ELSEVIER Biochimica et Biophysica Acta 1302 (1996) 153-158

BB Biochi~ic~a et Biophysica AEta

Age-related changes in mRNA, protein and catalytic activity of hepatic neutral cholesterol ester hydrolase in male rats: evidence for

transcriptional regulation

Ramesh Natarajan, Shobha Ghosh, W. McLean Grogan * Department of Biochemistry and Molecular Biophysics, Medical College of Virginia, Virginia Commonwealth Universi~, Richmond, VA 23298-0614, USA

Received 25 January 1996; revised 12 March 1996; accepted 15 March 1996

Abstract

Messenger RNA, protein mass and catalytic activity of hepatic neutral cholesteryl ester hydrolase (CEH) were measured in male Sprague-Dawley rats, aged 6, 8, 9.5, 12 and 24 weeks (wks). CEH mRNA increased 101% from 6 to 9.5 wks, corresponding to onset of puberty, and declined by 52% from 12 to 24 wks. CEH mass was highly correlated with mRNA levels at all ages, increasing 170% from 6 to 9.5 wks and declining 61% from 12 to 24 wks. CEH activity was highly correlated with mass and mRNA from 8-24 wks, but was greater at 6 wks than the activity predicted by the measured mass. In all age groups, activity was consistently increased by activation of endogenous protein kinase A and consistently inhibited by alkaline phosphatase, suggesting that age-related differences in catalytic activity were not due to differences in the level of enzyme phosphorylation. These data suggest transcriptional regulation and indicate an important role for CEH in cholesterol homeostasis in the developing rat.

Keywords: Cholesteryl ester hydrolase; Cholesterol esterase; Developmental change; Protein kinase A; Enzyme phosphorylation; Transcriptional regulation; (Rat liver)

1. Introduction

Hepatic levels of f ree and esterified cholesterol are regulated by the combined activities of 3-hydroxy-3- methylglutaryl-coenzyme A (HMGCoA) reductase, choles- terol 7c~-hydroxylase (C7aH), acyl-CoA:cholesterol acyl transferase (ACAT) and cholesterol ester hydrolase (CEH). Although the role of hepatic CEH in regulation of choles- terol homeostasis has received relatively little attention, a number of studies have indicated that the rat hepatic neutral cytosolic CEH is highly regulated in concert with the other three enzymes [1-3]. Whereas earlier studies have indicated that this enzyme is activated by protein

Abbreviations: HMGCoA, 3-hydroxy-3-methylglutaryl coenzyme A; C7c~H, cholesterol 7a-hydroxylase; ACAT, acyl-CoA:cholesterol acyl- transferase; CEH, cholesterol ester hydrolase; cAMP, adenosine 3',5'- cyclic monophosphate; SSC, 3.0 M sodium chloride and 0.3 M sodium citrate (pH 7.0); TCA, trichloroacetic acid; S.E.M., standard error of the mean,

Corresponding author. Fax: + 1 (804) 8281473; e-mail: grogan@gems.vcu.edu.

kinase [4], other mechanisms of regulation have not been reported. Extensive changes in synthesis, degradation and excretion of cholesterol occur during aging [5-10]. These changes are mediated by altered activities of HMGCoA reductase, C7aH, ACAT and CEH. Story et al. [6] showed that the activity of HMGCoA reductase decreases from 2 to 9 months of age, and then remains stable through 24 months of age. Choi et al. [8] reported that the activity of HMGCoA reductase was lower in 8 month-old rats than in 5 week-old rats. Stahlberg et al. [10] reported a higher activity in 1 month-old rats, than in 6 or 24 month-old rats. They also reported a decline in activity during sexual maturation. In general, HMGCoA reductase activities were lower in adult rats than in younger rats [11-14]. A gradual decrease with advancing age has also been reported in the activity of C7o~H [6,8,10], a rate-limiting enzyme in bile acid formation. Earlier studies revealed no age related changes in activity of ACAT, which catalyzes the esterifi- cation of cholesterol [15,16], although more recent reports indicate that ACAT activity increases slightly with age [ 10,17]. Erickson et al. have recently reported measuring very low levels of cytosolic CEH activity during postpar-

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154 R. Natarajan et al. / Biochimica et Biophysica Acta 1302 (1996) 153 158

tum development of the rat, using assay conditions opti- mized for measurement of microsomal CEH activity [18]. However, there are no reports of any similar study under conditions appropriate for measurement of cytosolic CEH.

