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INTRODUCTION |
Mevalonic aciduria (MA,1 MIM
251170) and hyper-IgD and periodic fever
syndrome (HIDS, MIM 260920) are two autosomal recessive disorders both
caused by a deficient activity of the enzyme mevalonate kinase (MK,
ATP:mevalonate-5-phosphotransferase, EC 2.7.1.36) due to
functional significant mutations in the encoding gene (MVK) (1-5). MA is a severe and often fatal multisystemic disease, characterized by psychomotor retardation, failure to thrive,
hepatosplenomegaly, anemia, and recurrent febrile episodes. HIDS is a
more benign condition, in which patients suffer, as in MA, from
recurrent fever episodes associated with lymphadenopathy, arthralgia,
gastrointestinal problems, and skin rash.
MK enzyme activity in MA is usually below detection levels when
measured in cultured skin fibroblasts of MA patients (2). In HIDS,
however, a residual MK activity ranging between 1 and 7% of the
control value can be measured both in fibroblasts and leukocytes from
patients (4, 6). As a result of the MK deficiency, excretion of
mevalonic acid in urine occurs, which correlates with disease activity
in both syndromes. The basal level of excreted mevalonic acid is 100- to 1000-fold higher in MA when compared with HIDS and in plasma may
reach concentrations of over 500 µM (2, 7).
MK is the first enzyme to follow the highly regulated
3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (HMGR, EC 1.1.1.34) in the isoprenoid/cholesterol biosynthesis pathway and converts mevalonate into 5-phosphomevalonate. This pathway provides cells with
isoprenoids that are vital for diverse cellular processes. The main end
products include isoprenylated proteins, heme A, dolichol,
ubiquinone-10, isopentenyl tRNAs, and sterols. Feedback regulation of
isoprenoid biosynthesis by cholesterol is achieved predominantly
through repression of transcription of genes that govern the synthesis
of cholesterol (HMG-CoA synthase and HMGR) and its receptor-mediated
uptake from plasma lipoproteins (LDL receptor) (8). This regulation is
performed by a class of transcription factors called sterol regulatory
element-binding proteins (SREBPs) (9). The SREBPs are conditional
positive transcription factors that enhance transcription when sterols
are absent but are not required for basal transcription when sterols
are present (8). HMGR, which performs the rate-limiting enzyme step in
isoprenoid biosynthesis, is also subject to several
post-transcriptional regulation mechanisms. These include translational
efficiency of the HMGR mRNA and turnover of the HMGR protein (8).
The rate of translation of HMGR mRNA is dictated by the cell's
demand for non-sterol isoprenoids, whereas the degradation rate of the HMGR protein is regulated by the cell's demand for both sterol and
non-sterol isoprenoids (8). The sterols probably act via the
membrane-spanning domain of HMGR, which is not necessary for catalytic
activity, but has a so-called "cholesterol sensing domain" (8).
Farnesol (FOH) has been implicated to be a non-sterol regulator of HMGR
degradation (10-16), however, this is still a matter of debate (17).
Combined, these different regulatory mechanisms can induce a
200-fold increase in HMGR protein in response to statins (8), potent
competitive inhibitors of HMGR that are used widely to treat
atherosclerosis, and familial hypercholesterolemia. These drugs block
the synthesis of mevalonate and, as a consequence, lower the endogenous
synthesis of isoprenoids.
Although the MK enzyme activity in fibroblasts from MA patients is
hardly detectable with the standard enzyme assays, the biosynthesis of
cholesterol from radiolabeled precursors can be (near) normal in these
cells (18-20). Thus, it appears that MA fibroblasts are able to
compensate for their defect in MK and that the flux through the
cholesterol biosynthesis pathway may be rather normal. This is due to
increased activity of HMG-CoA reductase and the LDL receptor pathway in
such cells (19, 20). It has been reported that this increased activity
of HMG-CoA reductase is insuppressible by exogenous LDL cholesterol and
can be up-regulated further under cholesterol-free culture conditions
(19). We have extended these studies by measuring the effect of
non-sterol and sterol end products on HMGR activity and determining the
effect of HMGR inhibition on protein isoprenylation in fibroblasts from both MA and HIDS patients. Our data indicate that MK-deficient cells
maintain the flux through the isoprenoid biosynthesis pathway by
elevating the intracellular mevalonate levels.
