Regulation of Melanopsins and Per1 by α-MSH and Melatonin in Photosensitive Xenopus laevis Melanophores

Moraes, Maria Nathália de Carvalho Magalhães; Santos, Luciane Rogéria dos; Mezzalira, Nathana; Poletini, Maristela Oliveira; Castrucci, Ana Maria de Lauro

doi:https://doi.org/10.1155/2014/654710

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Volume 2014 | Article ID 654710 | https://doi.org/10.1155/2014/654710

Regulation of Melanopsins and Per1 by α-MSH and Melatonin in Photosensitive Xenopus laevis Melanophores

Maria Nathália de Carvalho Magalhães Moraes,¹Luciane Rogéria dos Santos,¹Nathana Mezzalira,¹Maristela Oliveira Poletini,²and Ana Maria de Lauro Castrucci¹

Academic Editor: Mario Guido

Received26 Jan 2014

Revised27 Mar 2014

Accepted30 Mar 2014

Published13 May 2014

Abstract

α-MSH and light exert a dispersing effect on pigment granules of Xenopus laevis melanophores; however, the intracellular signaling pathways are different. Melatonin, a hormone that functions as an internal signal of darkness for the organism, has opposite effects, aggregating the melanin granules. Because light functions as an important synchronizing signal for circadian rhythms, we further investigated the effects of both hormones on genes related to the circadian system, namely, Per1 (one of the clock genes) and the melanopsins, Opn4x and Opn4m (photopigments). Per1 showed temporal oscillations, regardless of the presence of melatonin or α-MSH, which slightly inhibited its expression. Melatonin effects on melanopsins depend on the time of application: if applied in the photophase it dramatically decreased Opn4x and Opn4m expressions, and abolished their temporal oscillations, opposite to α-MSH, which increased the melanopsins’ expressions. Our results demonstrate that unlike what has been reported for other peripheral clocks and cultured cells, medium changes or hormones do not play a major role in synchronizing the Xenopus melanophore population. This difference is probably due to the fact that X. laevis melanophores possess functional photopigments (melanopsins) that enable these cells to primarily respond to light, which triggers melanin dispersion and modulates gene expression.

1. Introduction

Color change is an important strategy for animals’ camouflages, which allows them to match their body color with the environment [1]. In ectothermic vertebrates, pigment granule aggregation or dispersion, within the cytoplasm of cutaneous pigment cells, are responsible for the physiological color changes resulting in skin lightening or darkening, respectively [2, 3]. Notably, pigment translocation can be induced by light [4–7] and by several neurohumoral signals [7, 8].

α-Melanocyte stimulating hormone (α-MSH), which is produced in the pars intermedia of the pituitary gland, is a darkening agent in all vertebrates. It induces a very rapid dispersion in dermal melanophores of ectothermic vertebrates [9–13], and it also plays a role in skin, fur, and feather darkening [14, 15], in cell proliferation, in cell differentiation [16], and immune activity [17, 18] of endothermic animals. Human α-MSH is produced in sites other than the pituitary, such as the skin itself and its systemic action promotes melanin production by the human skin in response to UV light [19]. In addition, α-MSH is produced in the mammalian hypothalamus affecting behaviors such as food intake and lordosis [20, 21].

Melatonin (5-methoxy-N-acetyl tryptamine) is an indoleamine synthesized by the pineal gland and released at night. Contrary to α-MSH, melatonin aggregates melanin granules acting as a lightening hormone [7, 22]. Many ectothermic vertebrates exhibit a circadian rhythm of color change, a night paling related with melatonin secretion [23, 24]. Melatonin integrates the endocrine system and the external environment, translating the photoperiod information into hormone signal, thus synchronizing various aspects of physiology and neuroendocrine functions with light-dark cycles [25]. This hormone plays a crucial role in the regulation of circadian and seasonal changes, influencing reproductive functions through the hypothalamic-pituitary-gonadal (HPG) axis [26]. In addition, melatonin may also act as an antioxidant agent [27], some of its antioxidative activities being exerted by its metabolite, cyclic 3-hydroxymelatonin, which completely prevents the cytochrome C degradation by hydrogen peroxide [28].

Studies on the photoresponses of Xenopus laevis melanophores led to the discovery of the photopigment melanopsin, which is present in the retina of all vertebrates, including humans [29, 30]. In mammals, melanopsin is present in a subpopulation of photosensitive ganglion cells [31], which convey light information to the hypothalamic suprachiasmatic nuclei (SCN) [32, 33], the master biological clock. In addition to adjusting the biological clock to light-dark cycles, these cells mediate nonvisual photic responses, such as pupillary constriction and melatonin suppression [34, 35]. In 2006, Bellingham and coworkers [36] demonstrated the existence of two Opn4 genes, encoding melanopsins in nonmammalian vertebrates. The second melanopsin, Opn4m, presents a much higher homology with the mammalian than with the X. laevis melanopsin, which was then named Opn4x. Despite the presence of melanopsins in nonmammalian retinas, their function as a photoentrainment pigment has yet to be defined.

The molecular basis for endogenous biological rhythm is a loop of transcriptional/translation of clock proteins. In general, CLOCK and BMAL1 dimers acting through E-box elements increase the transcription of Per, Cry, Reverb-α, and Ror which after being translated inhibit CLOCK/BMAL1 effect [37]. Additionally, CLOCK/BMAL1 dimers regulate other genes known as clock controlled genes [38–40]. Tyrosinase, a rate-limiting enzyme for melanin production, has E-box motif in its promoter, suggesting that it can be a clock-controlled gene [41].

