- Email: [email protected]
A model for farnesoid feedback control in the mevalonate pathway
ELSEVIER
HYPOTHESES A Model for Farnesoid Feedback Control in the Mevalonate Pathway Cary Weinberger
Mevalonate is the rate-limiting substrate leading to farnesyl pyrophosphate (FPP), the central intermediate for isoprenoids such as cholesterol, dolichols, ubiquinone, and carotenoids. One major challenge has been to identify the isoprenoid effector molecules and transcription factors mediating negative regulation in this metabolic pathway. A nuclear receptor called FXR has recently been characterized that is activated by famesyl pyrophosphate metabolites such as famesol, farnesal, famesoic acid, and methyl famesoate. FXR expression in isoprenoidogenic tissues suggests a hypothesis that these intracellular “famesoids” may be signals for transcriptional feedback control of cholesterol biosynthesis.
(Trends
How is cholesterol synthesis regulated? This question has been puzzling biochemists since 1933 when it was noted that dietary cholesterol depressed its endogenous production (Schoenheimer and Breusch 1933, Tomkins et al. 1953). Identification of 3-hydroxy-3-methylglutar-y1 coenzyme A (HMG CoA) reductase as the rate-limiting enzyme for this repression (Siperstein and Guest 1960) awaited the complete description of the enzymatic steps from acetate to cholesterol (Rilling and Chayet 1985). Although the isoprenoid signals for repressing HMG CoA reductase activity have long been recognized, their polypeptide transducers are still only incompletely defined (Wang et al. 1994). The problem of bridging the effecters and gene targets remains the largest gap in our understanding of cholesterol regulation. A model will be outlined that
Gary Weinberger is at the National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
TEA4 Vol. 7, No. 1, 1996
Endocrinol
Metab
1996;7:
l-6).
places metabolites of farnesyl pyrophosphate (FPP) at key control points in the mevalonate pathway. Identification of a nuclear receptor activated by these farnesoids is the foundation for this intracellular signaling hypothesis.
??
Isoprenoid
Synthesis
In unicellular organisms, metabolite levels are typically fixed by feedback control of biosynthetic enzymes or their activities. Higher metazoans exercise similar control, but must also integrate cellular biochemical processes through endocrine signals. One important biosynthetic route in eukaryotes is the mevalonate pathway leading to isoprenoid products such as cholesterol, bile acids, dolichol, ubiquinone, carotenoids, vitamin D, and steroid hormones (Goldstein and Brown 1990). These isoprenoids are essential for membrane assembly, lipid uptake, glycoprotein synthesis, electron transport, and hormonal regulation. Isoprenoid biosynthesis begins with
01996, Elsevier Science Inc., 1043-2760/96/$15.00
the precursor acetyl CoA derived from intermediary metabolism (Figure 1). Most of the acetate is converted to fatty acids for energy storage, and much of the remainder is diverted to mevalonate for isoprenoid synthesis (Rilling and Chayet 1985). Mevalonate production is irreversible, and this rate-limiting step is regulated by the enzyme HMG CoA reductase. Mevalonate is subsequently phosphoxylated, decarboxylated, and isomerized to isopentenyl pyrophosphate (IPP), the basic isoprenoid building block (Figure 1). Self-condensation of IPP produces geranyl pyrophosphate, and an additional IPP condensation step yields farnesyl pyrophosphate (FPP), the principal intermediate for all isoprenoids (Figure 1). Cholesterol homeostasis is sustained by three metabolic pathways (Dietschy and Wilson 1970). The first is an endogenous biosynthetic route from acetate to isoprenoids catalyzed by HMG CoA synthase and HMG CoA reductase. A second one salvages sterol ester remnants by endocytosis of low-density lipoproteins (LDLs) mediated by the LDL receptor (Goldstein and Brown 1990). The third pathway is a catabolic detour to bile acids for fat absorption and excess cholesterol removal, which is catalyzed by cholesterol 7a-hydroxylase (Russell and Setchell 1992). As a result, the intestinally absorbed dietary cholesterol intake is matched with endogenous sterol production. Although the liver and intestine are the main sites for acetate incorporation into sterols, suppression mechanisms prevail in the liver when animals are fed cholesterol-rich diets (Dietschy and Siperstein 1967).
??
Feedback Signals and Gene Targets
Both the signals and the regulated genes participating in the cholesterol regulatory network have been described. Cholesterol and hydroxylated cholesterol molecules (oxysterols), especially those
SSDI 1043-2760(95)00180-8
1
FARNESOID SHUNT
fatty acids
OCH, methyl famesoate
f OH
famesolc acid
f
HOuJL4 frans-3-methylglutaconyl CoA
1
1
JAM 3,3-dlmethylacrylyl CoA
J-O,,
/‘\
A,,,
gelanyl-PP
AAAAAY,, fame+PP
f’ lsopentenyl-PP
3,3dimethylallyl-PP
dolichol ubiquinone carotenoids cholesterol
x
3,3dlmethylallyl alcohol
PPI
Figure 1. Model for FXR/farnesoid-mediated feedback control of isoprenoid synthesis illustrating glutaconate and famesoid metabolic shunts coupled to isoprenoid biosynthetic pathway, The isoprenoid biosynthetic pathway from acetyl coenzyme A (CoA) to cholesterol is schematically outlined along with the linked glutaconate and farnesoid shunts. Acetyl CoA is condensed to form mevalonate, which may be diverted to isoprenoid biosynthesis or may be salvaged by the truns-3-methylglutaconate cycle hypothesized by Popjak (see the text). Farnesyl pyrophosphate (FPP) from the mevalonate pathway may be an intermediate for the synthesis of other isoprenoids such as cholesterol, carotenoids, dolichol, and ubiquinone, or FPP may be metabolized to farnesoids via the farnesoid shunt. The famesoid shunt begins with a phosphatase activity converting FPP to farnesol (Christophe and Popjak 1961). These biochemical transformations are analogous to the initial steps from isopentenyl pyrophosphate in the glutaconate shunt. Subsequent oxidation steps producing farnesal and farnesoic acid are catalyzed by liver alcohol and aldehyde dehydrogenases, whereas methyl esterification and epoxidation generate methyl famesoate and juvenile hormone III, respectively. FXR is a nuclear receptor, activated by FPP and its metabolites, that is exclusively expressed in the liver, intestinal villi, renal tubules, and adrenal cortex. It is hypothesized that famesoid metabolites activate FXR, which represses the transcription of genes in the mevalonate pathway. Other nuclear receptors such as RXR and its effector may act in concert with FXR to mediate these effects. Suppression of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase and FPP synthase are two potential gene targets. FPP synthase inhibition would create an IPP surplus, which would be diverted back to HMG CoA via the glutaconate shunt. HMG CoA reductase inhibition should also increase HMG CoA levels. Transcriptional repression of these two genes should, therefore, effectively inhibit the production of isoprenoid precursors. Similarly, fatty acid biosynthesis and degradation may be controlled by binding of intracellular fatty acids such as linoleic, linolenic, and arachidonic acids to the peroxisome proliferator-activated receptor (PPAR) (Keller and Wahli 1993). Farnesoids and fatty may be present within the cell at the micromolar concentrations required for activation of their respective receptors. Consequently, PPAR and FXR and their respective ligands may regulate several networks of genes for enzymes involved in lipid homeostasis.
2
OH
famesol
Isopentenyl-PP
* 3,3dimethylacrylic acid
t
J-o,,
GLUTACONATE SHUNT
01996,
Elsevier Science Inc., 1043-2760/96/$15.00
at the 7, 25, and 26 positions, be the major
effecters
HMG CoA reductase 1978). A cytoplasmic protein (OSBP) that with
affinities
appear
to
for the gene target (Kandutsch binds
matching
these
their
et al.
