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Department of Biochemistry and Molecular Biology, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1
(Requests for offprints should be addressed to X-L Zheng; Email: xlzheng{at}ucalgary.ca)
(X-L Zheng is a recipient of the new investigator award from the Heart & Stroke Foundation of Canada)
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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It has been of interest to many laboratories, including our own, to investigate hormonal control of apo AI expression and address how thyroid hormone, glucocorticoids, estradiol, androgens, retinoic acid, and insulin modulate activation of the gene (Soyal et al. 1995, Murao et al. 1998, Ness et al. 1998, Hargrove et al. 1999). These hormones, except insulin, stimulate intracellular receptors that act within the nucleus. However, our previous studies have indicated that the activation of protein kinase A (PKA) and protein kinase C (PKC) also stimulates apo AI gene expression through the Sp1-transcription factor and the insulin-responsive core element (Murao et al. 1998, Zheng et al. 2000, 2001). These studies suggest that the apo AI gene could be regulated by various growth factors and hormones in vivo and the clinical drugs that modulate the intracellular signaling network may also regulate apo AI gene expression.
Cyclosporin A (CsA) is an inhibitor of calcineurin, a calcium/calmodulin-activated protein phosphatase (Klee et al. 1998), and has been widely used as an immunosuppressant in organ transplant patients. One of the side effects of CsA is hyperlipidemia (Ballantyne et al. 1989), in which total cholesterols are high, and HDL and apo AI levels are low. It has been proposed in a mouse model that CsA causes dyslipoproteinemia through selective activation of sterol-regulatory element-binding protein-2, which leads to enhanced expression of lipid metabolism genes and hepatic secretion of VLDL triglyceride (Wu et al. 1999). In addition, CsA is reported to inhibit ABCA1-dependent cholesterol efflux with reduced HDL levels in mice (Le Goff et al. 2004). Therefore, it is also possible that CsA reduces serum apo AI to lower HDL levels (Lopez-Miranda et al. 1992, Kuster et al. 1994). To date, it is not known whether CsA inhibits apo AI gene expression, leading us to the studies in this report. Our results showed that CsA inhibited gene expression of apo AI in vivo. Additionally, in stably transfected Hep G2 cells harboring an apo AI-474-CAT (chloramphenicol acetyltransferase) reporter gene, CsA inhibited apo AI gene expression induced by intracellular calcium mobilization.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Male Sprague–Dawley (SD) rats (~350 g) were purchased from Charles River Laboratories (Saint Constant, QC, Canada). The animal study protocol was approved by the Animal Use and Care Committee at the University of Calgary. Human hepatoma, Hep G2, cells were purchased from the American Type Culture Collection. CsA and FK506 were purchased from Sigma-Aldrich. The pAI.474-CAT plasmid was constructed using PCR and rat genomic DNA as the template as previously described (Romney et al. 1992).
Animal treatment with CsA
Five rats were included in each group. Rats were s.c. injected with CsA (20 mg/kg per day) or olive oil (vehicle alone) for 7 days (Wu et al. 1999). Animals were then killed, and whole blood was collected for serum apo AI protein measurement. Liver tissues were sampled for apo AI mRNA detection.
SDS-PAGE and western immunoblots
An aliquot of each serum sample was separated by electrophoresis in 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membrane (Millipore, Waters Corp., Bedford, MA, USA). Apo AI protein was identified using a monoclonal antibody (Calbiochem, Hornby, Ontario, Canada) and the signal was detected using ECL reagents (Amersham Pharmacia Biotech) as previously described (Gui & Zheng 2003).
RNA preparation, reverse transcription (RT)-PCR and northern blotting
Liver tissues were homogenized using a polytron homogenizer and total RNA was extracted using TRI reagent (Molecular Research Center, Cincinnati, OH, USA). RNA was reverse transcribed with a first-strand cDNA synthesis kit using a pd(N)6 primer (Amersham Pharmacia Biotech) according to the manufacturer’s protocol. Three microliters of this solution were amplified using PCR with a primer pair (forward: 5'-GATGAAAGCTGCAGTGTTGGCTGT-3'; reverse: 5'-TCCTGTAGGCGACCAACAGTTGAA-3') designed from the rat apo AI gene (GI:55746). The number of PCR cycles used (35 cycles of a primer extension period of 1 min at 72 °C) was within the linear range for the PCR signals yielded for apo AI. RT-PCR signals of 255 bp fragments from apo AI mRNA transcripts were normalized with the signals obtained from glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Zheng et al. 2000). The mRNA levels were then determined using northern blotting analysis as we previously described (Murao et al. 1998).
Cell culture and treatment
Hep G2 cells have previously been used by ourselves and others for studying both signaling pathways and apo AI expression (Herbomel et al. 1984, Murao et al. 1998, Sakai et al. 2000). Cells were maintained in Dulbecco’s modified Eagle’s medium (Life Technologies, Inc.) supplemented with 10% bovine calf serum (Life Technologies, Inc.) and penicillin/streptomycin and cultured at 37 °C in 5% CO2. Cells were cultured overnight in serum-free medium (0% fetal bovine serum) prior to addition of the agent(s) of interest. Times for treatment with various drugs are indicated in the figure legends.