Although α-MSH and melatonin have opposite effects on pigment translocation, both mediate light responses, that is, α-MSH disperses melanin granules in the same manner as light does, whereas melatonin is the messenger of the darkness. α-MSH and light have the same effect on pigment translocation within X. laevis melanophores; however, the intracellular signaling pathways are different [42, 43], which may indicate plasticity in this response. α-MSH acts through MC1R receptor coupled to a Gs-protein, triggering the activation of the cAMP pathway [44], whereas light stimulates melanopsin leading to phospholipid hydrolysis and increase of cytosolic calcium [42]. On the other hand, melatonin triggers X. laevis melanin aggregation through the activation of Mel 1c receptor, which is coupled to a Gi-protein, thus leading to a decrease in cAMP [22].

X. laevis melanophores incorporate all components of a circadian system, as they express clock genes and photopigments. In addition, Per1 expression increases in response to a blue light stimulus, indicating the mediation of a blue sensitive pigment [45]. The fact that both hormones, α-MSH and melatonin, signal external environmental light conditions to the organisms in an opposite manner, and that light is an important zeitgeber in X. laevis melanophores, we hypothesized that these hormones may differentially affect the melanopsins Opn4x and Opn4m (blue sensitive photo-pigments) as well as Per1 expression.

2. Material and Methods

Immortalized cultures of embryonic X. laevis melanophores (a kind gift of Professor Mark Rollag, University of New Mexico, USA) were kept in 60% L-15 medium, supplemented with 5 mg/mL insulin/transferrin/selenium, 2X hypoxanthine/thymidine, 1% antibiotic/fungicide solution (10,000 U/mL penicillin/10,000 μg/mL streptomycin/25 μg/mL amphotericin B), and 10% non-inactivated fetal calf serum (all from Life Technologies, Carlsbad, CA, USA) at pH 7.5 and 25°C [46]. The culture medium was changed weekly and when the cells reached 80% confluence, they were removed with 60% Tyrode/EDTA solution and transferred to new flasks (the cells from one flask were split into 3 flasks).

For the experiments, the cells were plated 2 × 10⁶ per 25 cm² flask, the serum was reduced to 2%, and 2 × 10⁻⁷ M retinaldehyde was added. Cells were manipulated in the dark using Konex illuminator with a 7 W red light (~5 lux) and a Safe-Light GBX-2 filter (Kodak, SP, Brazil). Each experiment was repeated 2 to 3 times with duplicate flasks of cells ( number presented in the graphs).

The protocols described below were based on the fact that Xenopus melanophores aggregate the melanin granules in DD, or in response to melatonin, and disperses pigment granules in response to light or to α-MSH. Therefore, α-MSH was employed to mimic light signal in aggregated cells, that is, in DD, whereas melatonin was applied on already dispersed cells; there is LD.

2.1. Protocol 1: α-MSH Treatment

Cells were kept under constant darkness for 5 days. At 8:00 h of the fifth day, the flasks were divided into two groups: (1) cells were subject to a medium change without hormone (control); (2) cells were subject to a medium change containing 10⁻⁹ M α-MSH (Sigma-Aldrich, St. Louis, MO, USA). Twelve hours later, both groups received a new medium change without hormone. At 8:00 h of the sixth day (24 hours after the beginning of the treatment) total RNA was extracted every 4 hours (0, 4, 8, 12, 16, and 20 ZT).

2.2. Protocol 2: Melatonin Treatment

Cells were kept in 12 : 12 LD for 5 days and then treated for 6 hours with 10⁻⁹ M melatonin (Sigma-Aldrich, St. Louis, MO, USA) in one of the following conditions: (a) beginning of the light phase of the 6th day and (b) beginning of the dark phase of the 6th day. Control cells were kept in the absence of the hormone but were subject to similar light conditions and medium changes. Twenty-four hours after beginning of the treatment, total RNA was extracted every 4 hours (0, 4, 8, 12, 16, and 20 ZT). During the light phase, the flasks received irradiance of 87.85 µw/cm² (600 lux, 2.5 × 10¹⁴ photons·s⁻¹·cm⁻², full spectrum, 8-W cool white fluorescent tube, T5-8W, SCT, São Paulo, Brazil).

2.3. RNA Extraction, RT-PCR and Quantitative PCR

Total RNA was extracted with TRIzol reagent; the RNA pellet was then resuspended in DEPC water and treated with DNase (turbo-DNA-free), according to the manufacturer’s instructions (all reagents from Life Technologies, Carlsbad, CA, USA). RNA concentration was determined with a spectrophotometer (Nanodrop, Willmington, DE, USA) and 1 μg was used in reverse transcription reaction with SuperScript III and random primers (Life Technologies, Carlsbad, CA, USA).

Quantitative PCR reactions were carried out with primers and probes specific for the genes of interest or ribosomal 18S, designed with Primer Express (Life Technologies, CA, USA) based on Genbank sequences (http://www.ncbi.nlm.nih.gov/PubMed/) and synthesized by IDT (Coralville, IA, USA) (Table 1). Multiplex quantification was performed for Opn4x and 18S RNA and Per1 and 18S RNA; the solutions contained 1 µL of cDNA, primers (300 nM for each gene,50 nM for 18S RNA), probes (200 nM for each gene, 50 nM for 18S RNA), Platinum Supermix (Life Technologies, Carlsbad, CA, USA) supplemented to final concentrations of 6 mM MgCl₂, 0.4 mM dNTPS, 0.1 U/µL Platinum Taq DNA polymerase.

To normalize the results, 18S ribosomal RNA was used, as recommended for mammalian and nonmammalian species [47–51]. The efficiency of each pair of primers was calculated and varied between 80 and 104%. The assays were performed in i5 thermocycler (BioRad Laboratories, Hercules, CA, USA), with the following conditions: 7 min at 95°C, followed by 40 cycles of 30 s at 95°C and 30 s at 55°C.