binding
oxysterol
sterols
potentials
HMG CoA reductase activity was characterized (Taylor et al. 1984). Molecular cloning of the OSBP gene, however, revealed no DNA binding structural features in the encoded protein (Dawson et al. 1990). Equally confounding was OSBP sequestration in the Golgi apparatus following oxysterol addition (Ridgway et al. 1992). These findings may point to a cytoplasmic regulatory role for OSBP, perhaps for promoting HMG CoA reductase enzyme degradation or decreasing the stability of its RNA. Alternatively, OSBP may bind reductase gene promoter elements by forming a complex with an unidentified DNA binding factor. Several proteins that interact with prospective DNA regulatory elements in the HMG CoA reductase for repressing
SSDI 1043-2760(95)00180-S
TEM Vol. 7, No. 1, 1996
promoter have been identified. CTF binds to six DNA sites in the tase promoter, one overlapping a responsive element (SRE) found
NF-l/ reducsterolin the
promoters of many mevalonate pathway biosynthetic genes (Gil et al. 1988). Other polypeptides have been distinguished by their SRE binding features, including CNBP (Rajavashisth et al. 1989), SRE-BF (Stark et al. 1992), and red25 (Osborne et al. 1992), but their roles in HMG CoA reductase regulation are not understood. Cholesterol-mediated repression of the low-density lipoprotein receptor (LDLR) and HMG CoA synthase genes also controls the flux of isoprenoids. Control of their transcription is considered secondary relative to HMG CoA reductase, but many aspects of their regulation are better appreciated (Wang et al. 1994). Cholesterol in the endoplasmic reticulum (ER) adjusts the proteolytic conversion of specific transcription factors called sterol response element binding proteins (SREBPs). In the absence of cholesterol, SREBP is truncated to a product that stimulates synthase and LDLR gene transcription. Cholesterol suppresses SREBP proteolysis, locking the native protein in the ER, which prevents transcription. SREBP may therefore be a membrane-bound transcriptional sensor of cellular cholesterol. Nonsterol mevalonate-derived famesol metabolites also suppress cholesterol synthesis (Brown and Goldstein 1980, Nakanishi et al. 1988). Famesoic acid and some structural analogues have been shown to inhibit cholesterol synthesis in vitro (Popjak et al. 1960). More recently, famesol, famesyl acetate, and famesyl methyl ether have been implicated in HMG CoA reductase posttranscriptional regulation (Giron et al. 1994, Bradfute and Simoni 1994, Correll et al. 1994). Support for a nonsterol transcriptional effector also comes from the finding that mevalonate or a metabolite inhibits primary transcription from the liver HMG CoA reductase gene (Clarke et al. 1985).
??
A Nuclear Receptor Activated by Farnesoids
Nuclear receptors are transcription factors that bind to target genes and regulate primary RNA synthesis by specific ligand interactions (Evans 1988). The re-
TEA4 Vol. 7, No. 1, 1996
ceptor activators include steroid hormones, ecdysteroids, and vitamin D, as well as retinoids and thyroid hormones. Despite cholesterol’s structural similarity to some nuclear receptor ligands, an isoprenoid-responsive receptor has not yet been identified. However, 30 orphan nuclear receptors isolated by nucleic acid homology offer fertile ground for continued prospecting (O’Malley 1990). Matching ligands with orphan receptors has been a formidable task, but some have been uncovered by sampling chemical compound inventories (Giguere et al. 1987, Petkovich et al. 1987). A rat orphan nuclear receptor gene called OR2 was recently isolated (Forman et al. 1995). OR2 is structurally related to the insect ecdysteroid receptor (EcR) and binds to ecdysone-responsive DNA elements (EcRE) in the presence of RXR (Yao et al. 1992, Manglesdorf and Evans 1992). RXR is an obligate receptor partner that facilitates the DNA binding of many other nuclear receptors (Manglesdorf and Evans 1992). The OR2 DNA binding features offered a way to test the receptor’s ligand dependency. Plasmid DNAs for OR2 and RXR were transfected with an EcRE-linked reporter gene into OR2-deficient cells. Increasing amounts of serum extract enhanced the activity that was dependent on both OR2 and RXR. The OR2 activator is soluble in chloroform, suggesting that OR2 is a ligand-dependent transcription factor responsive to lipophilic molecules (S. Kitareewan, E. Goode, and C. Weinberger unpublished results). A number of steroids, retinoids, fatsoluble vitamins, fatty acids, and other lipophilic compounds were surveyed for OR2 activation (Forman et al. 1995). Surprisingly, micromolar amounts of juvenile hormone (JH) III were stimulatory. JH III is a sesquiterpenoid that regulates insect development in concert with ecdysteroids (Schneiderman and Gilbert 1964). In contrast, methoprene, a potent synthetic juvenile hormone, did not activate. Other isoprenoids were also tested, including substrates from the mevalonate pathway (Figure 1) (Forman et al. 1995). Famesol was equipotent with JH III, whereas famesyl acetate and famesoic acid had slightly reduced activities, as did the diterpenoid geranylgeraniol. The famesol precursor mevalonate was much less potent, but geranFPP iol was completely ineffective.
metabolites, including squalene, squalene oxide, cholesterol, oxygenated sterols, and bile acids, were all negative in this assay. Together, these results suggest that OR2 encodes a famesoid-activated nuclear receptor that has been named FXR. Steroid receptors are widely distributed in animal tissues. In contrast, FXR has a very limited pattern of expression. FXR hybridizes to a 2.3-kb RNA species found only in the liver, intestinal villi, renal tubules, and the adrenal cortex (Forman et al. 1995). These tissues are isoprenoidogenic or ones with a high metabolic flux through the mevalonate pathway. The notable and most perplexing exception is the kidney, however, which concentrates and metabolizes circulating mevalonate to sterols faster than the liver and gut-the two most prodigious cholesterol-generating organs (Righetti et al. 1976, Dietschy and Siperstein 1967). The kidneys also use a nonsterol pathway for salvaging mevalonate, which will be detailed later here (HellStrom et al. 1973, Raskin and Siperstein 1974). FXR RNA expression is similar to that for HNF-4, an orphan receptor required for activation of liver-specific genes involved in lipid metabolism and transport (Sladek 1993).
??
Isoprenoid Metabolic Shunts
The molecules activating FXR are derived from FPP. Because FPP is a precursor for isoprenoid production, a feedback loop might be expected to regulate its synthesis. One trivial possibility is that FPP hydrolysis reduces the intracellular concentrations of sterol synthetic substrates. Alternatively, the FPP concentration would be reduced by generating metabolites that act as feedback signals for rate-limiting enzymes such as HMG CoA reductase. Finally, a metabolic pathway that diverts the mevaisoprenoids back to lonate-derived acetyl CoA would salvage carbon atoms for fatty acid synthesis.
Glutaconate
Shunt
Two observations support the hypothesis that the carbon atoms in mevalonate might be recycled to HMG CoA (Figure 1) (Popjak 1971). First, radiolabeled mevalonate was incorporated into famesoic acid in rat liver, intestines, brain, and blood and also into the saponified
01996, Elsevier Science Inc., 1043-2760/96/$15.00SSDI 1043-2760(95)00180-S
3
fatty acid fraction in the skin and brain (Edmond and Popjak 1974). Circulating mevalonate must therefore be tmnsformed back to HMG CoA and fatty acids in these latter organs (Edmond 1974). Liver extracts only made famesoic acid, demonstrating the tissue-specific utilization of the mevalonate precursor. Second, a liver microsomal enzyme activity was characterized (Christophe and Popjak 1961) that produces dimethylallyl alcohol from dimethylallyl pyrophosphate (Figure 1). Oxidation steps yield dimethylacrylic acid, which may be ester&d with coenzyme A to make trans-3-methylglutaconyl CoA, an intermediate in the leucine catabolic pathway leading to acetyl CoA or acetoacetate. In the final step, glutaconyl CoA is hydrolyzed to regenerate HMG CoA and acetyl CoA. Farnesoid
Shunt
An analogous path for farnesoid synthesis is proposed that is based on this transmethylglutaconate shunt (Figure 1). In the first step, the liver phosphatase converts FPP to famesol (Christophe and Popjak 1961). Liver alcohol and aldehyde dehydrogenases catalyze the next series of transformations to farnesal and famesoic acid. Methylation of famesoic acid to methyl famesoate (MF) is accomplished with S-adenosylmethionine as a substrate, as outlined for juvenile hormone synthesis in insects and mollusks (Schooley and Baker 1985). Among the compounds that have been tested for FXR activation, MF is as potent as farnesyl acetate (S. Kitareewan, E. Goode, and C. Weinberger unpublished results). Finally, although JH III is the most efficacious FXR activator identified to date, its physiological significance is unclear, as the epoxidation step to .lH III is seen only in insects. Note that each of the molecules in the farnesoid shunt is an FXR activator (Figure 1).
??
