From the Department of Embryology, Carnegie
Institution of Washington, Baltimore, Maryland 21210,
School
of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
19104, and ** Department of Neurology, The Johns Hopkins
University, Baltimore, Maryland 21205
Received for publication, January 24, 2003, and in revised form, February 21, 2003
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ABSTRACT |
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The 3-O-sulfotransferases (3OSTs)
catalyze the addition of sulfate groups at the 3-OH site of glucosamine
in heparan sulfate proteoglycans, which serve as critical mediators of
various biological functions. We demonstrate that the
3OST2 isoform is expressed at high levels in the rat
pineal specifically during the daylight hours. The dramatic diurnal
rhythm of 3OST2 is regulated by central clock-controlled
activities of the superior cervical ganglion, persists in constant
darkness, and is inducible by light at nighttime. Importantly,
3OST2 transcription is blocked by Heparan sulfate proteoglycans are implicated as co-receptors in
various processes including adhesion, proliferation, differentiation, and morphogenesis (1-3). The 3-O-sulfotransferases
(3OSTs)1 perform the last
enzymatic modification of a large number of steps required to generate
a broad array of mature heparan sulfate proteoglycans that have
distinct biological activities (4, 5). Four 3OST isoforms have been
identified (6), three of which are shown to transfer sulfate to
distinct disaccharides (7). For instance, the conversion of
non-anticoagulant heparan sulfate to anticoagulant heparan sulfate
requires 3OST1 (6, 7), which sulfates N-sulfated glucosamine
groups (5). On the other hand, 3OST3 is responsible for generating
sites for the binding and initiation of herpes simplex virus type 1 entry (8).
The pineal gland is a midline neuroendocrine structure of the brain
that synthesizes and secretes melatonin specifically at night to inform
the body about environmental light and dark information (9). This
rhythmic production of melatonin is dependent on the suprachiasmatic
nucleus clock, which relays information to the pineal via the superior
cervical ganglion (SCG) in the form of circadian norepinephrine
secretion at night. Light dramatically reduces hormone output at night
(9, 10).
To understand the molecular mechanisms underlying the dramatic
oscillation and tight regulation of diurnal melatonin formation, we
performed differential analysis of pineal day and night RNAs using
subtractive hybridization (11) and identified a number of night- and
day-specific transcripts. Previously we reported three of the
night-specific molecules, namely serotonin
N-acetyltransferase (NAT) (12), which is a crucial enzyme in
melatonin synthesis, pineal night-specific ATPase (13) (PINA), an
alternatively spliced product of Wilson disease gene ATP7B, and Patched
1 (14), which is a receptor for hedgehog signaling. One of the common
features of these genes is that their transcriptions are all
up-regulated by In this paper we report the cloning and characterization of a
day-specific 3OST2 expression in the pineal gland. Northern blot analysis reveals a dramatic diurnal rhythm of 3OST2
expression that persists in constant darkness and is abolished in
constant light. We show that the rhythm is dependent on the superior
cervical ganglion via adrenergic inputs to the pineal and is absent
during early postnatal development. The night expression of
3OST2 is inducible by light, whereas the day expression is
suppressed by Animals--
All animal protocols were conducted in accordance
with the institutional animal care and use committee. Adult male
Sprague-Dawley rats purchased from Harlan Sprague-Dawley were housed in
a temperature-controlled room under 14:10 light/dark conditions with
lights-off at 1 a.m. for more than 1 week before experiments.
Cloning of Rat 3SOT2 and Northern Blot Analysis--
The
subtractive hybridization of pineal day and night mRNAs has been
previously described (Borjigin et al. (12). Individual clones in the pineal day-specific subtracted cDNA library were used
for PCR Southern and subsequently Northern blot analysis (11). The
clone PL22 was specifically expressed during the daytime. GenBankTM analysis of PL22 revealed that it corresponds to
the 3'-untranslated region (nucleotide 4386-4805,
GenBankTM accession number AF105374) of human
3OST2. PL22 was then used to isolate full-length cDNA
clones from a day pineal cDNA library using the GeneTrapper
technique (Invitrogen). Sequence analysis of the longest clone PL22.16
shows that it is the rat ortholog (r3OST2) of the human 3OST2 with 96%
amino acid identity. Northern blots were hybridized with labeled
full-length r3OST2 (GenBankTM accession number
AY240873), NAT, and GAPDH probes as described (12).