Stable transfection and CAT activity assay
Hep G2 cells were transfected using LipofectAMINE (Life Technologies, Inc.). Stably transfected Hep G2 cells were created by cotransfecting the plasmids pAI.474-CAT and pRc/CMV2 (Invitrogen) that carries neomycin resistance. Single colonies that grew in media containing 400 µg/ml geneticin (G418) were assayed for CAT activity as previously described (Romney et al. 1992, Murao et al. 1998, Zheng et al. 2001). The positive colonies were selected for further studies as previously described (Zheng et al. 2000, 2001).
Calcineurin phosphatase activity assay
To measure the phosphatase activity of calcineurin, cultured cells were lysed by repeated freezing and thawing in lysis buffer (50 mM Tris–HCl (pH 7.5), 0.1 mM EGTA, 1 mM EDTA, 0.5 mM dithiothreitol (DTT), 0.3 mM phenylmethylsulfonyl fluoride, 0.1% CHAPS, 50 µg/ml soybean trypsin inhibitor, 5 µg/ml leupeptin, and 5 µg/ml aprotinin). The calcineurin activity was determined photometrically (630 nm) at 30 °C after using the RII phosphopeptide substrate (150 µM, Calbiochem) as previously described (Dunn et al. 2000). Essentially, 30 µl cell lysates were mixed with 20 µl reaction solution containing 40 mM Tris (pH 8), 100 mM NaCl, 6 mM MgCl2, 500 µM CaCl2, 500 µM DTT, 750 nM okadoic acid, 150 µM peptide, 3 µM calmodulin, and incubated for 30 min at 30 °C in the presence or absence of CsA, FK605, or rapamycin. The detection of released phosphate is based on the molybdate: malachite green assay (Promega). Data are expressed as the number of picomolar of phosphate released per minute per milligram of protein.
Data analysis
Data are presented as the mean ± S.E.M., which represents three independent experiments. Statistical differences among groups were examined using ANOVA.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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CsA is known to induce hyperlipidemia and dyslipoproteinemia with low concentrations of HDL (Wu et al. 1999). Therefore, we hypothesize that CsA can reduce serum apo AI concentrations. To test this possibility, we used a rat model created by s.c. injection of CsA for 7 days. Western-blot analysis showed that CsA-treated rats had significantly lower levels of apo AI than control rats (Fig. 1A
). Apo AI can be synthesized in the liver followed by secretion into the bloodstream. Therefore, the observed reduction in serum apo AI protein could result from either inhibition of secretion or apo AI gene expression or both. Therefore, we measured apo AI mRNA levels in liver tissues using semi-quantitative RT-PCR. Our results showed that apo AI mRNA levels were significantly lower in CsA-treated rats than in controls (Fig. 1B). This was subsequently confirmed using northern-blotting analysis, in which the reduction of apo AI mRNA levels reached up to 29 ± 5% compared with the controls (n = 5, P < 0.01). This finding was consistent with the observed reduction in serum apo AI protein. Our data, therefore, suggested that CsA inhibited gene expression of apo AI. However, no significant change in total cholesterol, triglycerides, and HDL-cholesterol was observed in the rats after 7 days (data not shown). This observation was consistent with the previous finding that short-term treatments with low doses of CsA have little influence on lipid profile in the rat (Andrade et al. 1993).
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Our in vivo data suggested that CsA inhibits induction of the apo AI gene at the transcription level. The next question was how CsA inhibited apo AI gene transcription? It is known that various hormones and growth factors can regulate the expression of apo AI gene (Hargrove et al. 1999, Mooradian et al. 2004). CsA could inhibit apo AI gene expression through one of those identified mechanisms. To study the underlying mechanism, we took advantage of human hepatoma, Hep G2 cells, because this cell line has been widely utilized in apo AI gene research (Herbomel et al. 1984, Murao et al. 1998, Sakai et al. 2000). In our previous studies, we established the stably transfected Hep G2 cells harboring an apo AI-474-CAT reporter gene (Zheng et al. 2000), and identified that epidermal growth factor (EGF) stimulates apo AI gene expression. Therefore, we first examined whether CsA inhibits apo AI gene activation induced by EGF (100 ng/ml). As shown in Fig. 2, EGF-induced apo AI gene activation was not inhibited by CsA (1 µM; Fig. 2). Furthermore, CsA did not affect apo AI gene expression induced by PKC or PKA activation as we previously reported (Zheng et al. 2000, 2001; data not shown), suggesting that CsA inhibition was through a novel pathway.
