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First published online 21 May 2008
doi: 10.1242/dev.016972
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1 Department of Biological Structure, University of Washington, Seattle, WA,
USA.
2 Department of Molecular Biology, Massachusetts General Hospital, Boston, MA,
USA.
* Author for correspondence (e-mail: weiqing{at}u.washington.edu)
Accepted 1 May 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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5-4 isomerases
(3β-HSDs), which are key steroidogenic enzymes in vertebrates, and is
exclusively expressed in two neuron-like XXX cells that are crucial in
preventing dauer arrest, suggesting that it is involved in biosynthesis of
dauer-preventing steroid hormones. The hsd-1 null mutant displays
defects in inhibiting dauer arrest: it forms dauers in the deletion mutant
backgrounds of ncr-1 or daf-28/insulin; as a single mutant,
it is hypersensitive to dauer pheromone. We found that hsd-1 defects
can be rescued by feeding mutant animals with several steroid intermediates
that are either downstream of or in parallel to the 3β-HSD function in
the dafachronic acid biosynthetic pathway, suggesting that HSD-1 functions as
a 3β-HSD. Interestingly, sterols that rescued hsd-1 defects also
bypassed the need for the NCR-1 and/or -2 functions, suggesting that
HSD-1-mediated steroid hormone production is an important functional output of
the NCR transporters. Finally, we found that the HSD-1-mediated signal
activates insulin/IGF-I signaling in a cell non-autonomous fashion, suggesting
a novel mechanism for how these two endocrine pathways intersect in directing
development.
Key words: 3β-HSD, Steroid hormone, Dauer, Insulin/IGF-1, NPC1, Niemann-Pick C
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Three
types of endocrine signals are known to be involved: steroid hormones
(Rottiers and Antebi, 2006),
insulin/IGF-1-like peptides (Li et al.,
2003; Pierce et al.,
2001) and TGFβ-like ligands
(Ren et al., 1996;
Schackwitz et al., 1996).
These signals act in a non-redundant fashion, i.e. active signaling by their
respective hormone receptor is required to promote reproductive development
and prevent dauer arrest.
The role of steroid-signaling in dauer formation became apparent with the
identification of several protein components. These include DAF-12/nuclear
hormone receptor (Antebi et al.,
2000), two steroid-processing enzymes, DAF-9/cytochrome P450
(Gerisch et al., 2001;
Jia et al., 2002) and
DAF-36/mono-oxygenase (Rottiers et al.,
2006), and the probable intracellular cholesterol transporters
NCR-1 and NCR-2 (Li et al.,
2004). Recently, several steroids have been identified as DAF-12
ligands that act to prevent dauer arrest
(Motola et al., 2006;
Held et al., 2006).
4- and 7-dafachronic acids are two such
hormones (Motola et al.,
2006). Additional hormones may also exist that prevent dauer
arrest (Matyash et al.,
2004).
A two-branch pathway has been proposed for the biosynthesis of
4- and 7-dafachronic acids from
cholesterol (Rottiers et al.,
2006). However, the enzymes for several biosynthetic steps remain
unknown. Genetic identification of new enzymes will not only help
validate/modify the putative dafachronic acid biosynthetic pathway, but also
reveal new pathways/networks for the biosynthesis of additional steroid
hormones in the worm.
The steroid and insulin/IGF-1-like pathways are likely to converge for
coordinated regulation in vivo, as both pathways regulate dauer formation,
lifespan and stress resistance (Baumeister
et al., 2006; Finch and
Ruvkun, 2001). The two pathways also resemble each other in mode
of action, as both involve multiple ligands that target one receptor. There
are 40 insulin-like peptides in C. elegans and several, including
DAF-28, have been reported to have functional relationships with
DAF-2/insulin-like receptor (Kimura et
al., 1997; Pierce et al.,
2001; Li et al.,
2003). Although the current model is that steroid-signaling
functions downstream of or in parallel to insulin-signaling in governing dauer
formation (Rottiers and Antebi,
2006), genetic evidence suggests that the two pathways may
intersect in a complex manner (Gems et
al., 1998). Given that insulin/IGF-1 and steroid pathways also
function in concert to regulate development in flies and mice
(Tatar et al., 2003),
understanding potential crosstalk between the two endocrine pathways in the
worm will help reveal conserved mechanisms for hormonal coordination in
general.
Nematodes cannot synthesize steroid hormones de novo, but instead modify
sterols, such as cholesterol, acquired from their environment
(Chitwood, 1999). The C.
elegans NCR-1 and NCR-2 (Li et al.,
2004; Sym et al.,
2000) are both orthologs of human NPC1, an intracellular
cholesterol transporter that results in Niemann-Pick type C (NPC) disease when
mutated (Carstea et al., 1997).
ncr-1 and ncr-2 function redundantly in preventing dauer
arrest, as knockout of both but neither alone causes dauer arrest.
ncr-1 is thought to be more important for sterol trafficking as it is
more broadly expressed than ncr-2
(Li et al., 2004). We
hypothesized that both NCR proteins are involved in providing cholesterol
and/or other sterols for the production of dafachronic acids or related
hormones to promote reproductive development.