2.4. Statistical Analyses

The results were analyzed using the ΔΔCT method [52]. The threshold was established by the thermocycler software and crossed the amplification curves to determine the number of cycles, the CT. The difference between the CTs for 18S RNA and the CT for each gene at the same time point is the ΔCT. The maximal ΔCT was then subtracted from each ΔCT value to obtain ΔΔCT, used as a negative exponential of base 2 . The log values (at least four flasks of cells, from two independent experiments) were averaged and graphed as mean ± SEM relative to the minimal value expression for each protocol. The levels of significance of differences among time points were determined comparing the log data by one-way ANOVA, followed by Tukey; between time points in different protocols, as well as control versus hormone-stimulated groups, the log data were compared by two-way ANOVA followed by Bonferroni’s test (significant at ).

3. Results and Discussion

The vertebrate circadian system consists of an element that detects environmental light, an internal oscillator, and one or more output signals. The internal oscillator is comprised by a central clock and several peripheral clocks which, when in synchrony, display a time-accurate output [53]. Notably, the central clock remains rhythmic when in culture [54], whereas peripheral clocks lose this ability due to the loss of coupling among cells [55, 56].

Per1 expression in X. laevis melanophores kept in constant dark (DD) did not statistically vary throughout ZTs after medium changes (Figure 1(a)). Several data from literature have shown that a single medium change, serum shock [57, 58], phorbol esters (TPA) [59], or glucocorticoids [60] act as coupling factor in a variety of cultured cell types leading to a synchronization of clock genes. Unlike the literature reports, our data have shown that medium changes were unable to induce Per1 temporal oscillation in X. laevis melanophores in DD, similarly to previous results in cells which were kept undisturbed in DD, that is, did not receive medium changes [45, 61]. However, when these cells were kept in light-dark cycles (12 : 12 LD) and subject to medium changes, Per1 mRNA showed a temporal oscillation. Higher levels of mRNA were found at ZT4 comparing to all other ZTs (Figure 1(b), ), and at ZT8 when was compared to ZT12 and ZT20 (Figure 1(b), ). Per1 mRNA has also been reported to increase in undisturbed X. laevis melanophores in LD 14 : 10 [61] or LD 12 : 12 [45] cycles. The results of the present study together with previous data suggest a major role for light in the clock gene expression changes in these cells, as it had already been demonstrated in X. laevis eye [62].

(a) Medium changes

(b) Medium changes in photophase

(c) 10−9 M α-MSH

(d) 10−9 M melatonin in photophase

Figure 1

Quantitative PCR of Per1 in X. laevis melanophores after 2 medium changes in DD (a); 2 medium changes in LD 12 : 12 (b); 10⁻⁹ M α-MSH in DD (c); and 10⁻⁹ M melatonin in the photophase of LD 12 : 12 (d). Each bar represents the mean, ± SE, mRNA relative to the noted ZT. (a) Significantly different from (b); (c) significantly different from (d) by one-way ANOVA, Tukey’s post-test; means significantly different from the respective control at the same ZT, by two-way ANOVA and Bonferroni’s post-test. number shown on top of each bar in this and the following figures.

Because of the similar effects of light and of α-MSH on melanin granule dispersion, we evaluated the action of this hormone on one of the clock genes, Per1, as well as the effects of melatonin, an internal dark messenger for the organism. Melatonin was applied at the beginning of the photophase for 6 hours, whereas α-MSH was applied for 12 hours at the beginning of the subjective photophase in DD, thus mimicking the presence of light in causing melanin dispersion. One-way ANOVA analyses showed that there was no temporal variation in either 10⁻⁹M α-MSH treated cells or in cells subject to medium changes in DD. On the other hand, two-way ANOVA showed that the hormone treatment considerably affected the results (, , Figures 1(a) and 1(c)), though no effects were seen when each time-point comparison was analyzed by Bonferroni’s post-test.

The temporal oscillations of Per1 expression seen in control cells persisted after melatonin treatment. Higher levels of Per1 mRNA were seen at ZT4 when compared to ZT0, ZT12, ZT16, and ZT20 (, Figure 1(d)), and at ZT8 as compared to ZT12 and ZT20 (, Figure 1(d)). Melatonin and time exerted strong influences on the results, as shown by two-way ANOVA (, and , , resp.). Bonferroni’s post-test analysis of each time point indicated that Per1 mRNA levels were 2 times lower at ZT4 () and ZT12 () after 10⁻⁹ M melatonin treatment in the photophase (Figure 1(d)) than the control group (Figure 1(b)). Interestingly, the temporal oscillation of Per1 expression seen in the control was maintained. Bluhm and coworkers [61] found that 1 hour melatonin treatment of X. laevis melanophores, kept in DD, did not affect Per1 expression, except for an increase at 3 hours. In mammals, entrainment mechanisms involve light stimulation of melanopsin positive ganglion cells that release glutamate/PACAP in the SCN cells, ultimately increasing Per1 expression [63]. In undisturbed X. laevis melanophores, light at 460 nm wavelength, which maximally stimulates the melanopsins (or one of them) [42], also leads to enhancement of Per1 expression [45]. These results point to a role of a blue light sensitive pigment in the light entrainment mechanism of X. laevis melanophores. Thus, we hypothesized that melatonin decreasing effects on the amplitude of Per1 expression found here could be due to melatonin-induced reduction of the photopigment expression. In fact, 10⁻⁹ M melatonin treatment in the photophase dramatically decreased Opn4x and Opn4m expression during the light period, as compared to cells only subject to medium changes (Figures 2(a) and 2(b); Figures 3(a) and 3(b)) as described below.

