid-G1 phase of the cell cycle.4 Hyperlipidemia is a significant side effect of rapamycin treatment, which may lead to cardiovascular disease. It has been shown that during rapamycin treatment, total plasma cholesterol, triglycerides, and ApoC-III are increased by 50%, 95%, and 92%, respectively. These changes are dose-dependent and reversible.5 Adipocyte fatty acid binding protein (aP2) is a member of the cytoplasmic fatty acid binding protein (FABP) family, which are small proteins expressed in a tissue-specific manner and bind fatty acid ligands.6,7 The adipocyte FABP (aP2) that affects insulin sensitivity, lipid metabolism, and lipolysis has recently been implicated in the development of atherosclerosis.8,9 To understand the mechanism(s) of the hyperlipidemia caused by rapamycin, we have evaluated the expression of aP2 in THP1 monocyte/macrophage cells exposed to rapamycin by real-time polymerase chain reaction (TaqMan). Our data show that aP2 expression is up-
regulated by rapamycin in a dose- and time-dependent manner. METHODS Cell Culture All cell culture reagents were obtained from Gibco-BRL (Grand Island, NY, United States). Phorbol 12, 13-dibutyrate was obtained from Sigma. THP-1 cells obtained from ATCC were cultured in RPMI medium containing 10% fetal bovine serum (FBS). For gene expression analysis, THP1 cells were differentiated with 150 nmol/L Phorbol 12, 13-dibutyrate for 3 days followed by rapamycin treatment as indicated.
RNA Analysis Total RNA was isolated using QIAGEN RNA miniPrep kit (Qiagen, Valencia, Calif, United States). Quantitative RT-PCR assays were performed using an Applied Biosystems 7700 sequence detector. Briefly, each amplification mixture (50 uL) contained 50 ng total RNA, 400 nmol/L forward primer, 400 nmol/L reverse primer, 200 nmol/L dual-labeled fluorogenic probe (Applied Biosystems), 5.5 mmol/L MgCl2, 1 U RNase inhibitor, 1.25 U Gold Taq (Applied Biosystems), and 10 U Supscript II reverse transcriptase. The RT-PCR thermocycling parameters were 48°C for 30 From the Department of Cardiovascular and Metabolic Diseases Research, Wyeth Research, Collegeville, Pennsylvania, USA. Address reprint requests to Ponnal Nambi, PhD, Wyeth Research, RN-2275, 500 Arcola Road, Collegeville, PA 19426. E-mail: [email protected]
© 2004 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/04/$–see front matter doi:10.1016/j.transproceed.2004.10.086
Transplantation Proceedings, 36, 3229 –3231 (2004)
3229
3230
LIU AND NAMBI
minutes, 95°C for 10 minutes, and 40 cycles at 95°C for 15 seconds and 60°C for 1 minute together with the samples and no-RT controls; a serially diluted RNA standard was analyzed in parallel. All samples were analyzed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in parallel in the same run using probe and primers from predeveloped assays for GAPDH (Applied Biosystems). All target gene expressions were normalized to the expression of GAPDH. Quantitative analysis was performed using the threshold procedure (Perkin-Elmer protocol), with relative amounts calculated from the standard curve.
Primers and Probes for RT-PCR The primers and probes used in these studies were as follows: human aP2 (Genbank accession no. BC003672) aP2-forward (5=-GGGCTTTGCCACCAGGA-3=) aP2-reverse (5=-TCATCACATGGGGATTCACACT-3=), aP2 TaqMan probe (FAM-TGGCTGGCATGGCCAAACCTAACA-TAMRA). Following initial optimization of the PCR protocols, the aP2 primer pairs successfully amplified single products from complementary DNA (cDNA), of the expected sequence (as confirmed by sequencing data). No products were detected when no-RT controls were analyzed (data not shown), demonstrating that the assay was specific for the transcribed aP2 product.
Statistical Analysis Statistical significance of differences between the 2 groups was determined using a Student t test; P ⬍ .05 was considered significant.
RESULTS
Treatment of differentiated THP-1 cells with increasing concentrations (2 nmol/L to 80 nmol/L) of rapamycin for 24 hours resulted in a dose-dependent increase in aP2 expression (Fig 1). The maximum increase (3-fold over basal) was observed with 10 nmol/L of rapamycin, after which there was a decrease in the response. When the cells were treated for 48 hours with the same concentrations of rapamycin, the response profile was similar to that observed with 24-hour treatment except the fold induction was much higher (16fold increase over basal of 10 nmol/L rapamycin) after 48-hour treatment (Fig 2). Treatment of undifferentiated THP-1 cells with increasing concentrations of rapamycin also resulted in a dosedependent increase in aP2 expression (Fig 3). Although the maximal response in differentiated THP-1 cells occurred on exposure to 10 nmol/L rapamycin, the maximum response in undifferentiated THP-1 cells required 80 nmol/L rapamycin.