Famesoids and Intracrine Signaling
The micromolar famesoids required for FXR induction are greater than the nanomolar hormones necessary for steroid hormone receptor activation. These doses may be overestimates, as famesoids may not readily enter the cell. On the other hand, the pharmacologic doses of farnesoids may be achieved in the isoprenoidogenic tissues expressing FXR. This possibility is supported by the
4
01996,
micromolar substrate requirements for FPP synthase and similar inhibitory concentrations of FPP for isopentenyl pyrophosphate isomerase activity (Rilling and Chayet 1985). Unexpectedly, even FPP induces FXR, although with a much lowered potency when compared with the other famesoid metabolites (S. Kitareewan, E. Goode, and C. Weinberger unpublished results). Therefore, the effective ligand concentration for FXR, or the intracellular famesoid charge, may be the sum of concentrations for each famesoid shunt metabolite. Famesoids are the most potent FXR activators, but their binding to FXR has not yet been established. Ligand-induced FXR protection from proteolysis has failed to show direct interactions (B.M. Forman and R.M. Evans unpublished results) and famesoid binding measurements have not been practicable owing to the lipophilicity of these molecules. However, MF chromatographs with the serum FXR effector activity and that material isolated from human urine when fractionated by reverse phase HPLC (S. Kitareewan, E. Goode, and C. Weinberger unpublished results). The finding of MF in the hemolymph of insects and crustacea may support the notion that MF is an excreted metabolite. In summary, the inability to demonstrate famesoid binding to FXR could be due to the high concentrations needed for its activation. These results might also imply that famesoids are converted to more potent physiological FXR ligands. As an example, famesoids may be cyclized to the authentic FXR binding components by an enzyme similar to squalene cyclase, which catalyzes lanosterol ring formation (Stork and Burgstahler 1955). Alternatively, famesoids can be esterified to cholesterol or fatty acids by an acyl CoA: cholesterol acetyltransferase-like activity such as that linking plant triter-penes and retinol to fatty acids (Tabas et al. 1989, Ross 1990). Whether famesoid metabolites or distinct induced molecular species, isolation of the serum activators should ultimately lead to their molecular structures and their roles in FXR-regulated cell physiology.
??
Potential FXR Gene Targets
Synthetic DNA binding sites have been defined for FXR (Fox-man et al. 1995,
Elsevier Science Inc., 1043-2760/96/$15.00
Seol et al. 1995), but the FXR-regulated genes are still unknown. Previously however, famesoids have been implicated in the posttmnscriptional regulation of HMG CoA reductase activity (Correll et al. 1994). FXR now promotes an intracellular signaling transcriptional control strategy for these metabolites (Figure 1). The mevalonate-derived FXR activators point to genes for isoprenoid biosynthetic enzymes such as HMG CoA reductase and FPP synthase as compelling targets (Dorsey and Porter 1968). Interestingly, Popjak originally proposed that repression of FPP synthase and HMG CoA reductase would effectively regulate cholesterol synthesis (Popjak 1971). Nine FPP synthase-related loci (Fpsl) have been mapped in the mouse genome (Andalibi et al. 1993). Coincidentally, three are found on chromosome 10 (Andalibi et al. 1993) near the FXR gene (Kozak et al. [in press]). Finally, it is likely that FXR and famesoids control genes other than those for mevalonate pathway enzymes. Differential screening of cDNA libraries using labeled liver RNAs from famesol- and control-treated animals should help to identify these famesoid-regulated genes (Wang and Brown 1991). Transcriptional regulation of the HMG CoA reductase gene by sterols remains largely undefined. Assembly of a transcriptional repressor complex composed of NF-1, OSBP, FXR, or a sterol-binding orphan nuclear receptor may be required for reconstitution. The inability to identify the oxysterolresponsive factors may partly relate to the experimental methods used to approach this question. Most have relied on tumor cells growing in culture, where cholesterol feedback regulation is abrogated and lipid metabolism is aberrant (Bricker et al. 1972). Tumor cells cannot synthesize sterols from acetate; instead, they scavenge cholesterol from lipoproteins (Siperstein 1970). The effects of isoprenoids on reductase activity in these cancer cells may be masked because the normal metabolic restraints on the mevalonate pathway have been overridden by their aberrant growth in culture. Studying cholesterol feedback in the livers of intact animals may dissociate these growth and metabolic regulatory properties.
SSDI 1043-2760(95)00180-S
TEM Vol. 7, No. 1, 1996
??
Fatty
also thanks Paavo
Acids as MetaboliteLSignaIs
FXR may serve as a paradigm for many distinct intracellular signaling pathways modulated by lipophilic ligands and orphan nuclear receptors (O’Malley 1989). In particular, faatty acids are becoming increasingly attractive ligands for the peroxisome proliferator-activated receptor (PPAR). PPAR was originally defined by its responsiveness to the hypolipidemic clofibrates (Isseman and Green 1990). More recently, unsaturated fatty acids such as linolenic, linoleic, and arachidonic acid have been identified as PPAR activators (Gottlicher et al. 1992). Some of these fatty acids have also been isolated as serum components that bind to PPAR (Banner et al. 1994). Most of the PPAR-regulated genes are involved in long chain fatty acid oxidation (Keller and Wahli 1993). Therefore, the four PPAR species may define an intracellular
signaling
fatty
system
acids.
The
coordinated
free fatty
acids
by PPAR
and FXR
verge
responsive
to free
synthesis
and cholesterol
for lipid
might,
of
regulated
therefore,
cal reading
Honkakoski
for criti-
Edmond J: 1974. Ketone bodies as precursors of sterols
of the manuscript.
Edmond
References Andalibi A, Diep A, Quon D, Mohandas Taylor BA, Lusis ti
1993. Mapping
homeostasis.
T,
of mulpy-
mone
rophosphate
Ge-
889-895.
gene.
Mamm
nome 4:211-219. Banner
CD, Gottlicher
M, Widmark
J, Rafter JJ, Gustafsson atic
analytical
proach clear
activators
receptors
from
characterization tor-activated
assay
apnu-
biological
receptor
extracts:
DL,
Simoni
compounds
that
R:
in plasma.
1994.
regulate
Non-sterol
cholesterogene-
LA, Morris
HP, Siperstein
Loss of the cholesterol
the intact hepatoma-bearing
system
MS, Goldstein
JL:
synthesis
in
rat. J Clin In-
1980. Multivalent
and
cell
growth.
Although
previously
lonate
implicated
regulation
pathway,
famesoids
ing as transcriptional FXR
expression
large
isoprenoid
the D(R
is a distinct
J
in the tissues should
and famesoids loop
These
findings
the wide nals
??
expressing
in the
their
a
help clarify
tissues
offer
FXR.
Their D(R
between regulated
regu-
pathway.
some
hope
that
isoprenoid targets
sig-
can
be
soon.
Sutisak their
thanks
Kitareewan advice
the
3-hy-
ative
of mevalonic
accelerated
acid
required
degradation
of
coenzyme
Dawson
PA,
Ridgway
for the J CA,
Brown MS, Goldstein
JL: 1990. cDNA clon-
ing
of
and
protein, tine
expression an oligomer
zipper.
J
oxysterol-binding
with a potential
Biol
Chem
leu-
264:16,798-
JM, Siperstein
cholesterol synthesis
feeding
MD: 1967. Effect and fasting
in seventeen
Lipid Res 8:97-l
tissues
of
on sterol
of the rat. J
04.
and
Goode
and
of his laboratory
for
support.
TEM Vol. 7, No. I, 1996
The
author
01996,
valonate-mediated
1179-1183;
mevalonic
kinase
pyrophosphates.
1241-1249.
by geranyl J Biol
4670.
Elsevier Science Inc., 1043-2760/96/$15.00
243:4667-
JA: 1994. Me-
suppression
of
3-hy-
JL, Brown MS: 1990. Regulation
the mevalonate
pathway.
Nature
of
343:425-
430. Gottlicher
M, Widmark
E, Li Q, Gustafsson
J-A: 1992. Fatty acids activate the clofibric
Hellstrom
acid-activated receptor.
KH,
a chimera
receptor
of
and the
Proc Nat1 Acad Sci
Siperstein
Luby LJ:
1973. Studies
tabolism
of mevalonic
MD, Bricker
LA,
of the in vivo meacid in the normal
rat. J Clin Invest 52:1303-1313. I, Green
member
S:
1990.
of the steroid
superfamily
Activation
hormone
by peroxisome
of a
receptor
proliferators.
Nature 347:645-650. AA, Chen HW, Heiniger
Biological
activity
Keller
H-J: 1978.
of some oxygenated
20 1:498-50
H, Wahli W: 1993. Peroxisome receptors:
endocrinology
and nutrition?
crinol
prolif-
a link between ‘Trends Endo-
Metab 4:291-296. mapping
famesoid some
ste-
1.
erator-activated
MC, Weinberger
10. Mamm
Manglesdorf
C: Ge-
of the gene encoding
receptor,
the
Fxr, to mouse chromoGenome
DJ, Evans
(in press).
Rh4: 1992. Retinoid
as transcription
factors.
In Mc-
Knight SL, Yamamoto KR, Transcriptional Regulation. New York, Cold Spring Harbor Laboratory
of
and famesyl
Chem
for 3-hyA reduc-
droxy-3-methylglutaryl coenzyme A reductase function in a-toxin-perforated cells.
receptors
Dorsey JK, Porter JW: 1968. The inhibition
factor l-like
coenzyme
Giron MD, Have1 CM, Watson
netic
1128-1138;
Elizabeth
that bind to the promoter
Kozak CA, Adamson
16,803. Dietschy
JL, et al.: 1988.
proteins
rols. Science Slaughter
Goldstein
genes encode nuclear
Kandutsch
3-hydroxy-3-
A reductase.
