Division of Endocrinology, Diabetes, and Medical Genetics, Medical University of South Carolina, Charleston, South Carolina 29425-2222
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ABSTRACT |
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The use of cyclosporin A has contributed greatly to the success of organ transplantation. However, cyclosporin-associated side effects of hypertension, nephrotoxicity, and dyslipoproteinemia have tempered these benefits. Cyclosporin-induced dyslipoproteinemia may be an important risk factor for the accelerated atherosclerosis observed posttransplantation. Using a mouse model, we treated Swiss-Webster mice for 6 days with a daily dose of 20 µg/g body wt of cyclosporin and observed significant elevations of plasma cholesterol, triglyceride, and apolipoprotein B (apoB) levels relative to vehicle-alone treated control animals. Measurement of the rate of secretion of very low-density lipoprotein (VLDL) by the liver in vivo showed that cyclosporin treatment led to a significant increase in the rate of hepatic VLDL triglyceride secretion. Total apoB secretion was unaffected. Northern analysis showed that cyclosporin A treatment increased the abundance of hepatic mRNA levels for a number of key genes involved in cholesterol biosynthesis relative to vehicle-alone treated animals. Two key transcriptional factors, sterol regulatory element-binding protein (SREBP)-1 and SREBP-2, also showed differential expression; SREBP-2 expression was increased at the mRNA level, and there was an increase in the active nuclear form, whereas the mRNA and the nuclear form of SREBP-1 were reduced. These results show that the molecular mechanisms by which cyclosporin causes dyslipoproteinemia may, in part, be mediated by selective activation of SREBP-2, leading to enhanced expression of lipid metabolism genes and hepatic secretion of VLDL triglyceride.
very low-density lipoprotein; sterol regulatory element-binding protein; immunotherapy; heart disease; transcription; liver
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INTRODUCTION |
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CYCLOSPORIN A TREATMENT for the prevention of organ rejection has revolutionized the success of transplantation therapy (18). However, this success is associated with an increase in three important side effects, namely hypertension, nephrotoxicity, and dyslipoproteinemia (17, 27, 37). The subsequent increased morbidity from cardiovascular disease is of major concern (19). We have investigated the mechanism by which cyclosporin use may be responsible for one of these risk factors, namely dyslipoproteinemia (2, 9, 13, 14, 19, 25). This pathological change may be one of the many reasons for the increased cardiovascular morbidity and mortality associated with solid organ transplantation. Elucidation of the mechanism of cyclosporin-induced dyslipoproteinemia may allow the identification of pathways that are inappropriately activated by cyclosporin, and this knowledge may allow the development of better immunosuppressive agents. In humans, cyclosporin treatment is associated with an increase in both plasma low-density lipoprotein (LDL) cholesterol as well as triglycerides (2, 27). Although the concomitant use of other drug therapies, such as steroid use, complicates the issue of whether cyclosporin per se is responsible for causing the dyslipoproteinemia (10, 27), the majority of the clinical evidence strongly implicates cyclosporin as the causative agent. Clearly either an increase in production or a reduction in the clearance of lipoprotein particles from the plasma, or a combination of both, may be responsible for the dyslipoproteinemia. Studies of lipoprotein particle clearance in rats treated with cyclosporin show that the fractional catabolic rate of LDL was reduced by drug treatment, as was calculated LDL production rates (24). Interestingly, in the same study, in vitro analysis of LDL-receptor-mediated clearance of LDL isolated from cyclosporin-treated animals by cultured normal fibroblasts showed it to be increased, not reduced, suggesting that LDL containing cyclosporin was cleared at a faster rate (24). In vitro studies with Hep G2 cells show that cyclosporin can reduce LDL-receptor activity in a dose-dependent manner, although the precise mechanism may not involve reduced 27-hydroxycholesterol levels (1, 43). Because most LDL particles are cleared by the liver in vivo, and not by fibroblasts, the above observations are compatible.
Cyclosporin has also been shown to inhibit apolipoprotein B (apoB) secretion from permeabilized Hep G2 cells (26) and has been implicated in causing cholestasis, with reduced bile acid synthesis, based on in vitro studies of inhibition of sterol 27-hydroxylase (2, 21, 31). Furthermore, LDL is produced in the plasma from very low-density lipoprotein (VLDL) as a result of metabolic cascades, and it is unclear whether any overproduction of VLDL is caused by cyclosporin. If cyclosporin inhibits apoB secretion (26), the reduced VLDL secretion should lead to an amelioration of any loss of LDL-receptor activity.