The major cytosolic cholesterol ester hydrolytic enzyme in rat liver has been purified and characterized in this laboratory [2,4,19,20]. The cDNA for this hepatic CEH has also been identified, cloned and expressed [21]. In the current study we have measured hepatic neutral CEH specific activities, CEH protein mass and CEH mRNA levels from rats of different ages in order to evaluate the role of this enzyme in cholesterol homeostasis during development of the rat.

2. Materials and methods

2.1. Chemicals and supplies

Cholesterol[1-14C]oleate (56.6 mCi/mmol) was pur- chased from New England Nuclear (Boston, MA). All solvents were purchased from Fischer Scientific (Col- umbia, MD). ATP disodium salt, adenosine 3',5'-cyclic monophosphate (sodium salt), MgC12, and alkaline phos- phatase (Type IX from bovine liver) were purchased from Sigma (St. Louis, MO). GeneScreen membrane was pur- chased from NEN (Boston, MA). SDS-PAGE and Western blot supplies were purchased from Bio-Rad (Richmond, CA). All other chemicals used were of analytical grade.

Sprague-Dawley rats with certified dates of birth were purchased from Zivic Miller Laboratories (Zellenople, PA) and fed standard laboratory chow ad libitum until use. Rats were maintained on a uniform 12 h light/12 h dark cycle at all times.

2.2. Preparation of 104 000 X g supernatant (cytosol)

Rats were killed by decapitation and livers removed and washed in 20 mM Tris-HC1 buffer (pH 7.5), containing 5 mM 2-mercaptoethanol, 100 mM sucrose and 80 mM KCI. Livers were homogenized with a loose Teflon pestle in 2 ml buffer/g tissue buffer. The 104000 X g supernatant was prepared by differential centrifugation, as described elsewhere [4].

2.3. Enzyme assays

CEH activity was assayed in 104000 × g supernatants, as described previously [4]. Endogenous cAMP-dependent protein kinase was activated as described previously [4], by preincubating the reaction mixture for 1-2 min with 1 mM MgCI 2, 5 mM ATP and 100 p~M cAMP, followed by addition of substrate and an additional 30 min incubation. For alkaline phosphatase inactivation, the reaction mixture was preincubated with the phosphatase for 30 min, fol- lowed by addition of substrate and an additional 30 rain

incubation, as described previously [4]. All assays were done in triplicate for each of 3-5 rat livers per age group.

2.4. Western blot analysis

Proteins separated by SDS-PAGE were electroblotted onto Immobilon-PVDF membranes using the Bio-Rad Trans Blot Cell. Western blots were reacted with poly- clonal antibody to hepatic cytosolic CEH and developed as described previously [20], substituting 2% gelatin in TNT (20 mM Tris containing 500 mM NaCI and 0.05% Tween 20, pH 8.0) for 5% nonfat dry milk in the blocking solution. For quantitation, Western blots were scanned at 350 nm with a Shimadzu densitometer. A standard curve was constructed using different concentrations of hepatic CEH, purified to a single band on PAGE gel by the procedure of Ghosh and Grogan [19]. The standard curve was linear over the range of CEH protein concentrations measured in rat cytosol (r = 0.97). Three different concen- trations of cytosolic protein were analyzed from each of 3-5 rat livers per group. Each of these concentrations yielded CEH values in the linear range of the standard curve and equivalent values when normalized for total cytosolic protein.