NC,
JR,
Multiple
Isseman
Con-e11 CC, Ng L, Edwards PA: 1994. Identification of farnesol as the non-sterol deriv-
Dietschy JM, Wilson J: 1970. Regulation of cholesterol metabolism. N Engl J Med 282:
Acknowledgments
The author
of
PA: 1985.
Biol Chem 269:17,390-17,393.
whether
a feedback
regulation
for the morpho-
USA 89:4653-4657.
droxy-3-methylglutaryl coenzyme A reductase gene in rat liver. J Biol Chem 260:
methylglutaryl
working
mevalonate
may
chasm
and
bridged
of
farnesoid-responsive
comprise
latory
metabo-
Neverthe-
provides
isolating
Transcriptional
AM, Edwards
of a receptor
acid. Nature 330:624629.
glucocorticoid
J Lipid Res 2:244-
14,363-14,367.
of a famesoid-activated
receptor for
isolation
effecters. FXR-bind-
biological
acids from ally1 pyrophosphates
in liver enzyme systems.
687-693.
Proc Nat1 Acad Sci USA 91:6398-6402.
257.
the notion
insights.
of prenoic
Clarke CF, Fogelman a
determination
from
further
less, identification
genes
generating
famesoid
structure
activators
nuclear
signals.
that the authentic
provide
model
regulatory
flux supports
lite. Molecular
meva-
reemerg-
are intracellular
It is possible
may
the
are
in tissues
that famesoids ing ligand
in post-
in
that is Cell 81:
Giguere V, Ong ES, Segui D, Evans RM: 1987.
Goldstein
Christophe J, Popjak G: 1961. Studies on the biosynthesis of cholesterol: xiv. The origin
transcriptional
240:
tase. Proc Nat1 Acad Sci USA 85:8963-8967.
vest 51:197-205. Brown
receptor
metabolites.
droxy-3-methylglutaryl
MD: 1972.
feedback
Science
of a nuclear
by famesol
Gil G, Smith
sis. J Biol Chem 269~6645-6650. Bricker
activated
gen retinoic
J Lipid Res 34:1583-1591. Bradfute
1.
BM, Goode E, Chen J, et al.: 1995.
Identification
prolifera-
activators
of car-
to n-fatty ac-
superfamily.
Identification
of orphan
of peroxisome
receptor
Forman
E, Sjovall
J-A: 1993. A system-
chemistry/cell
to isolate
isoprenoid
Summary
ids. J Biol Chem 249:66-7
tiple mouse loci related to the famesyl synthetase
G: 1974. Transfer
from mevalonate
Evans RM: 1988. The steroid and thyroid hor-
Lipid Res 21:505-517. 0
J, Popjak
bon atoms
feedback regulation of HMG CoA reductase, a control mechanism coordinating
con-
and fatty acids in the developing
rat. J Biol Chem 249172-80.
Nakanishi
Press, pp 1137-l
M, Goldstein
Multivalent control glutaryl coenzyme
167.
JL, Brown MS: 1988. of 3-hydroxy-3-methylA reductase. J Biol
Chem 263:8929-8937.
SSDI 1043-2760(95)00180-S
5
O’Malley BW: 1989. Did eucaryotic ceptors
evolve from intracrine
tors?
[editorial].
steroid regene regula-
Endocrinology
125:1119-
steroid
Bennett
a protein
predicted
M,
Rhee
1987.
K:
1992. to
coenzyme
A
which belongs ceptors.
acid
P:
receptor
to the family of nuclear
re-
Nature 330:444-450.
Popjak G: 197 1. Specificity rol biosynthesis.
Harvey Lect 65: 127-l 56.
Popjak G, Comforth hibition
RH, Clifford K: 1960. In-
of cholesterol
biosynthesis
by fame-
soic acid and analogues. Lancet 1:1270-1273. Rajavashisth
TB, Taylor AK, Andalibi A, Sven-
son KL, Lusis fi 1989. Identification of a zinc finger protein that binds to the sterol regulatory
element. Science
P, Siperstein
metabolism
MD:
245640-643.
1974.
Mevalonate
by renal tissue in vitro. J Lipid
Res 15:20-25. Goldstein
JL: 1992. Translocation
binding
protein
to Golgi
triggered by ligand binding. 307-3 19. Righetti
M, Wiley MH, Murril
of oxysapparatus
J Cell Biol 116: PA, Siperstein of me-
G, Burgstabler
DR: 1992. Bile acid bio-
acyl-CoAtriterpene
Biochemistry HA, Gilbert
LI: 1964. Control
of growth and development
in insects.
Sci-
R, Breusch
F: 1933. Synthesis
of cholesterol
in the organ-
ism. J Biol Chem 103:439-448. Schooley
DA, Baker
mone
FC: 1985. Juvenile
biosynthesis.
In Kerkut
LI, eds. Comprehensive
Insect
hor-
GA, Gilbert Physiology,
Biochemistry, and Pharmacology. Pergamon, ~017, pp 363-389.
Oxford,
Seol W, Choi H-S, Moore DD: 1995. Isolation of proteins
that
interact
specifically
with
the retinoid X receptor: two novel orphan receptors. Mol Endocrinol 9:72-85. Siperstein
MD:
tissues.
1970.
Regulation
of choles-
in normal and malignant
Curr Top Cell Regul 2:65-100.
Siperstein MD, Guest MJ: 1960. Studies on the site of the feedback control of cholesterol synthesis. FM:
1993.
and liver-specific tor 3~223-232.
I, Beatini
J Clin Invest 39:642-652. Orphan
receptor
HNF4
gene expression.
Recep-
FOR COttEAqlJES
J Am
N, Chen L-L, et al.: 1989. and characterization acyltransferase tissues.
of an activity
J Lipid Res
32:1689-1698. Taylor FR, Saucier SE, Shows EP, Parish EJ, Kandutsch
ence 143:325-333. Schoenheimer
Tabas
in rabbit and human
3 1:4737-4749.
The stere-
cyclization.
Chem Sot 77:5068-5077. Identification
synthesis.
AW: 1955.
of polyene
189:442-445.
Sladek
MD: 1976. The in vitro metabolism
Stork
esterification
DW, Setchell
of retinol.
DNA
Proc Natl Acad Sci USA 892180-2184.
zyme A-dependent Enzymol
J: 1992.
binding factor for a sterol regulatory element.
ochemistry of acyl coen-
0, Weinberger
double- and single-stranded
Methods
terol biosynthesis
Ridgway ND, Dawson PA, Ho YK, Brown MS,
H, Sjovall J, eds.
Measurement
and destruction
of enzymes of ste-
of
pp l-39.
Schneiderman
M, Brand NJ, Krust A, Chambon retinoic
In Danielsson
AC: 1990.
Stark HC, Weinberger Common
and Bile Acids. New York, Elsevier
Science,
Russell
J Biol Chem 267:18,973-18,982.
A human
Sterols Ross
region in the promoter
for 3-hydroxy-3-methylglutaryl Petkovich
for
that binds specifically
the sterol regulatory reductase.
receptor
Mol Endocrinol4:363-369.
TF,
Red25
The
more excitement
the future. Osborne
1990.
pathways.
HC, Chayet LT: 1985. Biosynthesis
cholesterol. BW:
superfamily:
terol
by sterol and non-sterol
J Biol Chem 251:2716-2721. Rilling
1120. O’Malley
Raskin
valonate
oxysterol protein
AA: 1984. Correlation binding
to a cytosolic
and potency
between binding
in the repression
hydroxymethylglutaryl
coenzyme
of
A reduc-
tase. J Biol Chem 259:12,382-12,387. Tomkins
G, Sheppard
Cholesterol
H, Chaikoff IL: 1953.
synthesis by liver. III. Its regu-
lation by ingested cholesterol. 201:137-141.
J Biol Chem
Wang Z, Brown DD: 1991. A gene expression screen. Proc Nat1 Acad Sci USA 88: 11,5051 1,509. Wang X, Sato R, Brown stein
JL:
1994.
bound transcription rol-regulated
MS, Hua X, Gold-
SREBP-1,
a membrane-
factor released by ste-
proteolysis.
Cell 77:53-62.
Yao TP, Segraves WA, Oro AE, McKeown M, Evans
RM:
1992.
Drosophila
ultraspiracle
modulates ecdysone receptor function heterodimer formation. Cell 71:63-72.
via T-EM
AbRoAd
Consider giving a subscription to TEM to colleagues abroad where US dollars or other hard currency is not available. Simply complete the subscription order card bound into any issue, giving the recipient’s name and address labeled “send to”; after “signature,” give your own name and address and mark this “bill to.” Renewal notices will be sent to your address and the recipient will receive the journal. Please inform the recipient of your action.
