Molecular rhythms in the pineal gland

648

Molecular Xiaodong Recent

rhythms in the pineal gland Li*, Jimo Borjigin? and Solomon H Snyderl

findings

have clarified

night- and pineal-specific N-acetyltransferase, formation.

the rate-limiting

Norepinephrine,

acting

CAMP at night, stimulates protein,

the mechanisms

transcription

enzyme

the CAMP response

CAMP early repressor,

N-acetyltransferase expression

transcription.

homeobox

protein,

This regulatory

The tissue-specific

selective

enzymes,

O-methyl

transferase,

of

gene in part,

factor, cone-rod

binds to a pineal regulatory is present

binding

of N-acetyltransferase

transcription

which

element

and

the major inhibitor

in promoters

element.

of pineal-

such as N-acetyltransferase,

hydroxyindole-

and pineal night-specific

*i;Neuroscience, ipsychiatry, “e-mail: ie-mail:

University

School

‘Pharmacology

725 N Wolfe

of Medicine,

and Molecular

Street,

Baltimore,

Departments Sciences,

Maryland

21205,

of

and USA

[email protected] [email protected]

Current

address:

Washington,

Department

of Embryology,

115 West University

Parkway,

Carnegie Baltimore,

Institution Maryland,

of USA;

e-mail: [email protected] Current

Opinion

in Neurobiology

1998,8:648-651

http://biomednet.com/elecref/0959438800800648 s) Current

Biology

Ltd ISSN

then

ascend

0959-4388

Abbreviations CRE CAMP response element CRE-binding protein CREB CREM CRE modulator HIOMT hydroxyindole-0-methyltransferase ICER inducible CAMP early repressor NAT N-acetyltransferase pineal night-specific ATPase PINA PIRE pineal regulatory element suprachiasmatic nucleus SCN

Introduction ‘I‘he pineal gland is the rhythmic organ,pnre~fe~/~t~~~, of the body, with lOO-fold variations between day and night in the expression of certain genes. In some species, such as chickens, the pineal gland itself is a biological clock, as rhythms in melatonin formation persist in isolated organ cultures. In mammals, however, the pineal gland does not seem to have an intrinsic clock-like apparatus; instead, the master clock of the body is located in the suprachiasmatic nucleus (SCN) of the hypothalamus (see [l]). The circadian clock of the SCN is entrained to the light/dark cycle via the retino-hypothalamic pathway. Information about light reaches the pineal gland via a circuitous, multisynaptic route, proceeding from the SCN through the paraventricular nucleus of the hypothalamus, descending to the intermedial lateral cell column of the spinal cord, out to the from which postganglionic superior cervical ganglion,

to the pineal

gland.

‘l’hough

the pineal

to be located in the center of the brain, it has direct neuronal connections with the brain

[Z]; however, it can be regarded as an end organ of the s);mpathetic nervous system. The photoreceptors responsible for entraining the mammalian clock may not be the same cells that mediate vision, as loss of visual photoreceptors in the retinally degenerate mouse does not affect its circadian response to light [3]. Likewise. suppression of plasma melatonin levels by light exposure is retained in some blind individuals who have normal sleep patterns [-I]. Interestinglp in blind insomniacs. light exposure does not affect plasma melatonin, though light may reach the pineal gland

ATPase.

Addresses The Johns Hopkins

fibers

gland appears rather limited

element

within the pineal gland and retina derives,

from a pineaketina-specific

the

in melatonin

via P-adrenoceptors

which turns on the transcription

and inducible

regulating

of serotonin

through

a nonretinal

pathway

in humans

[.5].

The major biological function of the pineal gland is to entrain numerous biological rhythms to the light/dark cycle of the environment by generating a melatonin rhythm. This contrasts with the role of the pineal gland as a third eye in lower vertebrates, in which pinealoc);tes themselves contain well-defined, morphologically appropriate photoreceptors that respond to light very much like the photoreceptors of the retina. Neonatal rodents appear to retain photoreceptive properties in their pineal gland, as recent studies show that the neonatal rat pineal gland is enriched in numerous photoreceptive proteins, especially cone elements such as rhe blue visual pigment [6’]. hloreover, exposure regulates pineal serotonin eyes have been

in the neonatal rat, light levels in animals \\,hose

removed.