(a) Medium changes in photophase

(b) 10−9 M Melatonin in photophase

(c) Medium changes in scotophase

(d) 10−9 M Melatonin in scotophase

(e) Medium changes

(f) 10−9 M α-MSH

Figure 2

Quantitative PCR of Opn4x in X. laevis melanophores after 2 medium changes in the photophase of LD 12 : 12 (a); 10⁻⁹ M melatonin in the photophase of LD 12 : 12 (b); 2 medium changes in the scotophase of LD 12 : 12 (c); 10⁻⁹ M melatonin in the scotophase of LD 12 : 12 (d); 2 medium changes in DD (e); 10⁻⁹M α-MSH in DD (f). Each bar represents the mean, ± SE, mRNA relative to the noted ZT. (a) Significantly different from (b); (c) significantly different from (d), except in 2 C where (a) is different from (b) and (c) is different from (a) and (d) by one-way ANOVA and Tukey’s post-test; means significantly different from the respective control at the same ZT, by two-way ANOVA and Bonferroni’s post-test.

(a) Medium changes in photophase

(b) 10−9 M Melatonin in photophase

(c) Medium changes in scotophase

(d) 10−9 M melatonin in scotophase

(e) Medium changes

(f) 10−9 M α-MSH

Figure 3

Quantitative PCR of Opn4m in X. laevis melanophores after to 2 medium changes in the photophase of LD 12 : 12 (a); 10⁻⁹M melatonin in the photophase of LD 12 : 12 (b); 2 medium changes in the scotophase of LD 12 : 12 (c); 10⁻⁹M melatonin in the scotophase of LD 12 : 12 (d); 2 medium changes in DD (e); 10⁻⁹M α-MSH in DD (f). Each bar represents the mean, ± SE, mRNA relative to the noted ZT. (a) Significantly different from (b); (c) significantly different from (d), by one-way ANOVAandTukey’s post-test; means significantly different from the respective control at the same ZT, by two-way ANOVA and Bonferroni’s post-test.

Melatonin treatment in the photophase considerably affected Opn4x (, and , resp.) and Opn4m (, and , resp.) expressions. In addition, the temporal oscillation of Opn4x and Opn4m seen after medium changes in the photophase (Figures 2(a) and 3(a)) was abolished by the hormone treatment (Figures 2(b) and 3(b)). This reduction was statistically significant as shown by Bonferroni post-test for Opn4x and Opn4m at ZT0 (, resp.) and ZT4 (). Therefore, melatonin is affecting the sensitivity of X. laevis melanophores to an important light-wavelength (460–480 nm), which we believe is the entrainment factor in this model. Melatonin per se does not seem to participate in the entrainment mechanism of the biological clock, as demonstrated by its lack of effect on Per1 expression when applied either in the photophase (Figure 1(d)), or in the scotophase (data not shown).

If medium changes were performed in the photophase, Opn4x and Opn4m, showed a temporal profile with higher expression throughout the light period (Figures 2(a) and 3(a)). Opn4x expression was higher at ZT0 as compared to ZT16 () and at ZT4 as compared to ZT12, ZT16, and ZT20 (). Opn4m expression was higher at ZT0 () and at ZT4 () than at ZT12, 16 and 20. When medium changes were performed in the scotophase, the highest levels of Opn4x and Opn4m mRNA were found at ZT8 ( and resp.), that is, at the end of the light period (Figures 2(c) and 3(c)). The profile seen for Opn4x (Figure 2(a)) did not differ from what has been reported by Moraes and coworkers in X. laevis melanophores subject to 12 : 12 LD without further manipulation, whereas for Opn4m, LD cycles were not sufficient to induce temporal changes (unpublished data). So, Opn4m requires LD cycles plus medium changes to display a temporal oscillation as shown here (Figure 3(a)). Therefore, light seems to be the important zeitgeber for Opn4x, since it already showed an oscillatory pattern, which was not affected by medium changes, similarly to what was observed in Per1 expression.

Melatonin applied in the scotophase strongly affected Opn4x and Opn4m expressions throughout the time points (, and , resp.), which implied an altered temporal profile of both melanopsins. Opn4x mRNA levels decreased at ZT0 () and increased at ZT4 (, Figure 2(d)), as compared to cells only subject to medium changes in the scotophase (Figure 2(c)). Opn4m mRNA levels increased at ZT4 (, Figure 3(d)), as compared to cells in control group (Figure 3(c)). Melatonin seems to exert permissive actions on gene expression, allowing Opn4x and Opn4m to resume a smoother time-course variation (Figures 2(d) and 3(d)). In control cells subject to medium changes in LD, Opn4x mRNA levels were higher at ZT8 compared to all other ZTs (), and Opn4m mRNA levels were higher at ZT8 compared to ZT0, ZT4, and ZT16 (). After melatonin treatment, both melanopsin expressions gradually increased during the photophase.

Melatonin administration during the scotophase mimics pineal melatonin secretion in physiological conditions, so one would expect a more relevant effect of melatonin. However, this hormone was incapable of significantly altering melanopsin temporal oscillations. Both diurnal and nocturnal animals release pineal gland melatonin at night, indicating the duration of the dark phase of the day, thus melatonin is a crucial compound that sets circadian temporal system [64]. During the photophase, melatonin seems to promote a disruption of the light sensor —melanopsins in this model— represented by the dramatic inhibition of both photopigment expressions and loss of changes in the temporal profile (Figures 2(a), 2(b), 3(a), and 3(b)). This inhibitory effect of melatonin on gene expression was also seen after only 1 hour melatonin treatment of X. laevis melanophores in DD [61]. X. laevis melanophores, therefore, is an interesting model to study melatonin regulation of the circadian peripheral systems.

