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Keywords:

  • melanogenesis;
  • α-MSH;
  • melanocortin 1 receptor;
  • catalase;
  • melanosomes;
  • anti-oxidants;
  • photo-protection

Summary

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Materials and methods
  8. Acknowledgements
  9. References

We demonstrated a direct correlation between melanogenic and catalase activities on in vitro and ex vivo models. Here, we investigated whether the stimulation of Melanocortin-1 Receptor (MC1R) could influence catalase expression, activity and cellular localization. For this purpose, we treated B16-F0 melanoma cells with α-Melanocyte Stimulating Hormone (α-MSH) and we showed a rapid induction of catalase through a cAMP/PKA-dependent, microphthalmia-associated transcription factor (MITF) independent mechanism, acting at post-transcriptional level. Moreover, α-MSH promoted a partial re-distribution of catalase to the cell periphery and dendrites. This work strengthens the correlation between melanogenesis and anti-oxidants, demonstrating the induction of catalase in response to a melanogenic stimulation, such as α-MSH-dependent MC1R activation. Moreover, this study highlights catalase regulatory mechanisms poorly known, and attributes to α-MSH a protective role in defending melanocytes, and possibly keratinocytes, not only on the basis of its pigmentary action, but also for its capacity to stimulate a quick anti-oxidant defence.


Significance

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Materials and methods
  8. Acknowledgements
  9. References

The capacity of α-MSH to drive, in association with melanogenesis, other aspects related to melanocyte and melanoma cells physiology, is only partially known. We have identified a new α-MSH-mediated biological mechanism able to confer a rapid anti-oxidant protection against reactive species generation. This work attributes to α-MSH a protective role in defending melanocytes, and possibly keratinocytes, not only on the basis of its pigmentary action, but also for its capacity to stimulate a quick anti-oxidant defence represented by catalase. Finally, this study highlights novel catalase regulatory mechanisms that so far have not been known.

Introduction

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Materials and methods
  8. Acknowledgements
  9. References

The ultraviolet radiation (UV) on the skin is responsible for both direct and free radical mediated damages on cell structures, such as proteins, lipids and DNA (Tobi et al., 2002; Yamaguchi et al., 2006). Different defence strategies are involved in maintaining cellular integrity. Among them, melanins are able to prevent UV propagation (Kadekaro et al., 2003; Meredith and Sarna, 2006) and anti-oxidants are involved in the detoxification of reactive oxygen species (Valko et al., 2007). Moreover, anti-inflammatory agents and DNA repair mechanisms are also protective for the cells (Calder, 2001; Kastan, 2008).

In the past, some experimental evidence has shown a correlation between pigmentation levels and anti-oxidants. In fact, the activity of thioredoxin reductase was directly associated with the clinically defined cutaneous photo-type in vivo (Fitzpatrick, 1988; Schallreuter and Wood, 2001). Moreover, we found low levels of catalase activity in in vitro primary cultures of human melanocytes from low photo-type subjects (Picardo et al., 1999). We confirmed the same characteristic in an ex vivo three-dimensional model of epidermal reconstructs, which resulted in increased susceptibility to the deleterious effects of UV (Bessou-Touya et al., 1998; Cario-André et al., 1999, 2005). More recently, working on primary cultures of human melanocytes, we focused on the possible link between catalase and melanogenesis, and we showed that catalase specific mRNA, protein amount and enzymatic activity were all directly correlated with the total cellular melanin content (Maresca et al., 2008).

Melanocortin-1 Receptor (MC1R) could be envisaged as a key element in this pleiotropic control, for both its well-known role as a driving force of eu-melanogenesis (Abdel-Malek et al., 2008; Rouzaud et al., 2005; Zanna et al., 2008), and its capacity to promote other photo-protective effects, which go beyond quantitative and qualitative changes in melanin synthesis. Among them, some experimental data have underlined the role of MC1R in protecting DNA against UV-mediated damages, by decreasing downstream generated intracellular levels of hydrogen peroxide, and promoting nucleotide excision repair mechanisms. These beneficial effects contribute to reduce genomic instability and mutagenesis (Böhm et al., 2005; Hauser et al., 2006; Kadekaro et al., 2005). Moreover, further evidences on melanogenically active cells, have shown the ability of α-Melanocyte Stimulating Hormone (α-MSH) to activate immediate defence responses to UV-induced oxidative stress (Song et al., 2009) and to exert anti-inflammatory effects (Böhm et al., 2006; Catania et al., 2004; Getting, 2006) by counteracting free radical species generated in response to TNF-alpha (Eves et al., 2006; Haycock et al., 2000).