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), and the stimulation of apo AI gene expression was time-dependent (Fig. 3). Six-hour treatment induced a significant activation of apo AI gene promoter. Pretreatment of stable Hep G2 cells with actinomycin D, a gene transcription inhibitor, completely abolished the induction of apo AI gene by A23187
[GenBank]
(data not shown), further supporting that activation of apo AI gene by A23187
[GenBank]
is through transcriptional events. Importantly, CsA treatment prevented A23187
[GenBank]
from stimulating the apo AI gene (Figs 2 and 3), suggesting the involvement of calcineurin in apo AI gene regulation. To confirm this, we measured the levels of endogenous apo AI protein within cells and in the culture medium in response to various treatments. As anticipated, A23187
[GenBank]
increased apo AI in cells and culture medium as well, and this stimulation was inhibited by pretreatment with CsA (Fig. 2C). Taken together, our data have suggested a novel mechanism for stimulation of apo AI gene expression involving activation of calcineurin, and also suggested that CsA inhibits apo AI gene expression through inhibiting the calcineurin pathway.
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). However, A23187
[GenBank]
-induced apo AI gene expression was not affected by rapamycin (1 µM), another immunosuppressive agent (data not shown). Finally, we measured the phosphatase activity of calcineurin in Hep G2 cells in the presence of CsA, FK506, or rapamycin. As anticipated, our data in Fig. 5 showed that CsA and FK506, but not rapamycin, inhibited the activities of calcineurin phosphatase, providing a further correlation between reduction of apo AI expression and inhibition of calcineurin activity. In summary, our data indicate that the calcineurin pathway is involved in apo AI gene expression and CsA inhibits apo AI gene expression through inhibiting calcineurin.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Apo AI is an important functional constitute of HDL particles, which are key to transfer of excess peripheral cholesterol to the liver for disposal. Increasing evidence indicates that activation of various intracellular signaling pathways stimulates apo AI gene expression, but it is not completely understood which pathway, if inhibited, will contribute to the reduction of apo AI in vivo. CsA as an immunosuppressive drug has been widely used in organ transplant patients and other conditions as well. Unfortunately, CsA induces significant hyperlipidemia, and its use is associated with increased atherosclerotic disease. CsA, a calcineurin inhibitor, was found to form a complex with ciclophilin to bind calcineurin (Colgan et al. 2005). However, it is not clear how calcineurin inhibition by CsA contributes to the development of atherosclerosis. Our data have shown that 7-day treatment with CsA reduces apo AI gene expression as indicated by the reduction of serum apo AI concentrations and apo AI mRNA levels in liver tissues. Since a low HDL-cholesterol is a significant independent cardiovascular risk factor, this mechanism has powerful clinical implications.
Through investigation of the underlying mechanisms, we have found that calcium mobilization as a novel mechanism stimulates apo AI gene expression and this pathway is inhibited by CsA, a calcineurin inhibitor. We previously identified that growth factors and hormones, such as EGF and insulin, can stimulate apo AI gene expression (Murao et al. 1998, Zheng et al. 2001). We also showed that activation of PKA or PKC can upregulate apo AI gene expression through activation of the Sp1-transcription factor (Zheng et al. 2000). These mechanisms identified in vitro could contribute to the in vivo regulation of apo AI gene activity. However, CsA inhibits apo AI gene transcription in vivo, but does not affect the EGF-induced apo AI gene expression in Hep G2 cells, suggesting that EGF-induced apo AI expression, if present in vivo, may not be inhibited by CsA. Instead, CsA inhibits the stimulation of apo AI by A23187 [GenBank] , suggesting that calcium mobilization-induced apo AI gene activation could be a mechanism present both in vitro and in vivo. Therefore, our finding that calcium stimulates apo AI gene expression may open a new avenue to develop clinical approaches to increase serum apo AI protein levels.
In addition, A23187 [GenBank] , a calcium ionophore, is known to stimulate calcineurin in various cell systems (Tsuboi et al. 1994, Schaefer et al. 1996, Crabtree 2001). Activation of calcineurin stimulates the gene transcription through dephosphorylating various transcription factors (Crabtree 2001). Therefore, that A23187 [GenBank] -induced apo AI gene expression was inhibited by the calcineurin inhibitors (Flanagan et al. 1991), CsA and FK506, strongly suggests that calcineurin is a key molecule that stimulates apo AI gene expression in vivo. Although more studies will be required to elucidate how calcineurin induces apo AI gene activity, our data have pointed to a direction in which to develop drugs to activate liver and intestinal calcineurin to increase apo AI production.
In summary, our results have provided evidence that CsA inhibits apo AI gene expression through inhibition of the calcineurin pathway. We have also identified a novel mechanism for apo AI gene expression involving the activation of calcium/calmodulin-dependent calcineurin. These studies should lead to a newer focus on HDL metabolism in patients treated with CsA. However, it is still unclear whether and howphysiologicalstimuliactivatethe calcium/calmodulin-dependent calcineurin pathway to upregulate apo AI gene expression in vivo. Inaddition, the signalingmechanism(s) at the downstreamofcalcineurin, including the transcription factor(s) and the response element(s), has not been determined. Therefore, more studies will be required for further clarification.
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Acknowledgements |
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References |
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Received in final form 10 May 2006
Accepted 1 August 2006
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Significant difference when compared with the group treated with A23187 alone (n = 6, P < 0.01). (C) A representative western-blot analysis of apo AI protein in the lysates and corresponding culture medium of Hep G2 cells exposed to A23187 and EGF with and without CsA.