We identified HSD-1 from an ncr-1 enhancer screen. HSD-1 is
related to 3β-HSD enzymes that are essential for producing active steroid
hormones in vertebrates. Based on protein similarity and the role of
hsd-1 in promoting reproductive growth, we hypothesize that HSD-1 is
involved in the biosynthesis of 4-dafachronic acid. To
determine the function of hsd-1 and dissect the proposed dafachronic
acid-biosynthetic pathway, we performed a series of sterol-feeding experiments
on hsd-1 mutants. The results supported our initial hypothesis. In
addition, our sterol-feeding experiments also addressed substrate specificity
of the NCR transporters at the organismal level, which we believe will
contribute to greater understanding and subsequent treatment of NPC disease.
Furthermore, our findings suggest that the expression of hsd-1 is
exclusive to the neuron-like XXX(L/R) cells, and that the HSD-1-mediated
steroid signal intersects with the insulin/IGF-I pathway by globally
regulating the translocation of the DAF-16/FOXO transcription factor.
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MATERIALS AND METHODS |
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SUMMARY
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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).
The following parental strains were used in this report: N2 (wild type),
CB4856, hsd-1(mg433) (6x outcrossed), ncr-2(nr2023),
ncr-1(nr2022), daf-36(k114), unc-75(e950), daf-28(tm2308) (3x
outcrossed), zIs356[Pdaf-16::DAF-16::GFP;
rol-6(su1006)]. Molecular methods were used to construct strains, in
addition to mutant phenotypes.
ncr-1 enhancer screen and mg433
An ncr-1 enhancer screen was designed to identify mutants that
phenocopy ncr-2(nr2023); ncr-1(nr2022), which form transient, pale
dauers at all temperatures. A synchronized L4 larval population of
ncr-1(nr2022) (P0) was mutagenized with EMS
(Sulston and Hodgkin, 1988).
F1 and F2 animals were cultivated at 20°C. Candidate mutants from
synchronized F2 animals, which represented the progeny of 
9000 F1s, were
divided into 18 pools and then subjected to a 30 minute treatment in 0.5% SDS
solution. Pale dauers were then selected from surviving animals and recovered
at 20°C. Sixteen mutant isolates were obtained from 10/18 pools, of these
mg433 displayed the highest penetrance of dauer arrest and was
selected for further analysis. unc-75(e950) was used as a positional
marker to introduce a wild-type stretch of chromosome I to the left of
mg433 for crossing out a background mutation.
Constructs for hsd-1 expression and rescue
The expression pattern of hsd-1 was determined with gfp
(Chalfie et al., 1994) fused to
the hsd-1 promoter, which contained 10.5 kb 5' sequence of
hsd-1, including the entire lagr-1 gene. This design was
based on a previous prediction in WormBase that these genes form an operon.
Phsd-1::GFP was generated by in vivo homologous recombination
(Aroian et al., 1990) of two
injected PCR fragments: the first contains 3.2 kb of promoter and most of
lagr-1; the second includes the 3' region of lagr-1,
the intergenic region between lagr-1 and the ATG of hsd-1
fused with gfp from pPD95.70, using a PCR-fusion method
(Hobert, 2002). pha-1
DNA was used as the co-injection marker and a pha-1
temperature-sensitive mutant was used for injection.
Psdf-9/eak-5::RFP is as described by Hu et al.
(Hu et al., 2006).
Two hsd-1 transgenes were generated to test for rescue of
hsd-1 defects: Psdf-9::hsd-1 and Phsd-1::hsd-1.
Psdf-9::hsd-1 contains the 3.5 kb sdf-9 promoter fused to the
hsd-1-coding region and 740 bp downstream sequences.
Phsd-1::hsd-1 contains the 275 bp 5' sequence of
hsd-1, the coding region and 180 bp downstream sequence. The
co-injection marker was sur-5::gfp
(Yochem et al., 1998).
Dauer assays, sterol feeding and pheromone treatment
Two different methods were used to synchronize worm populations for dauer
assays. The first was hypochlorite treatment of gravid adults as described by
Sulston and Hodgkin (Sulston and Hodgkin,
1988). The second was timed egg-laying with seven to eight
hermaphrodites per plate, allowing them to lay eggs for 5-6 hours before
removing them. The window of time for scoring dauers versus bypassors (L4 or
adults) at 25°C is 48-72 hours after egg preparation or egg-laying (65-72
hours for strains containing the ncr-1 deletion and 48-55 hours for
other strains). For dauer assays at 20°C and 15°C, worms were scored
70-76 and 94-100 hours after egg preparation/laying, respectively. The
transgenic rescue of hsd-1; ncr-1 transient dauers was monitored
using the developmental timing of synchronized populations, and the
percentages of rescued transgenic animals were determined at a time point of
68 hours after egg preparation, when the control hsd-1; ncr-1 animals
were still virtually all dauers and N2 animals were adults.