2.5. Northern blot analysis

Total RNA was prepared from freshly isolated liver using TriReagent according to the manufacturer's instruc- tions. An aliquot estimated to contain 10 p~g of total RNA was electrophoresed on a 1% agarose gel in the presence of formaldehyde. The RNA was stained with Ethidium bromide and the integrity of the isolated RNA verified by presence of 28S and 18S rRNA bands. RNA was trans- ferred to GeneScreen membrane and hybridized with 32p_ labeled full-length cDNA for CEH [21] according to the manufacturer's instructions. After high stringency washes (0.2 X SSC+0 .1% SDS at 65°C for 15 rain), positive hybridization was detected by exposure to Kodak-2 films for 18 h at -70°C. Blots were stripped with 0.1% SDS at 100°C for 5 rain and reprobed with a 32p-labeled 0.8 kb probe for cyclophilin mRNA, used as a constitutive inter- nal standard [22]. Autoradiograms were scanned using a USB SciScan 5000 densitometer for quantitation and val- ues obtained for CEH mRNA were normalized to corre- sponding cyclophilin values in the same sample lane.

2.6. Protein estimation

Protein was TCA precipitated and estimated by the BCA procedure [23].

3. Results

Specific activity, protein mass and mRNA levels of hepatic cytosolic CEH were measured in rats at 6, 8, 9.5,

R. Natarajan et al. / Biochimica et Biophysica Acta 1302 (1996) 153-158 155

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Fig. 1. Hepatic cytosolic CEH mRNA, mass and catalytic activity in male rats at various ages. (A) CEH mRNA was measured (relative to constitu- tive cyclophilin mRNA) by quantitative Northern blotting with full-length CEH cDNA (see Fig. 5, for representative autoradiogram and Section 2 for experimental details); CEH protein (~g immunoreactive protein/mg cytosolic protein), by quantitative Western blotting with anti-liver CEH polyclonal antibodies (cf. Fig. 2, for representative Western blot). (B) CEH catalytic activity (hydrolysis of cholesteryl oleate) was measured radiometrically in 104000 × g supernatants, in the presence ( + PKA) and absence ( -PKA) of protein kinase A activators (l mM MgCI2, 5 mM ATP, 100 IxM cAMP). Broken lines extrapolate to values expected at age 6 wks, calculated from the amounts of immunoreactive protein, using the linear regression functions depicted in Fig. 3A. Each data point is the mean _+ S.E.M. for 3-5 rats, each rat assayed in triplicate. Where not visible, error bars are contained within symbols.

12 and 24 wks after birth. As shown in Fig. 1A, C EH protein mass, measured by quant i ta t ive Western blot analy- sis (see Fig. 2 for representat ive Western blot) increased 170% be tween 6 and 9.5 wks ( P < 0.001), remained at that level through 12 wks and decl ined thereafter. At 24 wks, CEH protein had decreased 61% from the level in 12 week-old rats ( P < 0.001). CEH specific activity fol lowed a s imilar pattern, peaking at 9.5 wks and decl ining as the

12 24 Wks 6 8 9.5

- C E H

Fig. 2. Representative Western blot analysis of rat l iver cytosols with polyclonal antibody to rat l iver CEH, depicting variation of CEH mass in 6, 8, 9.5, 12 and 24 week-old male rats. Blots were scanned densitometri- cal ly and values converted to corresponding masses by comparison with a standard curve obtained with varying concentrations of purified rat l iver CEH. Result ing values are depicted in Fig. 1A, 3A and 3C. See Section 2 for experimental details.

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Fig. 3. Correlations among CEH catalytic activity, immunoreactive pro- tein and mRNA in livers from male rats of various ages. Lines in Fig. 3A-B depict best-fit linear regression for 8-24 week-old rats (solid symbols; r > 0.99). Open symbols represent corresponding values for 6 week-old rats. Fig. 3C depicts linear regression line of protein and mRNA for all ages, 6-24 wks (r > 0.99). Each data point is the mean for 3-5 rats.

rats aged (Fig. 1B). As seen in Fig. 3A, catalytic activity was highly correlated with C EH mass from 8 - 2 4 weeks ( r > 0.99), but was substant ial ly higher at 6 weeks, than the value predicted by l inear regression analysis (see also Fig. 1B for extrapolat ion to predicted value).

Previous studies from this laboratory have shown 1 0 0 - 140% st imulat ion of adult rat l iver cytosolic CEH by c A M P [4], apparent ly media ted by the endogenous cAMP-dependen t protein kinase [24]. In order to evaluate the contr ibut ion of cAMP-dependen t protein kinase to the observed changes in hepatic CEH activity be tween 6 and 24 wks, CEH activity was assayed both in the presence and absence of cofactors for c A M P dependent protein kinase. As shown in Fig. 1B, C EH activity was consis- tently higher in all rats, of all age groups, in the presence of cofactors. Al though there appeared to be a trend toward greater act ivat ion with increasing age (Fig. 4), there were no statistically s ignif icant differences in magni tudes of act ivation among the various age groups and this trend did not correlate with catalytic activity or CEH mass.