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EXPERIMENTAL PROCEDURES |
Materials--
HMG-CoA, geraniol (GOH), FOH, geranylgeraniol
(GGOH), cholesterol, and 25-hydroxycholesterol were obtained from
Sigma. Mevalonolactone, 3-methyl-3-buten-1-ol, and
3-methyl-2-buten-1-ol were obtained from Fluka and simvastatin was a
gift from Merck Sharpe and Dohme. Radiolabeled
[14C]HMG-CoA was obtained from Amersham Biosciences. When
necessary, the obtained batch was purified further by ethyl acetate
extraction. The antibody against Ras was obtained from Transduction
Laboratories; the antibody against RhoA was obtained from Santa Cruz Biotechnology.
Cell Culture--
Fibroblast cell lines obtained from confirmed
MK-deficient patients (HIDS and MA) and a homozygous familial
hypercholesterolemia (FHC) patient were cultured in nutrient mixture
Ham's F-10 with L-glutamine and 25 mM HEPES
(Invitrogen) supplemented with 10% fetal calf serum (FCS, Invitrogen)
or 10% delipidated FCS (Roche Molecular Biochemicals) as indicated.
For each experiment, cells were seeded in a T-75 culture flask (Costar)
and grown until confluency. Two days prior to a particular treatment,
the cells received fresh culture medium. To treat cells, culture medium
with the indicated compounds or, as a control, solvent was added to the
cells and the incubation was continued for another 2 days. For enzyme
and immunoblot analysis, cells were harvested after trypsinization and
washed twice with PBS, and either used directly or snap-frozen in
liquid nitrogen and stored at
80 °C until use.
Mevalonolactone, GOH, FOH, and GGOH were dissolved in ethanol as 250×
stock solutions. A 250 mM solution of mevalonic acid was prepared by dissolving the mevalonolactone in a 0.1 N
NaOH solution. A 10 mM simvastatin stock solution was
prepared by dissolving the prodrug in pure ethanol, followed by
hydrolysis of the lactone by adding 0.1 N NaOH. After
neutralization with 50 mM HEPES, pH 7.4, and 0.1 N HCl, the solution was sterilized by filtration through a
0.2-µm filter and stored in aliquots at
20 °C (end concentration
ethanol 25% v/v).
Quantitative PCR--
The relative expression levels of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and HMGR mRNAs
were determined using the Lightcycler system (Roche Molecular
Biochemicals). To this end, total RNA (free of genomic DNA) was
isolated with the SV total RNA isolation system (Promega) after
which first-strand cDNA was prepared as described by IJlst et
al. (21). The relative mRNA expression levels of HMGR
and GAPDH were determined using a plasmid containing the corresponding
gene as the standard. The GAPDH fragment was amplified using the
following primer set: GAPDH Fw, 5'-ACC ACC ATG GAG AAG GCT GG-3', and
GAPDH Rev, 5'-CTC AGT GTA GCC CAG GAT GC-3'. The HMGR fragment was
amplified using primers: HRED Fw2, 5'-TCA AAG GGT ACA GAG AAA GCA C-3',
and HRED Rev2, 5'-TAT GCT CCC AGC CAT GGC AG-3'. In every sample the
expression of GAPDH and HMGR was determined in duplicate.