This study investigated the role of MC1R in the coordinated control of melanogenesis and anti-oxidant response in B16-F0 melanoma cells. In particular, catalase expression and activity were analysed, also considering its main role in the neutralization of hydrogen peroxide in melanogenically active cells (Yohn et al., 1991). This work strengthens our previous evidence of a correlation between melanogenesis and anti-oxidants (Maresca et al., 2008; Picardo et al., 1999) and demonstrates the induction of catalase in response to α-MSH-dependent MC1R activation. The induction of this anti-oxidant protection parallels but is independent of the melanogenic process, is downstream cAMP/PKA signal transduction pathway, and is post-transcriptionally regulated. Moreover, in response to α-MSH, catalase showed a cellular re-distribution and partial co-localization with tyrosinase, which suggests a possible role of this enzyme, together with melanin, in the protection of melanocytes themselves and possibly of the whole skin against UV. Finally, this work clarifies the regulatory mechanisms of catalase that so far have been poorly understood.

Results

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Materials and methods
  8. Acknowledgements
  9. References

α-MSH-mediated MC1R stimulation induces the activity of catalase

We demonstrated a strict correlation between melanogenesis and catalase activity on primary cultures of human melanocytes (Maresca et al., 2008). Using B16-F0 melanoma cells, we hereby investigated whether or not stimulation of MC1R by α-MSH would promote anti-oxidant effect, through the induction of catalase. α-MSH, at the dose of 10−7 M, induced melanin synthesis (Figure 1A), reduced proliferative rate (Figure 1B), and promoted catalase activity after 2 h, and maintained until 24 h (Figure 1C).

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Figure 1.  α-MSH-mediated MC1R stimulation induces the activity of catalase. (A) Spectrophotometrical analysis of intra-cellular melanin concentration on cell lysates, after 6–48 h of treatment with 10−7 M α-MSH. Data are mean ± SD of three independent experiments and results are expressed in per cent of control (*P < 0.05 and **P < 0.001, respect to control). (B) Trypan blue exclusion assay analysis of cell number and viability after 24–72 h of treatment with 10−7 M α-MSH. Data are mean ± SD of three independent experiments performed in duplicate (*P < 0.01, respect to control). (C) Spectrophotometrical analysis of catalase activity on cell lysates, after 2–48 h of treatment with 10−7 M α-MSH. Data are mean ± SD of three independent experiments and results are expressed in per cent of control (*P < 0.05 and **P < 0.01, respect to control).

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α-MSH-mediated MC1R stimulation induces the over-expression of catalase

To understand whether α-MSH treatment could promote catalase up-regulation, Western Blot analysis of the enzyme was performed. A rapid induction of catalase was observed soon after 2 h of treatment with 10−7 M α-MSH, and such an increase was maintained until 24 h. The up-regulation of tyrosinase was seen only after 6 h of incubation with the same dose of α-MSH, and such an induction remained significant after 48 h (Figure 2).

image

Figure 2.  α-MSH-mediated MC1R stimulation induces the over-expression of catalase. (A) Western blot analysis of tyrosinase and catalase expression after 2–48 h of treatment with 10−7 M α-MSH. Total cellular proteins (30 μg/lane) were subjected to 10% SDS–PAGE. Protein loading variations were determined by immuno-blotting with an anti-β-tubulin antibody. Representative blot is shown. (B) The histogram shows the densitometric quantification of the data with means ± SD of three independent experiments (*P < 0.05 and **P < 0.01, respect to control).

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α-MSH-mediated catalase induction is independent of the melanogenic process

The up-regulation of catalase mediated by α-MSH could be indirectly modulated, after the first hours of stimulation, by melanin biosynthesis itself, which generates free radicals as by products (Meredith and Sarna, 2006). To investigate this possible influence, catalase expression was analysed after stimulation with 10−7 M α-MSH for 6–24 h, in the presence or absence of 20 μM phenylthiourea (PTU), as a specific tyrosinase inhibitor (Dieke, 1947; Hall and Orlow, 2005) (Figure 3). PTU was able to inhibit significantly the α-MSH-mediated melanin synthesis (Figure 3A) and to decrease the expression of tyrosinase itself (Hall and Orlow, 2005), but it did not influence the α-MSH-mediated catalase up-regulation (Figure 3B/C).

image

Figure 3.  α-MSH-mediated catalase induction is independent from the melanogenic process. (A) Spectrophotometrical analysis of intra-cellular melanin concentration on cell lysates, after 48 h of treatment with 10−7 M α-MSH, alone or in combination with 20 μM PTU. Data are mean ± SD of three independent experiments and results are expressed in per cent of control (*P < 0.05 and **P < 0.001, respect to control; §P < 0.01 respect to α-MSH-treated samples). (B) Western blot analysis of tyrosinase and catalase expression after 6–24 h of treatment with 10−7 M α-MSH, alone or in combination with 20 μM PTU. Protein loading variations were determined by immuno-blotting with an anti-β-tubulin antibody. Representative blot is shown. (C) The histogram shows the densitometric quantification of the data with means ± SD of three independent experiments (*P < 0.05 and **P < 0.001, respect to control; §P < 0.05 and §§P < 0.001, respect to α-MSH-treated samples).