To investigate the mechanisms involved in cyclosporin-induced dyslipoproteinemia, we treated Swiss-Webster mice with cyclosporin or vehicle for 6 days. Cyclosporin A-treated mice showed significantly higher fasting plasma levels of cholesterol, triglyceride, and apoB. Measurement of the hepatic rates of secretion of VLDL triglyceride and apoB, as well as mRNAs for a number of hepatic genes involved in lipoprotein synthesis and secretion, implicated a transcriptional mechanism as the cause of the dyslipoproteinemia.
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METHODS |
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Materials. Cyclosporin was a kind gift from Dr. David Weinstein (Sandoz, NJ). Tyloxapol (WR1339) was obtained from Sigma (St. Louis, MO). All other reagents were purchased from commercial sources and were of reagent grade or better.
Measurement of hepatic VLDL secretion in
vivo. All animal protocols were approved by the
Institutional Animal Committee for Research and were in compliance with
Public Health Service guidelines. Outbred female ND4
Swiss-Webster mice (Harlan, Indianapolis, IN) were housed with a
12:12-h light-dark cycle and allowed free access to standard rodent
chow and water. Animals were injected subcutaneously once a day, at 5 PM, for 6 days with either olive oil (control animals, vehicle alone)
or cyclosporin (treatment group) dissolved in olive oil, at a dose of
20 mg/kg (8, 24, 30, 38). This regimen was chosen to minimize
nephrotoxicity and to ensure that cyclosporin levels were at steady
state (30, 38). To control for the anorectic effects of cyclosporin,
the serial weights of the animals were monitored. No differences in
weight gain over the 6 days of treatment were noted (data not shown).
Blood was drawn via tail vein sampling before the commencement of the
treatment, as well as every 3 days for the monitoring of plasma
triglyceride and cholesterol levels. On
day 7 at 7 AM, animals were fasted for 2 h and measurement of hepatic
secretion rates of VLDL with tyloxapol injection was performed as
previously described in detail (22, 23). Briefly, tyloxapol was
injected at a dose of 400-600 mg/kg, and blood was sampled
preinjection and at various time points thereafter (30, 90, 150, and
210 min) for triglyceride determination. At the end of 5 h, animals
were euthanized, and blood was obtained by exanguination and portions
of livers were flash-frozen in liquid nitrogen and stored at
80°C for subsequent RNA analyses. VLDL was isolated from
plasma of each animal obtained at the 5-h time point. Loss of VLDL
during ultracentrifugation and harvesting was corrected for by
determining the plasma triglyceride before centrifugation and the
triglyceride in the VLDL harvested, with the assumption that all the
plasma triglyceride is in the VLDL fraction (22). Aliquots
of VLDL obtained from each animal were analyzed by SDS-PAGE, and
apoB-100 and apoB-48 were quantitated by Coomassie blue staining and
laser densitometry (22). Triglyceride secretion rates were computed
from time vs. plasma triglyceride plots, and data were normalized per
gram liver weight for comparison. Cholesterol and triglyceride were
measured enzymatically as previously described (22).
RNA analysis. Total RNA was extracted from frozen or freshly harvested liver, with Trizol (GIBCO-BRL, Bethesda, MD), according to the conditions of the manufacturer. Ten micrograms of RNA were denatured and separated by size on 1% formaldehyde-agarose gels and transferred to Hybond-N with standard techniques. Northern analysis was performed with radiolabeled probes and quantitated by phosphorimage analysis (Molecular Dynamics, Sunnyvale, CA). All data were normalized to the signal obtained with a glyceraldehyde-3-phosphate dehydrogenase probe. Mouse cDNA probes for hydroxymethylglutaryl (HMG)-CoA reductase, HMG-CoA synthase, LDL receptor, squalene synthase, sterol regulatory element-binding protein (SREBP)-1 and -2, acetyl-CoA carboxylase, and fatty acid synthase have been previously described (36) and were gifts from Drs. Hitoshi Shimano and Jay Horton (University of Texas Southwestern Medical Center, Dallas, TX). Murine microsomal triglyceride transfer protein (MTP) cDNA was a gift from Dr. Narayan Hariharan (Bristol-Myers Squibb, Princeton, NJ), and the murine hepatic lipase probe was kindly provided by Dr. Jimin Gao (University of Texas Southwestern Medical Center).
Western blotting. Extractions of mouse hepatic membranes and nuclear fractions for the detection of SREBPs were performed as previously described (16, 36), except that ~100- mg portions of fresh liver from five control or five cyclosporin A-treated animals were pooled and homogenized in buffer A, containing protease inhibitors. Aliquots of liver were also taken for subsequent RNA analysis. Nuclear or membrane fractions were prepared as described, and the protein concentration was determined with a bicinchoninic acid kit (Pierce Chemical, Rockford, IL). Aliquots of nuclear or membrane extracts (30 µg) from control and cyclosporin A treatment groups were separated by 8% SDS-PAGE, blotted onto Hybond-C membranes, and stained with amido black for verification of protein loading. Blotted membranes were incubated with rabbit polyclonal antibodies to SREBP-1 or -2 and washed, and bound antibodies were detected with horseradish-peroxidase-labeled donkey anti-rabbit antibody and enhanced chemiluminescence detection (Amersham, Arlington Heights, IL). For detection of SREBP cleavage-activating protein (SCAP), membrane fractions were blotted and incubated with primary antibody to SCAP as previously described (35).