HMGR Enzyme Assay--
HMGR was measured essentially as
described by Brown et al. (22) with some modifications. The
fibroblast pellets were dissolved in HMGR assay buffer containing 100 mM KPi, 200 mM KCl, 5 mM EGTA, 5 mM EDTA, 10 mM DTT, and
10 µg/ml leupeptin (pH 7.1). The cells were disrupted by sonication
(twice at 8-watt output, 40 J, at room temperature). One-hundred
microliters of the resulting homogenate was preincubated for 10 min at
37 °C with 60 µl of cofactor-mix containing 66.7 mM
glucose 6-phosphate, 10 mM NADPH, 16.7 mM EDTA, and 25 units/ml glucose-6-phosphate dehydrogenase. The enzyme reactions
were started with the addition of 1.7 nmol of
[14C]HMG-CoA and 5.6 nmol of HMG-CoA in 40 µl of
H2O. After a 30-min incubation period at 37 °C,
reactions were terminated by adding 50 µl of 1.2 N HCl.
After 30 min, the product was extracted three times with 2 ml of ethyl
acetate. The extracts were evaporated to dryness and analyzed by silica
thin layer chromatography using a solvent system toluene:acetone (1:1)
dried with Na2SO4. The formed product was
quantified by phosphorimaging (Fuji FLA-3000) with the aid of the Aida
software package using samples with known amounts of
[14C]mevalonate. In every sample, the activity of HMGR
was determined in duplicate.
Membrane and Cytosol Separation--
Cell pellets were dissolved
in hypotonic buffer containing 5 mM Tris-Cl, pH 7.0, 5 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 2 mM EGTA, 2 mM DTT, and Complete protease inhibitor mixture (Roche
Molecular Biochemicals). The cells were lysed by sonication (twice at
8-watt output, 40 J, with cooling between the pulse periods). The
membranes were separated from the cytosolic fraction by a 30-min
ultracentrifugation step in an Airfuge (Beckman, 15 p.s.i.,
100,000 × g). The supernatant was designated as the
cytosolic fraction. The membrane pellet was dissolved in radioimmune
precipitation assay (RIPA++) buffer containing 20 mM
Tris-Cl, pH 8.0, 150 mM NaCl, 10 mM
NaH2PO4, 5 mM EDTA, 10% glycerol,
1% Nonidet P-40, 1% sodium deoxycholic acid, 0.1% SDS, 1 mM DTT, and Complete protease inhibitor mixture and
homogenized by sonication (once 7-watt output, 40 J). Both fractions
were boiled in Laemmli sample buffer and stored at
20 °C.
Immunoblot Analysis--
Equal amounts of protein (measured in
the sonicated lysates prior to ultracentrifugation) were resolved on a
15% SDS-polyacrylamide gel and transferred onto nitrocellulose by
semi-dry immunoblotting. As a control for equal transfer of protein,
the blots were stained reversibly with Ponceau S. Membranes were
blocked using blocking buffer containing 5% nonfat dry milk and 1%
bovine serum albumin in PBS with 0.1% Tween. Membranes were probed
with either the Ras antibody (1:1000) or the RhoA antibody (1:1000) in
blocking buffer, which was diluted 10 times in PBS with 0.1% Tween.
Detection of the antigen-antibody complexes was performed with
horseradish peroxidase-conjugated secondary antibody (DAKO) and using
the enhanced chemiluminescence kit (ECL, Amersham Biosciences).
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RESULTS |
Elevated HMGR Activity Is Suppressed by Isoprenoid Precursors and
Sterols--
In accordance with previous observations by Gibson
et al. (19), we observed an elevation of HMGR activity in
cultured skin fibroblasts of MA patients. In HIDS fibroblasts, however,
HMGR activity was within the normal range (Table
I). Gibson et al. (19) also
reported that the elevated HMGR enzyme activity in MA fibroblasts could
not be suppressed by exogenous LDL cholesterol and was up-regulated
further when the cells were cultured with LDL-cholesterol (lipid)
depleted FCS (19). We also observed this up-regulation in MA cells when
cultured under lipid-depleted conditions.2 In addition and
similar to data previously shown in normal cells (8, 23) and our
control cells, we observed efficient down-regulation of HMGR activity
when MA fibroblasts were grown in the presence of various mixtures of
25-hydroxycholesterol and cholesterol (Fig. 1). This down-regulation was
dose-dependent and occurred in fibroblasts of MA patients
with different genotypes. These include cells from mildly affected MA
patients with mutations resulting in a stable MK with an elevated
Km for mevalonate (A334T; M1) and from severe MA
patients with mutations mainly affecting the stability of the MK
protein (I268T; M2) (24-26).