In this review. we discuss the molecular mechanism lying the melatonin rhythm in the pineal specifically, the role of transcriptional regulation.

undergland,

Melatonin The pineal gland influences the rest of the body through its hormone melatonin, which was finally identified after nearly half a century of experimentation. In 1917, pineal extrwts \vere shown to lighten the skins of frogs. After much effort, Aaron Lerner identified the active ingredient in 1959 as .‘V-acet);l-S-methoxytryptaminc, a deril.ati1.e of serotonin, which he named melatonin. Julius Axelrod and colleagues demonstrated that melatonin is formed b) A\r-acetvlation of serotonin follo\ved by 5-O-methylation via an enzyme designated hydroxyindole-O-methyltransferase (HIORIT). Subsequently, a dramatic rhythm of serotonin levels in the pineal gland was discovered. with peak levels at noon, about lo-times nocturnal values. Klein and associates (see [7]) then demonstrated that serotonin depletion at night is caused by a lOO-fold nocturnal increase in the activity of the serotonin S-acetyltransferdse (NAT). Both NAT and HIOhlT activities are limited to the pineal gland, with trace amounts in the retina. NAT is the

Molecular rhythms in the pineal gland Li, Borjigin

649

and Snyder

Figure 1 A model for the temporally

and spatially CREB

specific expression of NAT and PINA. At night, the clock-driven adrenergic signals activate CAMP-dependent kinase (PKA), which phosphorylates CREB. Activated

0

/z-Y 00000000000

CREB turns on the transcription of pineal night genes, such as NATand P//VA. In

adrenergic

parallel, it activates

transcription

of the

negative

ICER, which

binds to the

regulator,

+

Clock-driven signals

same CRE sites as CREB to block transcription, thereby regulating the amplitude of NAT, PINA, and its own transcription. The tissue-specific homeobox expression

transcription

factor

pCREB

cone-rod

(CRX) is required for the of NAT and PINA. Conceivably,

other homeobox proteins act as CRX partners (indicated by a ?). The combined actions of night-specific CREB and tissue-specific CRX and its hypothesized

partner

PIRE

bind to PIRE in

the promoter region of pineal/retina-specific genes, including /VAT, PINA, and HlOlllT

PIRE

CRE

NAT//=//VA

(not

shown). CRX activities determine the temporal and spatial expression of NAT and PINA.

Current Op,mon ,n Neurobiology

rate-limiting enzyme in melatonin synthesis, with high activity observed only at night. nlelatonin is secreted by the pineal gland into the circulation so that the rhythmic changes of NAT in the pincal gland parallel rhythms of

melatonin levels in the pineal gland and in blood. The rhythm of NAT activity and melatonin formation is driven by the adrenergic innerv,ation of the pineal gland, where norepinephrine activates 1%adrenoceptors to augment cAhlP levels. Remo\,al of the superior ganglia blocks the regulation of NAT by light.

cervical

XIajor advances in understanding the regulation of melatonin formation have come from the recent molecular cloning of the cDNA for NAT by two independent groups ([8,0]; see [lo] for a review). \Ve [8] used a subtrarctive hybridization technique based on the polymerase chain reaction to isolate rat pineal gland messages that are differentially expressed during the day or night, enabling us to identify and clone NAT [8]. Independently, Klein and co-vvorkers [C,] employed expression cloning techniques to identify NA’I‘ in sheep pineal glands. In rat, the time course for NA’l’ mRNh expression and catalytic activity are essentially the same, with a sudden increase in expression between midnight and 0230 (three to five hours after the lights go off), and an abrupt decline from peak levels at Oh:00 to undetectable values at 08:OO (one hour after the lights are turned on) [8]. Interestingly, in sheep, the NAT mRNA level at night is only twice as high as the daytime value [C,], whereas in the rat, nocturnal peaks are about 100 times the daytime trough [8]. As NAT catalytic activity in the sheep pineal gland varies greatly between the day and night, post-transcriptional events appear to be important.

