Erik Maronde

A survey of molecular details in the human pineal gland in the light of phylogeny, structure, function and chronobiological diseases

Abstract:  The human pineal gland is a neuroendocrine transducer that forms an integral part of the brain. Through the nocturnally elevated synthesis and release of the neurohormone melatonin, the pineal gland encodes and disseminates information on circadian time, thus coupling the outside world to the biochemical and physiological internal demands of the body. Approaches to better understand molecular details behind the rhythmic signalling in the human pineal gland are limited but implicitly warranted, as human chronobiological dysfunctions are often associated with alterations in melatonin synthesis. Current knowledge on melatonin synthesis in the human pineal gland is based on minimally invasive analyses, and by the comparison of signalling events between different vertebrate species, with emphasis put on data acquired in sheep and other primates. Together with investigations using autoptic pineal tissue, a remnant silhouette of premortem dynamics within the hormone’s biosynthesis pathway can be constructed. The detected biochemical scenario behind the generation of dynamics in melatonin synthesis positions the human pineal gland surprisingly isolated. In this neuroendocrine brain structure, protein‐protein interactions and nucleo‐cytoplasmic protein shuttling indicate furthermore a novel twist in the molecular dynamics in the cells of this neuroendocrine brain structure. These findings have to be seen in the light that an impaired melatonin synthesis is observed in elderly and/or demented patients, in individuals affected by Alzheimer’s disease, Smith–Magenis syndrome, autism spectrum disorder and sleep phase disorders. Already, recent advances in understanding signalling dynamics in the human pineal gland have significantly helped to counteract chronobiological dysfunctions through a proper restoration of the nocturnal melatonin surge.

The genetic subtypes of cAMP-dependent protein kinase — Functionally different or redundant?

II. Fundamental properties shared by types I and II of cAMP protein kinase (cAKI, cAKII) . . . . . . . . 250 A. Domain structure. Relation to other cyclic nucleotide binding proteins and kinases . . . . . . . . . 250 B. Origin of the positive cooperativity of cAMP activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 C. cAMP-modulated contacts between R and C subunits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251

Activated protein kinase A is required for differentiation-dependent transcription of the decidual prolactin gene in human endometrial stromal cells.

Decidualization of human endometrial stromal (ES) cells in vitro is induced by cAMP analogs and ligands that elevate cellular cAMP levels. A marker of this differentiation process is the activation of the decidual PRL (dPRL) promoter. In a primary ES cell culture system we show that relaxin not only acutely but permanently elevates cellular cAMP levels and leads to induction of PRL secretion after 6 days. Northern and Western blot analyses revealed that all regulatory subunit isoforms (RIα, RIβ, RIIα, and RIIβ) and catalytic subunits Cα and Cβ of protein kinase A (PKA) are expressed in ES cells. Transcript levels of PKA subunit isoforms are not altered during decidualization, but in decidualized ES cells, exposed to relaxin for more than 6 days, a significant reduction of RIα protein level occurs, whereas levels of all other forms remain unchanged. Reduction of R subunits might result in a net increase in free C subunit activity. This alteration is not due to a change in the mitotic state of the cells, as...

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.

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.

Isolation and biochemical characterization of LEAP-2, a novel blood peptide expressed in the liver.

The human genome contains numerous genes whose protein products are unknown in terms of structure, interaction partner, expression, and function. To unravel the function of these orphan genes, it is of particular value to isolate native forms of protein and peptide products derived from these genes. From human blood ultrafiltrate, we characterized a novel gene‐encoded, cysteine‐rich, and cationic peptide that we termed liver‐expressed antimicrobial peptide 2 (LEAP‐2). We identified several circulating forms of LEAP‐2 differing in their amino‐terminal length, all containing a core structure with two disulfide bonds formed by cysteine residues in relative 1–3 and 2–4 positions. Molecular cloning of the cDNA showed that LEAP‐2 is synthesized as a 77‐residue precursor, which is predominantly expressed in the liver and highly conserved among mammals. This makes it a unique peptide that does not exhibit similarity with any known human peptide regarding its primary structure, disulfide motif, and expression. Analysis of the LEAP‐2 gene resulted in the identification of an alternative promoter and at least four different splicing variants, with the two dominating transcripts being tissue‐specifically expressed. The largest native LEAP‐2 form of 40 amino acid residues is generated from the precursor at a putative cleavage site for a furin‐like endoprotease. In contrast to smaller LEAP‐2 variants, this peptide exhibited dose‐dependent antimicrobial activity against selected microbial model organisms. LEAP‐2 shares some characteristic properties with classic peptide hormones and it is expected that the isolation of this novel peptide will help to unravel its physiological role.