Agar plates containing different sterols were prepared according to the
standard procedure for NGM plates (Sulston
and Hodgkin, 1988), except that cholesterol was replaced by
different sterols at the equivalent concentration of 12.9 µM (1x).
Stock solutions of sterols, dissolved in ethanol, were added into liquid agar
media at a high temperature to allow even distribution. Sterols used in this
study are as follows: cholesterol, 4-cholesten-3-one, 5-cholesten-3-one,
lathosterol, lathosterone, 7-dehydrocholesterol, β-sitosterol/sitosterin
and ergosterol (Sigma-Aldrich, St Louis, MO, USA; Steraloids, Newport, RI,
USA; Research Plus, Barnegat, NJ, USA).
Crude pheromone extract was obtained as described by Golden and Riddle
(Golden and Riddle, 1984).
Pheromone extract was diluted with 750 µl of OP50 culture and distributed
over the entire surface of a 3 ml NGM plate. Plates were then dried in a
tissue culture hood until no liquid was seen on the surface (40-70 minutes).
The egg preparation method was used to obtain synchronized populations for the
pheromone assay. Bacterial food was always in excess during experiments, which
were carried out at 25°C.
Analysis of GFP patterns and imaging
DAF-16::GFP transgenic strains were propagated at 15°C. Five to eight
young adults were allowed to lay eggs for 4 hours and develop at 20°C.
Animals were scored for GFP subcellular localization under a fluorescent
dissecting scope. The fluorescent images in
Fig. 1A-C were obtained using a
compound scope with 40x optics and
Fig. 1D-F using a 16x
lens.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). We performed an EMS screen for transient dauers resembling
ncr-1; ncr-2 mutant animals. From this screen we isolated
mg433 (for phenotypic analyses, see
Table 1) and subsequently
mapped it, using the SNP method (Wicks et
al., 2001). The analyses of the SNP markers of 55 recombinants on
chromosome I located mg433 to the interval between the cosmid M01E5
and the YAC Y71A12B, which are about 650 kb or 5 cM apart on the physical and
genetic maps, respectively. We sequenced three candidates in this interval and
found that mg433 is a mutation in the gene Y6B3B.11. This gene is
predicted to encode a protein with extensive similarity to the
3β-hydroxysteroid dehydrogenase/5-4
isomerase family of enzymes, thus we designated Y6B3B.11 as hsd-1.
mg433 has a G to A transition that results in an early amber stop codon
at Trp45, suggesting that mg433 is a molecular null.
Transgenic expression of hsd-1 fully rescued the constitutive dauer
formation phenotype (referred to as dauer arrest throughout this report) of
hsd-1(mg433); ncr-1(nr2022) (see below), verifying that
hsd-1 is involved in the inhibition of dauer formation and that
mg433 impairs this function.
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4-3-keto-sterols and two are implicated in human diseases
(Konig et al., 2000;
Schwarz et al., 2000). The
extensive similarity between HSD-1 and the human 3β-HSDs suggests that
HSD-1 is involved in producing 4-3-keto-steroids such as
4-dafachronic acid. HSD-1 also has two paralogs in C.
elegans, HSD-2 (ZC8.1) and HSD-3 (ZC449.6).
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;
Ohkura et al., 2003), but are
also thought to be involved in steroidogenesis, based on the expression
patterns of NCR-1, NCR-2 and DAF-9
(Gerisch and Antebi, 2004;
Jia et al., 2002;
Li et al., 2004;
Mak and Ruvkun, 2004). To
confirm this prediction, we examined whether the expression of
Phsd-1::GFP co-localizes with that of Psdf-9/eak-5::RFP. The
sdf-9 gene is expressed only in the XXX cells
(Hu et al., 2006;
Ohkura et al., 2003) and we
observed co-localization of both markers
(Fig. 1A-C). Consistent with
this, a transgene containing the hsd-1-coding sequence driven by the
sdf-9 promoter, rescued the dauer arrest of hsd-1(mg433);
ncr-1(nr2022) (Table 2).
The expression of Phsd-1::GFP was initially observed at the pretzel
stage of embryogenesis. This XXX cell-specific expression persists through all
four larval stages and becomes fainter in adults, which is consistent with the
view that the XXX cells are an important endocrine tissue in regulating C.
elegans larval development.