SCG Removal--
A group of rats were sympathectomized by
removal of both left and right SCG by surgical approach from between
the sternohyoideus omohyoid muscles.
Surgery--
Animals were deeply anesthetized with a combination
of ketamine (10 mg/ml, 0.5 ml/100-g weight, intraperitoneal) and
xylazine (2 mg/ml, 0.5 ml/100 g-weight, intraperitoneal). The shaved
head was positioned in a stereotaxic frame, and a 2-cm midline incision was made 10 mm posterior to the transverse suture extending to the
occipital ridge. A circular opening (6.8 mm in diameter), centered
midline 1.5 mm posterior to the confluence of the superior sagittal and
transverse sinuses, was created using a dental burr drill equipped with
a shank diamond wheel point (Dremel). The dura was exposed after
grinding away the top bone and was removed by first making a cross-cut
with a scalpel blade and then peeling away with fine forceps. The pia
matter that covers the surface of the pineal was then removed carefully
to visualize the pineal, which is connected to the confluence of the
sinuses via the pineal vein. Three stainless steel screws were
placed surrounding the opening on the skull to serve as anchors. The
tip of the guide cannula (CMA/microdialysis) was then positioned
immediately over the exposed pineal before closing the skull opening
with dental cement. The guide cannula with the stylet was mounted on
the skull with dental cement. After surgery, animals were housed in
individual cages in light-controlled rooms described above and were
allowed to recover for 24 h.
Microdialysis--
Pineal microdialysis was carried out as
follows. Immediately before sampling, the rat was anesthetized with
halothane briefly, the stylet (or dummy probe) was replaced with a
microdialysis probe (CMA12, 20-kDa cut-off, 3- or 4-mm length)
(CMA/microdialysis) and fixed with plastic glue. The dialysis
probe was continuously perfused via microbore PEEK tubing (inner
diameter, 0.12 mm; outer diameter, 0.65 mm) at a flow rate of 2 µl/min with artificial cerebral spinal fluid (Harvard) that was
pumped by a CMA/102 microdialysis pump. Samples were collected
every 10 min via the PEEK tube into the 20-µl loop of an automatic
injector (BAS, West Lafayette, IN), which is on-line with the HPLC
system. The sample loop was set to be retained in the load (or collect)
position during the 10 min and was automatically switched to the
injection position very briefly, after which the cycle was repeated.
The rats were linked to the apparatus for dialysis through a quartz
dual channel swivel (Harvard) to prevent the tubing from entanglement.
HPLC--
The analytical conditions for the detection of
melatonin are based on Drijfhout et al. (15) with some minor
modifications. A Shimadzu (Columbia, MD) pump was used in conjunction
with Shimadzu fluorescence detector (excitation, 280 nm; emission, 345 nm). Samples were injected into the system through a Valco injection valve with BAS pollen 8 controller and subsequently separated on a
reversed phase C18 column (250 × 4.6 mm, Supelco) set at a
constant temperature of 30 °C using Shimadzu column
heater controlled by the Shimadzu system controller. The mobile phase
consisted of a mixture of 10 mM sodium acetate adjusted to
a pH of 4.5 with concentrated acetic acid, 0.01 mM
Na2-EDTA, 500 mg/liter heptane-sulfonic acid, and 20%
(v/v) acetonitrile. The flow rate of the HPLC pump was set at 2 ml/min
throughout the experiment. The chromatogram was set to run for 9 min,
with the remaining 1 min for the system to be ready for the next
trigger signal, which was provided by the BAS autoinjector controller.
Standard solutions were used to calibrate the system. The automated
control of the HPLC system and the programming of the flow rate as well
as handling and storage of the chromatograms was done with an external
computer with the Shimadzu Class-vp 5.03 chromatography software.
In Situ Hybridization Analysis--
3OST2 (clone
PL22.7, 3'-untranslated region of rat 3OST2), rat NAT, and rat
tryptophan hydroxylase (the rate-limiting enzyme of serotonin
synthesis) full-length cDNA probes were used for in situ
studies as previously described (13). At the end of the microdialysis
analysis of individual rats, rats were taken off the microdialysis
devices and rapidly sacrificed. When the pineals were placed in
Tissue-Tek (Sakura) compound for later sectioning, care was
taken to keep the microdialysis membranes in the pineal for ease of orientation.