α-MSH exerts opposite effect on Opn4x expression when compared to melatonin, as it does on pigment translocation. After 10⁻⁹M α-MSH treatment, Opn4x expression increased 3-4 times at ZT0 () and ZT16 (). The stimulatory effect of α-MSH on the overall expression was considered very significant (, ) (Figures 2(e) and 2(f)). Opn4m, on the other hand, was less affected by α-MSH; its mRNA levels decreased at ZT4 () and increased at ZT12 () as compared to the control group (Figures 3(e) and 3(f)). After α-MSH or medium changes, both Opn4x and Opn4m showed temporal oscillation. In DD after medium changes, similar levels of Opn4x mRNA were found at ZT0, ZT4, and ZT16, compared to ZT8, ZT12, and ZT20 () (Figure 2(e)); but higher levels were seen at ZT0 and ZT16 compared to ZT20 after α-MSH treatment (, Figure 2(f)). In DD after medium changes, Opn4m expression was higher at ZT4 as compared to ZT0, ZT8, ZT12, and ZT20 () and lower at ZT8 as compared to ZT16 (Figure 3(e)); after α-MSH treatment, its expression was higher at ZT12 as compared to ZT8 and ZT20 () (Figure 3(f)). Therefore α-MSH effects on melanopsins expressions do not seem to involve the molecular clock machinery, since the hormone did not affect Per1 expression except for a sporadic modulation throughout the day.

In fact, the opposite effects of α-MSH and melatonin seem to be conserved, because they have previously been described in other groups of animals [65, 66]. This may be explained by the opposite actions of these hormones on cAMP production in X. laevis melanophores: α-MSH increases the nucleotide concentration [43], whereas melatonin reverses melanosome dispersion through the inhibition of adenylyl cyclase [67].

We would expect similar effects of α-MSH (Figures 2(f) and 3(f)) and light (Figures 2(a) and 3(a)) on Opn4x and Opn4m expression; however, unlike the hormone, light was able to induce temporal variation in both melanopsin expressions. This may be explained by the fact that the signaling pathway leading to light-stimulated responses of gene expression and pigment dispersion is distinct from the one triggering melanin dispersion by α-MSH. In fact, light stimulates PKC pathway leading to an increase of cytosolic Ca²⁺ and cGMP [42], whereas α-MSH promotes an increase of cAMP [43]. This response is decreased by preexposure of melanophores to light, because light-induced Ca²⁺ rise inhibits adenylyl cyclase [43]. Although mammalian Per1 promoter is known to possess cAMP responsive element [68], little is known about X. laevis Per1 promoter. In Danio rerio embryonic cells, we and others have demonstrated that the light-stimulated pathway increases both Per1 and Per2 expressions, what is blocked by MAP kinase inhibitors [69–71], whereas there is controversial evidence of cAMP involvement [71]. In NIH-3T3 fibroblasts, it has been shown that the circadian oscillation of clock gene expression is abolished by a MEK inhibitor [59], suggesting that transcription factors other than CREB may be the activators of Per1 promoter in vertebrate peripheral clocks.

In summary, Per1 is sensitive to light-dark cycles, regardless of the presence of melatonin or α-MSH, which slightly inhibited its expression. It is worth to mention, however, that the hormones may be affecting the expression of other clock genes, such as Per2 or Bmal1, or even exerting a posttranslational effect. Melatonin effects on melanopsins depend on the time of application. Melatonin applied in the photophase dramatically decreases Opn4x and Opn4m expressions and abolishes its temporal oscillation, opposite to α-MSH which increases the melanopsin expressions. Our results demonstrate that unlike what has been reported for other peripheral clocks and cultured cells, medium changes or hormones do not play a major role in synchronizing Xenopus melanophore cell population. This difference is most probably due to the fact that X. laevis melanophores possess functional photopigments, the melanopsins, enabling these cells to respond primarily to light, which triggers melanin dispersion and modulation of gene expression.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Authors’ Contribution

Maristela Oliveira Poletini and Ana Maria de Lauro Castrucci contributed equally to this work.

Acknowledgments

The authors acknowledge Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Tecnológico e Científico (CNPq) Grants. Maria Nathália de Carvalho Magalhães Moraes is a fellow of FAPESP, Nathana Mezzalira is a fellow of CAPES, and Luciane Rogéria dos Santos was a fellow of FAPESP.