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) and the
mutants of DAF-12 that are defective in steroid-ligand binding, such as
rh273 (Antebi et al.,
1998). hsd-1; ncr-1 dauers can resume reproductive growth
at all temperatures, including the relatively high 25°C. We compared the
dauer recovery rates for hsd-1; ncr-1 and ncr-2; ncr-1 at
25°C (Table 1) and found
that hsd-1; ncr-1 dauers appeared to recover slightly slower. We
found that hsd-1; ncr-1 dauers recovered poorly and had
abnormalities such as protruding-vulvas and `bagging' (matricide by internal
hatching) phenotypes (9/25 animals), similar to ncr-2; ncr-1 (8/25).
Like ncr-2, ncr-1, we also found that hsd-1; ncr-1 has a
reduced brood size (Fig. 2A).
In the absence of dauer-preventing steroids, such as in daf-9 or
ncr-2; ncr-1, the un-liganded form of DAF-12 receptor is known to be
required for dauer formation (Rottiers and
Antebi, 2006). Similarly, we found that hsd-1; ncr-1
dauer arrest at 25°C can be suppressed by RNAi of daf-12
(Table 1). Similar to mutants
of other steroid-signaling components, except for null mutants of daf-12,
hsd-1; ncr-1 is capable of forming natural dauers under the appropriate
physiological conditions. To conclude, in a similar manner to DAF-9, the
HSD-1-mediated steroid signal normally promotes reproductive growth.
The hsd-1(mg433) single mutant appears to develop normally;
however, we observed that it forms dauers more readily than the wild-type N2
under the high population densities present in continuous plate culture. Dauer
pheromone is constitutively produced and released by worms, serving as an
external signal for judging and regulating population density
(Golden and Riddle, 1982;
Golden and Riddle, 1984). In
order to determine whether the hsd-1 single mutant is more sensitive
to induction by dauer pheromone, we examined the response of hsd-1 to
a crude extract of dauer pheromone. We found that hsd-1 forms dauers
at 98.2% and 56.7% with 8 and 4 µl of pheromone extract per plate,
respectively (Fig. 2B). At the
same pheromone concentrations, N2 did not form dauers, indicating that
hsd-1 is hypersensitive to pheromone induction. In fact, it took
approximately 30x higher pheromone concentration to induce dauers in N2
than hsd-1 (Table 1).
We also tested whether ncr-1, for comparison with hsd-1, is
hypersensitive to pheromone and found that although it does form some dauers,
it is not as sensitive (Fig.
2B). This result suggests that hsd-1 prevents dauer
arrest at population densities lower than those that would normally trigger
dauer formation in wild-type worms, which is consistent with a role for
hsd-1 in preventing dauer arrest in the ncr-1 background.
However, in the range of population densities used in this report,
hsd-1 animals rarely form dauers without pheromone
(Fig. 3), suggesting that our
dauer assays were not influenced by population density.
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). We
found that hsd-1(mg433); daf-36(k114) displayed 100% dauer arrest at
25°C (Table 1). This result
suggests that, compared with HSD-1, DAF-36 functions in a parallel
biosynthetic pathway, a different tissue, or both. Rottiers et al.
(Rottiers et al., 2006) have
previously shown that DAF-36 is expressed in the intestine and may function in
the 7-branch (Fig.
4A), which is consistent with the observed synergism.
We also investigated possible synergism between hsd-1(mg433) and
daf-28(tm2308), a deletion mutant of an insulin. The transient dauer
phenotype of the dominant-negative mutant daf-28(sa191) suggests that
DAF-28 functions as an agonist of the DAF-2 receptor
(Li et al., 2003;
Malone and Thomas, 1994). We
found that 28.5% of daf-28(tm2308) undergo dauer arrest at 25°C
(Table 1). Unlike the single
mutants of either hsd-1 or daf-28, hsd-1(mg433);
daf-28(tm2308) animals form 89.6% transient dauers at 25°C
(Table 1). This synthetic
phenotype suggests that, although the insulin and steroid pathways are
non-redundant with respect to each other, a subset of ligands from each
pathway can be redundant in preventing dauer arrest. Interestingly, the
transient dauers of hsd-1(mg433); daf-28(tm2308) have mixed
characteristics of dauers induced by either reduced insulin or steroid
signaling. They are large dauers, resembling the mutants of the DAF-2
receptor, but also pale and active, resembling ncr-2; ncr-1 dauers
(data not shown).
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5-4 isomerase in 4-steroid signaling), have shown that they carry out a sequential two-step
reaction: (1) the dehydrogenase activity oxidizes the 3β-hydroxyl group
of a sterol; (2) the isomerase activity switches the position of the double
carbon bond on the sterol ring from 5 to 4
(Fig. 4A). Thus, we hypothesize
that HSD-1 functions in the 4-dafachronic acid branch by
converting cholesterol to 4-cholesten-3-one
(Fig. 4A).