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Table I
Relative mRNA expression levels of HMGR and HMGR enzyme activity in
control, HIDS, and MA cultured skin fibroblasts
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Fig. 1.
Suppression of HMGR activity by sterols.
HMGR specific activities in control (C) and two different MA
(M1 and M2) fibroblast cell lines were determined
after 48 h of culturing in the presence or absence of various
mixtures of 25-hydroxycholesterol (25-OH chol) and
cholesterol (chol). The error bars indicate 1 S.D.
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Next, we tested whether the elevated HMGR activity in MA fibroblasts
could be suppressed by non-sterol isoprenoids. To this end, MA, HIDS,
and control fibroblasts were cultured first for 2 days with 60 µM GOH, FOH, or GGOH (Fig.
2A). The treatment with GGOH
caused a decrease in HMGR enzyme activity in all cell lines, whereas
the treatment with FOH caused only a decrease in the MA cell line. GOH
was ineffective in all three tested cell lines at this concentration.
To determine the effective concentration range of GOH, FOH, and GGOH,
we cultured MA fibroblasts in the presence of different concentrations
of these compounds (Fig. 2B). This showed a
dose-dependent decrease of the HMGR enzyme activity for all
three compounds. GGOH was the most effective in suppressing HMGR enzyme
activity and consequently showed significant cytotoxicity at 75 µM leading to death of the cells within 2 days. FOH was
more effective than GOH. The suppression of HMGR enzyme activity by
these non-sterol isoprenoids was fast with an almost maximal effect
reached already after 2 h.2 Similar as the effect of
sterols, the effect was observed in cells with different genotypes
(A334T; M1, I268T; M2). The suppressive effect of these compounds
appeared specific, because the alcohol of the closely related
isoprenoid isopentenyl pyrophosphate (IPOH, 3-methyl-3-buten-1-ol) did
not have any effect on HMGR activity even up to a concentration of 1 mM (Fig. 2B). These findings indicate that the
regulatory mechanisms of the isoprenoid/cholesterol biosynthesis pathway are not disturbed in MA patients.

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Fig. 2.
Suppression of HMGR activity by isoprenoid
precursors. A, HMGR specific activities in control
(C), HIDS (H), and MA (M1) fibroblast
cell lines were determined after 48 h of culturing in the presence
or absence of 60 µM GOH, FOH, and GGOH. B,
HMGR specific activities in control (C) and two different MA
(M1 and M2) fibroblasts cell lines were
determined after 48 h of culturing in the presence or absence of
GOH, FOH, GGOH, and IPOH at different concentrations. The error
bars indicate ±1 S.D.
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To determine whether the elevated HMGR activity was due to an increase
in HMGR gene transcription, we analyzed HMGR mRNA by quantitative
PCR and determined the activity of HMGR in the same sample. This showed
that the elevated activity in the MA fibroblasts was not due to a
higher transcription rate of the HMGR gene, because HMGR mRNA
levels were similar in all cell lines, whereas HMGR activity was
elevated only in the MA cell line (Table I). When, as a control, the
same cell lines were cultured under lipid-depleted conditions, however,
we observed the expected increase in HMGR mRNA levels indicating
that HMGR gene transcription per se is not disturbed in
these cells.2
Differential Suppression of HMGR Activity in MA and FHC
Cells by GGOH and Sterols--
The finding of similar HMGR
mRNA levels in the MA cells suggest that the elevated HMGR activity
in the cells is not due to the sterol-dependent activation
of the SREBP pathway but to one of the post-transcriptional
non-sterol-dependent regulatory mechanisms. To obtain
additional support for this we studied the suppressive effect of
various mixtures of 25-hydroxycholesterol and cholesterol and of GGOH
on the HMGR activity in MA fibroblasts and in fibroblasts of a
homozygous familial hypercholesterolemia (FHC) patient. The latter cell
line also exhibits elevated HMGR activity, which is not due to MK
deficiency but to a complete deficiency of the LDL receptor,
responsible for LDL-cholesterol import. We observed that the
suppression of HMGR activity in the MA cell line was more sensitive to
supplementation with GGOH (which can only be used as precursor for
non-sterol isoprenoids) than in the FHC cell line (Fig.