156 R. Natarajan et al. / Biochimica et Biophysica Acta 1302 (1996) 153 158

Hepatic CEH was earlier shown to be inactivated by alkaline phosphatase, as well as by an endogenous Mg 2+- dependent phosphatase present in rat liver cytosol [4]. In the present study, alkaline phosphatase decreased CEH activity by 23-52% below levels in simultaneous controls, in rats of all age groups (Fig. 4). Although the magnitude of inactivation by alkaline phosphatase appeared to in- crease with age, there were no statistically significant differences among the various groups and this trend did not correlate with activity or CEH mass. Taken together, the trends toward increasing activation by protein kinase and inactivation by alkaline phosphatase may reflect in- creasing susceptibility to phosphorylation/dephosphoryla- tion with age after 6 wks.

Nevertheless, taken with the correlation between cat- alytic activity and protein mass (Fig. 3A), this data sug- gests that the observed variation in activity with age after 6 wks is primarily due to changes in protein mass, rather than differences in the level of enzyme phosphorylation. Moreover, even at 6 wks, neither activation by protein kinase nor inactivation by alkaline phosphatase was suffi- cient to account for the anomalously high catalytic activity in terms of protein phosphorylation. Lending further sup- port to these conclusions, catalytic activity with protein kinase cofactors (Fig. 1B) or alkaline phosphatase (data not shown) exhibited similar variation with age as activity without treatment and exhibited the same pattern of corre- lation with immunoreactive protein (Fig. 3A).

Moreover, as seen in Fig. 1A, mRNA levels, measured by quantitative Northern blot analysis (see Fig. 5) closely paralleled CEH mass, increasing 101% from 6 to 9.5 wks ( P < 0.01) and declining by 52% from 12 to 24 wks ( P < 0.01). As seen in Fig. 3C, CEH mRNA was highly correlated with mass at all ages. Consistent with the corre- lation of activity with mass, catalytic activity was also highly correlated with mRNA from 8-24 wks (Fig. 3B),

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Fig. 4. Comparison of the effects of alkaline phosphatase and protein kinase A on activity of hepatic CEH in male rats of various ages. Aliquots of cytosol were incubated with alkaline phosphatase for 30 min, or protein kinase cofactors (1 mM MgCI 2, 5 mM ATP, 100 IxM cAMP) for 1-2 rain, prior to measurement of CEH activity with cholesteryl oleate. Simultaneous controls were treated identically, with no additions. Values are % difference from corresponding controls _+ S.E.M. for 3-5 rats/group, each rat assayed in triplicate. Where not visible, error bars are contained within symbols. See Section 2 for experimental details.

Wks. 6 8 9.5 12 24

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Fig. 5. Representative Northern blot analysis of total RNA from male rat livers of different ages as indicated. Blots were probed with ~2 P-labeled full-length CEH cDNA under high stringency conditions (upper panel), exposed to film, stripped and reprobed for constitutive cyclophilin mRNA (lower panel). Intensities of individual bands were quantified by densito- metric scanning and values for CEH mRNA were normalized [o corre- sponding values for cyclophilin mRNA to generate data graphed in Fig. IA, Fig. 3B and Fig. 3C.

but deviated substantially from the linear regression line at 6 wks. These results suggest that alterations in catalytic activity and CEH mass reflect altered levels of mRNA in 8-24 week-old rats and provide strong evidence for tran- scriptional regulation of this enzyme.

4. Discussion

Corresponding changes in mRNA, protein mass and specific activity of rat hepatic neutral CEH with age (Figs. 1 and 3) suggest transcriptional regulation of this enzyme. Measurement of catalytic activity under conditions previ- ously determined to give minimum and maximum activa- tion by protein phosphorylation did not substantially alter the relative variation of CEH activity with age; i.e., under all assay conditions, specific activity declined from 6-8 wks, rose to a maximum at 10-12 wks and declined to very low levels at 24 wks. Thus, protein phosphorylation did not appear to play a significant role in the observed variation of activity with age.