Statistical analysis. Statistical analysis between control and treated groups was performed with unpaired two-tailed t-test and Prizm software (GraphPad Software, San Diego, CA) and expressed as means ± SD. Significance was set at P < 0.05.
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RESULTS |
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Cyclosporin treatment induces a combined
hyperlipidemia. Table 1
shows the baseline characteristics of the control and
cyclosporin-treated animals after 6 days of treatment. No significant
differences were observed in the body weights or liver weights between
control and treated animals. Cyclosporin-treated animals showed a
significant increase in fasting triglyceride (175 ± 56 vs. 116 ± 27 mg/dl, P < 0.05) and
cholesterol (107 ± 17 vs. 77 ± 11 mg/dl,
P < 0.05) levels. Additionally,
baseline plasma apoB-48 (16.5 ± 5.2 vs. 8.5 ± 1.4 pmol/l,
P < 0.05) and apoB-100
concentrations (3.4 ± 0.7 vs. 1.37 ± 0.5 pmol/l,
P < 0.05) were also higher. An
increase in the proportion of apoB-100 relative to apoB-48 was also
detected in the treatment group. Hence, treatment of mice with
cyclosporin resulted in dyslipoproteinemia, characterized by increased
cholesterol, triglyceride, and apoB levels.
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Cyclosporin results in an increased hepatic VLDL
triglyceride secretion rate. The Triton method was used
to measure hepatic VLDL triglyceride secretion rate in vivo (22).
Figure 1 shows the rates of VLDL
triglyceride secretion between control and cyclosporin-treated animals.
Cyclosporin treatment (open bar, Fig. 1), relative to vehicle alone,
resulted in a statistically significant increase in hepatic secretion
of VLDL triglyceride (6.65 ± 0.84 vs. 5.22 ± 0.34 mg · h1 · g
liver
1,
P < 0.05). No difference in liver or
body weights between the two groups of animals was present (see Table
1); thus triglyceride secretion rate, whether expressed per body weight
or per animal, shows significantly higher secretion of VLDL
triglyceride in the treated group (data not shown).
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Cyclosporin causes a change in the proportions of
apoB-48 and apoB-100 secreted by the liver. The murine
liver secretes both apoB-48 and apoB-100, a functional consequence of
apoB mRNA editing (6, 12). However, each VLDL particle secreted
contains only one apoB protein, either apoB-48 or apoB-100. Hence,
measurement of apoB secretion rates allows an estimation of the number
of VLDL particles secreted. Figure 2 shows
the rates of secretion of apoB-100 and apoB-48. Although apoB-100 was
significantly increased by cyclosporin treatment (open bars) relative
to vehicle alone (32.6 ± 7.3 vs. 26.5 ± 4.9 pmol · h1 · g
liver
1,
P < 0.05), apoB-48 was unchanged
relative to vehicle alone (212 ± 37 vs. 238 ± 52 pmol · h
1 · g
liver
1,
P = 0.22). Because the mouse liver
secretes predominantly apoB-48-containing VLDL (22), this increase in
apoB-100 represents a change in the type of the VLDL particle secreted
(Fig.
3A), as
the total amount of apoB secreted was not significantly different (Fig. 3B). Because there is only one apoB
molecule per VLDL particle, these results imply that the total number
of VLDL particles secreted by the liver is unchanged with treatment
with cyclosporin A but more apoB-100 is secreted. Although there is a
23% increase in apoB-100 secretion by cyclosporin treatment, the total
apoB rates are relatively unaffected by this increase (Fig.
3B) because murine hepatic secretion
of apoB-100 represents between 9 and 12% of total apoB secreted, the
predominant form secreted being apoB-48 (12, 22). The
method used in the current study does not allow the determination of
whether the increased triglyceride is accounted for by the increased
apoB-100 secretion or whether there is an increase in triglyceride
content of all of the individual particles.
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Cyclosporin treatment causes differential regulation
of genes for sterol and fatty acid biosynthesis and
metabolism. To further characterize the mechanism
underlying the increased hepatic triglyceride secretion, we examined
mRNA levels by Northern analysis for a number of key genes in the
cholesterol biosynthesis pathway. The mRNA for the rate-limiting
enzyme, HMG-CoA reductase, as well as LDL receptor, stearoyl-CoA
desaturase, and squalene synthase mRNAs, was increased in abundance in
the cyclosporin-treated group (Fig. 4).