In order to test whether dietary supply of 4-cholesten-3-one could
compensate for the lack of hsd-1 activity, we first examined the
hypersensitivity of hsd-1(mg433) to dauer pheromone when grown on
agar plates containing 4-cholesten-3-one instead of cholesterol. We observed
substantial rescue of dauer arrest with 4-cholesten-3-one, at two different
pheromone concentrations (Fig.
5A). We investigated whether 4-cholesten-3-one could rescue
synthetic dauer arrest associated with the double mutants of
hsd-1(mg433) with daf-28(tm2308), daf-36(k114) and
ncr-1(nr2022), and we observed robust rescue
(Fig. 5B;
Fig. 6A,B). As a control, we
also tested the predicted product of the first step of the HSD-1 reaction,
5-cholesten-3-one, which contains the C-3 ketone group but still has the
5-ring configuration
(Fig. 4A). We found that
5-cholesten-3-one did not rescue hsd-1; ncr-1 as well as
4-cholesten-3-one (Fig. 6A,B),
suggesting that both structural characteristics of 4-cholesten-3-one
contribute to the rescue of hsd-1(mg433). The above results are
consistent with the hypothesis that HSD-1 converts cholesterol to
4-cholesten-3-one in vivo, or alternatively, HSD-1 modifies a derivative of
cholesterol into a 4-3-keto-sterol.
7-Dafachronic acid, another dauer-preventing hormone, is
thought to be produced in parallel to 4-dafachronic acid
from cholesterol (Fig. 4A)
(Motola et al., 2006). If
hsd-1 is required by the 4-biosynthetic branch,
raising the level of 7-signaling by increasing the levels of
7-intermediates may compensate for impaired
4-dafachronic acid biosynthesis. We found lathosterol and
lathosterone, two 7-intermediates, can suppress both
hsd-1(mg433) dauer pheromone hypersensitivity and synthetic dauer
arrest in the double mutants of hsd-1 with daf-28(tm2308),
daf-36(k114) and ncr-1(nr2022)
(Fig. 5A,B;
Fig. 6A,B). Higher
concentrations of lathosterol were required for full suppression of hsd-1;
ncr-1 dauer arrest (Fig.
6A). By contrast, cholesterol was unable to suppress this
phenotype at any of the concentrations tested
(Fig. 6A). In a titration
experiment to compare the efficacy of the sterols in alleviating
hsd-1-associated dauer phenotypes, we found that, like
4-cholesten-3-one, lathosterone is quite potent at a quarter of the standard
12.9 µM concentration of cholesterol. Lathosterone still displayed rescue
at an eighth this concentration (Fig.
6B). The physiological concentration for
4-dafachronic acid is estimated to be 200 nM
(Motola et al., 2006), and our
experiment showed that lathosterone at a concentration 8-fold greater
than this can be effective. Interestingly, 7-dehydrocholesterol, the first
intermediate in the 7-branch, did not suppress
hsd-1-associated dauer arrest
(Fig. 5A,B;
Fig. 6A), except in hsd-1;
daf-36, where the suppression is probably due to the rescue of
daf-36, as this is the predicted end product of the DAF-36 reaction
(Fig. 4A). The above results
suggest that some sterol intermediates in the parallel
7-branch can compensate for the lack of
HSD-1-mediated 4-dafachronic acid production.
The requirement for the NCR-1 and/or -2 transporter function can be bypassed by dietary supply of certain sterol intermediates
hsd-1(mg433) was identified as an enhancer of
ncr-1(nr2022), with the double mutant forming transient dauers when
grown on cholesterol-containing media. We considered the possibility that one
role of NCR-1 or -2 transporters is to mobilize and display cholesterol for
HSD-1-mediated 3β-dehydrogenation. According to this hypothesis,
sterol intermediates beyond the point of the HSD-1 reaction, or those in the
parallel 7-branch, should be able to bypass the requirement
for the NCR-1 and NCR-2 transporters in preventing dauer arrest.
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;
Hieb and Rothstein, 1968).
Given that the sterols that alleviated hsd-1-associated defects also
appeared to suppress ncr-2; ncr-1, HSD-1-mediated hormone production
appears to be an important functional output of the NCR transporters.