CEH mRNA, mass and activity increased markedly from ages 6-9.5 wks, coinciding with onset of puberty, as defined by hormonal changes and initiation of spermatoge- nesis [25]. In contrast, others have reported that HMGCoA reductase exhibits a dramatic decrease in mRNA, mass and activity [10-14,26,27] over the same age range, whereas ACAT exhibits little or no change in activity [10,15,16]. Thus, CEH apparently mediates a compensatory shift in the balance between synthesis and hydrolysis of cholesteryl esters in response to a decrease in de novo cholesterol biosynthesis.

Although both HMGCoA reductase and C7c~H activi- ties are reported to decrease from 1-6 months, the balance between cholesterol synthesis and degradation shifts to-

R. Natarajan et aL /Biochimica et Biophysica Acta 1302 (1996) 153-158 157

ward synthesis, increasing hepatic total cholesterol levels with age [5-7,10]. Inasmuch as most of this increase occurs in cholesteryl ester and there is little or no change in A C A T activity [10], the decline in CEH mRNA, mass and activity observed from 12-24 wks in the current study probably reflects a mechanism which stabilizes hepatic free cholesterol levels in aging rats.

The large variations in CEH mRNA, mass and activity observed here may reflect changes in hormonal balance which occur during puberty. Among the other enzymes of cholesterol homeostasis, progesterone is reported to stimu- late H M G C o A reductase and C7e~ H [27-29] and to inhibit ACAT activity [30,31], whereas estrogen inhibits C 7 ~ H activity [29]. CEH is reported to be stimulated by proges- terone [3,32], whereas estradiol inhibits CEH activity [32], when administered in vivo, but activates CEH in primary cultured hepatocytes [33,34]. Although these earlier studies are consistent with transcriptional regulation of CEH and other enzymes of cholesterol homeostasis by gonadal hor- mones, the mechanisms for the observed effects are not known. Moreover, the effects of androgens on these en- zymes have not been reported.

These studies support the conclusion from earlier stud- ies that this cytosolic CEH is the predominant cholesteryl ester hydrolytic activity in rat liver. Ghosh and Grogan earlier reported that 90% of CEH activity is found in the cytosolic compartment [20], as previously proposed by Deykin and Goodman [35]. Moreover, they reported that hepatic CEH activity is neutralized by the antibodies to purified CEH used for immunoassay in the current study [20]. In the current study, the cytosolic hydrolytic activity was highly correlated with immunoreact ive protein ( r > 0.99) at ages 8 - 2 4 wks (Fig. 3A). Moreover, extrapolation of the regression line to zero activity indicates that this protein accounted for essentially all of the activity at ages greater than 6 wks. Consistent with this observation, mRNA coding for this CEH was also highly correlated with CEH activity at 8 - 2 4 wks (Fig. 3B) and with CEH protein at all ages (Fig. 3C).

In contrast, at 6 wks, hydrolytic activity was much higher than predicted by linear regression with either immunoreact ive protein or mRNA (Fig. 3A and B), indi- cating that either the specific activity or the active fraction of the enzyme was much greater at 6 wks than at greater ages. Although this suggests post-translational regulation, it is unlikely that the anomaly can be explained by differ- ing levels of enzyme phosphorylation, since the anomaly persisted with either protein kinase or alkaline phosphatase treatment and susceptibili ty to protein kinase activators or phosphatase at 6 weeks did not differ significantly from that at any other age (Fig. 4). Mechanisms involving modification of enzyme specific activity or substrate avail- ability by other endogenous factors, such as inhibitory proteins, can not be ruled out [36].

These results provide further evidence for the impor- tance of hepatic cytosolic CEH in the maintenance of

cholesterol homeostasis in the rat. Whereas this study strongly suggests that this CEH is regulated at the tran- scriptional level, identification and characterization of the gene encoding this enzyme and its regulatory elements is essential to unders tanding its role in cholesterol metabolism.

Acknowledgements

This work was supported by a grant from the National Institutes of Health (HD44613).

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