ELSEVIER
Atherosclerosis
journal homepage: www.elsevier.com/locate/atherosclerosis
HE
atherosclerosis
Paraoxonase 1 deficiency in mice is associated with reduced steroid biosynthesis: Effects on HDL binding, cholesteryl ester accumulation and scavenger receptor type BI expression
Aviva Gamliel-Lazarovicha, Anna Gantmana, Maayan Shinerª, Raymond Colemanb, Michael Aviramª, Shlomo Keidara,*
a Lipid Research Laboratory, The Rappaport Family Institute for Research in the Medical Sciences, Rappaport Faculty of Medicine, Technion and Rambam Medical Center, Haifa 31096, Israel
b Department of Anatomy and Cell Biology, Rappaport Faculty of Medicine, Technion - Israel Institute of Technology, Haifa, Israel
ARTICLE INFO
Article history: Received 2 October 2009 Received in revised form 14 January 2010
Accepted 28 January 2010 Available online 6 February 2010
Keywords: Paraoxonase 1 (PON1) Corticosterone HDL SR-BI Adrenal
ABSTRACT
Objective: Selective uptake of high density lipoprotein (HDL) cholesteryl ester (CE) is considered as the major source of cholesterol for production of steroids in the adrenal gland in rodents. As paraox- onase 1 (PON1) is an HDL-associated lipo-lactonase that has been shown to increase binding of HDL to macrophages, we used PON1 knock-out (PON1KO) mice to test the possible role of PON1 in corticosterone (CS) biosynthesis.
Methods and results: PON1 deficiency was associated with reduced serum CS concentration. Adrenal glands obtained from PON1KO mice had significantly lower CE content compared to adrenals from C57Bl6 control mice. Binding of HDL obtained from PON1KO mice to human adrenocortical carcinoma cell line was found to be significantly lower than that of control HDL, and was associated with decreased CS biosynthesis. Addition of purified PON1 to HDL from PON1KO mice increased HDL binding and CS synthe- sis. Furthermore, the expression of the HDL receptor, SR-BI, protein and mRNA, was reduced in adrenals from PON1KO mice compared to control mice. When challenged with low salt diet, PON1KO mice demon- strated an increase in adrenal SR-BI gene expression and in serum corticosterone which reached levels similar to those obtained in control mice.
Conclusion: PON1 regulates adrenal CS biosynthesis at two levels: (a) via an accessory role in HDL binding properties, and (b) a supportive role in SR-BI expression and CE supply to the cells.
@ 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Synthesis of steroidal hormones in the adrenal gland requires a supply of cholesterol which is the precursor of the steroidogenic axis and production of corticosteroids (CS). The major source of cholesterol for steroid production in rodents is cholesteryl ester (CE) from HDL [1-3]. In vitro and in vivo studies have demonstrated that the majority of CE is delivered from HDL to the cells without particle uptake [4-8]. The delivery of HDL-CE involves binding of HDL to the specific HDL scavenger receptor class B type I (SR-BI) which allows for cholesterol flux down a concentration gradient [9]. SR-BI was shown to be the major route for the selective uptake of HDL-CE in cultured adrenal cells [10]. Specific apolipoprotein com- ponents on HDL were shown to play a role in the HDL-CE selective cellular uptake. Whereas apolipoprotein A-I (apo A-I) was shown
to be essential for CE accumulation, and steroid production under normal or stimulated conditions, apo E and apo A-II deficiency had only modest effect on cellular CE accumulation [11].Paraoxonase 1 (PON1) is an HDL-associated lipo-lactonase and its activity is inversely related to the risk for cardiovascular diseases [12,13]. The major apolipoprotein on HDL, apoA-I, was shown to stabilize PON1 activity [14-16] and deficiency of apo-AI resulted in a signif- icant reduction in PON1 mass and activity [17]. The protective role of PON1 against atherosclerosis development was demonstrated in studies, which used mice lacking PON1 (PON1KO) [18,19] or overexpressing PON1 [20]. The PON1 antiatherogenic properties include protection of LDL, HDL and macrophages against oxida- tive stress, attenuation of oxidized-LDL uptake by macrophages and inhibition of macrophage cholesterol biosynthesis (reviewed in ref- erence [21]). PON1 was previously shown also to stimulate HDL binding and HDL-mediated macrophage cholesterol efflux [22].
Since HDL is the major source of cholesterol for steroid synthe- sis and as PON1 stimulates HDL binding to cells, we hypothesized that PON1 might play an important role in CS biosynthesis in the
* Corresponding author. Tel .: +972 4 8542518; fax: +972 4 852721. E-mail address: skeidar@rambam.health.gov.il (S. Keidar).
adrenal. Using PON1KO mice and adrenal cell line, we show that PON1 plays an important role in adrenal function expressed in HDL binding, SR-BI expression, CE accumulation and CS biosynthesis in the adrenal.