3A). The reverse was true for
supplementation with the sterol mixtures, which showed that the
suppression of HMGR activity in the FHC cell line was more sensitive
than in the MA cell line (Fig. 3B).

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Fig. 3.
Differential suppression of HMGR activity in
MA and FHC cells by GGOH and sterols. A, suppression of
the elevated HMGR activity in MA fibroblasts and in fibroblasts of a
homozygous familial hypercholesterolemia (FHC) patient by
various concentrations of the non-sterol isoprenoid precursor GGOH.
B, suppression of the elevated HMGR activity in MA
fibroblasts and in fibroblasts of a homozygous familial
hypercholesterolemia (FHC) patient by various mixtures of
25-hydroxycholesterol (25-OH) and cholesterol
(Chol). The error bars indicate ±1 S.D.
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Exogenous Mevalonate Suppresses HMGR Activity in MA
Fibroblasts--
The fact that fibroblasts from MA patients are still
capable of synthesizing cholesterol and several other isoprenoids from radiolabeled precursors, like acetate, octanoate, and mevalonate (18-20),2 implies that MK activity is not entirely
deficient in these cells, although it is below detection level in our
MK enzyme assay. To test whether exogenous mevalonate is capable of
normalizing HMGR enzyme activity in MA fibroblasts, these cells were
cultured for 48 h in the presence of different concentrations of
mevalonolactone or sodium mevalonate (Fig.
4). This treatment caused a
dose-dependent decrease in HMGR enzyme activity. This
down-regulation occurred not only in fibroblasts of an MA patient with
a mutation affecting the Km of MK for mevalonate
(A334T; M1) but also in an MA fibroblast cell line in which the
stability of the MK protein is decreased (I268T; M2). Sodium mevalonate
was more effective than mevalonolactone in suppressing HMGR enzyme
activity. In controls, treatment with mevalonolactone reduced the HMGR
activity to undetectable levels.

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Fig. 4.
Suppression of HMGR activity by exogenous
mevalonate. HMGR specific activity in control (C) and
two different MA (M1 and M2) fibroblast cell
lines after 48 h of culturing in the presence or absence of
mevalonate or mevalonolactone at different concentrations. The
error bars indicate ±1 S.D.
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HIDS and MA Skin Fibroblasts Have Increased Sensitivity for
Simvastatin--
From our results we hypothesized that MA and HIDS
fibroblasts compensate for their depressed MK activity by raising
intracellular mevalonate levels. To test this hypothesis we determined
the sensitivity of a control, an HIDS, and an MA fibroblast cell line
to inhibition of HMGR activity by simvastatin. This was done by
culturing these cell lines in the presence of different concentrations
simvastatin for 48 h. After incubation, the cells were
fractionated into a membrane and a cytosolic fraction, which were
subjected to immunoblotting with antibodies against two isoprenylated
proteins (Fig. 5). Most isoprenylated
proteins function in the membrane and need farnesyl or geranylgeranyl
moieties to become associated with the membrane. As a consequence of
normal protein turnover, inhibition of HMGR by simvastatin will lead to
an increase in non-isoprenylated (and non-functional) proteins in the
cytosolic fraction and a decrease in isoprenylated proteins in the
membrane fraction (27, 28). We used antibodies to Ras, which is a
farnesylated protein, and RhoA, which is a geranylgeranylated protein.