References

D. Stuart-Fox and A. Moussalli, “Camouflage, communication and thermoregulation: lessons from colour changing organisms,” Philosophical Transactions of the Royal Society B: Biological Sciences, vol. 364, no. 1516, pp. 463–470, 2009.
View at: Publisher Site | Google Scholar
J. T. Bagnara and M. E. Hadley, Chromatophores and Color Change: The Comparative Physiology of Animal Pigmentation, Prentice-Hall, Englewood Cliffs, NJ, USA, 1973.
H. Nilsson-Sköld, S. Aspengren, and M. Wallin, “Rapid color change in fish and amphibians—function, regulation, and emerging applications,” Pigment Cell Melanoma Research, vol. 26, no. 1, pp. 29–38, 2013.
View at: Google Scholar
J. T. Bagnara, “Hypophysectomy and the tail darkening reaction in Xenopus,” Proceedings of the Society for Experimental Biology and Medicine, vol. 94, no. 3, pp. 572–575, 1957.
View at: Google Scholar
T. Iga and I. Takabatake, “Melanophores of Zacco temmincki (Teleostei) are light sensitive,” Journal of Experimental Zoology, vol. 227, no. 1, pp. 9–14, 1983.
View at: Google Scholar
M. D. Rollag, “Pertussis toxin sensitive photoaggregation of pigment in isolated Xenopus tail-fin melanophores,” Photochemistry and Photobiology, vol. 57, no. 5, pp. 862–866, 1993.
View at: Google Scholar
R. Fujii, “The regulation of motile activity in fish chromatophores,” Pigment Cell Research, vol. 13, no. 5, pp. 300–319, 2000.
View at: Publisher Site | Google Scholar
L. E. M. Nery and A. M. D. L. Castrucci, “Pigment cell signalling for physiological color change,” Comparative Biochemistry and Physiology A: Physiology, vol. 118, no. 4, pp. 1135–1144, 1997.
View at: Publisher Site | Google Scholar
R. Fujii and Y. Miyashita, “Receptor mechanisms in fish chromatophores V. MSH disperses melanosomes in both dermal and epidermal melanophores of a catfish (Parasilurus asotus),” Comparative Biochemistry and Physiology C: Pharmacology Toxicology and Endocrinology, vol. 71, no. 1, pp. 1–6, 1982.
View at: Google Scholar
L. A. M. De Castrucci, E. Hadley Mac, and V. J. Hruby, “Melanotropin bioassays: in vitro and in vivo comparisons,” General and Comparative Endocrinology, vol. 55, no. 1, pp. 104–111, 1984.
View at: Google Scholar
W. C. Sherbrooke, M. E. Hadley, and A. M. L. Castrucci, “Melanotropic peptides and receptors: an evolutionary perspective in vertebrate physiological color change,” in The Melanotropic Peptides, M. E. Hadley, Ed., vol. 2, pp. 175–190, CRC Press, Washington, DC, USA, 1988.
View at: Google Scholar
H. Vaudry, N. Chartrel, L. Desrues et al., “The pituitary-skin connection in amphibians. Reciprocal regulation of melanotrope cells and dermal melanocytes,” Annals of the New York Academy of Sciences, vol. 885, pp. 41–56, 1999.
View at: Google Scholar
C. R. Camargo, M. A. Visconti, and A. M. L. Castrucci, “Physiological color change in the bullfrog, Rana catesbeiana,” Journal of Experimental Zoology, vol. 283, pp. 160–169, 1999.
View at: Google Scholar
W. C. Chou, M. Takeo, P. Rabbani et al., “Direct migration of follicular melanocyte stem cells to the epidermis after wounding or UVB irradiation is dependent on Mc1r signaling,” Nature Medicine, vol. 19, no. 7, pp. 924–929, 2013.
View at: Google Scholar
X. H. Zhang, Y. Z. Pang, S. J. Zhao et al., “The relationship of plumage colours with MC1R (Melanocortin 1 Receptor) and ASIP (Agouti Signaling Protein) in Japanese quail (Coturnix coturnix japonica),” British Poultry Science, vol. 54, no. 3, pp. 306–311, 2013.
View at: Google Scholar
A. Slominski and J. Wortsman, “Neuroendocrinology of the skin,” Endocrine Reviews, vol. 21, no. 5, pp. 457–487, 2000.
View at: Google Scholar
T. Brzoska, M. Böhm, A. Lügering, K. Loser, and T. A. Luger, “Terminal signal: anti-inflammatory effects of α-melanocyte- stimulating hormone related peptides beyond the pharmacophore,” Advances in Experimental Medicine and Biology, vol. 681, pp. 107–116, 2010.
View at: Publisher Site | Google Scholar
W. Chen, J. Li, H. Qu et al., “The melanocortin 1 receptor (MC1R) inhibits the inflammatory response in Raw 264.7 cells and atopic dermatitis (AD) mouse model,” Molecular Biology Reports, vol. 40, no. 2, pp. 1987–1996, 2013.
View at: Google Scholar
B. Iyengar, “The melanocyte photosensory system in the human skin,” SpringerPlus, vol. 1, pp. 158–188, 2013.
View at: Google Scholar
A. Bertolini, R. Tacchi, and A. V. Vergoni, “Brain effects of melanocortins,” Pharmacological Research, vol. 59, no. 1, pp. 13–47, 2009.
View at: Publisher Site | Google Scholar
E. Keen-Rhinehart, K. Ondek, and J. E. Schneider, “Neuroendocrine regulation of appetitive ingestive behavior,” Frontiers in Neuroscience, vol. 7, pp. 213–243, 2013.
View at: Google Scholar
D. Sugden, K. Davidson, K. A. Hough, and M.-T. Teh, “Melatonin, melatonin receptors and melanophores: a moving story,” Pigment Cell Research, vol. 17, no. 5, pp. 454–460, 2004.
View at: Publisher Site | Google Scholar
S. Binkley, Pineal: Endocrine and Nonendocrine Function, Prentice-Hall, Englewood Cliffs, NJ, USA, 1988.
A. M. C. Filadelfi and A. M. D. L. Castrucci, “Comparative aspects of the pineal/melatonin system of poikilothermic vertebrates,” Journal of Pineal Research, vol. 20, no. 4, pp. 175–186, 1996.
View at: Google Scholar
D. Hazlerigg, “The evolutionary physiology of photoperiodism in vertebrates,” Progress in Brain Research, vol. 199, pp. 413–422, 2012.
View at: Google Scholar
L. Shi, N. Li, L. Bo, and Z. Xu, “Melatonin and hypothalamic-pituitary-gonadal axis,” Current Medicinal Chemistry, vol. 20, no. 15, pp. 2017–2031, 2013.
View at: Google Scholar