However, the abundance of MTP mRNA was not increased. MTP-mediated
transfer of triglyceride to the nascent apoB-containing lipoprotein in
the endoplasmic reticulum is thought to be a rate-limiting step in VLDL
secretion (11, 42). The abundance of hepatic triglyceride
lipase mRNA was also not affected by cyclosporin treatment (Fig. 4).
Northern analysis for two genes in the fatty acid biosynthesis pathway,
acetyl CoA carboxylase (×0.93-fold) and fatty acid synthase
(×2.4-fold), showed that only the fatty acid synthase mRNA was
increased (Fig. 4). There was no change in the mRNA abundance for
cholesterol 7-hydroxylase, the rate-limiting enzyme for bile acid
synthesis (Fig. 4).
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Two transcriptional factors that regulate cholesterol and fatty acid
biosynthetic pathways (4), SREBPs, were also analyzed. SREBP-1 mRNA was
reduced in abundance (×0.45-fold), and mRNA for SREBP-2 was
increased (×1.6-fold) in the cyclosporin-treated group (Fig. 4).
SREBPs are activated from membrane-bound precursor forms by proteolytic
cleavage and nuclear translocation. To establish that the increased
abundance of the various mRNAs was mediated by increased SREBP
activation, we performed semiquantitative Western blot analysis of
SREBP-1 and -2 in liver homogenates from control and treated animals
(Fig. 5). Equal amounts of liver
homogenates from control and cyclosporin-treated animals were separated
by SDS-PAGE and blotted for SREBPs (see
METHODS). SREBP-1 active forms
(nuclear extract, Fig. 5A) appeared
to be marginally reduced by cyclosporin treatment. In contrast, there
was an increase in the active form of SREBP-2 in the nuclear extracts
from the cyclosporin-treated livers (Fig.
5B). The protein detected at ~84
kDa in Fig. 5A is an intermediate
cleaved form of SREBP that has been previously described (39). Figure
5B shows that the nuclear fraction for SREBP-2 has some precursor forms present. Although the precursor form
of SREBP is thought to be localized to the endoplasmic reticulum membrane, nuclear preparations with some contamination of this form
have been noted previously (39). One of the key proteins that has been
identified as responsible for activation of cleavage of membrane-bound
SREBPs is SCAP (5). Western blot analysis showed that the amounts of
this protein were not significantly altered by cyclosporin treatment
(Fig. 5C).
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DISCUSSION |
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The clinical use of cyclosporin A as a powerful immunosuppressive agent has contributed greatly to the success of transplantation as a therapy, a success that is associated with an increase in cardiovascular morbidity and mortality observed in patients treated with cyclosporin.
We have examined the mechanism of how cyclosporin may cause dyslipoproteinemia, with a mouse model. Cyclosporin A treatment of Swiss-Webster mice for 6 days resulted in a significant elevation of plasma cholesterol, triglyceride, and apoB levels, resulting in a dyslipoproteinemia. Cyclosporin treatment can be hypothesized to cause this dyslipoproteinemia by 1) increased hepatic secretion of lipoproteins, 2) decreased clearance of lipoproteins, or 3) a combination of both. Our study shows that a combination of both overproduction of triglyceride and reduced clearance is present in cyclosporin-treated animals. Measurement of the rate of hepatic VLDL secretion showed that cyclosporin treatment caused a 27% increase in triglyceride secretion but no increase in the amount of total apoB secreted. Because each VLDL particle contains only one apoB molecule, the number of VLDL particles secreted does not appear to be increased by cyclosporin treatment. However, plasma levels of apoB are increased by cyclosporin treatment, suggesting that the dyslipoproteinemia induced by cyclosporin is a combination of both overproduction of triglyceride and reduced clearance of apoB-containing lipoproteins. This interpretation is complicated in that the mouse liver secretes predominantly apoB-48 (~90%) and only a small amount of apoB-100 (~10%) (12). Hence, although cyclosporin treatment resulted in a 23% increase in hepatic apoB-100 secretion, this increased contribution is somewhat attenuated by the disproportionate secretion of apoB-48 and apoB-100. Because human liver secretes only apoB-100 (12), it is possible that cyclosporin may result in overproduction of apoB in humans. Our data suggest that in the mouse, the cyclosporin-mediated dyslipoproteinemia is complex, as both an element of overproduction (measured directly) and a defect in clearance (inferred) are present.
