Melatonin as a central molecule connecting neural ... - Springer Link

Funct Integr Genomics (2011) 11:383–388 DOI 10.1007/s10142-011-0221-8

REVIEW

Melatonin as a central molecule connecting neural development and calcium signaling Joice de Faria Poloni & Bruno César Feltes & Diego Bonatto

Received: 5 December 2010 / Revised: 16 March 2011 / Accepted: 17 March 2011 / Published online: 5 April 2011 # Springer-Verlag 2011

Abstract Melatonin (MEL) is a neuroendocrine hormone secreted by the pineal gland in association with the suprachiasmatic nucleus and peripheral tissues. MEL has been observed to play a critical role in the reproductive process and in the fetomaternal interface. Extrapineal synthesis has been reported in mammalian models during pregnancy, especially by the placenta tissue. MEL can regulate intracellular processes (e.g., G-proteins) and the activity of second messengers (e.g., cAMP, IP3, Ca2+). During neurodevelopment, these activities regulated by melatonin have an important role as an intracellular signaling for gene expression regulation. To review the role of MEL in neurodevelopment, we built interactome networks of different proteins that act in these processes using systems biology tools. The analyses of interactome networks revealed that MEL could modulate neurodevelopment through the regulation of Ca2+ intracellular levels and influencing BMP/SMAD signaling, thus affecting neural gene responses and neuronal differentiation. Keywords Melatonin . Calcium signaling . Neurodevelopment . Systems biology Electronic supplementary material The online version of this article (doi:10.1007/s10142-011-0221-8) contains supplementary material, which is available to authorized users. J. de Faria Poloni : B. C. Feltes Instituto de Biotecnologia, Universidade de Caxias do Sul (UCS), Caxias do Sul, Rio Grande do Sul, Brazil D. Bonatto (*) Centro de Biotecnologia da UFRGS—Sala 219, Departamento de Biologia Molecular e Biotecnologia, Universidade Federal do Rio Grande do Sul—UFRGS, Avenida Bento Gonçalves 9500—Prédio 43421, Caixa Postal 15005, Porto Alegre, Rio Grande do Sul, Brazil 91509–900 e-mail: [email protected]

Introduction Melatonin (MEL) is a lipophilic indoleamine that, in mammals, is synthesized by the pineal gland in association with the suprachiasmatic nucleus and peripheral tissues (Foulkes et al. 1997; Lanoix et al. 2008). MEL synthesis begins with the acetylation of serotonin by the enzyme arylalkylamine Nacetyltransferase to generate N-acetylserotonin, which is subsequently methylated by hydroxyindole O-methyltransferase, finally generating the extrapineal MEL (Foulkes et al. 1997). MEL has a key function in the regulation of circadian rhythms, and studies have reported an important role for MEL in mammalian reproduction, as shown by the increase of spontaneous abortions in rats that have undergone a pinealectomy (Iwasaki et al. 2005). However, the physiological function of extrapineal MEL and its role in the fetal–maternal interface are not well documented. During pregnancy, the level of MEL in the placenta remains constant due to the local presence of receptors and enzymes that synthesize MEL during the first trimester (Iwasaki et al. 2005). It has been shown that MEL is one of the few maternal hormones able to cross the placenta without prior modification (Tamura et al. 2008). Moreover, in zebrafish embryos, it was observed that MEL could act as an intracellular signaling molecule, regulating processes such as proliferation, differentiation, and cell migration (Danilova et al. 2004). In mammals, the MEL receptor subtypes MT1 and MT2 are members of the seven-transmembrane G proteincoupled receptor family and mediate adenylyl cyclase and guanylyl cyclase inhibition, decreasing intracellular levels of cAMP and cGMP (von Gall et al. 2002). The expression of these receptors is greater in embryos than in adults. In addition, the expression of MT1 and MT2 follows the organogenesis process (Danilova et al. 2004). MEL also