Fig 1. THP-1 cells were incubated in RPMI medium supplemented with 10% FBS and were differentiated for 72 hours with phorbol ester. Various concentrations of rapamycin were added to the medium and were incubated for an additional 24 hours. Total RNA of the cells were isolated and analyzed using TaqMan as described under Materials and Methods. Data are expressed as the fold increase above vehicle treatment and are expressed as the mean ⫹ SEM of triplicate samples.
addition to adipocytes, aP2 is also expressed in macrophages, playing a critical role in the development of atherosclerosis. ApoE knockout mice that are devoid of aP2 were protected from the development of atherosclerosis.9 Therefore, the present investigation was undertaken to delineate the mechanism(s) involved in rapamycin-mediated triglyceride increase. As shown in these studies, exposure of differentiated THP-1 cells (human macrophage cell line) to increasing concentrations of rapamycin resulted in a timedependent and dose-dependent increase in aP2 expression. The maximal expression was observed with 10 nmol/L rapamycin, at higher concentrations, the increase in aP2 expression was smaller. Because aP2 is a marker of differentiation, it was not detectable in undifferentiated macrophages (monocytes). Treatment of undifferentiated cells with rapamycin also resulted in a dose-dependent increase in aP2 expression, although the dose-response curve was shifted to the right compared with that observed in differentiated cells. Thus, the present data indicate that a rapamycin-mediated increase in triglyceride accumulation
DISCUSSION
Although rapamycin has been shown to be an effective immunosuppressant, its side effects include increases in plasma cholesterol and triglycerides and the development of atherosclerosis. Fatty acid binding protein, especially aP2 (originally identified in adipocytes), has been shown to be involved in the differentiation of adipocytes, lipolysis, insulin resistance, and obesity.10,11 Recent data indicate that in
Fig 2. Differentiated THP-1 cells (as explained in the legend of Fig 1) were treated with increasing concentrations of rapamycin for 48 hours. aP2 mRNA levels were quantitated using TaqMan. Data are expressed as explained in Fig 1.
SIROLIMUS UP-REGULATES aP2
3231
to regulate aP2 expression in undifferentiated THP-1 cells. As shown in Fig 3, and in agreement with the previous studies, there was no detectable aP2 messenger RNA (mRNA) expression in undifferenteated THP-1 cells. aP2 expression was induced in undifferentiated THP-1 cells after treatment with rapamycin for 48 hours (Fig 3). Thus, the data presented herein suggest that rapamycin induces aP2 accumulation, which may be responsible for the triglyceride accumulation. REFERENCES Fig 3. Undifferentiated THP-1 cells were incubated in RPMI medium containing 10% FBS plus rapamycin (2 nmol/L– 80 nmol/L) for 48 hours. The aP2 mRNA expression levels were analyzed using TaqMan. Because there was no aP2 expression in the vehicle-treated undifferentiated THP-1 cells, the data are represented as percent maximum. In this experiment, 80 nmol/L treatment gave the maximum response and this was kept as 100%.
might be mediated through the up-regulation of aP2 expression. It has been shown that aP2 was not expressed in resting monocytes and a significant increase in its expression was observed following treatment of cells with Phorbol 12, 13-dibutyrate. We then addressed the ability of rapamycin
1. Sehgal SN, Molnar-Kimber K, Ocain TO, et al: Med Res Rev 14:1, 1994 2. Wood MA, Bierer BE: Rapamycin. Persp Drug Disc Des 2:163, 1994 3. Sehgal SN, Camardo JS, Scarola JA, et al: Hypertension 4:482, 1995 4. Aagaard-Tillery KM, Jelinek D: Cell Immunol 156:493, 1994 5. Morrisett JD, Abdel-Fattah G, Hoogeveen R, et al: J Lipid Res 43:1170, 2002 6. Glatz JF, and van der Vusse GJ: Prog Lipid Res 35:243, 1996 7. Cae NR, Bernlohr DA: Biochim Biophys Acta 1391:287, 1998 8. Biird HB, Fazio S, Linton MF: Curr Opin Lipidol 13:141, 2002 9. Kakowski L, Boord JB, Maeda K, et al: Nat Med 7:699, 2001 10. Hotamisligil GS, Johnson RS, Distel RJ, et al: Science 274:1377, 1996 11. Uysal KT, Scheja L, Wiesbrock SM, et al: Endocrinology 141:3388, 2000