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α-MSH-mediated catalase induction is downstream cAMP/PKA pathway

We investigated whether forskolin (FSK), a cAMP elevating agent (Bertolotto et al., 1996; Buscà and Ballotti, 2000), could influence catalase expression (Figure 4). Treatment for 24 h with 1 μM FSK was able to induce an over-expression for both tyrosinase and catalase proteins to levels comparable to those promoted in response to 24 h treatment with 10−7 M α-MSH (Figure 4A, B). Treatment for 48 h with 1 μM FSK stimulated a significant increase in the intracellular melanin to levels similar to those seen after treatment with 10−7 M α-MSH (Figure 4C). The possible involvement of protein kinase A (PKA), downstream cAMP elevation, on the over-expression of catalase, was also investigated. To this purpose, the expression of catalase was analysed after 6 h stimulation with 10−7 M α-MSH in the presence of 10 μM H89, as a potent inhibitor of PKA (Davies et al., 2000; Leemhuis et al., 2002). H89 was able to significantly inhibit α-MSH-mediated catalase over-expression (Figure 4D, E).

image

Figure 4.  α-MSH-mediated catalase over-expression is downstream cAMP elevation. (A) Western blot analysis of tyrosinase and catalase expression after 24 h of treatment with 10−7 M α-MSH or 1 μM FSK. Protein loading variations were determined by immuno-blotting with an anti-β-tubulin antibody. Representative blot is shown. (B) The histogram on the right side shows the densitometric quantification of the data with means ± SD of three independent experiments (*P < 0.001, respect to control). (C) Spectrophotometrical analysis of intra-cellular melanin concentration on cell lysates, after 48 h of treatment with 10−7 M α-MSH or 1 μM FSK. Data are mean ± SD of three independent experiments and results are expressed in per cent of control (*P < 0.001, respect to control). (D) Western blot analysis of catalase expression after 6 h of treatment with 10−7 M α-MSH, in cells pretreated or not for 1 h with 10 μM H-89. Protein loading variations were determined by immuno-blotting with an anti-β-tubulin antibody. Representative blot is shown. (E) The histogram below shows the densitometric quantification of the data with means ± SD of three independent experiments (*P < 0.05, respect to control; §P < 0.01, respect to α-MSH-treated samples).

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Moreover, we investigated whether FSK could influence the activity of catalase. Results showed that 1 μM FSK was able to stimulate catalase activity soon after 2 h of treatment, maintaining it up-regulated until 24 h (Figure 5A). The possible involvement of PKA on the control of catalase activity was also investigated. To this purpose, the activity of the enzyme was analysed after 6 h stimulation with 10−7 M α-MSH in the presence of 10 μM H89. H89 was able to significantly counteract the increase of activity mediated by α-MSH (Figure 5B). As catalase activity can be regulated through direct phosphorylation both in cell free systems and in cultured cells (Yano and Yano, 2002), we investigated the presence of phosphorylated catalase in response to α-MSH exposure. For this purpose, cells were incubated with10−7 M α-MSH for 6 h before immuno-precipitation with an anti-catalase antibody and immuno-blotting with an anti-phosphoserine antibody. A protein reacting with anti-phosphoserine antibody and corresponding to the molecular weight of catalase was visible in α-MSH-treated cells, and no reactivity was detected in control cells (Figure 5C). PKA is capable of increasing catalase activity through direct phosphorylation both in cell free systems and in cultured cells (Yano and Yano, 2002). To examine the effects of PKA catalytic subunit on catalase activity in our experimental model, a B16 native cell lysate (partially purified from melanins and low molecular weight components with catalase like activity, see Materials and Methods) was incubated at 30°C for upto 6 h, in the presence of an excess of both PKA (200 mU) catalytic subunit and/or ATP (150 μM), and catalase activity measured in the time. When cell lysate was incubated with PKA or ATP alone, no changes of catalase activity occurred in the time. By contrast, when cell lysate was put in the presence of both PKA and ATP, catalase activity increased significantly starting from 4 h of treatment (Figure 5D).

image

Figure 5.  α-MSH-mediated catalase activity induction is downstream cAMP/PKA-mediated pathway. (A) Spectrophotometrical analysis of catalase activity on cell lysates, after 2–24 h of treatment with 1 μM FSK. Data are mean ± SD of three independent experiments and results are expressed in per cent of control (*P < 0.001, respect to control). (B) Spectrophotometrical analysis of catalase activity on cell lysates, after 6 h of treatment with 10−7 M α-MSH, alone or in combination with 10 μM H-89. Data are mean ± SD of three independent experiments and results are expressed in per cent of control (*P < 0.05, respect to control; §P < 0.05, respect to α-MSH-treated samples). (C) Western blot analysis of immunoprecipitated catalase-pSer (top panel) and catalase (bottom panel) in cell lysates obtained after 6 h of treatment with 1 μM FSK (see Materials and Methods). (D) Spectrophotometrical analysis of catalase activity on cell lysates. An aliquot taken at the indicated time points (30 min to 6 h) was incubated with PKA catalytic subunit (see Materials and Methods) and catalase activity was assayed. Data are mean ± SD of three independent experiments and results are expressed in per cent of t0 value (*P < 0.05 and **P < 0.01, respect to t0).