6
01996,
Elsevier Science Inc., 1043-2760/96/$15.00
SSDI 1043-2760(95)00180-8
TEM Vol.7,No, 1,1996
HYPOTHESES A Model for Farnesoid Feedback Control in the Mevalonate Pathway Cary Weinberger
Mevalonate is the rate-limiting substrate leading to farnesyl pyrophosphate (FPP), the central intermediate for isoprenoids such as cholesterol, dolichols, ubiquinone, and carotenoids. One major challenge has been to identify the isoprenoid effector molecules and transcription factors mediating negative regulation in this metabolic pathway. A nuclear receptor called FXR has recently been characterized that is activated by famesyl pyrophosphate metabolites such as famesol, farnesal, famesoic acid, and methyl famesoate. FXR expression in isoprenoidogenic tissues suggests a hypothesis that these intracellular “famesoids” may be signals for transcriptional feedback control of cholesterol biosynthesis.
(Trends
How is cholesterol synthesis regulated? This question has been puzzling biochemists since 1933 when it was noted that dietary cholesterol depressed its endogenous production (Schoenheimer and Breusch 1933, Tomkins et al. 1953). Identification of 3-hydroxy-3-methylglutar-y1 coenzyme A (HMG CoA) reductase as the rate-limiting enzyme for this repression (Siperstein and Guest 1960) awaited the complete description of the enzymatic steps from acetate to cholesterol (Rilling and Chayet 1985). Although the isoprenoid signals for repressing HMG CoA reductase activity have long been recognized, their polypeptide transducers are still only incompletely defined (Wang et al. 1994). The problem of bridging the effecters and gene targets remains the largest gap in our understanding of cholesterol regulation. A model will be outlined that
Gary Weinberger is at the National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
TEA4 Vol. 7, No. 1, 1996
Endocrinol
Metab
1996;7:
l-6).
places metabolites of farnesyl pyrophosphate (FPP) at key control points in the mevalonate pathway. Identification of a nuclear receptor activated by these farnesoids is the foundation for this intracellular signaling hypothesis.
??
Isoprenoid
Synthesis
In unicellular organisms, metabolite levels are typically fixed by feedback control of biosynthetic enzymes or their activities. Higher metazoans exercise similar control, but must also integrate cellular biochemical processes through endocrine signals. One important biosynthetic route in eukaryotes is the mevalonate pathway leading to isoprenoid products such as cholesterol, bile acids, dolichol, ubiquinone, carotenoids, vitamin D, and steroid hormones (Goldstein and Brown 1990). These isoprenoids are essential for membrane assembly, lipid uptake, glycoprotein synthesis, electron transport, and hormonal regulation. Isoprenoid biosynthesis begins with
01996, Elsevier Science Inc., 1043-2760/96/$15.00
the precursor acetyl CoA derived from intermediary metabolism (Figure 1). Most of the acetate is converted to fatty acids for energy storage, and much of the remainder is diverted to mevalonate for isoprenoid synthesis (Rilling and Chayet 1985). Mevalonate production is irreversible, and this rate-limiting step is regulated by the enzyme HMG CoA reductase. Mevalonate is subsequently phosphoxylated, decarboxylated, and isomerized to isopentenyl pyrophosphate (IPP), the basic isoprenoid building block (Figure 1). Self-condensation of IPP produces geranyl pyrophosphate, and an additional IPP condensation step yields farnesyl pyrophosphate (FPP), the principal intermediate for all isoprenoids (Figure 1). Cholesterol homeostasis is sustained by three metabolic pathways (Dietschy and Wilson 1970). The first is an endogenous biosynthetic route from acetate to isoprenoids catalyzed by HMG CoA synthase and HMG CoA reductase. A second one salvages sterol ester remnants by endocytosis of low-density lipoproteins (LDLs) mediated by the LDL receptor (Goldstein and Brown 1990). The third pathway is a catabolic detour to bile acids for fat absorption and excess cholesterol removal, which is catalyzed by cholesterol 7a-hydroxylase (Russell and Setchell 1992). As a result, the intestinally absorbed dietary cholesterol intake is matched with endogenous sterol production. Although the liver and intestine are the main sites for acetate incorporation into sterols, suppression mechanisms prevail in the liver when animals are fed cholesterol-rich diets (Dietschy and Siperstein 1967).
??
Feedback Signals and Gene Targets
Both the signals and the regulated genes participating in the cholesterol regulatory network have been described. Cholesterol and hydroxylated cholesterol molecules (oxysterols), especially those
SSDI 1043-2760(95)00180-8
1
FARNESOID SHUNT
fatty acids
OCH, methyl famesoate
f OH
famesolc acid
f
HOuJL4 frans-3-methylglutaconyl CoA
1
1
JAM 3,3-dlmethylacrylyl CoA
J-O,,
/‘\
A,,,
gelanyl-PP
AAAAAY,, fame+PP
f’ lsopentenyl-PP
3,3dimethylallyl-PP
dolichol ubiquinone carotenoids cholesterol
x
3,3dlmethylallyl alcohol
PPI
Figure 1. Model for FXR/farnesoid-mediated feedback control of isoprenoid synthesis illustrating glutaconate and famesoid metabolic shunts coupled to isoprenoid biosynthetic pathway, The isoprenoid biosynthetic pathway from acetyl coenzyme A (CoA) to cholesterol is schematically outlined along with the linked glutaconate and farnesoid shunts. Acetyl CoA is condensed to form mevalonate, which may be diverted to isoprenoid biosynthesis or may be salvaged by the truns-3-methylglutaconate cycle hypothesized by Popjak (see the text). Farnesyl pyrophosphate (FPP) from the mevalonate pathway may be an intermediate for the synthesis of other isoprenoids such as cholesterol, carotenoids, dolichol, and ubiquinone, or FPP may be metabolized to farnesoids via the farnesoid shunt. The famesoid shunt begins with a phosphatase activity converting FPP to farnesol (Christophe and Popjak 1961). These biochemical transformations are analogous to the initial steps from isopentenyl pyrophosphate in the glutaconate shunt. Subsequent oxidation steps producing farnesal and farnesoic acid are catalyzed by liver alcohol and aldehyde dehydrogenases, whereas methyl esterification and epoxidation generate methyl famesoate and juvenile hormone III, respectively. FXR is a nuclear receptor, activated by FPP and its metabolites, that is exclusively expressed in the liver, intestinal villi, renal tubules, and adrenal cortex. It is hypothesized that famesoid metabolites activate FXR, which represses the transcription of genes in the mevalonate pathway. Other nuclear receptors such as RXR and its effector may act in concert with FXR to mediate these effects. Suppression of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase and FPP synthase are two potential gene targets. FPP synthase inhibition would create an IPP surplus, which would be diverted back to HMG CoA via the glutaconate shunt. HMG CoA reductase inhibition should also increase HMG CoA levels. Transcriptional repression of these two genes should, therefore, effectively inhibit the production of isoprenoid precursors. Similarly, fatty acid biosynthesis and degradation may be controlled by binding of intracellular fatty acids such as linoleic, linolenic, and arachidonic acids to the peroxisome proliferator-activated receptor (PPAR) (Keller and Wahli 1993). Farnesoids and fatty may be present within the cell at the micromolar concentrations required for activation of their respective receptors. Consequently, PPAR and FXR and their respective ligands may regulate several networks of genes for enzymes involved in lipid homeostasis.
2
OH
famesol
Isopentenyl-PP
* 3,3dimethylacrylic acid
t
J-o,,
GLUTACONATE SHUNT
01996,
Elsevier Science Inc., 1043-2760/96/$15.00
at the 7, 25, and 26 positions, be the major
effecters
HMG CoA reductase 1978). A cytoplasmic protein (OSBP) that with
affinities
appear
to
for the gene target (Kandutsch binds
matching
these
their
et al.