D.-X. Tan, L. C. Manchester, M. P. Terron, L. J. Flores, and R. J. Reiter, “One molecule, many derivatives: a never-ending interaction of melatonin with reactive oxygen and nitrogen species?” Journal of Pineal Research, vol. 42, no. 1, pp. 28–42, 2007.
View at: Publisher Site | Google Scholar
D. X. Tan, R. Hardeland, L. C. Manchester, A. Galano, and R. J. Reiter, “Cyclic-3-hydroxymelatonin (C3HOM), a potent antioxidant, scavenges free radicals and suppresses oxidative reactions,” Current Medicinal Chemistry, vol. 21, no. 13, pp. 1557–1565, 2014.
View at: Google Scholar
I. Provencio, G. Jiang, W. J. De Grip, W. Pär Hayes, and M. D. Rollag, “Melanopsin: an opsin in melanophores, brain, and eye,” Proceedings of the National Academy of Sciences of the United States of America, vol. 95, no. 1, pp. 340–345, 1998.
View at: Publisher Site | Google Scholar
I. Provencio, I. R. Rodriguez, G. Jiang, W. P. Hayes, E. F. Moreira, and M. D. Rollag, “A novel human opsin in the inner retina,” Journal of Neuroscience, vol. 20, no. 2, pp. 600–605, 2000.
View at: Google Scholar
I. Provencio, M. D. Rollag, and A. M. Castrucci, “Photoreceptive net in the mammalian retina. This mesh of cells may explain how some blind mice can still tell day from night,” Nature, vol. 415, no. 6871, p. 493, 2002.
View at: Google Scholar
D. M. Berson, F. A. Dunn, and M. Takao, “Phototransduction by retinal ganglion cells that set the circadian clock,” Science, vol. 295, no. 5557, pp. 1070–1073, 2002.
View at: Publisher Site | Google Scholar
S. Hattar, H.-W. Liao, M. Takao, D. M. Berson, and K.-W. Yau, “Melanopsin-containing retinal ganglion cells: architecture, projections, and intrinsic photosensitivity,” Science, vol. 295, no. 5557, pp. 1065–1070, 2002.
View at: Publisher Site | Google Scholar
S. Panda, T. K. Sato, A. M. Castrucci et al., “Melanopsin (Opn4) requirement for normal light-induced circadian phase shifting,” Science, vol. 298, no. 5601, pp. 2213–2216, 2002.
View at: Publisher Site | Google Scholar
S. Panda, I. Provencio, D. C. Tu et al., “Melanopsin is required for non-image-forming photic responses in blind mice,” Science, vol. 301, no. 5632, pp. 525–527, 2003.
View at: Publisher Site | Google Scholar
J. Bellingham, S. S. Chaurasia, Z. Melyan et al., “Evolution of melanopsin photoreceptors: discovery and characterization of a new melanopsin in nonmammalian vertebrates,” PLoS Biology, vol. 4, no. 8, article e254, 2006.
View at: Google Scholar
U. Albrecht, “Timing to perfection: the biology of central and peripheral circadian clocks,” Neuron, vol. 74, no. 2, pp. 246–260, 2012.
View at: Publisher Site | Google Scholar
S. Panda, M. P. Antoch, B. H. Miller et al., “Coordinated transcription of key pathways in the mouse by the circadian clock,” Cell, vol. 109, no. 3, pp. 307–320, 2002.
View at: Publisher Site | Google Scholar
K.-F. Storch, O. Lipan, I. Leykin et al., “Extensive and divergent circadian gene expression in liver and heart,” Nature, vol. 417, pp. 78–83, 2002.
View at: Google Scholar
B. E. McIntosh, J. B. Hogenesch, and C. A. Bradfield, “Mammalian Per-Arnt-Sim proteins in environmental adaptation,” Annual Review of Physiology, vol. 72, pp. 625–645, 2010.
View at: Publisher Site | Google Scholar
C. Murre, P. Schonleber McCaw, H. Vaessin et al., “Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence,” Cell, vol. 58, no. 3, pp. 537–544, 1989.
View at: Google Scholar
M. C. Isoldi, M. D. Rollag, A. M. De Lauro Castrucci, and I. Provencio, “Rhabdomeric phototransduction initiated by the vertebrate photopigment melanopsin,” Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 4, pp. 1217–1221, 2005.
View at: Publisher Site | Google Scholar
M. C. Isoldi, I. Provencio, and A. M. D. L. Castrucci, “Light modulates the melanophore response to α-MSH in Xenopus laevis: an analysis of the signal transduction crosstalk mechanisms involved,” General and Comparative Endocrinology, vol. 165, no. 1, pp. 104–110, 2010.
View at: Publisher Site | Google Scholar
F. Rouzaud, A. L. Kadekaro, Z. A. Abdel-Malek, and V. J. Hearing, “MC1R and the response of melanocytes to ultraviolet radiation,” Mutation Research: Fundamental and Molecular Mechanisms of Mutagenesis, vol. 571, no. 1-2, pp. 133–152, 2005.
View at: Publisher Site | Google Scholar
M. N. C. M. Moraes, M. O. Poletini, B. C. R. Ramos, L. H. R. G. Lima, and A. M. L. Castrucci, “Effect of light on expression of clock genes in Xenopus laevis melanophores,” Photochemistry and Photobiology, 2013.
View at: Publisher Site | Google Scholar
M. D. Rollag, I. Provencio, D. Sugden, and C. B. Green, “Cultured amphibian melanophores: a model system to study melanopsin photobiology,” Methods in Enzymology, vol. 316, pp. 291–309, 2000.
View at: Google Scholar
J. L. Aerts, M. I. Gonzales, and S. L. Topalian, “Selection of appropriate control genes to assess expression of tumor antigens using real-time RT-PCR,” BioTechniques, vol. 36, no. 1, pp. 84–91, 2004.
View at: Google Scholar
D. Goidin, A. Mamessier, M.-J. Staquet, D. Schmitt, and O. Berthier-Vergnes, “Ribosomal 18S RNA prevails over glyceraldehyde-3-phosphate dehydrogenase and β-actin genes as internal standard for quantitative comparison of mRNA levels in invasive and noninvasive human melanoma cell subpopulations,” Analytical Biochemistry, vol. 295, no. 1, pp. 17–21, 2001.
View at: Publisher Site | Google Scholar
A. T. McCurley and G. V. Callard, “Characterization of housekeeping genes in zebrafish: male-female differences and effects of tissue type, developmental stage and chemical treatment,” BMC Molecular Biology, vol. 9, pp. 102–119, 2008.
View at: Publisher Site | Google Scholar