Adenoviral Vector-mediated 3OST2 Expression in Living
Pineals--
Recombinant adenovirus expressing full-length rat 3OST2
was generated by using the AdEasy system (16). The shuttle plasmid pAdTrack-CMV-3OST2 was transfected into HEK 293 cells with AdEasy-1 vector using LipofectAMINE (Invitrogen). The recombinant adenovirus AdCMV-3OST2:GFP, generated by homologous recombination in human embryonic kidney cells, was isolated, amplified, and titrated as
described (16).
The surgical procedures used for exposing the pineals before injection
of recombinant adenovirus were identical to those used for
microdialysis probe implantation (see above). Immediately after the
pineals were exposed, AdCMV-3OST2:GFP virus was injected into the
pineal using a thin pulled glass pipette and nanojector II (Drummond
Scientific Co.) with the aid of a stereotaxic instrument. A total of 1 to 2 µl (1011 plaque-forming units/ml) of the
AdCMV-3SOT2:GFP virus were injected into each pineal. In cases
where the injected animals needed to be analyzed by microdialysis,
dummy probes were inserted into the pineal (as described above) right
after the injection was done.
3OST2 Is Diurnally Expressed in the Pineal--
In a first round
of screening of the subtracted day-specific pineal cDNA library, we
found that 3OST2 expression is day-specific with no
expression during the late night. Tissue distribution studies suggest
that 3OST2 expression is the highest in the day pineal than
in any other tissues examined (Fig.
1A), and no diurnal rhythm of
3OST2 expression was found in other tissues (data not shown). In the pineal significant expression was first detectable during the first hour after the lights-on and remained high throughout the daylight period and initial 2-3 h of the dark period (Fig. 1B). Transcript levels fell coincident with NAT
activation and were dramatically down-regulated to undetectable levels
during peak period of NAT gene expression (middle
panel) and melatonin production (data not shown). The
daytime-specific expression of 3OST2 was developmentally
regulated (Fig. 1C) and first appeared between postnatal
days 5 and 10, coinciding with the developmental onset of melatonin
daily rhythms (17) and lagging behind the appearance of the
NAT RNA rhythm (postnatal day 2 or earlier (14)).
3OST2 Expression Is Controlled by a Central Clock via the SCG and
Is Light-inducible at Night--
Temporal expression of
3OST2 was examined in pineals of rats maintained in constant
light or dark conditions. The circadian expression of 3OST2
was maintained in constant dark, suggesting that it is under a central
clock control (Fig. 2A).
Continuous light exposure, which abolishes melatonin production and
expression of all pineal night-specific genes including NAT,
PINA, and Patched 1, up-regulates the
3OST2 transcription in the rat pineal (Fig. 2B).
The time course of 3OST2 light induction (Fig.
2C) indicates that the 3OST2 expression is
detectable within 2 h of light exposure and peaks at 8 h. The
induction time course of 3OST2 expression corresponds with
the inactivation of NAT transcription by light (middle
panel in Fig. 2C). Bilateral ablation of the SCG, which suppresses NAT, restores 3OST2 expression at
night (Fig. 2D).
Night Pineal Expression of 3OST2 Is Compatible with NAT Expression,
Melatonin Production, and Light Responsiveness of Melatonin
Formation--
The expression patterns and regulatory mechanisms of
3OST2 are the exact opposite of those of NAT and melatonin
production, suggesting that 3OST2 expression might be
incompatible with melatonin synthetic pathways. To test this, rat
pineals injected with AdCMV-3OST2:GFP were isolated 4 days after the
injection during dark hours and sectioned for in situ
analysis. As shown in Fig. 4A,
NAT and TPH gene expression was not affected in
areas expressing 3OST2. The effect of 3OST2 gene
expression in the night pineals on melatonin secretion was monitored
continuously in living rats for 5 days with or without light
stimulation at night. At the end of the 5th day, rats were sacrificed
at night, and pineals were sectioned and analyzed for expression of the
recombinant 3OST2-expressing adenovirus using fluorescent
microscopy and in situ hybridization (Fig. 4B).