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modulates intracellular levels of [Ca2+] by regulating the high-voltage-activated calcium channel, and it opens Ca2+ intracellular stores (Wronka et al. 2008). The Ca2+ signaling system includes transmembrane calcium influx modulated by receptors and voltage-dependent calcium channels, pumps, exchangers, and binding proteins. Ca2+sensing proteins interpret calcium oscillations, modulating an intracellular signal in individual cells and between connected cells, where calcium signaling has an important role in embryonic development (Webb and Miller 2000). Neurodevelopment begins when the dorsal cells produce the Nieuwkoop signal, which is necessary for establishing the Spemann–Mangold organizer in the dorsal mesoderm (Vonica and Gumbiner 2007). The notochord then induces the transition of ectoderm to neuroectorderm by secreting the factors noggin, chordin, and follistatin (Rogers et al. 2009). These molecules act by inhibiting bone morphogenetic protein 4 (BMP4), which belongs to the transforming growth factor β (TGF-β) superfamily and is a negative neural regulator that blocks the formation of the neural plate and promotes epidermal differentiation in dissociated cells (Rogers et al. 2009). Studies show that in ribbed newts (Pleurodeles genus), activated L-type channels participate in neural induction via a calcium signaling cascade that is related to the transduction of neuralizing signals (Duprat, 1996). Neurodevelopment also shows a correlation with calcium signaling in the upregulation of the nuclear protein encoded by c-fos; this transcript is necessary for neural development (Leclerc et al. 1999). Considering the apparent connection between MEL and calcium in development, we used systems biology tools to review the role of MEL in the induction of neurodevelopment through calcium signaling during embryogenesis in Homo sapiens (see Supplementary material 1 to a description of materials and methods). In this sense, the analyses of the interactomic data using different literature data mining and STITCH 2.0 softwares (Supplementary material 1) concerning MEL and calcium signaling and their interplay within neurodevelopmental processes generated an interactome network with 440 different proteins and 5,360 edges (data not shown). From gene ontology (GO) analyses using Molecular Complex Detection and Biological Network Gene Ontology softwares (Supplementary material 1) performed within this interactome network, a subnetwork (Fig. 1) composed of 272 proteins and 1,596 edges was gathered that contained overrepresented biologic processes (Table 1). The systems biology results obtained show an interesting interplay between MEL/calcium signaling and response proteins in neurodevelopment. MEL is one of many diverse signals generated by the mother during pregnancy that establish an intrauterine communication between the fetus and the mother (Tamura

Funct Integr Genomics (2011) 11:383–388

et al. 2008), and it has a role in the coordination of intracellular and signaling molecules during embryonic development (Kawashima et al. 2008). MEL may indirectly exert transcriptional and differentiation control of neurodevelopment through interaction with the signals mediated by calcium and elements sensitive to calcium. A subgraph was obtained by choosing the closest neighbor of MEL and its receptors (Fig. 2). From this subgraph, with 31 proteins and 136 edges directly connected to MEL, we performed gene ontology analyses that indicated different but statistically significant biological processes, shown in Table 2. In this network, it can be observed that MEL interacts with proteins that have an essential role in neurodevelopment and calcium signaling (Fig. 2), e.g., calmodulin (CALM1), G-protein (GNAI1, GNAI3, GNA11), protein kinase α (PRKACG, PRKACA, PRKACB, PRKAR1B, TSE1), and adenylyl cyclase (ADCY2, ADCYAP1); these proteins are involved in calcium signaling and the cAMP signaling cascade. MEL also interacts with the neural stem cell marker nestin (NES) (Sharma et al. 2008) and c-fos (FOS), which collaborates with c-Jun (AP-1 complex) in BMP and fibroblast growth factor (FGF) signaling and also has an important role in the transcription of neural genes (Peng et al. 2002). MEL is also connected with BCL2-associated X protein (BAX), which is an apoptotic activator (Figs. 2 and 3) (Baydas et al. 2005). Furthermore, MEL interacts with protein kinase B (AKT1), which activates neuroprotective signaling pathways (Lee et al. 2006), and with the transcription factors histone acetyltransferase p300 and cAMP-response element-binding protein-1 (CREB1). In addition, the melatonin receptor (MTNR1B) interacts with solute carrier family 26, member 6 (WISH; Figs. 2 and 3), which has a role in intracellular pH modulation (Aust et al. 2006).