To investigate the molecular mechanism(s) by which cyclosporin causes an increase in secretion of triglyceride and apoB-100 by the mouse liver, we examined the mRNA levels for MTP. MTP, in a tight complex with protein disulfide isomerase, shuttles triglyceride to the nascent apoB, and this activity is essential for normal VLDL secretion (41, 42). Mutations of MTP that inactivate lipid transfer prevent secretion of VLDL both in vivo (40) and in vitro (11, 33). Hence, an increase in MTP could account for the increased triglyceride and apoB-100 secretion seen in cyclosporin-treated animals. However, Northern analysis did not support this hypothesis; the mRNA for MTP was unchanged, if not slightly reduced by cyclosporin treatment (Fig. 4). Instead, a number of mRNAs encoding cholesterol biosynthesis enzymes, including the rate-limiting enzyme, HMG-CoA reductase, were significantly elevated by cyclosporin treatment, suggesting that this pathway was upregulated. To explore these results further, we examined mRNAs for two transcriptional factors known to regulate the transcription of many genes involved in lipid metabolism, namely SREBP-1 and SREBP-2. SREBP-1 mRNA was reduced in abundance, and this decline was further reflected in a reduction of the active, nuclear form of the protein. In contrast, both SREBP-2 mRNA and its active nuclear form were increased by cyclosporin treatment (Fig. 5). SREBP-1 and -2 are potent transcriptional activators of genes along the cholesterol and fatty acid biosynthesis pathways (4). In mice that transgenically overexpress the active forms of either SREBP-1 or SREBP-2, hepatic synthesis of cholesterol and fatty acids is increased, as is triglyceride storage, although there are differences in the resultant gene expression patterns between SREBP-1 and SREBP-2 transgenic mice (16, 36). SREBP-2 transgenic animals had a greater induction of the sterol biosynthesis pathway relative to the SREBP-1 transgenic mice, which induce the fatty acid biosynthesis pathway (16). More recently, the same investigators have now shown that there is increased hepatic cholesterol and fatty acid secretion in the SREBP-1a transgenic mice when unmasked on a background of LDL-receptor deficiency (15). Thus increased SREBP-2 active form in cyclosporin-treated animals observed in the present study is likely to lead to increased free fatty acid and triglyceride synthesis and secretion. The precise mechanism by which SREBP-2 activation leads to increased VLDL secretion has not been characterized. Although MTP contains sterol regulatory elements (29), in our study we observed no changes in MTP mRNA abundance, although activity for this protein was not examined. Pathways other than direct MTP involvement may be important for the link between SREBP and VLDL secretion.
Much of our understanding of the molecular mechanisms by which cyclosporin mediates its biological actions stems from characterization of its actions in activated T cells. Cyclosporin specifically binds to a ubiquitous cytoplasmic protein, cyclophilin A (an immunophilin), which in turn inhibits calcineurin, a serine-threonine protein phosphatase (20, 32, 34). The latter protein is required for the activation of a number of transcriptional pathways. One well-characterized pathway is the dephosphorylation and activation of the nuclear factor of activated T cells, after stimulation by calmodulin and increases in intracellular calcium, mediated by cell surface receptor activation (32). The actions of cyclosporin on tissues other than lymphoid cells are also now beginning to be unraveled (3, 28). Although the expression of the nuclear factor of activated T cells is thought to be confined mainly to lymphoid cells, tissues other than those involved in immune regulation may also express this factor. Another mechanism by which cyclosporin may express biological activity is by modulation of the peptidyl-proly cis/trans isomerase activity of cyclophilins (34). Although all of the immunophilins possess this activity, inhibition of this activity by cyclosporin has not yet been shown to lead to a disruption of a biological pathway.
Finally, cyclosporin A has also been shown to inhibit directly an
enzyme in the bile acid synthesis pathway, sterol 27-hydroxylase, both
in rat liver and a human hepatoma cell line (7, 21, 31). Sterol
27-hydroxylase is a multifunctional enzyme, catalyzing oxidation steps
of a variety of sterol substrates. Cyclosporin A inhibits
27-hydroxylation of cholesterol but not hydroxylation of
5-cholestane-3
, 7
, 12
-triol. The latter substrate is an intermediate in the formation of cholic acid, whereas in some species,
including humans, the former is an intermediate in chenodeoxycholic acid synthesis. Inhibition of 27-hydroxylation cholesterol is therefore
predicted to lead to a decrease in the cellular levels of the oxysterol
27-hydroxycholesterol. One possibility is that 27-hydroxycholesterol is
a potent inhibitor of SREBP cleavage; thus a fall in its intracellular
concentration, in response to cyclosporin treatment, may lead to an
activation of SREBP.
We propose three speculative mechanism(s) by which cyclosporin may
increase SREBP-2 activation: 1) if
activation of the nuclear form of SREBP-2 was also dependent on
phosphorylation of SREBP-2, and its inactivation, by dephosphorylation
mediated by calcineurin, then cyclosporin-mediated inhibition of this
pathway would lead to an increase in active SREBP-2, which is also
known to stimulate its own transcription (Fig.