2. Materials and methods
2.1. Animals
PON1 knock-out (PON1KO) mice on the background of C57BL6 mice were obtained as previously described [23]. C57BL6 mice were used as wild type controls (C57). Mice aged 6-8 weeks were fed with a normal or a low salt diet (LSD) (Harlan Teklad, Madison, WI, USA) for 4 weeks. The animal study protocol was approved by the Committee for the Supervision of Animal Experiments of the Technion - Israel Institute of Technology and was conducted in accordance with Israel laws for animal care.
2.2. Lipoprotein preparation
HDL was separated from serum by sequential ultracentrifuga- tion [24]. The yields of two different preparations of PON1KO- and C57-HDL were similar and the ratio of cholesterol to protein did not differ significantly (2.37 ± 0.05 and 2.41 ± 0.05, respectively). FITC- conjugated HDL was prepared from human serum as described by Smythe et al. [25].
HDL enriched with recombinant PON1 was prepared as previ- ously described [26].
2.3. Human adrenocortical carcinoma (H295R) cell culture
Human adrenocortical carcinoma cells NCI-H295R were pur- chased from ATCC (Manassas, VI, USA) and grown in complete medium according to ATCC recommendation (NuSerum IV from BD Biosciences, medium and other supplements from Biological Indus- tries, Bet HaEmek, Israel). For steroid production and cholesterol content, cells were seeded in 24-well plates and for HDL binding, in 96-well plates. Experiments were done in confluent cultures in complete growth medium with additives as indicated.
2.4. Steroid analysis
Cortisol was measured by Immulite (Siemens Healthcare Diag- nostics Deerfield, IL, USA), and corticosterone was measured using an ELISA assay kit (AssayPro, St. Charles, MO, USA).
2.5. Adrenal cholesterol content
Lipids were extracted from adrenals or H295R cells with hex- ane:isopropanol (3:2, v/v), and the upper phase was dried under nitrogen. Dry lipids were dissolved in cholesterol reaction buffer and subjected to cholesterol/CE quantification according to assay protocol (BioVision, Mountain View, CA, USA). Fluorescence inten- sity, ex535/em590, was measured in the Fluostar Galaxy plate reader (BMG Lab Technologies, Offenburg, Germany).
2.6. HDL binding to H295R cells
Confluent 96-well plate cultures were placed on ice and washed three times with cold phosphate-buffered saline (PBS). Cells were then incubated with serum free medium with mice HDL (from PON1KO or C57) at the indicated concentrations for 1h at 4℃. Twenty µg protein/ml of FITC-labeled human HDL were added to the cells for a further 30 min, followed by three washes with PBS. Fluorescence intensity, ex490/em520, was measured in the Fluostar Galaxy plate reader (BMG Lab Technologies, Offenburg,
Germany). Total binding was determined in the presence of labeled HDL alone and nonspecific binding was determined in the presence of 200 µg unlabeled HDL. Specific binding was calculated by sub- tracting the nonspecific binding from the total binding. Results are presented as percent of specific fluorescence bound in the presence of mice HDL to specific binding in the absence of mice HDL.
2.7. Adrenal gene expression
RNA was extracted from tissue or cells using MasterPure™ RNA purification kit (Epicentre Biotechnologies, Madison, WI, USA). cDNA was prepared using Verso™ cDNA kit (Thermo Scientific, Epsom, UK). Primers and probes for GAPDH were designed by Primer Design, Southampton, UK. Using ABsolute Blue QPCR ROX mix (Thermo Scientific), expression was determined by quantita- tive real-time PCR with Rotor-Gene 6000 amplification detection system. SR-BI expression was measured as described previously [27].
2.8. SR-BI immunohistochemistry
Frozen sections of freshly dissected adrenals were fixed with ice cold acetone for 10 min. After blocking with 5% BSA in PBS, sections were incubated for 1 h with rabbit anti-mouse SR-BI anti- body (gift from Dr. M Krieger, MIT, Cambridge, MA, USA), diluted 1:50 (v/v) followed by 30 min incubation with FITC-labeled donkey anti-rabbit antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Nuclei were counterstained with propidium iodide (Sigma-Aldrich, St. Louis, MO, USA). Photographs were captured in Radiance2000 Bio-Rad confocal microscope with Plan Apo 60x/1.4 oil DIC objective. Intensity analysis was done using Image Pro Plus® 5.0 software (Media Cybernetics, Silver Spring, MD, USA).
2.9. Statistical analysis
Results are expressed as mean ±SEM. Two-tailed Student’s t- test was used to determine statistical significance when comparing two arrays of data.
3. Results
3.1. Decreased serum CS concentration in PON1KO mice compared to wild type C57 mice
Basal serum corticosterone levels were significantly reduced by 50% in PON1KO in comparison to control mice (p<0.001) (Fig. 1A).