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α-MSH-mediated catalase induction is not-transcriptionally regulated

The involvement of microphthalmia-associated transcription factor (MITF), downstream cAMP/PKA pathway, in mediating catalase induction, was investigated. A 24-h treatment with 10−7 M α-MSH failed to promote the over-expression of tyrosinase, in cells which were selectively silenced for the expression of MITF (siMITF). However, at the same dose and time of incubation, α-MSH was able to up-regulate catalase expression in siMITF (+78% versus siMITF untreated cells, P < 0.01) as well as in control cells (siCtr) (+72% versus siCtr untreated cells, P < 0.01) (Figure 6A). Further experiments excluded any other regulative transcriptional control on catalase, as a consequence of cAMP elevation; in fact the treatment of cells with 10−7 M α-MSH did not influence the mRNA expression profile for the anti-oxidant enzyme (Figure 6B), and the co-treatment for 12 h with 4 μg/ml actinomycin D (actD), as a transcription inhibitor (Hurwitz, 1963), did not modify either catalase induction, observed in response to α-MSH alone, or its basal expression level (Figure 6C, D).

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Figure 6.  α-MSH-mediated catalase induction is not-transcriptionally regulated. (A) Western blot analysis of catalase, tyrosinase and MITF expression, after 24 h of treatment with 10−7 M α-MSH, in cells transfected with siRNA Ctr or siMITF. β-Tubulin was used as loading control. Representative blot is shown. Results are relative to three independent experiments. (B) Semi-quantitative RT-PCR was performed to measure the expression of catalase mRNA at various time points of 10−7 M α-MSH treatment. PCR amplification of catalase representative data is shown. Results are relative to three independent experiments. (C) Western blot analysis of catalase and tyrosinase expression, after 12 h of treatment with 10−7 M α-MSH, in cells pretreated for 1 h or not with 4 μg/ml actD. Proteins from equal amount of cells (1 × 106 cells) were subjected to 10% SDS–PAGE. Representative blot is shown. (D) The histogram on the right side shows the densitometric quantification of the data with means ± SD of three independent experiments (*P < 0.05 and **P < 0.01, respect to control; §P < 0.01, respect to α-MSH-treated samples).

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α-MSH-mediated catalase induction is post-transcriptionally regulated

The mechanisms modulating catalase expression and activity are poorly known. However, data in the literature underline the existence of post-transcriptionally regulative strategies able to quickly modulate the expression levels of this enzyme (Schmidt et al., 2002). On the basis of the above described experimental data, we analysed whether cycloheximide (CHX), a well-known selective inhibitor of translation (Obrig et al., 1971), could influence catalase protein expression after stimulation with α-MSH. Actually, we observed that the pretreatment for 12 h with 1 μg/ml CHX, and subsequent co-treatment for 4 h with 10−7 M α-MSH, was able to inhibit significantly catalase induction mediated by α-MSH alone (Figure 7A, B). Moreover, it was noteworthy that this prolonged treatment with CHX, which dramatically influenced tyrosinase expression, did not modify basal expression level of catalase. A mRNA 5′ Cap ribose reversible methylation is known to increase protein translational efficiency of different proteins in several experimental models by improving mRNA capacity to interact with ribosomes to be translated (Gillian-Daniel et al., 1998; Hausmann et al., 2005; Kuge et al., 1998). This mechanism was identified also for catalase from rye leaves (Schmidt et al., 2002). On this basis, we analysed if cycloleucine (CL), as a non-metabolizable inhibitor of S-adenosylmethionine synthetase, commonly used as an inhibitor of RNA methylation (Tuck et al., 1999), could influence catalase protein expression, in response to α-MSH. Treatment with 10 mM CL did not influence either cell viability or total protein content even after 48 h of exposure. Pretreatment for 1 h with 10 mM CL and subsequent co-treatment for 6 h with 10−7 M α-MSH, did not influence tyrosinase over-expression induced by 10−7 M α-MSH alone. In contrast, the same co-treatment was able to significantly inhibit catalase over-expression induced by α-MSH (Figure 7C, D). Moreover, to investigate whether α-MSH could determine catalase over-expression by accelerating the translation machinery or through alternative mechanisms able to act directly on the protein product, the level of catalase protein expression was evaluated following 10−7 M α-MSH exposure in cells treated with 1 μg/ml CHX in the presence or absence of 10 mM CL. Western blot analyses showed that catalase expression was not modified by any treatment performed (Figure 7E, F), strengthening the role of the mechanism acting on translation rate efficiency.