The NCR-independent sterols block crosstalk between steroid and insulin signaling
In order to detect any potential crosstalk between steroid and
insulin/IGF-1-like signaling, we examined the subcellular localization of
DAF-16::GFP in hsd-1(mg433); ncr-1(nr2022) and ncr-2(nr2023);
ncr-1(nr2022). Upon downregulation of DAF-2, DAF-16/FOXO, a transcription
factor in the insulin pathway, is activated after translocating from the
cytoplasm to the nucleus (Henderson and
Johnson, 2001; Lee et al.,
2001; Lin et al.,
2001). In wild-type animals, DAF-16::GFP is localized to the
cytoplasm, thus displaying a diffuse pattern
(Fig. 1D). When DAF-16::GFP is
in the nucleus, it displays a punctate pattern. We found that 29% of
hsd-1; ncr-1 and 60.6% of ncr-2; ncr-1 animals displayed
punctate DAF-16::GFP at the L2d stage, a preparatory stage prior to dauer
formation (Table 3). The
difference between the two double mutants suggests that the downregulation of
steroid signaling is more severe in ncr-2; ncr-1 than in hsd-1;
ncr-1, although dauer arrest between the two is comparable. We observed
variable degrees of DAF-16 nuclear localization amongst individuals of both
hsd-1; ncr-1 and ncr-2; ncr-1 mutants. For example,
Fig. 1E shows a mix of diffuse
and punctate DAF-16::GFP, whereas Fig.
1F displays a punctate pattern. Interestingly, we observed that in
both double mutants, DAF-16::GFP is often more punctate in the posterior of
the worm, as though insulin signaling is more active in the head compared with
the tail (Fig. 1E, see
Discussion). In addition, we observed that a complete punctate pattern is
often found among the double mutants close to or during the molting period
prior to dauer formation (Fig.
1F). However, DAF-16::GFP is primarily diffuse amongst dauers
(Table 3). These results
suggest that the insulin pathway is partially, or transiently, downregulated
in both double mutant backgrounds prior to dauer formation.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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5-4 isomerase4-3-keto-sterols, which is thought to be required
for the formation of active vertebrate steroid hormones
(Payne and Hales, 2004).
Although vertebrate steroid hormones lack the long side chain that is present
in the dafachronic acids, the presence of a C-3 ketone group appears to be a
shared characteristic (Motola et al.,
2006). The genetic identification of HSD-1 in C. elegans
has reinforced this notion.
Our sterol-feeding experiments showed that 4-cholesten-3-one rescued
hsd-1- associated defects more effectively than 5-cholesten-3-one,
suggesting that both structural changes enacted by the dehydrogenase and
isomerase activities of 3β-HSD contribute to rescuing hsd-1.
However, our in vivo experiments did not identify the true or preferred
substrate(s) of HSD-1. Conceivably, the substrate(s) of HSD-1 is cholesterol
or its derivatives. Our experiments also did not address why 5-cholesten-3-one
could rescue the hsd-1 defects. There are several possible reasons
why 5-cholesten-3-one can substantially rescue hsd-1; ncr-1:
(1) this 5-sterol may be converted to
4-sterol by enzymes with ketosteroid isomerase activity,
such as GSTs with a similar function to GST A3-3 in humans
(Johansson and Mannervik,
2001); (2) this 5-sterol may be processed into a
5-steroid similar to 5-cholesten-3β-ol-(25S)-carboxylic
acid, which has dauer-preventing activity
(Held et al., 2006); (3) this
5-sterol may be modified into dauer-preventing hormones that
remain unidentified. Therefore, it is possible that the in vivo metabolic
pathways/networks are more complex and plastic than the proposed pathway for
dafachronic acids, in both protein and sterol/steroid contents. Nevertheless,
our results are consistent with the hypothesis that HSD-1 functions as a
3β-HSD in vivo.
hsd-1-mediated 4 steroid signaling in the XXX cells
In C. elegans, steroid signaling is thought to be present in
multiple tissues. Besides the XXX cells, steroids are believed to be produced
in the hypodermis, intestine and somatic gonads
(Gerisch et al., 2001;
Jia et al., 2002; Mak and
Ruvkun; Rottiers et al.,
2006). hsd-1 is the first steroidogenic enzyme that
displays expression exclusive to the XXX cells. This expression pattern is not
all that surprising: it is known that the XXX cells are where ncr-2
is predominantly expressed in young larvae
(Li et al., 2004), when the
dauer decision is made. hsd-1 and ncr-2 both enhance
ncr-1, resulting in dauer arrest and thus may share a similar focus
in their functions, which may be to promote the generation of steroids in XXX
cells. The molecular identity of HSD-1 and our sterol-feeding results suggest
that HSD-1 is likely to function in 4-dafachronic acid
biosynthesis (Fig. 4A). Thus,
we speculate that the phenotypes associated with the hsd-1(mg433)
null in this report represent the impairment of 4-signaling
in the XXX cells.
hsd-1-mediated 4-signaling in XXX cells
contributes to promoting reproductive growth, which is also indicated by the
fact that hsd-1 becomes crucial when the population density reaches a
certain level. Dauer pheromone is a surrogate measure of population density.