The novel β-defensin DEFB123 prevents lipopolysaccharide-mediated effects in vitro and in vivo

Defensins are a family of secreted antimicrobial peptides proposed to directly interfere with bacterial membranes. Here we show a functional analysis of the novel β‐defensin DEFB123. A peptide comprising the β‐defensin core region was synthesized and used for our analysis. Like other β‐defensins, DEFB123 exerted antimicrobial activity against a broad spectrum of Gram‐positive and Gram‐negative bacteria, which was assessed by microbroth dilution assay and radial diffusion zone assay. In addition, the peptide showed lipopolysaccharide (LPS)‐binding activity in a Limulus amoebocyte lysate (LAL) assay. Moreover, DEFB123 prevented LPS‐induced tumor necrosis factor (TNF)‐alpha secretion in a murine monocyte cell line (RAW264.7). Accordingly, DEFB123 abolished LPS‐mediated MAPK induction in these cells. Protection against LPS‐mediated effects was then investigated in a murine model of acute sepsis. Our experiments show that synthetic β‐defensin DEFB123 prevents LPS‐induced mortality in C57BL/6 mice in a therapeutic approach. We propose that the physiological role of β‐defensins may include interference with LPS‐action on macrophages, a function formerly thought to be restricted to the family of cathelicidins, a structurally unrelated group of antimicrobial peptides.—Motzkus, D., Schulz‐Maronde, S., Heitland, A., Schulz, A., Forssmann, W.‐G., Jübner, M., and Maronde, E. The novel β ‐defensin DEFB123 prevents lipopolysaccharide‐me‐diated effects in vitro and in vivo. FASEB J. 20, E997‐E1004 (2006)

The human PER1 gene is transcriptionally regulated by multiple signaling pathways.

The mammalian period (Per) genes are components of the circadian clock and appear to be regulated via an autoregulatory feedback loop. Here we show that the human PER1 (hPER1) gene is synergistically activated by protein kinases A and C (PKA, PKC) and cAMP responsive element binding protein. Activators and inhibitors of PKA as well as PKC modulate endogenous hPER1 expression and hPER1 promoter‐driven reporter gene activity in a dose‐dependent manner. Our results suggest that the hPER1 promoter acts as a sensor for multiple signaling molecules thereby integrating different physiological parameters. This regulation of hPER1 appears to be significant for rapid adaptation to changing environmental conditions.

The human PER1 gene is inducible by interleukin-6.

The mammalian period (Per) genes which are components of the circadian clock are mainly regulated via an autoregulatory feedback loop. Here we show that a human PER1 (hPER1) reporter gene activity is stimulated by interleukin-6 (IL-6), a member of the large cytokine gene family and an inducer of the acute phase reaction, in human hepatoma (HuH-7) cells. Our results confirm and extend the view that the hPER1 promoter acts as a sensor for multiple signaling molecules thereby integrating different physiological parameters.

The Clock Genes Period 2 and Cryptochrome 2 Differentially Balance Bone Formation

BACKGROUND Clock genes and their protein products regulate circadian rhythms in mammals but have also been implicated in various physiological processes, including bone formation. Osteoblasts build new mineralized bone whereas osteoclasts degrade it thereby balancing bone formation. To evaluate the contribution of clock components in this process, we investigated mice mutant in clock genes for a bone volume phenotype. METHODOLOGY/PRINCIPAL FINDINGS We found that Per2(Brdm1) mutant mice as well as mice lacking Cry2(-/-) displayed significantly increased bone volume at 12 weeks of age, when bone turnover is high. Per2(Brdm1) mutant mice showed alterations in parameters specific for osteoblasts whereas mice lacking Cry2(-/-) displayed changes in osteoclast specific parameters. Interestingly, inactivation of both Per2 and Cry2 genes leads to normal bone volume as observed in wild type animals. Importantly, osteoclast parameters affected due to the lack of Cry2, remained at the level seen in the Cry2(-/-) mutants despite the simultaneous inactivation of Per2. CONCLUSIONS/SIGNIFICANCE This indicates that Cry2 and Per2 affect distinct pathways in the regulation of bone volume with Cry2 influencing mostly the osteoclastic cellular component of bone and Per2 acting on osteoblast parameters.