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Figure 7.  α-MSH-mediated catalase induction is post-transcriptionally regulated. (A) Western blot analysis of catalase and tyrosinase expression, after 4 h of treatment with 10−7 M α-MSH, in cells pretreated overnight or not with 1 μM CHX. Proteins from equal amount of cells (1 × 106 cells) were subjected to 10% SDS–PAGE. Representative blot is shown. (B) The histogram on the right side shows the densitometric quantification of the data with means ± SD of three independent experiments (*P < 0.01 and **P < 0.001, respect to control; §P < 0.001, respect to α-MSH-treated samples). (C) Western blot analysis of catalase expression, after 6 h of treatment with 10−7 M α-MSH, in cells pretreated for 1 h or not with 10 mM CL. Protein loading variations were determined by immuno-blotting with an anti-β-tubulin antibody. Representative blot is shown. (D) The histogram on the right side shows the densitometric quantification of the data with means ± SD of three independent experiments (*P < 0.05 and **P < 0.01, respect to control). (E) Western blot analysis of catalase expression, after 6 h of treatment with 10−7 M α-MSH, in cells pretreated initially with 1 μM CHX alone (30 min) and then in co-incubation (30 min) with 1 μM CHX and 10 mM CL. Proteins from equal amount of cells (1 × 106 cells) were subjected to 10% SDS–PAGE. Representative blot is shown. (F) The histogram on the right side shows the densitometric quantification of the data with means ± SD of three independent experiments.

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α-MSH-induced catalase shows a partial re-distribution toward the cell periphery associated with tyrosinase

α-Melanocyte stimulating hormone treatment evoked evident modifications of the cell shape that correlated with the differentiation process. On this basis, we investigated whether the increased catalase protein expression, observed after α-MSH treatment (see above), could be associated with cellular re-distribution of the enzyme. To this purpose, confocal immunofluorescence analysis was performed before and after 10−7 M α-MSH treatment for 2–24 h (Figure 8). In untreated cells, the enzyme was distributed over all the cytoplasm, with a preferential localization in the peri-nuclear area (Figure 8A, F). In α-MSH-stimulated cells, catalase not only significantly increased, in agreement with western blot results (see above), but also showed a partial re-distribution toward the cell periphery, below the plasma membrane and to the dentrites (Figure 8B/C/D/I). Moreover, the treatment for 24 h with 1 μM FSK evoked a response similar to that observed in response to 10−7 M α-MSH (Figure 8E).

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Figure 8.  α-MSH-induced catalase shows a partial re-distribution toward the cell periphery associated with melanosomes. (A–E) Immunofluorescence confocal microscopy of catalase localization in B16 melanoma cells untreated (A) and treated for the indicated time points with 10−7 M α-MSH (B–D) or with 1 μM FSK (E). Scale bar: 10 μm. (F–M) Double immunofluorescence staining with anti-catalase (red) (F/I) and anti-tyrosinase (green) (G/J) antibodies to detect melanosomes, in B16 melanoma cells untreated (F–H) or treated for 24 h with 10−7 M α-MSH (I–K) analysed by confocal microscopy. Co-localization of the two signals is shown in yellow (H/K, arrows and insert in K). Scale bar: 10 μm.

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The clear re-distribution of catalase to the cell periphery, in response to α-MSH, prompted us to investigate if this anti-oxidant enzyme could be carried to the dendrites in association with tyrosinase. To this purpose, double immunolabelling with anti-catalase and anti-tyrosinase antibodies was performed. Immunostaining of untreated cells confirmed the preferential distribution of catalase on the peri-nuclear area, with a low co-localization with tyrosinase (Figure 8F, G, H). Immunostaining of 10−7 M α-MSH-stimulated cells showed a clear co-localization of the two signals mainly beneath the plasma membrane and dendrites (Figure 8I, J, K, arrows and insert in K). However, a large amount of catalase, not co-localizing with tyrosinase, regularly distributed within cytoplasm was also observed.

Discussion

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Materials and methods
  8. Acknowledgements
  9. References

The epidermal melanin unit represents a structural and functional integration between melanocytes and surrounding keratinocytes (Nordlund, 2007) to ensure the photo-protection of the skin against harmful solar radiation (Costin and Hearing, 2007). This interaction is regulated by paracrine hormones and cytokines, mainly released from keratinocytes in response to UV, and able to act on melanocytes, promoting their differentiation (Imokawa, 2004). Among these mediators, α-MSH, stimulating MC1R receptor on the membrane of melanocytes, plays a crucial role both in the promotion of melanogenesis, and in other protective functions which include the reduction of intracellular levels of hydrogen peroxide (Kadekaro et al., 2005).