6A);
2) that cyclosporin leads to increased cleavage activation of SREBP-2 (perhaps mediated by a
phosphorylation-dephosphorylation cycle, Fig.
6B); and
3) cyclosporin causes a fall in the
concentration of a regulatory molecule, such as 27-hydroxycholesterol
via inhibition of the sterol 27-hydroxylase, leading to activation of
SREBP-2 (Fig. 6C). We note that the
effect of cyclosporin in vivo is to selectively activate SREBP-2 but not SREBP-1. Hence, it is likely that the mechanism leading to this
selective activation may involve more than the explanations put forward
above.
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ACKNOWLEDGEMENTS |
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We thank the Goldstein and Brown labs, and in particular, Dr. Yuriv Bashmakov, for the help with the Western blot analysis for SREBPs, and Drs. Hitoshi Shimano and Jay Horton and Jimin Gao for the generous gifts of the cDNA probes. We also thank Dr. Hariharan Narayan for the mouse MTP cDNA, Dr. David Weinstein at Sandoz (now Novartis) for the gift of cyclosporin A, John Whitaker for technical assistance, and Dr. David Russell for critical reading of the manuscript.
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FOOTNOTES |
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This work was initiated at University of Texas Southwestern Medical Center, Dallas and was supported by a Grant-in-Aid from the American Heart Association, Texas Affiliate, Inc. and by unrestricted grants from Merck & Co., Inc., West Point, PA, Bristol-Myers Squibb, New Brunswick, NJ, the Southwestern Medical Foundation, Dallas, TX, and the Moss Heart Foundation, Dallas, TX.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. B. Patel, Medical Univ. of South Carolina, Division of Endocrinology, Diabetes and Medical Genetics, STR Rm. 541, 114 Doughty St., Charleston, SC 29403 (E-mail: patelsb{at}musc.edu).
Received 26 March 1999; accepted in final form 13 July 1999.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Al Rayyes, O.,
A. Wallmark,
and
C. H. Floren.
Additive inhibitory effect of hydrocortisone and cyclosporine on low-density lipoprotein receptor activity in cultured HepG2 cells.
Hepatology
26:
967-971,
1997[Medline].
2.
Ballantyne, C. M.,
E. J. Podet,
W. P. Patsch,
Y. Harati,
V. Appel,
A. M. Gotto, Jr.,
and
J. B. Young.
Effects of cyclosporine therapy on plasma lipoprotein levels.
JAMA
262:
53-56,
1989[Abstract].
3.
Boss, V.,
K. L. Abbott,
X. F. Wang,
G. K. Pavlath,
and
T. J. Murphy.
The cyclosporin A-sensitive nuclear factor of activated T cells (NFAT) proteins are expressed in vascular smooth muscle cells. Differential localization of NFAT isoforms and induction of NFAT-mediated transcription by phospholipase C-coupled cell surface receptors.
J. Biol. Chem.
273:
19664-19671,
1998
4.
Brown, M. S.,
and
J. L. Goldstein.
The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor.
Cell
89:
331-340,
1997[Medline].
5.
Brown, M. S., and J. L. Goldstein.
Sterol regulatory element binding proteins (SREBPs): controllers
of lipid synthesis and cellular uptake. Nutr.
Rev. 56: S1-S3 and S54-S75, 1998.
6.
Chan, L.
RNA editing: exploring one mode with apolipoprotein B mRNA.
Bioessays
15:
33-41,
1993[Medline].
7.
Dahlback-Sjoberg, H.,
I. Bjorkhem,
and
H. M. Princen.
Selective inhibition of mitochondrial 27-hydroxylation of bile acid intermediates and 25-hydroxylation of vitamin D3 by cyclosporin A.
Biochem. J.
293:
203-206,
1993[Medline].
8.
Emeson, E. E.,
and
M. L. Shen.
Accelerated atherosclerosis in hyperlipidemic C57BL/6 mice treated with cyclosporin A.
Am. J. Pathol.
142:
1906-1915,
1993[Abstract].
9.
Ettinger, W. H.,
W. L. Bender,
A. P. Goldberg,
and
W. R. Hazzard.
Lipoprotein lipid abnormalities in healthy renal transplant recipients: persistence of low HDL2 cholesterol.
Nephron
47:
17-21,
1987[Medline].
10.
Fernandez-Miranda, C.,
C. Guijarro,
A. de la Calle,
C. Loinaz,
I. Gonzalez-Pinto,
T. Gomez-Izquierdo,
S. Larumbe,
E. Moreno,
and
A. del Palacio.
Lipid abnormalities in stable liver transplant recipientseffects of cyclosporin, tacrolimus, and steroids.
Transpl. Int.
11:
137-142,
1998[Medline].
11.
Gordon, D. A.
Recent advances in elucidating the role of the microsomal triglyceride transfer protein in apolipoprotein B lipoprotein assembly.