binding
oxysterol
sterols
potentials
HMG CoA reductase activity was characterized (Taylor et al. 1984). Molecular cloning of the OSBP gene, however, revealed no DNA binding structural features in the encoded protein (Dawson et al. 1990). Equally confounding was OSBP sequestration in the Golgi apparatus following oxysterol addition (Ridgway et al. 1992). These findings may point to a cytoplasmic regulatory role for OSBP, perhaps for promoting HMG CoA reductase enzyme degradation or decreasing the stability of its RNA. Alternatively, OSBP may bind reductase gene promoter elements by forming a complex with an unidentified DNA binding factor. Several proteins that interact with prospective DNA regulatory elements in the HMG CoA reductase for repressing
SSDI 1043-2760(95)00180-S
TEM Vol. 7, No. 1, 1996
promoter have been identified. CTF binds to six DNA sites in the tase promoter, one overlapping a responsive element (SRE) found
NF-l/ reducsterolin the
promoters of many mevalonate pathway biosynthetic genes (Gil et al. 1988). Other polypeptides have been distinguished by their SRE binding features, including CNBP (Rajavashisth et al. 1989), SRE-BF (Stark et al. 1992), and red25 (Osborne et al. 1992), but their roles in HMG CoA reductase regulation are not understood. Cholesterol-mediated repression of the low-density lipoprotein receptor (LDLR) and HMG CoA synthase genes also controls the flux of isoprenoids. Control of their transcription is considered secondary relative to HMG CoA reductase, but many aspects of their regulation are better appreciated (Wang et al. 1994). Cholesterol in the endoplasmic reticulum (ER) adjusts the proteolytic conversion of specific transcription factors called sterol response element binding proteins (SREBPs). In the absence of cholesterol, SREBP is truncated to a product that stimulates synthase and LDLR gene transcription. Cholesterol suppresses SREBP proteolysis, locking the native protein in the ER, which prevents transcription. SREBP may therefore be a membrane-bound transcriptional sensor of cellular cholesterol. Nonsterol mevalonate-derived famesol metabolites also suppress cholesterol synthesis (Brown and Goldstein 1980, Nakanishi et al. 1988). Famesoic acid and some structural analogues have been shown to inhibit cholesterol synthesis in vitro (Popjak et al. 1960). More recently, famesol, famesyl acetate, and famesyl methyl ether have been implicated in HMG CoA reductase posttranscriptional regulation (Giron et al. 1994, Bradfute and Simoni 1994, Correll et al. 1994). Support for a nonsterol transcriptional effector also comes from the finding that mevalonate or a metabolite inhibits primary transcription from the liver HMG CoA reductase gene (Clarke et al. 1985).
??
A Nuclear Receptor Activated by Farnesoids
Nuclear receptors are transcription factors that bind to target genes and regulate primary RNA synthesis by specific ligand interactions (Evans 1988). The re-
TEA4 Vol. 7, No. 1, 1996
ceptor activators include steroid hormones, ecdysteroids, and vitamin D, as well as retinoids and thyroid hormones. Despite cholesterol’s structural similarity to some nuclear receptor ligands, an isoprenoid-responsive receptor has not yet been identified. However, 30 orphan nuclear receptors isolated by nucleic acid homology offer fertile ground for continued prospecting (O’Malley 1990). Matching ligands with orphan receptors has been a formidable task, but some have been uncovered by sampling chemical compound inventories (Giguere et al. 1987, Petkovich et al. 1987). A rat orphan nuclear receptor gene called OR2 was recently isolated (Forman et al. 1995). OR2 is structurally related to the insect ecdysteroid receptor (EcR) and binds to ecdysone-responsive DNA elements (EcRE) in the presence of RXR (Yao et al. 1992, Manglesdorf and Evans 1992). RXR is an obligate receptor partner that facilitates the DNA binding of many other nuclear receptors (Manglesdorf and Evans 1992). The OR2 DNA binding features offered a way to test the receptor’s ligand dependency. Plasmid DNAs for OR2 and RXR were transfected with an EcRE-linked reporter gene into OR2-deficient cells. Increasing amounts of serum extract enhanced the activity that was dependent on both OR2 and RXR. The OR2 activator is soluble in chloroform, suggesting that OR2 is a ligand-dependent transcription factor responsive to lipophilic molecules (S. Kitareewan, E. Goode, and C. Weinberger unpublished results). A number of steroids, retinoids, fatsoluble vitamins, fatty acids, and other lipophilic compounds were surveyed for OR2 activation (Forman et al. 1995). Surprisingly, micromolar amounts of juvenile hormone (JH) III were stimulatory. JH III is a sesquiterpenoid that regulates insect development in concert with ecdysteroids (Schneiderman and Gilbert 1964). In contrast, methoprene, a potent synthetic juvenile hormone, did not activate. Other isoprenoids were also tested, including substrates from the mevalonate pathway (Figure 1) (Forman et al. 1995). Famesol was equipotent with JH III, whereas famesyl acetate and famesoic acid had slightly reduced activities, as did the diterpenoid geranylgeraniol. The famesol precursor mevalonate was much less potent, but geranFPP iol was completely ineffective.
metabolites, including squalene, squalene oxide, cholesterol, oxygenated sterols, and bile acids, were all negative in this assay. Together, these results suggest that OR2 encodes a famesoid-activated nuclear receptor that has been named FXR. Steroid receptors are widely distributed in animal tissues. In contrast, FXR has a very limited pattern of expression. FXR hybridizes to a 2.3-kb RNA species found only in the liver, intestinal villi, renal tubules, and the adrenal cortex (Forman et al. 1995). These tissues are isoprenoidogenic or ones with a high metabolic flux through the mevalonate pathway. The notable and most perplexing exception is the kidney, however, which concentrates and metabolizes circulating mevalonate to sterols faster than the liver and gut-the two most prodigious cholesterol-generating organs (Righetti et al. 1976, Dietschy and Siperstein 1967). The kidneys also use a nonsterol pathway for salvaging mevalonate, which will be detailed later here (HellStrom et al. 1973, Raskin and Siperstein 1974). FXR RNA expression is similar to that for HNF-4, an orphan receptor required for activation of liver-specific genes involved in lipid metabolism and transport (Sladek 1993).
??
Isoprenoid Metabolic Shunts
The molecules activating FXR are derived from FPP. Because FPP is a precursor for isoprenoid production, a feedback loop might be expected to regulate its synthesis. One trivial possibility is that FPP hydrolysis reduces the intracellular concentrations of sterol synthetic substrates. Alternatively, the FPP concentration would be reduced by generating metabolites that act as feedback signals for rate-limiting enzymes such as HMG CoA reductase. Finally, a metabolic pathway that diverts the mevaisoprenoids back to lonate-derived acetyl CoA would salvage carbon atoms for fatty acid synthesis.
Glutaconate
Shunt
Two observations support the hypothesis that the carbon atoms in mevalonate might be recycled to HMG CoA (Figure 1) (Popjak 1971). First, radiolabeled mevalonate was incorporated into famesoic acid in rat liver, intestines, brain, and blood and also into the saponified
01996, Elsevier Science Inc., 1043-2760/96/$15.00SSDI 1043-2760(95)00180-S
3
fatty acid fraction in the skin and brain (Edmond and Popjak 1974). Circulating mevalonate must therefore be tmnsformed back to HMG CoA and fatty acids in these latter organs (Edmond 1974). Liver extracts only made famesoic acid, demonstrating the tissue-specific utilization of the mevalonate precursor. Second, a liver microsomal enzyme activity was characterized (Christophe and Popjak 1961) that produces dimethylallyl alcohol from dimethylallyl pyrophosphate (Figure 1). Oxidation steps yield dimethylacrylic acid, which may be ester&d with coenzyme A to make trans-3-methylglutaconyl CoA, an intermediate in the leucine catabolic pathway leading to acetyl CoA or acetoacetate. In the final step, glutaconyl CoA is hydrolyzed to regenerate HMG CoA and acetyl CoA. Farnesoid
Shunt
An analogous path for farnesoid synthesis is proposed that is based on this transmethylglutaconate shunt (Figure 1). In the first step, the liver phosphatase converts FPP to famesol (Christophe and Popjak 1961). Liver alcohol and aldehyde dehydrogenases catalyze the next series of transformations to farnesal and famesoic acid. Methylation of famesoic acid to methyl famesoate (MF) is accomplished with S-adenosylmethionine as a substrate, as outlined for juvenile hormone synthesis in insects and mollusks (Schooley and Baker 1985). Among the compounds that have been tested for FXR activation, MF is as potent as farnesyl acetate (S. Kitareewan, E. Goode, and C. Weinberger unpublished results). Finally, although JH III is the most efficacious FXR activator identified to date, its physiological significance is unclear, as the epoxidation step to .lH III is seen only in insects. Note that each of the molecules in the farnesoid shunt is an FXR activator (Figure 1).
??