Neurodevelopment may be regulated by calcium signals mediated by melatonin Through examination of the data gathered by the systems biology analyses, a model of the action of MEL in calcium signaling and neurodevelopment could be drawn (Fig. 3). It was observed that MEL could alter the intracellular concentration of Ca2+ via stimulation of L-type voltagedependent channels (Mei et al. 2001; Wronka et al. 2008). The activation of the MEL receptor by MEL binding resulted in the stimulation of G-proteins GNAI1 and GNAI3, which stimulated the phospholipases PLCB1, PLCB2, and PLCB4 (Figs. 2 and 3). These phospholipases mediated the breakdown of phosphatidylinositol 4,5bisphosphate to the second messengers diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). It should be noted that IP3 interacts with its receptors ITPKA and


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Fig. 1 Major interactome subnetwork present in the large melatonin-associated network containing the most statistically significant biologic processes (see the text for more details). Melatonin and its receptors are indicated in gray shadow

ITPKB (Fig. 3) (Bach et al. 2005), leading to the opening of Ca2+ stores. In addition, MEL interacted with calmodulin (CALM1; Fig. 3) with high affinity by binding to a single site on the calmodulin molecule, thus changing the Ca2+-calmodulin electrophoretic mobility and inhibiting calmodulin-dependent phosphodiesterase activity (Benítez-King et al. 1993). Moreover, MEL can stimulate calmodulin phosphorylation by activating protein kinase C α (Soto-Vega et al. 2004)

and, in MDCK cells, MEL can induce calmodulin compartmentalization (Benítez-King et al. 1996). In this sense, calmodulin-dependent processes can be inhibited through MEL interactions (Fig. 3). Many enzymes and proteins are regulated by the Ca2+-calmodulin complex, among them protein kinases such as Ca2+/CaM-dependent protein kinases (CAMK2G, CAMK4, CAMK2B, CAMKD, CAMKA; Fig. 2), whose activity and autophosphorylation are inhibited by MEL (Benítez-King et al. 1996).

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Table 1 Major GO classes obtained from the interactions observed between melatonin, calcium signaling, and neuronal development Description

GO number

p value

Corrected p valuea

kb

fc

Proteins

Pattern specification process

7389

3.35×10−23

3.46×10−21

29

131

Intracellular signaling cascade

7242

5.10×10−15

1.96×10−13

68

1,323

Nervous system development

7399

4.24×10−11

9.67×10−10

40

659

Cellular calcium ion homeostasis

6874

3.31×10−6

2.82×10−5

11

102

NOG; WNT3A; FST; GLI2; DWFC; SHH; G13; CTNNB; BHF-1; GBX2; FGF1; FGF2; FGF3; BMP4; NODAL; P300; EMX2; OTX2; SMAD4; EN1; SMAD2; RS; ACVR2A; ACVR2B; PTCH1; ASH1; BMPR1B; BMP7; ACVR1 PLCZ1; ADCY4; ADCY1; ADCY2; GNA16; ADCY7; ADCY8; ADCY5; ADCY6; PRKCB1; AKT1; G13; PLCB4; PIK3CA; PRKACA; PRKACB; PLCB1; PLCB2; FGF2; EGFR; PIK3CB; PRKCH; PRKCG; DGK; PRKCE; PRKCD; PRKCQ; ADRB2; PLCE1; GNAQ; TSE1; P53; MAPK8; JNK3; PRKCZ; GNAI3; NFATC; MEKK; ADCYAP1; PKCI; DGKA; KRAS; V; DGKE; CTNNB; DGKD; DGKG; RASGRP2; RAC1; ACH; PLCD3; PLCD4; EGF; RASA1; DGKQ; ERK; RAF1; SMAD2; DGKH; DGKI; PKCA; ADCY9; BAX; MKK4; MTNR1B; DGKZ; GRP3; MTNR1A NOG; WNT3A; PAX6; GLI2; SHH; TGFB2; FOS; ATP2B2; WNT1; CTNNB; LHX1; RAC1; BHF-1; GBX2; WISH; EGF; FGF2; BMP4; NES; BMP2; GNAO1; TRPC5; ERK; P300; OTX2; EMX2; MEK1; NEUROG2; EN1; EN2; WS1; HDAC4; MOP4; NHBA; MSX1; GNAQ; AKA; FHM; ASH1; BMP7 PLCE1; GNA16; PIK3CB; RYR3; GRIN1; FHM; TRPV4; RYR2; CACN2; PRKCB1; MHS