All fibroblast cell lines cultured in the absence of simvastatin had
similar levels of Ras and RhoA protein in the membrane fraction,
indicating that MA and HIDS fibroblasts are capable of synthesizing
isoprenylated proteins as efficiently as control fibroblasts (Fig. 5).
Similar results were obtained in cultured lymphoblasts of MA and HIDS patients.2 It appears that in confluently grown fibroblasts
the majority of the RhoA protein is localized in the cytosol fraction,
whereas the majority of Ras is localized in the membrane fraction.

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Fig. 5.
Effect of simvastatin on protein
isoprenylation in HIDS and MA skin fibroblasts. A,
immunoblot analysis of Ras in cytosol and membrane fractions of
fibroblast lysates (15 µg of protein/lane). The control, HIDS, and MA
cell lines were cultured in the presence of different concentrations of
simvastatin. The concentrations are indicated in the figure.
B, immunoblot analysis of RhoA in cytosol and membrane
fractions of fibroblast lysates (12.5 µg of protein/lane and 25 µg
of protein/lane, respectively). The control, HIDS, and MA cell lines
were cultured in the presence of different concentrations of
simvastatin in medium containing FCS and medium containing lipid-free
FCS. The concentrations are indicated in the figure. Shown are the
results from one representative experiment from three independently
performed experiments.
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Depletion of cellular mevalonate levels induced by simvastatin
treatment of the cells showed a marked difference. First, in all cell
lines, total RhoA protein levels are increased in response to
simvastatin treatment, a previously reported compensatory response to
the lowered levels of functional RhoA in the membrane (27-30). In
addition, the MA cell line had more RhoA in the cytosolic fraction than
the control cell line. Moreover, in MA and HIDS fibroblasts, the
increase of Ras (Fig. 5A) and RhoA (Fig. 5B) in
the cytosolic fraction occurs at lower concentrations of simvastatin
than in controls. The concomitant decrease of RhoA in the membrane
fraction is much stronger than for Ras, suggesting a higher turnover
rate for RhoA. In fibroblasts from an MA patient an effect was already visible at a simvastatin concentration as low as 8 nM. Also
fibroblasts from an HIDS patient were more sensitive to simvastatin
than a control cell line. Although both the control and the HIDS cell line start to accumulate RhoA in the cytosol at 40 nM
simvastatin, this process is faster in the HIDS cell line. In addition,
quantification of the immunoblots revealed that this process was
already maximal at 200 nM in the HIDS cell line, whereas in
the control cell line it was only half-maximal at this concentration.
Thus, the extent of the MK deficiency reflects the sensitivity to
simvastatin, with the cells displaying the lowest MK activity (and
highest HMGR activity) being the most sensitive.
To study the effect of an increased pathway flux on the sensitivity to
simvastatin, MA, HIDS, and control fibroblasts were cultured in
lipid-free FCS. The sensitivity to inhibition of HMGR by simvastatin
decreased in all cell lines, which most probably reflects the
up-regulation of HMGR enzyme activity (Fig. 5B). In MA and
HIDS fibroblasts, however, RhoA appears in the cytosolic fraction at
lower concentrations of simvastatin than controls. Without addition of
simvastatin, lipid-free FCS did not induce an apparent difference in
isoprenylation between MA, HIDS and control fibroblast cell lines.
FOH and GGOH Can Rescue Deficient Isoprenylation in MA--
FOH
and GGOH can be utilized by cells for isoprenoid biosynthesis when
added to the culture medium (31-33). We tested whether fibroblasts
from an MA patient were also able to use FOH and GGOH for the rescue of
protein isoprenylation when HMGR was inhibited by simvastatin. FOH
rescued farnesylation of Ras (Fig.