Curr. Opin. Lipidol.
8:
131-137,
1997[Medline].
12.
Greeve, J.,
I. Altkemper,
J. H. Dieterich,
H. Greten,
and
E. Windler.
Apolipoprotein B mRNA editing in 12 different mammalian species: hepatic expression is reflected in low concentrations of apoB-containing plasma lipoproteins.
J. Lipid Res.
34:
1367-1383,
1993[Abstract].
13.
Harris, K. P.,
G. I. Russell,
S. D. Parvin,
P. S. Veitch,
and
J. Walls.
Alterations in lipid and carbohydrate metabolism attributable to cyclosporin A in renal transplant recipients.
Br. Med. J.
292:
16,
1986[Medline].
14.
Hess, M. L.,
A. Hastillo,
T. Mohanakumar,
M. J. Cowley,
G. Vetrovac,
S. Szentpetery,
T. C. Wolfgang,
and
R. R. Lower.
Accelerated atherosclerosis in cardiac transplantation: role of cytotoxic B-cell antibodies and hyperlipidemia.
Circulation
68:
1194-1101,
1983[Abstract].
15.
Horton, J. D.,
H. Shimano,
R. L. Hamilton,
M. S. Brown,
and
J. L. Goldstein.
Disruption of LDL receptor gene in transgenic SREBP-1a mice unmasks hyperlipidemia resulting from production of lipid-rich VLDL.
J. Clin. Invest.
103:
1067-1076,
1999
16.
Horton, J. D.,
I. Shimomura,
M. S. Brown,
R. E. Hammer,
J. L. Goldstein,
and
H. Shimano.
Activation of cholesterol synthesis in preference to fatty acid synthesis in liver and adipose tissue of transgenic mice overproducing sterol regulatory element-binding protein-2.
J. Clin. Invest.
101:
2331-2339,
1998
17.
Kahan, B. D.
Cyclosporine.
N. Engl. J. Med.
321:
1725-1738,
1989[Medline].
18.
Kahan, B. D.
Cyclosporine: the base for immunosuppressive therapypresent and future.
Transplant. Proc.
25:
508-510,
1993[Medline].
19.
Kasiske, B. L.
Risk factors for accelerated atherosclerosis in renal transplant recipients.
Am. J. Med.
84:
985-992,
1988[Medline].
20.
Klee, C. B.,
H. Ren,
and
X. Wang.
Regulation of the calmodulin-stimulated protein phosphatase, calcineurin.
J. Biol. Chem.
273:
13367-13370,
1998
21.
Levy, J.,
K. Budai,
and
N. B. Javitt.
Bile acid synthesis in HepG2 cells: effect of cyclosporin.
J. Lipid Res.
35:
1795-1800,
1994[Abstract].
22.
Li, X.,
F. Catalina,
S. M. Grundy,
and
S. Patel.
Method to measure apolipoprotein B-48 and B-100 secretion rates in an individual mouse: evidence for a very rapid turnover of VLDL and preferential removal of B-48- relative to B-100-containing lipoproteins.
J. Lipid Res.
37:
210-220,
1996[Abstract].
23.
Li, X.,
S. M. Grundy,
and
S. B. Patel.
Obesity in db and ob animals leads to impaired hepatic very low density lipoprotein secretion and differential secretion of apolipoprotein B-48 and B-100.
J. Lipid Res.
38:
1277-1288,
1997[Abstract].
24.
Lopez-Miranda, J.,
E. Vilella,
F. Perez-Jimenez,
A. Espino,
J. A. Jimenez-Pereperez,
L. Masana,
and
P. R. Turner.
Low-density lipoprotein metabolism in rats treated with cyclosporine.
Metabolism
42:
678-683,
1993[Medline].
25.
Luke, D. R.,
J. E. Beck,
K. Vadiei,
M. Yousefpour,
C. F. LeMaistre,
and
J. C. Yau.
Longitudinal study of cyclosporine and lipids in patients undergoing bone marrow transplantation.
J. Clin. Pharmacol.
30:
163-169,
1990
26.
Macri, J.,
and
K. Adeli.
Studies on intracellular translocation of apolipoprotein B in a permeabilized HepG2 system.
J. Biol. Chem.
272:
7328-7337,
1997
27.
Markell, M. S.,
V. Armenti,
G. Danovitch,
and
N. Sumrani.
Hyperlipidemia and glucose intolerance in the post-renal transplant patient.
J. Am. Soc. Nephrol.
4, Suppl. 8:
S37-S47,
1994[Abstract].
28.
Molkentin, J. D.,
J. R. Lu,
C. L. Antos,
B. Markham,
J. Richardson,
J. Robbins,
S. R. Grant,
and
E. N. Olson.
A calcineurin-dependent transcriptional pathway for cardiac hypertrophy.