a

Values calculated from p value after FDR application.

b

Total number of proteins found in the network that belong to a specific GO.

c

Total number of proteins belonging to a specific GO.

MEL also regulates cAMP levels via adenylyl cyclase (ADCY2, ADCYAP1; Figs. 2 and 3) and acts through cAMP-dependent protein kinase (PRKACG, PRKACA, PRKACB, PRKAR1B; Figs. 2 and 3). In addition, MEL activates protein kinase c (PRKCE) via stimulation of DAG. The protein kinases and CaMKinases have an important role in calcium signaling and phosphorylation of a variety of protein targets, such as cAMP-responsive element-binding protein (CREB1), MAP kinase family proteins (ERK, ERK1), p300, c-fos and AKT1 (Fig. 2). All of these proteins play an important role in the transcription of genes involved in neuronal development and plasticity (Figs. 2 and 3) (Barbado et al. 2009; Ha and Redmond 2008; Leclerc et al. 1999; Lee et al. 2006).

Melatonin can exert a direct influence on neuronal development The antagonist molecules of BMP4 signaling (e.g., chordin, noggin, and follistatin) prevent the interaction of BMP4 with its receptor (Rogers et al. 2009). In the absence of antagonists, BMP4 induces mothers against decapentaplegic homolog 1 (SMAD) proteins through the phosphorylation of

Fig. 2 A motif formed by proteins associated with embryonic development and calcium signaling mechanisms that are closest to melatonin (indicated in black shadow) and its receptors (indicated in gray shadow)


387

Table 2 Major GO classes obtained from the interactions observed between melatonin and its neighboring proteins Description

GO number

p value

Corrected p valuea

kb

fc

Proteins

Cell communication

7154

9.10×10−9

2.57×10−6

23

3,697

System development

48731

8.36×10−8

1.24×10−5

15

1,504

Second-messenger-mediated signaling

19932

5.42×10−6

3.06×10−4

6

215

Cell differentiation

30154

2.43×10−4

5.97×10−3

11

1,568

Embryonic development Nervous system development

9790 7399

1.41×10−3 2.45×10−3

1.90×10−2 2.67×10−2

4 6

230 660

GNAI3; ADCY2; GNAI1; CREB1; GNA11; P300; ADCYAP1; AKT1; MOP4; ADRB2; G13; BAX; TSE1; PRKAR1B; RAC1; MTNR1B; GPR50; WISH; WNT11; PRKACA; PRKACB; RASA1; MTNR1A NES; GNA11; P300; PRKACG; AKT1; FOS; MOP4; G13; ADRB2; BAX; TSE1; RAC1; WISH; BOC; RASA1 ADRB2; GNAI3; MTNR1B; PRKACB; MTNR1A; ADCYAP1 AKT1; ADRB2; G13; BAX; P300; GNA11; RAC1; WISH; PRKACA; BOC; RASA1 G13; TSE1; P300; RASA1 MOP4; FOS; NES; P300; RAC1; WISH

a

Values calculated from p value after FDR application.

b

Total number of proteins found in the network that belong to a specific GO.