Melatonin secretion patterns of AdCMV-3OST2:GFP-injected rats
(upper panel) are indistinguishable with that of animals injected with GFP-expressing virus (AdCMV-GFP, lower panel).
A light pulse of 10 min, a known disrupter of melatonin formation at
night, resulted in precipitous decline of melatonin secretion, which
recovered shortly after, in both 3OST2- and GFP-expressing rat pineals
(Fig. 4B). These in vivo studies indicate that
3OST2 does not antagonize melatonin synthesis.
Molecular mechanisms of circadian rhythm generation have been the
subject of intense investigation. In this study we characterized the
expression of 3OST2, a pineal daytime-specific transcript identified in the same subtractive screening that permitted isolation of NAT, PINA, and Patched 1 as
night-specific genes. In the pineal, Melatonin is a circadian hormone whose daily cycle is under strict
clock control. Cyclic AMP signaling resulted from activation of pineal
Analysis of mammalian gene function has traditionally been accomplished
through germ line manipulation of mice via either gene knockout or
transgenesis. Although these types of analysis typically provide useful
information, they are not ideal for pineal studies due to the small
size of the pineal gland and the fact that most mouse strains do not
produce detectable levels of melatonin. Transgenic rat studies,
although valuable for analyzing a couple of genes in detail, appear to
be time-consuming and costly. In this study, we demonstrated the
successful gene transfer into the living pineals by direct delivery of
recombinant adenovirus that expresses genes of interests via surgery.
Using a novel surgical approach, we were able to target viral vectors
to the rat pineals with more than 90% accuracy. To delineate
functional consequences of expressed genes on melatonin production
in vivo, we integrated the pineal adenoviral gene expression
technique with in vivo microdialysis approach, which
permitted us to probe the functional relevance of 3OST2 to the
melatonin output in living animals in real time. Final validation of
accurate and high level infection is possible as shown by in
situ hybridization at the conclusion of in vivo monitoring. Traditionally, pineal gene expression analysis and functional studies are performed in separate cohorts of animals. In
this paper we integrated molecular analysis of pineal gene expression
with physiological measurement of in vivo pineal melatonin output in the same individuals, which enabled us to obtain a
comprehensive molecular understanding of pineal 3OST2
regulation. This novel approach can be particularly useful in
situations where gene expression patterns need to be correlated with
physiology in the same animals. We believe that these newly developed
approaches should accelerate further understanding of functions of
pineal diurnally expressed gene as well as mechanisms of their in
vivo regulation.
3OST2 is the first day-specific transcript identified in the
pineal gland. Because all the night-specific genes identified thus far
are up-regulated by Recently, accumulating insights from genetic studies in
Drosophila and mice have made it apparent that heparan
sulfate proteoglycans formed by enzymes including sulfotransferases are
critical in the interactions between specific extracellular ligands and
their signal-transducing receptors (3). 3OST2 has been shown to sulfate precursor heparan sulfate at selective sites and, therefore, may generate glycosaminoglycans with targeting specificities different from
that of 3OST1 or 3OST3 (7). It remains to be determined what
receptor-ligand interactions are mediated by the enzymatic activities
of 3OST2 and how it contributes to the pineal circadian physiology. Our
demonstration that in vivo 3OST2 overexpression fails to
affect melatonin production argues strongly for the existence of novel
diurnal functions of the pineal.
-adrenergic agonists that activate the pineal melatonin formation and is induced by
-adrenergic antagonists, which block melatonin production in vivo. Because of the inverse expression and regulation patterns of 3OST2 with serotonin N-acetyltransferase,
the enzyme controlling the melatonin rhythm in the pineal, we tested
the effects of forced expression of 3OST2 in the night pineals on
N-acetyltransferase gene expression and melatonin
production and found that, surprisingly, 3OST2 expression
at night fails to interfere with melatonin synthesis. These data
suggest 3OST2 may serve a unique function in the pineal that may be
independent of melatonin formation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-adrenergic signaling and suppressed by light, which
terminates adrenergic inputs to the pineal.