In this work, we focused mainly on tyrosinase and catalase expression and activity in response to α-MSH, with the former being a crucial regulator of melanogenesis and the latter involved in the detoxification against hydrogen peroxide inside melanocytes (Yohn et al., 1991). α-MSH treatment promoted a melanogenic response, as expected, and induced catalase. A quick stimulation of this anti-oxidant enzyme, both in its activity and protein expression, was observed soon after 2 h of treatment, although tyrosinase was first up-regulated after 6 h. This result clearly showed that in the first 2 h of α-MSH treatment, catalase induction was independent of the melanogenic process and from free radicals generated as melanin synthesis by products. However, this observation did not exclude that a possible indirect influence of melanogenic process could be responsible for maintaining the induction of such detoxifying activity later in time. On this purpose we have stimulated our cells with α-MSH in the presence of PTU as a specific inhibitor of tyrosinase and melanogenesis (Dieke, 1947; Hall and Orlow, 2005). In this condition, catalase was still up-regulated. Thus, α-MSH was able to promote in parallel two independent protective strategies: melanin synthesis and catalase activity, respectively. PTU was also able to influence tyrosinase expression, possibly through the degradation of the enzyme itself, in accordance with a previous study (Hall and Orlow, 2005), contributing to clarify the action mechanism of this tyrosinase inhibitor, at biological level, whose effects on the enzyme processing have not been elucidated yet.

To clarify the signal transduction pathway responsible for the α-MSH-mediated catalase induction, we first focused on cAMP/PKA pathway, a main signalling pathway for the regulation of melanogenesis in melanocytes and melanoma cells (Buscà and Ballotti, 2000). The involvement of this pathway, downstream the α-MSH-mediated MC1R stimulation, in promoting pigmentation is indisputable (Abdel-Malek et al., 2008; Rouzaud et al., 2005) and implies the activation of MITF transcription factor (Buscà and Ballotti, 2000). The capacity of MC1R, when stimulated, to drive other aspects related to melanocyte and melanoma cell physiology, is only partially known and needs to be clarified better at molecular level. The capacity of activated MC1R to modulate inflammation induced by TNF-alpha was demonstrated in melanogenically active cells (Haycock et al., 2000). Recent works, also on B16 melanoma cells, focused on the capacity of α-MSH to suppress specifically NF-kB transcription factor, which is mainly activated in response to oxidative imbalance (Liu et al., 2006; Luger and Brzoska, 2007). The ability of activated MC1R to down-regulate inflammation and redox environment, by acting on NF-kB, is intriguing and deserves to be investigated in association with catalase. However, on the basis of the results we described, we have to exclude any type of transcriptional control for the α-MSH-mediated catalase induction. In fact, we were unable to alter the α-MSH-mediated induction of catalase, either by silencing MITF transcription factor, and by blocking the gene transcription mechanism with actD. Even the mRNA expression profile for catalase in response to α-MSH was not modified, compared to that observed in the untreated control, at any time of treatment. Instead, when stimulation with α-MSH was done in the presence of CHX, as a translation inhibitor, the catalase over-expression mediated by α-MSH was totally counteracted. We obtained similar results in co-treatment experiments with α-MSH and CL, a commonly used inhibitor of mRNA 5′ Cap ribose reversible methylation, known to increase protein translation efficiency (Gillian-Daniel et al., 1998; Hausmann et al., 2005; Kuge et al., 1998). CL did not influence either total protein content (even after prolonged exposure) or tyrosinase over-expression induced by α-MSH alone, suggesting the specificity of such a type of control on catalase in response to α-MSH. These results are in agreement with previous studies showing the 5′ Cap ribose reversible methylation as a regulative mechanism for catalase from rye leaves (Schmidt et al., 2006). Moreover, to investigate whether α-MSH could determine catalase over-expression through alternative mechanisms acting directly on the protein product, catalase protein expression levels were evaluated after α-MSH exposure in cells treated with both CHX and CL. Results we obtained showed that catalase expression was not modified by any treatment performed, strengthening the role of the mechanism acting on translation rate efficiency in the α-MSH-mediated catalase up-regulation.

Catalase contains conserved amino-acid residues that are potential target for PKA, and it has been reported that catalase activity can be regulated through PKA-dependent phosphorylation, both in cell free systems and in cultured cells (Yano and Yano, 2002). Therefore, we investigated the possibility of such post-translational control for catalase in response to α-MSH exposure. Our results showed that α-MSH induced an activating phosphorylation on Serine residues, on the 60 kDa catalase monomer. We cannot exclude that this phosphorylation was the only one possible for catalase. In fact, sporadic studies underlined the complexity of this enzyme which exists as multiple molecular weight iso-forms that can be phosphorylated also on alternative residues (Yano and Yano, 2002; Yano et al., 2004).