c

Total number of proteins belonging to a specific GO.

the serine threonine kinase receptor. Once phosphorylated, SMADs translocate to the nucleus and repress the transcription of genes associated with neurodevelopment (Rogers et al. 2009). In the interactome subnetwork, SMAD2 and SMAD4 are connected with CALM1 (Fig. 1). Interestingly, calmodulin has been described to regulate SMAD protein function in vitro (Zimmerman et al. 1998). The c-jun/c-fos protein complex (AP-1 complex) was observed cooperate functionally and physically with SMAD proteins in transmitting the TGF-β signal, and the AP-1 complex interacted with BMP4, which inhibited the formation of neural ectoderm (Fig. 3) (Peng et al. 2002). However, several studies showed that upregulation of c-fos protein is associated with the regulation of genes involved in neural specification, and c-fos activation is related to L-type calcium channel activation (Moreau et al. 1994). In this sense, MEL interacts with FOS in our interactome subnetwork (Figs. 2 and 3) and may suppress the induction of the AP-1 complex, thus interrupting BMP/SMAD signaling (Ross et al. 1996). Moreover, the interruption of BMP/SMAD signaling allowed neural induction (Rogers et al. 2009). Additionally, our model shows that MEL interacts with the neural stem cell (NSC) marker nestin (NES; Fig. 3). Previous studies suggested that MEL induces a neural progenitor phenotype through an increase in nestin gene expression (Sharma et al. 2008) and the stimulation of protein kinase B (AKT1; Figs. 2 and 3), which has been reported to induce nestin (Sharma et al. 2008) and activate the neuroprotective signaling pathway (Lee et al. 2006).

Fig. 3 Schematic model for the role played by melatonin in neural development via modulation of calcium signaling. The dotted box refers to the complexes formed by AP-1 and calmodulin-Ca2+. Melatonin participates in calcium modulation via stimulation of L-type channels and a signaling cascade triggered by melatonin receptors 1 and 2 (MT1/2), leading to an increase in intracellular calcium concentration (Ca↑2+). Both MT1 and 2 are connected to G-proteins, PLCB (phospholipases), DAG (diacylglycerol), IP3 (inositol 1,4,5-trisphosphate), PKC (protein kinase c), and PKA (cAMP-dependent protein kinase), which control the phosphorylation of a variety of protein targets. The increase in IP3 synthesis (IP3↑) is also related to the accumulation of calcium in the cytoplasm. The regulation of cAMP is indicated by the connection of melatonin with AC (adenylyl cyclase) and results in regulation of the activity of PKA (cAMP-dependent protein kinase). In addition, there is a connection between melatonin and FOS, which acts in neural gene regulation, as well the AP-1 complex (FOS/JUN), which is associated with BMP/SMAD signaling. The model indicates the relationship of NES (increased expression), BAX (lowered expression), and AKT1 with melatonin and its role in neurogenesis

388


Conclusion Through the systems biology analysis performed in this work, it is clear that MEL is an essential regulator and modulator of early embryonic neurodevelopment. Interestingly, it has been reported that MEL is necessary for stimulation of adult neurogenesis during sleep; in in vitro tests, MEL promoted cell survival, increasing the number of new neurons derived from adult hippocampal neural precursor cells (Ramírez-Rodríguez et al. 2009). Additionally, neurogenesis was promoted in the rat dentate gyrus after pinealectomy when subjected supplementation with melatonin via the drinking water (Rennie et al. 2009). In this sense, it is imperative to develop more biochemical experiments to elucidate how MEL acts not only in embryonic neurodevelopment but also in adult neurogenesis induced by the differentiation of neuron stem cells (NSCs) present in the CNS. Acknowledgments This work was supported by research grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; Grant number 471769/2007-0) and Programa Institutos Nacionais de Ciência e Tecnologia (INCT de Processos Redox em BiomedicinaREDOXOMA; Grant number 573530/2008-4). Conflict of interest competing interests.

The authors declare that they have no

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