Cell
93:
215-228,
1998[Medline].
29.
Navasa, M.,
D. A. Gordon,
N. Hariharan,
H. Jamil,
J. K. Shigenaga,
A. Moser,
W. Fiers,
A. Pollock,
C. Grunfeld,
and
K. R. Feingold.
Regulation of microsomal triglyceride transfer protein mRNA expression by endotoxin and cytokines.
J. Lipid Res.
39:
1220-1230,
1998
30.
Nooter, K.,
B. Meershoek,
W. Spaans,
P. Sonneveld,
R. Oostrum,
and
J. Deurloo.
Blood and tissue distribution of cyclosporin A after a single oral dose in the rat.
Experientia
40:
559-561,
1984[Medline].
31.
Princen, H. M.,
P. Meijer,
B. G. Wolthers,
R. J. Vonk,
and
F. Kuipers.
Cyclosporin A blocks bile acid synthesis in cultured hepatocytes by specific inhibition of chenodeoxycholic acid synthesis.
Biochem. J.
275:
501-505,
1991[Medline].
32.
Rao, A.,
C. Luo,
and
P. G. Hogan.
Transcription factors of the NFAT family: regulation and function.
Annu. Rev. Immunol.
15:
707-747,
1997[Medline].
33.
Rehberg, E. F.,
M. E. Samson-Bouma,
B. Kienzle,
L. Blinderman,
H. Jamil,
J. R. Wetterau,
L. P. Aggerbeck,
and
D. A. Gordon.
A novel abetalipoproteinemia genotype. Identification of a missense mutation in the 97-kDa subunit of the microsomal triglyceride transfer protein that prevents complex formation with protein disulfide isomerase.
J. Biol. Chem.
271:
29945-29952,
1996
34.
Ruhlmann, A.,
and
A. Nordheim.
Effects of the immunosuppressive drugs CsA and FK506 on intracellular signalling and gene regulation.
Immunobiology
198:
192-206,
1997[Medline].
35.
Sakai, J.,
A. Nohturfft,
D. Cheng,
Y. K. Ho,
M. S. Brown,
and
J. L. Goldstein.
Identification of complexes between the COOH-terminal domains of sterol regulatory element-binding proteins (SREBPs) and SREBP cleavage-activating protein.
J. Biol. Chem.
272:
20213-20221,
1997
36.
Shimano, H.,
J. D. Horton,
R. E. Hammer,
I. Shimomura,
M. S. Brown,
and
J. L. Goldstein.
Overproduction of cholesterol and fatty acids causes massive liver enlargement in transgenic mice expressing truncated SREBP-1a.
J. Clin. Invest.
98:
1575-1584,
1996
37.
Sturrock, N. D.,
and
A. D. Struthers.
Hormonal and other mechanisms involved in the pathogenesis of cyclosporin-induced nephrotoxicity and hypertension in man.
Clin. Sci. (Colch.)
86:
1-9,
1994[Medline].
38.
Ueda, C. T.,
M. Lemaire,
G. Gsell,
P. Misslin,
and
K. Nussbaumer.
Apparent dose-dependent oral absorption of cyclosporin A in rats.
Biopharm. Drug Dispos.
5:
141-151,
1984[Medline].
39.
Wang, X.,
R. Sato,
M. S. Brown,
X. Hua,
and
J. L. Goldstein.
SREBP-1, a membrane-bound transcription factor released by sterol-regulated proteolysis.
Cell
77:
53-62,
1994[Medline].
40.
Wetterau, J. R.,
L. P. Aggerbeck,
M. E. Bouma,
C. Eisenberg,
A. Munck,
M. Hermier,
J. Schmitz,
G. Gay,
D. J. Rader,
and
R. E. Gregg.
Absence of microsomal triglyceride transfer protein in individuals with abetalipoproteinemia.
Science
258:
999-1001,
1992[Medline].
41.
Wetterau, J. R.,
K. A. Combs,
L. R. McLean,
S. N. Spinner,
and
L. P. Aggerbeck.
Protein disulfide isomerase appears necessary to maintain the catalytically active structure of the microsomal triglyceride transfer protein.
Biochemistry
30:
9728-9735,
1991[Medline].
42.
Wetterau, J. R.,
K. A. Combs,
S. N. Spinner,
and
B. J. Joiner.
Protein disulfide isomerase is a component of the microsomal triglyceride transfer protein complex.
J. Biol. Chem.
265:
9801-9807,
1990
43.
Winegar, D. A.,
J. A. Salisbury,
S. S. Sundseth,
and
R. L. Hawke.
Effects of cyclosporin on cholesterol 27-hydroxylation and LDL receptor activity in HepG2 cells.
J. Lipid Res.
37:
179-191,
1996[Abstract].