All these results delineate the ability of α-MSH to modulate catalase expression with different modalities, which all act at post-transcriptional level (Schmidt et al., 2002; Yano and Yano, 2002; Yano et al., 2004). Post-transcriptional and post-translational controls guarantee a rapid modulation of catalase and a quick neutralization of reactive species, generated in response to UV or as side products of melanogenesis itself. Such quick regulation strategies were associated with the high stability of catalase. In fact the expression levels of this protein appeared unmodified, either in the presence of transcription or translation inhibitors, differently from tyrosinase which was rapidly degraded, in the same experimental conditions.

Previous studies showed that catalase was localized not only in the peroxisomes but also in other cellular districts, such as in the nucleus, sometimes in covalent association with other proteins (Ballinger et al., 1994; Bulitta et al., 1996; Yano et al., 2004). These studies attribute to catalase an unusual localization, and suggest new possible functions, beside its canonical role as an anti-oxidant enzyme (Bulitta et al., 1996; Sun, 1997; Yano et al., 2004). On this basis, we investigated if catalase up-regulation, in response to α-MSH, was associated with its partial re-distribution inside the cell. Confocal immunofluorescence analysis of this enzyme, in response to α-MSH, showed its significant increase over all the cytoplasm, with a well visible re-distribution toward the cell periphery, below the plasma membrane and to the dendrites. This result suggests a potential protection exerted by catalase against free radicals, generated both inside and outside the cells by melanogenesis itself and UV exposure. As FSK treatment induced a response similar to that observed after α-MSH incubation, this result suggests the involvement of cAMP/PKA pathway even in mediating the morphological modifications responsible for the re-distribution of catalase inside the cells. Our attention was mainly focused on catalase that was localized on cell dendrites. Currently, we cannot exclude that this localization could be partially attributed to a possible function of catalase, as a structural protein in association with the integrins and their signal pathways, as described in a different experimental model (Yano et al., 2004). Localization of catalase in dendrites we describe here could be explained also on the basis of a possible re-distribution inside the melanosomes, before their transfer to the surrounding keratinocytes. A catalase activity was found inside the melanosomes, as a key element of some specific step of melanogenesis (Shibata et al., 1993). Here, we showed this anti-oxidant enzyme co-localizing with tyrosinase, suggesting its association with melanosomes as a catalyst element of some specific steps of melanogenesis and as a protective enzyme also for keratinocytes against UV.

In conclusion, this work attributes to α-MSH a protective role in defending melanocytes, and perhaps keratinocytes, not only on the basis of its pigmentary action, but also for its capacity to stimulate catalase. This integrated synergy could represent a main strategy against stress sources, such as those related to UV-induced oxidative damage.

Materials and methods

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Materials and methods
  8. Acknowledgements
  9. References

Cell culture and treatments

Mouse melanoma cells B16-F0 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 7% heat-inactivated foetal bovine serum (FBS) and antibiotics, all purchased by Gibco (Gibco, Life Technologies Italia, Milan, Italy). For all the treatments, cells were plated, and 24 h later stimulated with chemicals in completed replaced fresh medium. α-MSH (Sigma-Aldrich Srl, Milan, Italy) was employed at the dose of 10−7 M; PTU (Sigma-Aldrich Srl), as a specific inhibitor of tyrosinase activity (Dieke, 1947; Hall and Orlow, 2005), was used at the concentration of 20 μM; Forskolin (FSK) (Sigma-Aldrich Srl), as a cAMP-stimulating agent, was employed at the dose of 1 μM (Bertolotto et al., 1996); H-89 (Calbiochem, Merck KGaA, Darmstadt, Germany), as a selective and potent inhibitor of PKA, was used at the dose of 10 μM (Davies et al., 2000; Leemhuis et al., 2002). Actinomycin D (actD) (Sigma-Aldrich Srl), as a transcription inhibitor (Hurwitz, 1963), was employed at the dose of 4 μg/ml; CHX (Sigma-Aldrich Srl), as a selective inhibitor of protein synthesis in eukaryotic cells (Obrig et al., 1971), was employed at the dose of 1 μg/ml. Cycloleucine (Sigma-Aldrich Srl), as a non-metabolizable inhibitor of S-adenosylmethionine synthetase, commonly used as an inhibitor of RNA methylation (Tuck et al., 1999) was employed at the dose of 10 mM (Schmidt et al., 2002).

Cell proliferation analysis

Cells were plated in a 12-well plate at a density of 1 × 104 cells/well and left to grow overnight. Cells were treated with α-MSH 10−7 M for 24–72 h. Cell proliferation was determined by the Trypan blue exclusion test (final concentration 0.1% w/v). Cells were harvested by incubation in 0.5% trypsin and 0.2% EDTA for 10 min at 37°C, and counted using a hemocytometer. Results represent the mean of three experiments in triplicate.