The degree of hsd-1(mg433) hypersensitivity to pheromone implies
that, without hsd-1, a growing worm culture would begin to form
dauers at a much lower population density
(Fig. 3,
Table 1). The involvement of
hsd-1 in promoting growth is also indicated by its synergism with
several genes in preventing dauer arrest: ncr-1, daf-28 and
daf-36. Together, we conclude that HSD-1 becomes crucial in the
growth versus dauer decision, when population density is relatively high,
cholesterol trafficking is attenuated, insulin-signaling is reduced or a
parallel steroid-signaling event is compromised.
The expression of both hsd-1 and ncr-2 is limited to the
head at the stage when the dauer versus growth decision is made. Given that
the formation of dauers requires remodeling of multiple tissues
(Cassada and Russell, 1975),
including the pharynx and hypodermis, the XXX cell-expressed HSD-1 can
apparently function from a distance. Consistent with this prediction, we found
that the influence of hsd-1/ncr-2-mediated steroid signaling on
DAF-16::GFP, which is ubiquitously expressed, appears to affect many cells
(Fig. 1E,F). This is consistent
with the cell non-autonomous nature of steroid hormone action. Under an
epifluorescent dissecting scope, we observed that DAF-16::GFP is often found
fully confined to the nucleus in the posterior of the worm, but gradually
becomes more cytoplasmic towards the anterior end in both hsd-1;
ncr-1 and ncr-2; ncr-1 (Fig.
1E). This implies that there is a decreasing gradient of hormones
that prevent the downregulation of insulin signaling or DAF-16 nuclear
localization, running in an anterior-posterior direction. Alternatively, as a
recipient tissue for hormonal signals, posterior cells maybe more prone to
downregulation of insulin signaling than their anterior neighbors in
hsd-1; ncr-1 or ncr-2; ncr-1 animals.
Our finding that DAF-16::GFP undergoes nuclear translocation upon the
reduction of steroid signaling suggests a novel mechanism for the cell
non-autonomous coordination of these two endocrine pathways in directing
development. It has been previously shown that a steroid/lipophilic signal
induced by the loss of germline cells extends lifespan by promoting nuclear
translocation of DAF-16, indicating that steroid-signaling negatively
regulates the insulin pathway (Gerisch et
al., 2001; Hsin and Kenyon,
1999; Lin et al.,
2001). Our finding suggests a comparable epistatic relationship
between the two endocrine pathways in dauer regulation, except that in the
latter case, the presence of steroid hormones upregulates insulin signaling.
Previously, steroid signaling was thought to function downstream of or in
parallel to insulin signaling in regulating development
(Rottiers and Antebi, 2006).
We believe that our findings are not contradictory to the previous model;
instead, it reflects that these two endocrine pathways interact in a complex
manner.
Redundancy of steroid signaling in C. elegans
Based on comparisons between the null mutant phenotypes, expression
patterns and biochemical roles of daf-9 and hsd-1, we
predict that hsd-1 functions in the daf-9 pathway, but is
not the sole input to daf-9. daf-9 worms display a severe dauer
arrest phenotype (Gerisch et al.,
2001; Jia et al.,
2002), whereas hsd-1 worms only show hypersensitivity to
dauer pheromone; daf-9 is expressed in multiple tissues
(Gerisch and Antebi, 2004;
Mak and Ruvkun, 2004), whereas
hsd-1 is expressed only in the XXX cells. DAF-9 is involved in
biosynthesis of both 7- and 4-dafachronic
acids (Motola et al., 2006),
whereas HSD-1 is likely only to be involved in the latter. We speculate that
HSD-1 provides a C-3 keto-sterol precursor for the production of
4-dafachronic acid by DAF-9 in XXX cells, whereas the role
of producing C-3 keto-sterols in other pathways or tissues may be carried out
by other 3β-HSDs or sterol dehydrogenases, such as HSD-2 and HSD-3. We
speculate that a complex sterol metabolic network involving multiple branches
or alternative pathways exists to allow versatile regulation of development in
response to different environmental conditions. It is known that brassinolide
signaling in plants, which regulates development in response to light,
comprises such a network (Thummel and
Chory, 2002).
If 7- and 4-dafachronic acids function
redundantly in preventing dauer arrest, raising the level of
7-signaling should compensate for the lack of HSD-1-mediated
4-signaling. We found that two predicted
7-intermediates, lathosterol, a 3β-hydroxysterol, and
lathosterone, could suppress hsd-1-associated defects. Conceptually,
suppression is no different from rescue. Therefore, one possible
interpretation of this result is that hsd-1 functions upstream of the
generation of lathosterol. However, based on protein similarity, HSD-1 is
likely to produce 4-3-keto-sterols
(Fig. 4A). Therefore, it is
more likely that increased 7-biosynthesis accounts for the
ability of lathosterol to suppress hsd-1 defects. By contrast,
feeding of 7-dehydrocholesterol does not compensate for the impairment of
4-signaling (Fig.