-adrenergic signaling that activates melatonin
formation. Finally we demonstrate that forced night expression of
3OST2 does not prevent NAT gene expression nor
does it affect melatonin secretion and its suppression by light
in vivo.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
3OST2 expression in the pineal
displays marked circadian rhythm that is developmentally
regulated. A, tissue distribution of 3OST2
gene expression. Each lane is loaded with 5 µg of total rat tissue
RNA, except for the last two lanes, which were loaded with
10 µg of pineal day or night RNAs. The blot was first hybridized with
a full-length 3OST2 probe, which was subsequently hybridized
with GAPDH probe. B, temporal expression profiles
of 3OST2 in the pineal. Five µg of pineal total RNA taken
at the indicated times were loaded in each lane. Identical blots were
hybridized with 3OST2 (upper panel),
NAT (middle panel), and GAPDH
(lower panel) probes. C, 3OST2 rhythm
is developmentally regulated. Day (2-4 p.m., D) and night
(7-8 a.m., N) pineal RNA was taken from animals at birth
(P0), on postnatal days 2 (P2), 5 (P5), 10 (P10), 20 (P20), and 30 (P30). Identical blots were hybridized with 3OST2
and GAPDH.
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Fig. 2.
3OST2 rhythm is controlled by a
clock, light, and superior cervical ganglion innervation.
A, diurnal rhythm of 3OST2 expression persists in
constant darkness. Rats were placed in constant darkness for 48 h
before tissue harvesting. Five µg of total pineal RNA taken from
animals sacrificed at the indicated times were loaded in each lane.
Identical blots were probed with 3OST2 and GAPDH.
B, constant light elevates 3OST2 gene expression
during the subjective night. Rats were placed in constant light
conditions for 48 h before RNA was collected. Each lane was loaded
with 5 µg of total pineal RNA. The sample collected at 3 a.m.
was lost during the RNA preparation. Blots were hybridized with the
probes indicated. C, light stimulates 3OST2
transcription. Rats were stimulated with light for hours indicated
before harvesting of pineal RNA at 9 a.m. Three identical blots
were hybridized with probes indicated. D, 3OST2
night expression was restored by removal of SCG. Bilateral superior
cervical ganglia (SCGX) of adult rats were removed, and
pineal RNAs isolated at 3 p.m. (D) or 7 a.m.
(N) were analyzed with indicated probes after
blotting.
-Adrenergic Signaling Blocks 3OST2 Expression--
We next
correlated variations in 3OST2 gene expression with
melatonin production in vivo in the presence of
-adrenergic agonist and antagonist. We first performed microdialysis
on individual rats until their melatonin secretion patterns stabilized,
which normally took 24 h after the beginning of on-line sampling
(18). Rats were then given isoproterenol (ISO) or phosphate-buffered saline through either intraperitoneal injection or via the
microdialysis probe. Rats injected with intraperitoneal
phosphate-buffered saline showed no effect on melatonin formation or
daytime expression of 3OST2, NAT, or
TPH (tryptophan hydroxylase (a gene that does not diurnally
vary) during the day (Fig.
3B). In contrast,
intraperitoneal ISO injection abolished daytime 3OST2
expression, produced large increases of melatonin secretion, and
increased NAT expression through the entire pineal (Fig.
3A). To demonstrate that ISO acts directly on pineal, we
delivered the drug into the pineal through the microdialysis probe.
Direct infusion of ISO into the pineal gland elevated melatonin
production (Fig. 3C, upper panel) and blocked
3OST2 expression in areas immediately around the
microdialysis membrane. This correlated spatially with areas of
NAT transcriptional activation (Fig. 3C,
lower panels). Propranolol (PROP), a potent
-adrenergic receptor antagonist infused directly into the pineal (Fig. 3D), activated 3OST2, suppressed NAT gene
expression (lower panels), and completely abolished
melatonin production (upper panel) in the same pineal
gland.
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Fig. 3.
Pineal 3OST2 expression is
suppressed by adrenergic signaling. A and B,
systemic injection of isoproterenol (ISO) suppresses pineal
3OST2 expression. In vivo pineal microdialysis
was performed with individual rats for 3 days. On day 3 at 2 p.m.,
ISO (1 mg/kg) (A) or phosphate-buffered saline
(B) was injected intraperitoneally, the rats were sacrificed
3.5 h later, and the pineals were collected for in situ
analysis with indicated ribo-probes. In the lower panels,
asterisks indicate the areas of microdialysis probe
penetration, whereas arrows indicate positions of the
microdialysis probe membranes. C, ISO acts directly on the
pineal to suppress 3OST2 expression. ISO (1 µM) was delivered directly into the pineals through the
microdialysis probes for 4 h (2 p.m. to 6 p.m.). Rats were
taken off the microdialysis apparatus immediately after the
microdialysis, and pineals were isolated immediately, serially
sectioned, and analyzed using probes indicated. D,
3OST2 expression is activated by a -adrenergic blocker.