H. Schmid, C. D. Cohen, A. Henger, S. Irrgang, D. Schlöndorff, and M. Kretzler, “Validation of endogenous controls for gene expression analysis in microdissected human renal biopsies,” Kidney International, vol. 64, no. 1, pp. 356–360, 2003.
View at: Publisher Site | Google Scholar
L.-J. Zhu and S. W. Altmann, “mRNA and 18S-RNA coapplication-reverse transcription for quantitative gene expression analysis,” Analytical Biochemistry, vol. 345, no. 1, pp. 102–109, 2005.
View at: Publisher Site | Google Scholar
K. J. Livak and T. D. Schmittgen, “Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method,” Methods, vol. 25, no. 4, pp. 402–408, 2001.
View at: Publisher Site | Google Scholar
D. A. Golombek and R. E. Rosenstein, “Physiology of circadian entrainment,” Physiological Reviews, vol. 90, no. 3, pp. 1063–1102, 2010.
View at: Publisher Site | Google Scholar
S. Liu, Y. Cai, R. B. Sothern, Y. Guan, and P. Chan, “Chronobiological analysis of circadian patterns in transcription of seven key clock genes in six peripheral tissues in mice,” Chronobiology International, vol. 24, no. 5, pp. 793–820, 2007.
View at: Publisher Site | Google Scholar
A.-J. F. Carr and D. Whitmore, “Imaging of single light-responsive clock cells reveals fluctuating free-running periods,” Nature Cell Biology, vol. 7, no. 3, pp. 319–321, 2005.
View at: Publisher Site | Google Scholar
D. K. Welsh, T. Imaizumi, and S. A. Kay, “Real-time reporting of circadian-regulated gene expression by luciferase imaging in plants and mammalian cells,” Methods in Enzymology, vol. 393, article 11, pp. 269–288, 2005.
View at: Publisher Site | Google Scholar
A. Balsalobre, F. Damiola, and U. Schibler, “A serum shock induces circadian gene expression in mammalian tissue culture cells,” Cell, vol. 93, no. 6, pp. 929–937, 1998.
View at: Publisher Site | Google Scholar
S. Yamazaki, R. Numano, M. Abe et al., “Resetting central and peripheral circadian oscillators in transgenic rats,” Science, vol. 288, no. 5466, pp. 682–685, 2000.
View at: Google Scholar
M. Akashi and E. Nishida, “Involvement of the MAP kinase cascade in resetting of the mammalian circadian clock,” Genes and Development, vol. 14, no. 6, pp. 645–649, 2000.
View at: Google Scholar
A. Balsalobre, S. A. Brown, L. Marcacci et al., “Resetting of circadian time in peripheral tissues by glucocorticoid signaling,” Science, vol. 289, no. 5488, pp. 2344–2347, 2000.
View at: Publisher Site | Google Scholar
A. P. C. Bluhm, N. N. Obeid, A. M. L. Castrucci, and M. A. Visconti, “The expression of melanopsin and clock genes in Xenopus laevis melanophores and their modulation by melatonin,” Brazilian Journal of Medical and Biological Research, vol. 45, pp. 730–736, 2012.
View at: Google Scholar
M. Zhuang, Y. Wang, B. M. Steenhard, and J. C. Besharse, “Differential regulation of two period genes in the Xenopus eye,” Molecular Brain Research, vol. 82, no. 1-2, pp. 52–64, 2000.
View at: Publisher Site | Google Scholar
S. M. Reppert and D. R. Weaver, “Coordination of circadian timing in mammals,” Nature, vol. 418, no. 6901, pp. 935–941, 2002.
View at: Publisher Site | Google Scholar
R. J. Reiter, “The pineal gland: an intermediary between the environment and the endocrine system,” Psychoneuroendocrinology, vol. 8, no. 1, pp. 31–40, 1983.
View at: Publisher Site | Google Scholar
A. M. C. Filadelfi and A. M. L. Castrucci, “Melatonin desensitizing effects on the in vitro responses to MCH, alpha-MSH, isoproterenol and melatonin in pigment cells of a fish (S. marmoratus), a toad (B. ictericus), a frog (R. pipiens), and a lizard (A. carolinensis), exposed to varying photoperiodic regimens,” Comparative Biochemistry and Physiology A: Physiology, vol. 109, no. 4, pp. 1027–1037, 1994.
View at: Publisher Site | Google Scholar
A. L. Kadekaro, L. N. S. Andrade, L. M. Floeter-Winter et al., “MT-1 melatonin receptor expression increases the antiproliferative effect of melatonin on S-91 murine melanoma cells,” Journal of Pineal Research, vol. 36, no. 3, pp. 204–211, 2004.
View at: Publisher Site | Google Scholar
B. H. White, R. D. Sekura, and M. D. Rollag, “Pertussis toxin blocks melatonin-induced pigment aggregation in Xenopus dermal melanophores,” Journal of Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology, vol. 157, no. 2, pp. 153–159, 1987.
View at: Google Scholar
S. A. Tischkau, J. W. Mitchell, S.-H. Tyan, G. F. Buchanan, and M. U. Gillette, “Ca^2+/cAMP response element-binding protein (CREB)-dependent activation of Per1 is required for light-induced signaling in the suprachiasmatic nucleus circadian clock,” Journal of Biological Chemistry, vol. 278, no. 2, pp. 718–723, 2003.
View at: Publisher Site | Google Scholar
N. Cermakian, M. P. Pando, C. L. Thompson et al., “Light induction of a vertebrate clock gene involves signaling through blue-light receptors and MAP kinases,” Current Biology, vol. 12, no. 10, pp. 844–848, 2002.
View at: Publisher Site | Google Scholar
J. Hirayama, L. Cardone, M. Doi, and P. Sassone-Corsi, “Common pathways in circadian and cell cycle clocks: light-dependent activation of Fos/AP-1 in zebrafish controls CRY-1a and WEE-1,” Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 29, pp. 10194–10199, 2005.
View at: Publisher Site | Google Scholar
N. Cermakian, M. P. Pando, C. L. Thompson et al., “Light induction of a vertebrate clock gene involves signaling through blue-light receptors and MAP kinases,” Current Biology, vol. 12, no. 10, pp. 844–848, 2002.
View at: Publisher Site | Google Scholar

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Copyright © 2014 Maria Nathália de Carvalho Magalhães Moraes et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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