5A,B; Fig. 6A),
which could result from a rate-limiting step in the trafficking/processing of
7-dehydrocholesterol into later steroid intermediates, as discussed later.
Steroidogenesis, cholesterol intracellular trafficking and Niemann-Pick type C disease
hsd-1 is an enhancer of ncr-1, suggesting that
steroidogenesis and NCR-mediated cholesterol transport are interdependent. The
following are three non-mutually exclusive models that interpret why the
hsd-1 mutant only displays dauer arrest when ncr-1 is
deleted. (1) When both NCR transporters are present, steroid signaling other
than hsd-1-mediated 4-signaling in XXX cells,
including 7, may be sufficient to prevent dauer arrest;
however, when NCR-1 is removed, 7-signaling, for example,
may be attenuated due to insufficient sterol substrates, and thus HSD-1
activity becomes crucial. (2) The broadly expressed NCR-1 may be involved in
sterol absorption, in addition to intracellular trafficking; thus, a lower
level of cholesterol absorption in the ncr-1 mutant makes HSD-1
activity necessary in preventing dauer arrest. In mice and flies, the
NPC1-related proteins NPC1L1 and dNPCb are known to be involved in dietary
sterol absorption in the intestine (Altmann
et al., 2004; Voght et al.,
2007). In worms, although the route for initial sterol absorption
remains unknown, the intestinal expression of NCR-1 would be consistent with a
possible role in sterol absorption. (3) Perhaps 3β-hydroxyl steroids can
prevent dauer arrest when produced in sufficient amounts, which may be
permitted by the presence of both NCR transporters. This model is supported by
findings showing that a 3β-hydroxyl steroid similar to dafachronic acids,
5-cholesten-3β-ol-(25S)-carboxylic acid, transactivates the DAF-12
receptor in vitro (Held et al.,
2006). These models are not mutually exclusive.
NPC1 is thought to mobilize cholesterol out of endo/lysosomes and
redistribute it to other cellular compartments through vesicular transport
(Ko et al., 2001). Mutations
in NPC1 are causal to 95% cases of the fatal neurodegenerative NPC disease
(Carstea et al., 1997).
NPC1-related transporters potentially are involved in the transport of other
sterols/lipids as well. However, little is known about substrate specificity
of these transporters in general. Our sterol-feeding studies suggest that
intermediates that are either beyond the point of the HSD-1 reaction, such as
4-cholesten-3-one, or the ones that do not need HSD-1 for further processing,
such as lathosterol, are independent of the NCR-1 and/or NCR-2 transporters
for their trafficking amongst cellular compartments/organelles. Our data
further suggest that the function of the NCR transporters is likely to be
limited to trafficking common dietary sterols, or those used for storage in
vivo. We demonstrated that, besides cholesterol, the NCR transporters are
indispensable when worms are fed with either ergosterol or β-sitosterol,
which may well be the main dietary sterols available to C. elegans in
the wild. To control the rate of hormone production accurately, it would be
appropriate if restrictive regulation were applied to intracellular
trafficking of common dietary sterols, because the intake of these sterols
would most probably fluctuate in quantity. We speculate that the NPC1 pathway
may serve as one such regulatory `hurdle'. This potential regulation also
appears to apply to 7-dehydrocholesterol, which is known to be a major sterol
component of worms (Chitwood,
1999) and thus may be a storage sterol.
The mechanisms resulting in NPC neurodegeneration remain unclear. Although
NPC1 was thought to have only general cell biological roles, recent studies in
animal NPC models suggest that steroidogenesis could be an important
functional output of NPC1-related transporters. Emerging evidence from
invertebrate models of NPC disease, including this study, suggests that NCR
transporters have a conserved role in supplying sterol substrates for the
biosynthesis of dauer-preventing hormones in worms
(Li et al., 2004;
Motola et al., 2006) and of
molting hormones in flies (Fluegel et al.,
2006; Huang et al.,
2005).
Currently, there are no effective therapeutic treatments for NPC disease.
In mice, it has been demonstrated that a combination of the neurosteroid
allopregnanolone and a synthetic oxysterol can promote neuronal survival and
mildly suppress the lethality of NPC1-deficient mice
(Griffin et al., 2004;
Langmade et al., 2006).
Neurosteroids have thus become a possible direction for development of drugs
to treat NPC disease (Mellon et al.,
2008). However, the therapeutic effects of the reported treatment
in the mouse model are quite mild. As defects in the NPC1 protein impact the
production of multiple known neurosteroids
(Mellon et al., 2008) and
potentially others awaiting identification, an effective treatment may rely on
the use of steroid intermediates that are trafficked independently of NPC1 and
can still be processed into a diverse range of hormones. Our results showing
effective bypassing of the NCR transporters in the worm suggests that a
similar steroid intermediate approach may also work in mammals.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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