Famesoids and Intracrine Signaling
The micromolar famesoids required for FXR induction are greater than the nanomolar hormones necessary for steroid hormone receptor activation. These doses may be overestimates, as famesoids may not readily enter the cell. On the other hand, the pharmacologic doses of farnesoids may be achieved in the isoprenoidogenic tissues expressing FXR. This possibility is supported by the
4
01996,
micromolar substrate requirements for FPP synthase and similar inhibitory concentrations of FPP for isopentenyl pyrophosphate isomerase activity (Rilling and Chayet 1985). Unexpectedly, even FPP induces FXR, although with a much lowered potency when compared with the other famesoid metabolites (S. Kitareewan, E. Goode, and C. Weinberger unpublished results). Therefore, the effective ligand concentration for FXR, or the intracellular famesoid charge, may be the sum of concentrations for each famesoid shunt metabolite. Famesoids are the most potent FXR activators, but their binding to FXR has not yet been established. Ligand-induced FXR protection from proteolysis has failed to show direct interactions (B.M. Forman and R.M. Evans unpublished results) and famesoid binding measurements have not been practicable owing to the lipophilicity of these molecules. However, MF chromatographs with the serum FXR effector activity and that material isolated from human urine when fractionated by reverse phase HPLC (S. Kitareewan, E. Goode, and C. Weinberger unpublished results). The finding of MF in the hemolymph of insects and crustacea may support the notion that MF is an excreted metabolite. In summary, the inability to demonstrate famesoid binding to FXR could be due to the high concentrations needed for its activation. These results might also imply that famesoids are converted to more potent physiological FXR ligands. As an example, famesoids may be cyclized to the authentic FXR binding components by an enzyme similar to squalene cyclase, which catalyzes lanosterol ring formation (Stork and Burgstahler 1955). Alternatively, famesoids can be esterified to cholesterol or fatty acids by an acyl CoA: cholesterol acetyltransferase-like activity such as that linking plant triter-penes and retinol to fatty acids (Tabas et al. 1989, Ross 1990). Whether famesoid metabolites or distinct induced molecular species, isolation of the serum activators should ultimately lead to their molecular structures and their roles in FXR-regulated cell physiology.
??
Potential FXR Gene Targets
Synthetic DNA binding sites have been defined for FXR (Fox-man et al. 1995,
Elsevier Science Inc., 1043-2760/96/$15.00
Seol et al. 1995), but the FXR-regulated genes are still unknown. Previously however, famesoids have been implicated in the posttmnscriptional regulation of HMG CoA reductase activity (Correll et al. 1994). FXR now promotes an intracellular signaling transcriptional control strategy for these metabolites (Figure 1). The mevalonate-derived FXR activators point to genes for isoprenoid biosynthetic enzymes such as HMG CoA reductase and FPP synthase as compelling targets (Dorsey and Porter 1968). Interestingly, Popjak originally proposed that repression of FPP synthase and HMG CoA reductase would effectively regulate cholesterol synthesis (Popjak 1971). Nine FPP synthase-related loci (Fpsl) have been mapped in the mouse genome (Andalibi et al. 1993). Coincidentally, three are found on chromosome 10 (Andalibi et al. 1993) near the FXR gene (Kozak et al. [in press]). Finally, it is likely that FXR and famesoids control genes other than those for mevalonate pathway enzymes. Differential screening of cDNA libraries using labeled liver RNAs from famesol- and control-treated animals should help to identify these famesoid-regulated genes (Wang and Brown 1991). Transcriptional regulation of the HMG CoA reductase gene by sterols remains largely undefined. Assembly of a transcriptional repressor complex composed of NF-1, OSBP, FXR, or a sterol-binding orphan nuclear receptor may be required for reconstitution. The inability to identify the oxysterolresponsive factors may partly relate to the experimental methods used to approach this question. Most have relied on tumor cells growing in culture, where cholesterol feedback regulation is abrogated and lipid metabolism is aberrant (Bricker et al. 1972). Tumor cells cannot synthesize sterols from acetate; instead, they scavenge cholesterol from lipoproteins (Siperstein 1970). The effects of isoprenoids on reductase activity in these cancer cells may be masked because the normal metabolic restraints on the mevalonate pathway have been overridden by their aberrant growth in culture. Studying cholesterol feedback in the livers of intact animals may dissociate these growth and metabolic regulatory properties.
SSDI 1043-2760(95)00180-S
TEM Vol. 7, No. 1, 1996
??
Fatty
also thanks Paavo
Acids as MetaboliteLSignaIs
FXR may serve as a paradigm for many distinct intracellular signaling pathways modulated by lipophilic ligands and orphan nuclear receptors (O’Malley 1989). In particular, faatty acids are becoming increasingly attractive ligands for the peroxisome proliferator-activated receptor (PPAR). PPAR was originally defined by its responsiveness to the hypolipidemic clofibrates (Isseman and Green 1990). More recently, unsaturated fatty acids such as linolenic, linoleic, and arachidonic acid have been identified as PPAR activators (Gottlicher et al. 1992). Some of these fatty acids have also been isolated as serum components that bind to PPAR (Banner et al. 1994). Most of the PPAR-regulated genes are involved in long chain fatty acid oxidation (Keller and Wahli 1993). Therefore, the four PPAR species may define an intracellular
signaling
fatty
system
acids.
The
coordinated
free fatty
acids
by PPAR
and FXR
verge
responsive
to free
synthesis
and cholesterol
for lipid
might,
of
regulated
therefore,
cal reading
Honkakoski
for criti-
Edmond J: 1974. Ketone bodies as precursors of sterols
of the manuscript.
Edmond
References Andalibi A, Diep A, Quon D, Mohandas Taylor BA, Lusis ti
1993. Mapping
homeostasis.
T,
of mulpy-
mone
rophosphate
Ge-
889-895.
gene.
Mamm
nome 4:211-219. Banner
CD, Gottlicher
M, Widmark
J, Rafter JJ, Gustafsson atic
analytical
proach clear
activators
receptors
from
characterization tor-activated
assay
apnu-
biological
receptor
extracts:
DL,
Simoni
compounds
that
R:
in plasma.
1994.
regulate
Non-sterol
cholesterogene-
LA, Morris
HP, Siperstein
Loss of the cholesterol
the intact hepatoma-bearing
system
MS, Goldstein
JL:
synthesis
in
rat. J Clin In-
1980. Multivalent
and
cell
growth.
Although
previously
lonate
implicated
regulation
pathway,
famesoids
ing as transcriptional FXR
expression
large
isoprenoid
the D(R
is a distinct
J
in the tissues should
and famesoids loop
These
findings
the wide nals
??
expressing
in the
their
a
help clarify
tissues
offer
FXR.
Their D(R
between regulated
regu-
pathway.
some
hope
that
isoprenoid targets
sig-
can
be
soon.
Sutisak their
thanks
Kitareewan advice
the
3-hy-
ative
of mevalonic
accelerated
acid
required
degradation
of
coenzyme
Dawson
PA,
Ridgway
for the J CA,
Brown MS, Goldstein
JL: 1990. cDNA clon-
ing
of
and
protein, tine
expression an oligomer
zipper.
J
oxysterol-binding
with a potential
Biol
Chem
leu-
264:16,798-
JM, Siperstein
cholesterol synthesis
feeding
MD: 1967. Effect and fasting
in seventeen
Lipid Res 8:97-l
tissues
of
on sterol
of the rat. J
04.
and
Goode
and
of his laboratory
for
support.
TEM Vol. 7, No. I, 1996
The
author
01996,
valonate-mediated
1179-1183;
mevalonic
kinase
pyrophosphates.
1241-1249.
by geranyl J Biol
4670.
Elsevier Science Inc., 1043-2760/96/$15.00
243:4667-
JA: 1994. Me-
suppression
of
3-hy-
JL, Brown MS: 1990. Regulation
the mevalonate
pathway.
Nature
of
343:425-
430. Gottlicher
M, Widmark
E, Li Q, Gustafsson
J-A: 1992. Fatty acids activate the clofibric
Hellstrom
acid-activated receptor.
KH,
a chimera
receptor
of
and the
Proc Nat1 Acad Sci
Siperstein
Luby LJ:
1973. Studies
tabolism
of mevalonic
MD, Bricker
LA,
of the in vivo meacid in the normal
rat. J Clin Invest 52:1303-1313. I, Green
member
S:
1990.
of the steroid
superfamily
Activation
hormone
by peroxisome
of a
receptor
proliferators.
Nature 347:645-650. AA, Chen HW, Heiniger
Biological
activity
Keller
H-J: 1978.
of some oxygenated
20 1:498-50
H, Wahli W: 1993. Peroxisome receptors:
endocrinology
and nutrition?
crinol
prolif-
a link between ‘Trends Endo-
Metab 4:291-296. mapping
famesoid some
ste-
1.
erator-activated
MC, Weinberger
10. Mamm
Manglesdorf
C: Ge-
of the gene encoding
receptor,
the
Fxr, to mouse chromoGenome
DJ, Evans
(in press).
Rh4: 1992. Retinoid
as transcription
factors.
In Mc-
Knight SL, Yamamoto KR, Transcriptional Regulation. New York, Cold Spring Harbor Laboratory
of
and famesyl
Chem
for 3-hyA reduc-
droxy-3-methylglutaryl coenzyme A reductase function in a-toxin-perforated cells.
receptors
Dorsey JK, Porter JW: 1968. The inhibition
factor l-like
coenzyme
Giron MD, Have1 CM, Watson
netic
1128-1138;
Elizabeth
that bind to the promoter
Kozak CA, Adamson
16,803. Dietschy
JL, et al.: 1988.
proteins
rols. Science Slaughter
Goldstein
genes encode nuclear
Kandutsch
3-hydroxy-3-
A reductase.