3.2. PON1KO mice express lower adrenal cholesteryl ester content than control C57 mice
To test whether the diminished steroid production is a result of decreased accumulation of adrenal CE, cholesterol profile of the adrenal lipid extracts were examined. Adrenal CE was 50% (p <0.05) lower in PON1KO compared to C57 mice (Fig. 1B), whereas total cholesterol (TC) in the adrenals did not differ significantly (Fig. 1C).
3.3. PON1 has an accessory role in HDL cellular binding and steroidogenesis in human adrenal carcinoma cells (H295R)
As PON1 is an HDL-associated enzyme, the decreased CE accu- mulation in the adrenals from PON1KO mice may reflect a direct effect of PON1 on HDL binding. To examine this point, we deter- mined binding of HDL obtained from PON1KO and from control C57 mice to H295R cells. The ability of HDL lacking PON1 to bind to H295R cells is significantly (p <0.05) reduced compared to con- trol, PON1-containing HDL (Fig. 2A). At a concentration of 50 µg/ml
Serum Corticosterone, ng/ml
80
(A)
3.5
(B)
30.0
(C)
70
3.0
1
T
60
Adrenal CE, ug/mg
Adrenal TC, µg/mg
25.0
2.5
50
**
20.0
2.0
40
*
15.0
30
T
1.5
10.0
20
1.0
10
0.5
5.0
0
C57
PON1KO
0.0
C57
PON1KO
0.0
C57
PON1KO
of HDL cholesterol, C57-HDL inhibited binding of labeled HDL by 50%, whereas PON1KO-HDL at the same concentration inhibited the binding by about 20% of the binding. The contribution of PON1 to binding of HDL was examined in another set of experiments where purified recombinant PON1 was added to HDL obtained from PON1KO mice. Whereas 50 µg/ml HDL from PON1KO inhibited binding of FITC-HDL to H295R cells by 14% only, addition of purified recombinant PON1 to HDL obtained from PON1KO mice increased HDL binding to a level similar to HDL obtained from C57, with inhibition of FITC-HDL of 34% and 46%, respectively (Fig. 2B). Corti- sol biosynthesis in H295R cells supplemented with 50 µg/ml HDL cholesterol obtained from PON1KO mice was only 64% (p<0.05) of that observed with HDL obtained from C57 control mice. Enrich- ment of HDL from PON1KO mice with purified PON1 increased cortisol synthesis to 85% of that measured in HDL obtained from C57 mice (Fig. 2C). These results point to the direct involvement of
PON1 in mediating HDL binding to the cells and consequently the steroid hormone synthesis.
3.4. Expression of SR-BI is lower in adrenals from PON1KO vs. control C57 mice
The observation that PON1 contributed to CS biosynthesis in H295R adrenal cells, suggests that the HDL receptor SR-BI may be involved. Immunohistochemistry of cryostat sections of the adrenal cortices from control C57 mice shows indeed prominent staining of cellular SR-BI, whereas in PON1KO mice, SR-BI is almost absent (Fig. 3A). The intensity of SR-BI labeling itself or the ratio of SR-BI to nucleus number is significantly lower in PON1KO compared to C57 control mice adrenals (Fig. 3B). SR-BI mRNA expression in adrenals from PON1KO mice was less than 50% of that observed in control mice (Fig. 3C).
100%
% FTIC-HDL specific binding
% FTIC-HDL specific binding
(B)
*
110%
(A)
80%
100%
60%
*
40%
90%
*
*
20%
80%
0%
70%
HDL C57
HDL
HDL PON1KO +repon1
PON1KO
60%
240.0
(C)
50%
Cortisol, nmol/L
200.0
40%
160.0
*
0
20
40
60
HDL, mg/ml
120.0
80.0
40.0
0.0
HDL C57
HDL PON1KO
HDL PON1KO +rePON1
(A)
Adrenal SR-BI intensity/nuclei, AU
400
(B)
Adrenal SR-BI mRNA expression Normalized to GAPDH, AU
(C)
PON1KO
1000
C57
C57
350
900
SR-BI
300
800
GAPDH
250
700
600
200
500
**
PON1ko
150
400
*
100
300
200
50
100
0
C57
PON1KO
0
C57
PON1KO
3.5. Dietary sodium restriction increased CS biosynthesis in PON1KO mice
When challenged with low salt diet (LSD), which was previously shown [28] to stimulate the key molecule in regulating cholesterol transfer across the mitochondrial membrane, the steroidogenic acute regulatory protein (StAR), serum CS levels in PON1KO sig- nificantly increased, reaching a level similar to that observed in C57 control mice (Fig. 4A). Serum CS levels in C57 mice were not significantly different under normal or low sodium diet.