Melatonin secretion was monitored in individual rats for three cycles.
Cycles 2 (solid circles) and 3 (open circles) are
shown (upper panel). During the last circadian cycle, the
pineals were infused with propranolol (PROP) for 8 h
from 11 p.m.. Rats were sacrificed at 7 a.m.
(arrow), and pineals sectioned and analyzed for
3OST2, NAT, and TPH gene expression
(lower panels).
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Fig. 4.
Night expression of 3OST2 in
the pineal does not antagonize NAT
transcription, melatonin formation, or melatonin light
responsiveness. A, NAT expression is normal
in rats expressing 3OST2 at night. One to two µl of
AdCMV-3OST2:GFP virus was injected directly into the living rat pineals
using the surgical technique described (see "Experimental
Procedures"). Four days after the procedure, the injected pineals
were harvested at night (7 a.m.) for in situ analysis using
3OST2, NAT, and TPH ribo-probes. An
adjacent section was also analyzed using fluorescent microscopy to
demonstrate co-expressed GFP. B, 3OST2 expression
does not disrupt melatonin production or its response to light
stimulation at night. Rats injected with AdCMV-3OST2:GFP (upper
panel) or AdCMV-GFP (lower panel) viruses were
monitored for their melatonin output with or without a light pulse. A
light pulse of 10 min was given to rats at 4 a.m.
(arrows). Rats were sacrificed during the following night at
7 a.m., and pineals were analyzed for 3OST2 RNA
(upper panel inset) or GFP (lower panel inset)
expression.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-adrenergic signaling
suppresses and light activates 3OST2 gene expression.
Although 3OST2 message displays an opposite mode of regulation with all known night-specific pineal transcripts, 3OST2 night expression does not affect NAT gene expression and
melatonin production.
-adrenergic receptor has been shown to be the primary local force
behind the dynamic oscillation of melatonin formation. Because cAMP
signaling in cell types other than the pineolocytes does not activate
NAT and melatonin
production,2 it is evident
that other signaling molecules in the pineal gland are important to the
generation of melatonin rhythm. Because melatonin formation itself is
dynamically regulated at transcriptional level, we postulated that
other factors that are crucial for melatonin rhythm also are controlled
by rhythmic circadian gene activity. We therefore performed large scale
screening of our subtracted pineal day and night cDNA library (12)
and identified an additional few dozen night-specific and several
day-specific clones.2 Here we demonstrate the utility of
using an in vivo system to systematically analyze the
function of one of these genes, 3OST2, a day-specific
transcript in its natural contests.
-adrenergic signaling, we hypothesized that the
day-specific 3OST2 regulation could be achieved by
-adrenergic receptor-controlled repression. We demonstrated that
this prediction was indeed correct and that 3OST2 is
activated by light stimulation and suppressed by
-adrenergic
signaling. The identification of diurnally regulated genes whose
expression and regulation complement those of pineal night-specific
genes such as NAT, PINA, and patched 1 will undoubtedly facilitate understanding of the molecular mechanisms that govern the pineal circadian gene expression and melatonin production.
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FOOTNOTES |
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* This work was supported by a National Institutes of Health grant (to J. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of the John Merck Scholars Award. To whom correspondence should be addressed: Dept. of Embryology, Carnegie Institution of Washington, 115 West University Pkwy., Baltimore, MD 21210. Tel.: 410-554-1231; Fax: 410-243-6311; E-mail: borjigin@ciwemb.edu.
¶ Current address: Dept. of Surgery, Shanghai Pudong New Area People's Hospital, Shanghai 201200, People's Republic of China.
Published, JBC Papers in Press, February 22, 2003, DOI 10.1074/jbc.M300828200
2 J. Borjigin, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: 3OST, 3-O-sulfotransferase; SCG, superior cervical ganglion; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPLC, high performance liquid chromatography; GFP, green fluorescent protein; ISO, isoproterenol; TPH, tryptophan hydroxylase gene.
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