Jörg H. Stehle

Mammalian melatonin receptors: molecular biology and signal transduction

Abstract. The pineal hormone, melatonin, is an important regulator of seasonal reproduction and circadian rhythms. Its effects are mediated via high-affinity melatonin receptors, located on cells of the pituitary pars tuberalis (PT) and suprachiasmatic nucleus (SCN), respectively. Two subtypes of mammalian melatonin receptors have been cloned and characterized, the MT1 (Mel1a) and the MT2 (Mel1b) melatonin receptor subtypes. Both subtypes are members of the seven-transmembrane G protein-coupled receptor family. By using recombinant melatonin receptors it has been shown that the MT1 melatonin receptor is coupled to different G proteins that mediate adenylyl cyclase inhibition and phospholipase Cβ activation. The MT2 receptor is also coupled to inhibition of adenylyl cyclase and additionally it inhibits the soluble guanylyl cyclase pathway. In mice with a targeted deletion of the MT1 receptor, the acute inhibitory effects of melatonin on SCN multiunit activity are completely abolished, while the phase-shifting responses to melatonin (given in physiological concentrations) appear normal. Furthermore, melatonin inhibits the phosphorylation of the transcription factor cyclic AMP response element binding protein, induced by the pituitary adenylate cyclase-activating polypeptide in SCN cells predominantly via the MT1 receptor. However, a functional MT2 receptor in the rodent SCN is partially able to compensate for the absence of the MT1 receptor in MT1 receptor-deficient mice. These findings indicate redundant and non-redundant roles of the receptor subtypes in regulating SCN function. In the PT, a functional MT1 receptor is essential for the rhythmic synthesis of the clock gene product mPER1. Melatonin produces a long-lasting sensitization of adenylyl cyclase and thus amplifies cyclic AMP signaling when melatonin levels decline at dawn. This action of melatonin amplifies gene expression rhythms in the PT and provides a mechanism for reinforcing rhythmicity in peripheral tissues which themselves lack the capacity for self-sustained oscillation. Mice with targeted deletion of melatonin receptor subtypes provide an excellent model to understand cellular mechanisms through which melatonin modulates circadian and photoperiodic rhythmicity.

Rhythmic gene expression in pituitary depends on heterologous sensitization by the neurohormone melatonin

In mammals, many daily cycles are driven by a central circadian clock, which is based on the cell-autonomous rhythmic expression of clock genes. It is not clear, however, how peripheral cells are able to interpret the rhythmic signals disseminated from this central oscillator. Here we show that cycling expression of the clock gene Period1 in rodent pituitary cells depends on the heterologous sensitization of the adenosine A2b receptor, which occurs through the nocturnal activation of melatonin mt1 receptors. Eliminating the impact of the neurohormone melatonin simultaneously suppresses the expression of Period1 and evokes an increase in the release of pituitary prolactin. Our findings expose a mechanism by which two convergent signals interact within a temporal dimension to establish high-amplitude, precise and robust cycles of gene expression.

Targeted disruption of the mouse Mel(1b) melatonin receptor.

ABSTRACT Two high-affinity, G protein-coupled melatonin receptor subtypes have been identified in mammals. Targeted disruption of the Mel1a melatonin receptor prevents some, but not all, responses to the hormone, suggesting functional redundancy among receptor subtypes (Liu et al., Neuron 19:91-102, 1997). In the present work, the mouse Mel1b melatonin receptor cDNA was isolated and characterized, and the gene has been disrupted. The cDNA encodes a receptor with high affinity for melatonin and a pharmacological profile consistent with its assignment as encoding a melatonin receptor. Mice with targeted disruption of the Mel1b receptor have no obvious circadian phenotype. Melatonin suppressed multiunit electrical activity in the suprachiasmatic nucleus (SCN) in Mel1b receptor-deficient mice as effectively as in wild-type controls. The neuropeptide, pituitary adenylyl cyclase activating peptide, increases the level of phosphorylated cyclic AMP response element binding protein (CREB) in SCN slices, and melatonin reduces this effect. The Mel1a receptor subtype mediates this inhibitory response at moderate ligand concentrations (1 nM). A residual response apparent in Mel1a receptor-deficient C3H mice at higher melatonin concentrations (100 nM) is absent in Mel1a-Mel1b double-mutant mice, indicating that the Mel1b receptor mediates this effect of melatonin. These data indicate that there is a limited functional redundancy between the receptor subtypes in the SCN. Mice with targeted disruption of melatonin receptor subtypes will allow molecular dissection of other melatonin receptor-mediated responses.