6A) as judged from the
decreased level of this protein in the cytosol, whereas GGOH rescued
geranylgeranylation of RhoA (Fig. 6B) as judged from the
increased level of this protein in the membrane fraction. A combination
of both compounds rescued isoprenylation of both proteins. Also the
addition of 1 mM mevalonate to the medium rescued
isoprenylation of RhoA, again indicating that MA fibroblasts can use
mevalonate when concentrations are sufficiently high (Fig.
6B). In accordance with a rescue of protein isoprenylation, we observed that in response to simvastatin treatment the total RhoA
protein levels decreased after treatment with FOH, GGOH, and mevalonate
(Fig. 6B).

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Fig. 6.
Rescue of deficient protein isoprenylation in
HIDS and MA skin fibroblasts by FOH and GGOH. A,
immunoblot analysis of Ras in cytosol and membrane fractions of
fibroblast lysates of an MA patient (10 µg of protein/lane and 20 µg of protein/lane, respectively) showing the effect of 20 µM FOH and 20 µM GGOH alone or in
combination on the treatment with simvastatin. B, immunoblot
analysis of RhoA in cytosol and membrane fractions of fibroblast
lysates of an MA patient (10 µg of protein/lane and 20 µg of
protein/lane, respectively) showing the effect of 20 µM
FOH and 20 µM GGOH alone or in combination on the
treatment with 40 nM simvastatin (lanes 2-5)
and the effect of 1 mM mevalonate on the treatment with 100 nM simvastatin (lanes 6 and 7). Shown
are the results from one representative experiment from three
independently performed experiments.
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To verify if the rescue of protein isoprenylation by FOH and GGOH was
specific, we tested whether the alcohols of the isoprenoids isopentenyl
pyrophosphate and dimethylallyl pyrophosphate (3-methyl-2-buten-1-ol) could rescue also the deficient isoprenylation in the MA cells when
HMGR was inhibited by simvastatin. No rescue was observed with any of
the tested concentrations or with combinations of these
compounds.2
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DISCUSSION |
MA and HIDS are syndromes both caused by a deficiency of MK enzyme
activity but to variable degrees. Despite this deficiency, cholesterol
biosynthesis in fibroblasts derived from MA patients can be near normal
depending on the culture condition (18-20).2 Thus, it
appears that MA fibroblasts are able to compensate for their defect in
MK. Previously, it has been postulated that this is due to an increased
activity of HMGR and the LDL receptor pathway (18-20). HMGR that
catalyzes the conversion of HMG-CoA into mevalonate, which is the
enzyme step preceding the one catalyzed by MK, is believed to perform
the main rate-limiting step in isoprenoid biosynthesis and is among the
most highly regulated enzymes in nature (8). The increased activity of
HMGR in MA fibroblasts had been reported as insuppressible by exogenous
LDL cholesterol and was up-regulated further under cholesterol-free
culture conditions (19). This suggests that the LDL receptor pathway is
saturated and that the high basal HMGR activity in MA is not due to a
shortage of sterol end products. Apparently, the lipoproteins normally present in the FCS provide the cells with sufficient cholesterol, thus
preventing SREBP activation. In accordance with this, we found that
HMGR mRNA levels in MA fibroblasts are similar to the levels in
control cells, indicating that under standard growth conditions the
sterol-dependent SREBP pathway, involved in transcriptional regulation, is not activated. We also found that the increased HMGR
activity was down-regulated when the medium of MA cells was supplied
with FOH, GGOH, sterols, or extra mevalonate. Moreover, we noted that
the HMGR activity in an MA cell line was more sensitive to GGOH
suppression than the HMGR activity in an FHC cell line, whereas the
HMGR activity in the FHC cell line was more sensitive to sterol
suppression than the HMGR activity in the MA cell line. Together, these
findings indicate that the regulation mechanisms of the
isoprenoid/cholesterol biosynthesis pathway are still functional in MA
fibroblasts and that under normal growth conditions one of the
non-sterol-dependent regulatory mechanisms causes the
increase in HMGR activity. These mechanisms are post-transcriptional
and involve higher mRNA translation efficiency and decreased
protein turnover. FOH has been implicated to be a non-sterol regulator of HMGR activity, because it accelerates HMGR protein turnover (10-16). In our experiments, however, GGOH was more effective than FOH
in down-regulating HMGR enzyme activity. Because Correll et al. (13) reported that FOH was more effective than GGOH in
promoting HMGR protein degradation, the effect by GGOH may be related
to mRNA translation efficiency. The difference in efficacy of
mevalonate and mevalonolactone is probably a reflection of the activity
of specific hydrolases (esterases) to activate the mevalonolactone in
fibroblasts and the different diffusion coefficients of both molecules
for crossing the cellular membrane.