NC,
JR,
Multiple
Isseman
Con-e11 CC, Ng L, Edwards PA: 1994. Identification of farnesol as the non-sterol deriv-
Dietschy JM, Wilson J: 1970. Regulation of cholesterol metabolism. N Engl J Med 282:
Acknowledgments
The author
of
PA: 1985.
Biol Chem 269:17,390-17,393.
whether
a feedback
regulation
for the morpho-
USA 89:4653-4657.
droxy-3-methylglutaryl coenzyme A reductase gene in rat liver. J Biol Chem 260:
methylglutaryl
working
mevalonate
may
chasm
and
bridged
of
farnesoid-responsive
comprise
latory
metabo-
Neverthe-
provides
isolating
Transcriptional
AM, Edwards
of a receptor
acid. Nature 330:624629.
glucocorticoid
J Lipid Res 2:244-
14,363-14,367.
of a famesoid-activated
receptor for
isolation
effecters. FXR-bind-
biological
acids from ally1 pyrophosphates
in liver enzyme systems.
687-693.
Proc Nat1 Acad Sci USA 91:6398-6402.
257.
the notion
insights.
of prenoic
Clarke CF, Fogelman a
determination
from
further
less, identification
genes
generating
famesoid
structure
activators
nuclear
signals.
that the authentic
provide
model
regulatory
flux supports
lite. Molecular
meva-
reemerg-
are intracellular
It is possible
may
the
are
in tissues
that famesoids ing ligand
in post-
in
that is Cell 81:
Giguere V, Ong ES, Segui D, Evans RM: 1987.
Goldstein
Christophe J, Popjak G: 1961. Studies on the biosynthesis of cholesterol: xiv. The origin
transcriptional
240:
tase. Proc Nat1 Acad Sci USA 85:8963-8967.
vest 51:197-205. Brown
receptor
metabolites.
droxy-3-methylglutaryl
MD: 1972.
feedback
Science
of a nuclear
by famesol
Gil G, Smith
sis. J Biol Chem 269~6645-6650. Bricker
activated
gen retinoic
J Lipid Res 34:1583-1591. Bradfute
1.
BM, Goode E, Chen J, et al.: 1995.
Identification
prolifera-
activators
of car-
to n-fatty ac-
superfamily.
Identification
of orphan
of peroxisome
receptor
Forman
E, Sjovall
J-A: 1993. A system-
chemistry/cell
to isolate
isoprenoid
Summary
ids. J Biol Chem 249:66-7
tiple mouse loci related to the famesyl synthetase
G: 1974. Transfer
from mevalonate
Evans RM: 1988. The steroid and thyroid hor-
Lipid Res 21:505-517. 0
J, Popjak
bon atoms
feedback regulation of HMG CoA reductase, a control mechanism coordinating
con-
and fatty acids in the developing
rat. J Biol Chem 249172-80.
Nakanishi
Press, pp 1137-l
M, Goldstein
Multivalent control glutaryl coenzyme
167.
JL, Brown MS: 1988. of 3-hydroxy-3-methylA reductase. J Biol
Chem 263:8929-8937.
SSDI 1043-2760(95)00180-S
5
O’Malley BW: 1989. Did eucaryotic ceptors
evolve from intracrine
tors?
[editorial].
steroid regene regula-
Endocrinology
125:1119-
steroid
Bennett
a protein
predicted
M,
Rhee
1987.
K:
1992. to
coenzyme
A
which belongs ceptors.
acid
P:
receptor
to the family of nuclear
re-
Nature 330:444-450.
Popjak G: 197 1. Specificity rol biosynthesis.
Harvey Lect 65: 127-l 56.
Popjak G, Comforth hibition
RH, Clifford K: 1960. In-
of cholesterol
biosynthesis
by fame-
soic acid and analogues. Lancet 1:1270-1273. Rajavashisth
TB, Taylor AK, Andalibi A, Sven-
son KL, Lusis fi 1989. Identification of a zinc finger protein that binds to the sterol regulatory
element. Science
P, Siperstein
metabolism
MD:
245640-643.
1974.
Mevalonate
by renal tissue in vitro. J Lipid
Res 15:20-25. Goldstein
JL: 1992. Translocation
binding
protein
to Golgi
triggered by ligand binding. 307-3 19. Righetti
M, Wiley MH, Murril
of oxysapparatus
J Cell Biol 116: PA, Siperstein of me-
G, Burgstabler
DR: 1992. Bile acid bio-
acyl-CoAtriterpene
Biochemistry HA, Gilbert
LI: 1964. Control
of growth and development
in insects.
Sci-
R, Breusch
F: 1933. Synthesis
of cholesterol
in the organ-
ism. J Biol Chem 103:439-448. Schooley
DA, Baker
mone
FC: 1985. Juvenile
biosynthesis.
In Kerkut
LI, eds. Comprehensive
Insect
hor-
GA, Gilbert Physiology,
Biochemistry, and Pharmacology. Pergamon, ~017, pp 363-389.
Oxford,
Seol W, Choi H-S, Moore DD: 1995. Isolation of proteins
that
interact
specifically
with
the retinoid X receptor: two novel orphan receptors. Mol Endocrinol 9:72-85. Siperstein
MD:
tissues.
1970.
Regulation
of choles-
in normal and malignant
Curr Top Cell Regul 2:65-100.
Siperstein MD, Guest MJ: 1960. Studies on the site of the feedback control of cholesterol synthesis. FM:
1993.
and liver-specific tor 3~223-232.
I, Beatini
J Clin Invest 39:642-652. Orphan
receptor
HNF4
gene expression.
Recep-
FOR COttEAqlJES
J Am
N, Chen L-L, et al.: 1989. and characterization acyltransferase tissues.
of an activity
J Lipid Res
32:1689-1698. Taylor FR, Saucier SE, Shows EP, Parish EJ, Kandutsch
ence 143:325-333. Schoenheimer
Tabas
in rabbit and human
3 1:4737-4749.
The stere-
cyclization.
Chem Sot 77:5068-5077. Identification
synthesis.
AW: 1955.
of polyene
189:442-445.
Sladek
MD: 1976. The in vitro metabolism
Stork
esterification
DW, Setchell
of retinol.
DNA
Proc Natl Acad Sci USA 892180-2184.
zyme A-dependent Enzymol
J: 1992.
binding factor for a sterol regulatory element.
ochemistry of acyl coen-
0, Weinberger
double- and single-stranded
Methods
terol biosynthesis
Ridgway ND, Dawson PA, Ho YK, Brown MS,
H, Sjovall J, eds.
Measurement
and destruction
of enzymes of ste-
of
pp l-39.
Schneiderman
M, Brand NJ, Krust A, Chambon retinoic
In Danielsson
AC: 1990.
Stark HC, Weinberger Common
and Bile Acids. New York, Elsevier
Science,
Russell
J Biol Chem 267:18,973-18,982.
A human
Sterols Ross
region in the promoter
for 3-hydroxy-3-methylglutaryl Petkovich
for
that binds specifically
the sterol regulatory reductase.
receptor
Mol Endocrinol4:363-369.
TF,
Red25
The
more excitement
the future. Osborne
1990.
pathways.
HC, Chayet LT: 1985. Biosynthesis
cholesterol. BW:
superfamily:
terol
by sterol and non-sterol
J Biol Chem 251:2716-2721. Rilling
1120. O’Malley
Raskin
valonate
oxysterol protein
AA: 1984. Correlation binding
to a cytosolic
and potency
between binding
in the repression
hydroxymethylglutaryl
coenzyme
of
A reduc-
tase. J Biol Chem 259:12,382-12,387. Tomkins
G, Sheppard
Cholesterol
H, Chaikoff IL: 1953.
synthesis by liver. III. Its regu-
lation by ingested cholesterol. 201:137-141.
J Biol Chem
Wang Z, Brown DD: 1991. A gene expression screen. Proc Nat1 Acad Sci USA 88: 11,5051 1,509. Wang X, Sato R, Brown stein
JL:
1994.
bound transcription rol-regulated
MS, Hua X, Gold-
SREBP-1,
a membrane-
factor released by ste-
proteolysis.
Cell 77:53-62.
Yao TP, Segraves WA, Oro AE, McKeown M, Evans
RM:
1992.
Drosophila
ultraspiracle
modulates ecdysone receptor function heterodimer formation. Cell 71:63-72.
via T-EM
AbRoAd
Consider giving a subscription to TEM to colleagues abroad where US dollars or other hard currency is not available. Simply complete the subscription order card bound into any issue, giving the recipient’s name and address labeled “send to”; after “signature,” give your own name and address and mark this “bill to.” Renewal notices will be sent to your address and the recipient will receive the journal. Please inform the recipient of your action.
6
01996,
Elsevier Science Inc., 1043-2760/96/$15.00
SSDI 1043-2760(95)00180-8
TEM Vol.7,No, 1,1996