Temporal regulation cAMP signaling

of NAT transcription

by

In rodents, the rhythmic NAT activity is regulated primarily at the transcriptional level. Elevated nocturnal cAhlP levels activate CAMP-dependent protein kinase (PKA), the catalytic subunits of which then translocate into the nuclei and phosphorylate the cAhlP response element binding protein (CREB), which, in turn, transactivates the NAT gene. A family of leucine-zipper transcription factors, including both activators and repressors, act through the cAhlP response element (CRE). The CAMP response element modulator (CRERI) gene is related to CREB but encodes a family of alternatively spliced isoforms, among which is the inducible CAMP early repressor (ICER) [ll]. The ICER protein is small; it contains only a DNA-binding domain, comprising a leucine zipper and basic region, and functions as a dominant repressor of cAhlP-induced transcription. Like NAT, ICER expression is activated by CREB [ 121, and ICER mRNA manifests dramatic diurnal variations in the pineal gland, with a peak during the second part of the night [ll]. Surprisingly, levels exhibit no marked day/night rhythm.

ICER

protein

ICER binds to the same CRE sites in the NAT promoter region as CREB. The physiologic relevance of this binding was established in mice by knocking out the CREM gene [13]. At virtually all time points examined throughout the day and night, NAT expression was substantially higher in CREhl knockouts than in controls, with the night-time induction beginning earlier, reaching a higher peak of expression and persisting longer than in wild-type controls. By contrast, the kinetics and magnitude of expression for a Fos-related antigen 2 (Fra-2) [14], which undergoes

650

Molecular clocks

marked diurnal \.ariations, are unaltered in mutant animals. The selective augmentation of NAT expression in CREM knockouts indicates that ICER normally inhibits NAT expression. /N eltf~ studies utilizing a reporter construct attached regulates

to the NAT promoter showed NAT expression [ 131.

that ICER

down-

These results fit with a mode1 for diurnal regulation of NAT in which increased phosphorylation of CREB at night turns on NAT transcription, with a balance between CREB phosphorylation and dephosphorylation being a critical regulator [15] (Figure 1). Thus far, regulatory mechanisms and enzymes involved in CREB dephosphorylation have not yet been clarified. The sustained ICER protein levels in the day and night may modulate the rate and magnitude of melatonin induction throughout the 24 h cycle. By binding to the CRE element in the NAT promoter. ICER ma)- modulate the threshold for cAhIP to stimulate melatonin formation. The threshold would be fairly stable under typical lighting cycles, changing ICER protein levels

Spatial regulation

under [lb].

extreme

cycles

of NAT transcription

that

displays three-fold changes. much less than the N.4’1’ rhythm. CRX protein exhibits no marked diurnal \.ariation and appears to act synergistically with CREB (X Li. unpublished data).

alter

by CRX

‘hlaster’ transcription factors account for selective expression of tissue-specific proteins. We sought pineal-specific transcription factors by looking for unique pineal regulatory elements in the promoter regions of pineal-specific proteins [17**]. The two best-characterized pineal-specific proteins are NAT and HIORIT. In addition, in the nightsubtracted pineal cDNA library employed to discover NAT, we identified an alternatively spliced form of Atp7b (J Borjigin, unpublished data), the human homologue of which is disrupted in Wilson’s disease, a disorder of copper metabolism [18]. We named this novel protein PINA for pineal night-specific ATPase [17”]. PINA is uniquely expressed in the pineal gland and displays dramatic diurnal variations essentially identical to those of NAT (J Borjigin, unpublished data), suggesting a common mechanism of transcription regulation for NAT and PINA. LJsing electrophoresis mobility shift assay (ELlSA), we identified in the PINA promoter region a consensus TAATC/T, that is recognized by a tissue-specific nuclear factor in the pineal gland and retina, but not in a wide range of other tissues [17”]. This pineal regulatory element (PIRE) is present in the promoter regions of NAT and HIOhIT, as well as PINA, with multiple copies in NAT and PINA promoters. sequence,