As reported for cholesterol biosynthesis from radiolabeled precursors
(18-20),2 we now report that under normal conditions
protein isoprenylation in HIDS and MA fibroblasts is normal. In cells
from both disorders, the farnesylated protein Ras and the
geranylgeranylated protein RhoA were present in the membrane fraction
as shown by cellular subfractionation followed by immunoblotting. From
these observations it can be concluded that MA and HIDS cells are able
to compensate for reduced MK activity by elevating their intracellular
mevalonate levels. This was illustrated also by the fact that addition
of extra mevalonate to the medium down-regulated the HMGR activity in
MA fibroblasts. This implies that the elevated HMGR activity observed
in MA fibroblasts mainly serves to compensate for the leakage of
mevalonate (or mevalonolactone) out of the cell. This is inevitable
because a higher mevalonate concentration in the cell will lead to an
increased leakage. Indeed, MA fibroblasts are more sensitive to
simvastatin than HIDS fibroblasts, whereas HIDS fibroblasts are more
sensitive to HMGR inhibition than control fibroblasts as demonstrated
by the variable accumulation of non-isoprenylated proteins in the
cytosol after treatment of cells with different concentrations of
statins. Furthermore, this is reflected by the 100- to 1000-fold higher
mevalonic acid excretion in MA patients when compared with HIDS
patients (2, 7).
The elevation of intracellular mevalonate concentrations is expected to
promote a normal flux through the isoprenoid/cholesterol biosynthesis
pathway in the MK-deficient cells when the following three criteria are
met: 1) MK is not saturated with substrate. (When MK is saturated, any
elevation in mevalonate levels would have no effect.); 2) HMGR is able
to generate mevalonate levels that are high enough for MK to function
at a normal rate. (HMGR has to compensate for the leakage of mevalonate
out of the cell.); 3) HMGR is not subjected to non-competitive product
inhibition. (This is not the case because HMGR is insensitive to
any form of product inhibition (34).2)
When MA and HIDS fibroblasts are able to compensate largely for their
MK deficiency by elevation of intracellular mevalonate levels, one
could imagine a pathogenetic mechanism in which toxic levels of
mevalonate are the cause of the observed symptoms in HIDS and MA.
Hoffmann et al. (2), however, reported that a trial with
lovastatin in two MA patients, used in an attempt to lower their
presumed toxic mevalonate levels, resulted in severe clinical crises.
This indicates that an excess of mevalonate itself may not be the major
pathogenic factor in MA but instead a shortage of one of the isoprenoid
end products. In fact, the outcome of this trial illustrates the
importance of maintaining elevated mevalonate levels. However, at this
moment, it cannot be excluded that few of the symptoms, exclusively
observed in the far more severe MA, are induced by toxic mevalonate levels.
In conclusion, our results suggest that supplementation of isoprenoid
precursors, such as mevalonate, FOH, and GGOH, may be beneficial in the
abortion and prevention of fever episodes in HIDS and MA. Because it is
known that, in vitro, FOH and GGOH have substantial
cytotoxicity and are able to down-regulate HMGR enzyme activity,
studies to the in vivo effects of isoprenoid precursor
supplementation will be needed.