Melatonin: A clock-output, a clock-input

In mammals, the circadian system is comprised of three major components: the lateral eyes, the hypothalamic suprachiasmatic nucleus (SCN) and the pineal gland. The SCN harbours the endogenous oscillator that is entrained every day to the ambient lighting conditions via retinal input. Among the many circadian rhythms in the body that are driven by SCN output, the synthesis of melatonin in the pineal gland functions as a hormonal message encoding for the duration of darkness. Dissemination of this circadian information relies on the activation of melatonin receptors, which are most prominently expressed in the SCN, and the hypophyseal pars tuberalis (PT), but also in many other tissues. A deficiency in melatonin, or a lack in melatonin receptors should therefore have effects on circadian biology. However, our investigations of mice that are melatonin‐proficient with mice that do not make melatonin, or alternatively cannot interpret the melatonin message, revealed that melatonin has only minor effects on signal transduction processes within the SCN and sets, at most, the gain for clock error signals mediated via the retino‐hypothalamic tract. Melatonin deficiency has no effect on the rhythm generation, or on the maintenance of the oscillation. By contrast, melatonin is essential for rhythmic signalling in the PT. Here, melatonin acts in concert with adenosine to elicit rhythms in clock gene expression. By sensitizing adenylyl cyclase, melatonin opens a temporally‐restricted gate and thus lowers the threshold for adenosine to induce cAMP‐sensitive genes. This interaction, which determines a temporally precise regulation of gene expression, and by endocrine–endocrine interactions possibly also pituitary output, may reflect a general mechanism by which the master clock in the brain synchronizes clock cells in peripheral tissues that require unique phasing of output signals.

The mammalian pineal gland: known facts, unknown facets.

In the mammalian pineal gland, information on environmental lighting conditions that is neuronally encoded by the retina is converted into nocturnally elevated synthesis of the hormone melatonin. Evolutionary pressure has changed the morphology of vertebrate pinealocytes, eliminating direct photoreception and the endogenous clock function. Despite these changes, nocturnally elevated melatonin synthesis has remained a reliable indicator of time throughout evolution. In the photo-insensitive mammalian pineal gland this message of darkness depends on the master circadian pacemaker in the hypothalamic suprachiasmatic nuclei. The dramatic change in vertebrate pinealocytes has received little attention; here, we therefore link the known evolutionary morphodynamics and well-investigated biochemical details responsible for rhythmic synthesis of melatonin with recently characterized patterns of gene expression in the pineal gland. We also address the enigmatic function of clockwork molecules in mammalian pinealocytes.

Temporal dynamics of mouse hippocampal clock gene expression support memory processing

Hippocampal plasticity and mnemonic processing exhibit a striking time‐of‐day dependence and likely implicate a temporally structured replay of memory traces. Molecular mechanisms fulfilling the requirements of sensing time and capturing time‐related information are coded in dynamics of so‐called clock genes and their protein products, first discovered and described in the hypothalamic suprachiasmatic nucleus. Using real‐time PCR and immunohistochemical analyses, we show that in wildtype mice core clock components (mPer1/PER1, mPer2/PER2, mCry1/CRY1, mCry2/CRY2, mClock/CLOCK, mBmal1/BMAL1) are expressed in neurons of all subregions of the hippocampus in a time‐locked fashion over a 24‐h (diurnal) day/night cycle. Temporal profiling of these transcriptional regulators reveals distinct and parallel peaks, at times when memory traces are usually formed and/or consolidated. The coordinated rhythmic expression of hippocampal clock gene expression is greatly disordered in mice deficient for the clock gene mPer1, a key player implicated in both, maintenance and adaptative plasticity of circadian clocks. Moreover, Per1‐knockout animals are severely handicapped in a hippocampus‐dependent long‐term spatial learning paradigm. We propose that the dynamics of hippocampal clock gene expression imprint a temporal structure on memory processing and shape at the same time the efficacy of behavioral learning.

Transcription Factors in Neuroendocrine Regulation: Rhythmic Changes in pCREB and ICER Levels Frame Melatonin Synthesis

Neurotransmitter-driven activation of transcription factors is important for control of neuronal and neuroendocrine functions. We show with an in vivo approach that the norepinephrine cAMP-dependent rhythmic hormone production in rat pineal gland is accompanied by a temporally regulated switch in the ratio of a transcriptional activator, phosphorylated cAMP-responsive element–binding protein (pCREB), and a transcriptional inhibitor, inducible cAMP early repressor (ICER). pCREB accumulates endogenously at the beginning of the dark period and declines during the second half of the night. Concomitant with this decline, the amount of ICER rises. The changing ratio between pCREB and ICER shapes thein vivo dynamics in mRNA and, thus, protein levels of arylalkylamine-N-acetyltransferase, the rate-limiting enzyme of melatonin synthesis. Consequently, a silenced ICER expression in pinealocytes leads to a disinhibited arylalkylamine-N-acetyltransferase transcription and a primarily enhanced melatonin synthesis.