The finding that the pineal gland and retina utilize the transcription fidctor fits with the same ‘tissue-specific’ expression of NAT, PINA and HIOhlT in both the retina and the pineal gland. Furthermore, a number of presumed retina-specific proteins are expressed in the pineal gland, including rhodopsin, transducin, arrestin, visual pigments. rod cyclic nucleotidc channels, interphotoreceptor retinoid binding protein, and rod and cone phosphodiesterases [f)‘]. The multiple CRX-binding sites in the promoter regions of several pineal/retina-specific genes retlcct a general role for CRX as a ‘master’ transcription factor (Figure 1). Homeobox genes have been studied extensively over the past decade, and they have a well-established crucial role in differentiation, cell fate determination, pattern formamorphogenesis. CRX is first tion and the photoreceptor-specific transcription factor to be identified as playing a crucial role in the differentiation and maintenance of photoreceptor cells. \lutations in (:RX appear to account for the arltosomal-donlinant disease \\hich is associated \\.ith cone-rod dystrophy (CORD), major loss of vision [20”,22’]. RI’in d ne\s:: 111:\ome patients with Leber congenital amaurosis (LCA) is also associated with recessive mutations in CRX [23’]. Given the rcsemand photorecepror cells, blance bet\veen pinealocytes CRX probably also regulates pincalocyte differentiation. Homeobox proteins bind DNA i/l ~it/~ with rather lo\\ affinity and specificity. Ho\vel.er, homeobox proteins can form heterodimers with much greater DNA-binding specificity, as illustrated by yeast hl.AT gent products [24]. Conceivably, other homeobox proteins could act as CRX partners in the pineal gland, contributing to tissuespecific

regulation.

Conclusions The conservation of diurnal variation in melatonin le\.els throughout vertebrate evolution indicates that it is of a fundamental importance. Interestingly, both diurnal and nocturnal animals ha\z increased melatonin levels at night, suggesting a divergence in dowiistream signaling pathways. Transcriptional regulation is the primary mechanism used by rodents to achieve rhythmic NAT activit); whereas post-transcriptional mechanisms are important in some other

Other researchers, seeking retino-specific transcription factors, have identified CRX, a member of the OTX family of the homeobox class of transcription factors, which is selectively expressed in the retina and the pineal gland [19”-Zl”]. We have demonstrated that CRX accounts for the PIRE-binding activity of pineal and retina extracts. CRX mRNA displays a diurnal variation in the pineal gland, with its peak preceding that of NAT by 1-2 h. From peak to trough, the CRX rhythm

gene

species.

The temporally and spatially specific expression patterns of pineal proteins may reflect combined actions of CREB and CRX (Figure 1). Phosphorylation of CREB by PK;i may serve as the switch of transcription that sets the time of NAT transcription. ICER appears to be the major downregulator of NAT transcription. CRX. on the other hand, accounts for the pineal gland and retinal selectivity for the expression of NAT, HIOhlT and PINA.

Molecular

Acknowledgements

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1,

in the pineal gland Li, Borjigin and Snyder

17. ..

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..

20. *

Freund CL, Gregory-Evans CY, Furukawa T, Papaioannou M, Looser J, Ploder L, Bellingham J, Ng D, Herbrick JA, Duncan A et a/.: Cone-rod dvstroohv due to mutations in a novel ohotoreceotor-soecific horneobox gene (CRX) essential for maintenance of the photoreceptor. Cell 1997, 91:543-553. See annotation [2 1“1

l

21. l0

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Swain PK, Chen S, Wang QL, Affatigato LM, Coats CL, Brady KD, Fishman GA, Jacobson SG, Swaroop A, Stone E et a/.: Mutations in the cone-rod homeobox gene are associated with the conerod dystrophy photoreceptor degeneration. Neuron 1997, 19:1329-I 336. See annotation (23’1. 23. .

Freund CL, Wang QL, Chen S, Muskat BL, Wiles CD, Sheffield VC, Jacobson SG, Mclnnes RR, Zack DJ, Stone EM: De novo mutations in the CRX homeobox gene associated with Leber congenital amaurosis. Nat Genet 1998, 18:31 l-31 2. Both cone-rod dystrophy (CORD) [20”,22’] and Leber congenital amaurosis (LCA) [23-l were mapped to mutations in CRX. The autosomal-dominant form of CORD has been assigned to four loci, among which CRX is so far the only identified gene related to this disease. LCA is a clinically heterogenous group of childhood retinal degeneration inherited in an autosomal-recessive manner. These findings indicate that CRX IS not only essential for photoreceptor development [19”,21”], but also required for the maintenance of normal cone and rod function. See also annotatlon [21*-l. 24.

Goutte C, Johnson AD: al protein alters the DNA binding specificity of a2 repressor. Cell 1998, 52:875-882.