Expression of SR-BI in adrenals from PON1KO mice were also significantly increased and reached the same levels as measured in control C57 mice (Fig. 4B).
4. Discussion
The results of the present study show, for the first time, that PON1 deficiency is associated with attenuated steroidogenegis. This study provides evidence that the absence of HDL-associated PON1 resulted in reduced HDL binding to adrenal cells and decreased adrenal CE accumulation. Moreover, in the absence of PON1, cellular SR-BI (an HDL receptor) expression was suppressed in the adrenal under normal diet, and it was re-activated under LSD stimulation. These results indicate that PON1 is involved at least in
two levels of adrenal regulation, i.e. an accessory role in HDL lig- and binding properties to adrenal cells, and a stimulatory effect on the cellular expression of HDL receptor through which adrenal cholesteryl ester channeling occurs.
Although it was previously shown that serum cholesterol con- centration and distribution did not differ significantly between PON1KO and wild type mice [23], in the present study we have demonstrated that adrenal CE content and steroidal production, are severely blunted in PON1KO mice. Binding of HDL, which is the first event in the selective uptake of CE from HDL to adrenal cells, was significantly lower in the absence of PON1 and this phenomenon was consistent with the reduced CS biosynthesis. Addition of recombinant PON1 to HDL from PON1KO miced improved both, HDL binding and CS synthesis. However, PON1 contribution to CS production was only a partial one, suggesting that PON1 has an accessory and probably regulatory, rather than a major role in HDL binding, HDL-dependent CE delivery to the cells, and CS biosynthe- sis.
The observed differences in steroidogenesis under a normal diet vs. LSD stimulated conditions, suggest that the effects of PON1 defi- ciency extend beyond a direct ligand-receptor interaction (HDL binding to the adrenal SR-BI) and involve cellular mechanisms that regulate adrenal CE supply.
In apo-AI deficient mice, alternative cellular cholesterol supply mechanisms were reported to be up-regulated under non-
90
(A)
1000
(B)
Serum Corticosterone, ng/ml
Adrenal SR-BI mRNA expression normalized to GAPDH
80
900
I
70
800
T
60
700
600
50
500
40
400
30
300
20
200
10
100
0
0
T
1
C57
T
PON1KO
C57
PON1KO
stimulated, as well as under stimulated conditions [11]. However, in PON1 deficiency, SR-BI, which is considered as the major recep- tor mediating CE transport from the HDL particles to the adrenal cells [9], is down-regulated in PON1KO adrenals at both, the pro- tein and the mRNA levels. Thus, not only that the HDL of PON1KO mice have a lower ability to bind to SR-BI, but also the amount of the receptors is reduced.
Serum corticosterone levels in control mice were similar under normal or sodium restricted diet and this result is in agreement with previous reports in rats [29,30]. The observation that in PON1KO mice LSD treatment increased CS levels which paralleled the increased SR-BI expression might reflect, at least in part, a role for PON1 in regulating cellular CE transfer process required for ade- quate steroidogenesis. A possible role for PON1 in this respect may be related to its hydrolytic activity on oxidized lipids [31] and the release of arachidonic acid, which was shown to be essential for steroidogenesis [32]. As cAMP, known to be activated by arachi- donic acid and other polyunsaturated fatty acids, is increased in response to dietary sodium restriction [33], LSD could compensate for the absence of PON1 in order to achieve normal steroidogene- sis rate. Alternatively, the anti-oxidative properties of PON1 could also contribute to reverse cholesterol transport as it was recently shown that specific oxidized phospholipids inhibit SR-BI-mediated selective uptake of CE [34].
Mutual relations between PON1 and SR-BI have also been shown in SR-BI knockout mice where reduced expression of PON1 was noted [35]. As SR-BI was shown to play a protective role against atherosclerosis [36], if the phenomenon of reduced SR-BI in the absence of PON1 extends to tissues other than the adrenal, such as arterial macrophages, PON1 anti-atherogenicity could be attributed, at least in part, to the beneficial regulation of SR- BI.
Epidemiological studies have reported conflicting data on the role of PON1 in cardiovascular disease (CVD) [37,38]. Neverthe- less, the genetic variant of PON1, the R phenotype, with the amino acid substitution of glycine to arginine at position 192, with a higher catalytic activity was associated with increased risk for CVD. The contradiction between the higher activity and the increased risk for CVD could be explained, at least in part, by the results of the present study which indicate possible opposing roles for PON1; on the one hand, PON1 is associated with higher CS, a known inflammatory and atherosclerotic risk factor, but on the other, it increases SR-BI which protects against atherosclerosis [39].
In summary, PON1 may enable normal steroidogenesis by its regulatory role on HDL binding properties to the adrenal on one hand, as well as its role on SR-BI expression on the other hand, which results in cellular CE accumulation and corticosteroids pro- duction.
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