Melatonin Plays a Crucial Role in the Regulation of Rhythmic Clock Gene Expression in the Mouse Pars Tuberalis

Abstract: Circadian rhythms in physiology and behavior are driven by a central clock residing within the hypothalamic suprachiasmatic nucleus (SCN). Molecularly, the biological clock is based on the transcriptional/translational feedback loop of clock genes (mPer, mCry, Clock, and Bmal1). Circadian expression of clock genes is not limited to the SCN, but is found in many peripheral tissues. Peripheral rhythms depend on neuroendocrine/neuronal output from the SCN. Melatonin, the hormone of darkness, represents an important neuroendocrine output of the circadian clock. The hypophyseal pars tuberalis (PT) is one of the main target regions for melatonin. The aim of the study was to test whether mPer, mCry, Clock, and Bmal1 are rhythmically expressed in the mouse PT and how the absence of melatonin receptors affects clock gene expression. We analyzed clock gene expression by in situ hybridization and compared wild‐type (WT), melatonin 1 receptor knockout (MT1 ko), and melatonin 2 receptor knockout (MT2 ko) mice. mPer1, mCry1, Clock, and Bmal1, but not mPer2 and mCry2, were rhythmically expressed in the PT of WT and MT2 ko mice. In the PT of MT1 ko mice, expression of mPer1, mCry1, Clock, and Bmal1 was dramatically reduced. We conclude that melatonin, acting through the MT1 receptor, is an important regulator of rhythmic clock gene expression in the mouse PT.

Of Rodents and Ungulates and Melatonin: Creating a Uniform Code for Darkness by Different Signaling Mechanisms

Melatonin synthesis in the mammalian pineal gland is one of the best investigated output pathways of the circadian clock because it can be readily measured and is tightly regulated by a clearly defined input, the neurotransmitter norepinephrine. In this system, a regulatory scenario was deciphered that is centered around the cyclic AMP pathway but shows peculiar species-specific differences. In rodents, the cyclic AMP–mediated, temporally sequential up-regulation of two transcription factors, the activator CREB (cyclic AMP–responsive elementbinding protein) and the inhibitor ICER (inducible cyclic AMP–dependent early repressor), is the core mechanism to determine rhythmic accumulation of the mRNA encoding for the rate-limiting enzyme in melatonin synthesis, the arylalkylamine N-acetyltransferase (AA-NAT). Thus, in rodents, the regulation of melatonin synthesis bears an essential transcriptional component, which, however, is flanked by posttranscriptional mechanisms. In contrast, in ungulates, and possibly also in primates, AA-NAT appears to be regulated exclusively on the posttranscriptional level. Here, increasing cyclic AMP levels inhibit the breakdown of constitutively synthesized AA-NAT protein by proteasomal proteolysis, leading to an elevated enzyme activity. Thus, self-restriction of cellular responses, as a reaction to external cues, is accomplished by different mechanisms in pinealocytes of different mammalian species. In such a temporally gated cellular adaptation, transcriptionally active products of clock genes may play a supplementary role. Their recent detection in the endogenously oscillating nonmammalian pineal organ and, notably, also in the slave oscillator of the mammalian pineal gland underlines that the mammalian pineal gland will continue to serve as an excellent model system to understand mechanisms of biological timing.

Transcription factor dynamics and neuroendocrine signalling in the mouse pineal gland: a comparative analysis of melatonin-deficient C57BL mice and melatonin-proficient C3H mice

In rodents, the nocturnal rise and fall of arylalkylamine N‐acetyltransferase (AANAT) activity controls the rhythmic synthesis of melatonin, the hormone of the pineal gland. This rhythm involves the transcriptional regulation of the AANAT by two norepinephrine (NE)‐inducible transcription factors, e.g. the activator pCREB (phosphorylated Ca2+/cAMP‐response element binding protein) and the inhibitor ICER (inducible cAMP early repressor). Most inbred mouse strains do not produce melatonin under standard laboratory light/dark conditions. As melatonin‐deficient mice are often the founders for transgenic animals used for chronobiological experimentations, molecular components of neuroendocrine signalling in the pineal gland as an integral part of clock entrainment mechanisms have to be deciphered. We therefore compared calcium signalling, transcriptional events and melatonin synthesis in the melatonin‐deficient C57BL mouse and the melatonin‐proficient C3H mouse. Pineal glands and primary pinealocytes were cultured and stimulated with NE or were collected at various times of the light/dark (LD) cycle. Changes in intracellular calcium concentrations, the phosphorylation of CREB, and ICER protein levels follow similar dynamics in the pineal glands of both mouse strains. pCREB levels are high during the early night and ICER protein shows elevated levels during the late night. In the C57BL pineal gland, a low but significant increase in melatonin synthesis could be observed upon NE stimulation, and, notably, also when animals were exposed to long nights. We conclude that the commonly used C57BL mouse is not completely melatonin‐deficient and that this melatonin‐deficiency does not affect molecular details involved in regulating transcriptional events of melatonin synthesis.