Melanin content determination

Cells were plated in a 12-well plate at a density of 1 × 104 cells/well and left to grow overnight. Cells were treated with α-MSH 10−7 M for 6–48 and 72 h. For intracellular melanin content determination, cell pellets were dissolved in 1 M NaOH and incubated at 40°C for 1 h. Total melanin in the cell suspension was determined spectrophotometrically by a Lambda 25 UV/Vis spectrophotometer (Perkin-Elmer, London, UK), by reading the absorbance at 405 nm. Melanin content was calculated by interpolating the results with a standard curve, generated by the absorbance of known concentrations of synthetic melanin, as previously described (Barker et al., 1995), and corrected for protein content in the supernatant of the cell lysates. Three determinations were performed in duplicate, the results were expressed as μg of total melanin/mg proteins and values are reported as % of control.

Catalase activity

Supernatants of native cell lysates were filtered on Microcon Amicon YM-100 centrifugal filter devices (Millipore, Bedford, MA, USA) able to remove particles, (such as the bulk of melanin) and molecules with molecular weight under 100 KDa. Then, on filtered supernatants, catalase activity was determined by a Lambda 25 UV/Vis spectrophotometer (Perkin-Elmer), evaluating the disappearance of hydrogen peroxide in the buffer used (10 mM) (Claiborne, 1985). After setting the baseline at 240 nm against air, 2 ml of a solution of 10 mM H2O2 (Merck KGaA, Darmstadt, Germany) in 0.2 M phosphate buffer pH 7.4, were put into a quartz cuvette. Afterwards, 10–50 μl of the supernatant was gently mixed with the buffer for 10 s with a tip. The kinetics of H2O2 consumption by catalase was then started and monitored at 240 nm for 2 min at 25°C. The H2O2 consumption/min in the buffer was converted to units of enzymatic activity on the basis of a standard curve obtained testing scalar units of bovine catalase (Sigma-Aldrich Srl). Units were corrected for protein content in the supernatant of cell lysate. Protein concentration was determined on the supernatants of cell lysates by Bradford reagent (Sigma-Aldrich Srl). Data were obtained from three different experiments performed in duplicate.

Western blot analysis

Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Harlow and Lane, 1988) supplemented with protease inhibitors cocktail (Roche, Mannheim, Germany). The protein concentration of extracts was estimated with Bradford reagent (Bio-Rad, Milan, Italy). Equal amounts of proteins (30 μg), or proteins from equal amount of cells (1 × 106 cells, in the experiments with actD or CHX) were separated on acrylamide SDS–PAGE, transferred onto nitrocellulose (Amersham Biosciences, Milan, Italy) and then treated overnight at 4°C with the appropriate antibodies. Goat anti-tyrosinase antibody (1:1000) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), mouse anti-catalase antibody (1:1000) (Sigma-Aldrich Srl), mouse anti-MITF (C5) antibody (1:1000) (Abcam, Cambridge, UK) were used. For tyrosinase detection, a secondary bovine anti-goat IgG HRP-conjugated antibody (1:1000) (Santa Cruz Biotechnology Inc) was used, for catalase and MITF a secondary goat anti-mouse IgG HRP-conjugated antibody (1:3000) (Dako Cytomation, Glostrup, Denmark) was used. Membranes were then washed and specific bands visualized by enhanced chemiluminescence reagent (ECL) (Santa Cruz Biotechnology Inc). A subsequent hybridization with anti-tubulin (1:10 000) (Sigma-Aldrich Srl) was used as a loading control. Densitometric analysis was performed using a GS-800 Calibrated Image Densitometer (Bio-Rad Laboratories Srl). Results are relative to three independent experiments.

Immuno-precipitation analysis

Cells were lysed in RIPA buffer (Harlow and Lane, 1988) supplemented with protease inhibitors cocktail (Roche) and phosphatase inhibitors (1 mM sodium orthovanadate, 1 mM NaF) (both from Sigma-Aldrich Srl). The protein concentration of extracts was estimated with Bradford reagent (Bio-Rad). Equal amounts of protein (1 mg) were then immunoprecipitated overnight at 4°C with a sheep anti-human Cat antibody (1:150) (Biodesign International, Industrial Park Saco, Maine). Immunocomplexes, aggregated with 70 μl of GammaBindTM G SepharoseTM (Amersham Biosciences Europe, Gmbh, Cologno Monzese, Milan, Italy), were collected by centrifugation and washed three times with lysis buffer. For the detection of catalase-pSer, immunoprecipitated Cat was resolved under reducing conditions by 10% SDS–PAGE and processed for immuno-blotting as described above, incubating the membranes with mouse anti-phosphoserine (pSer) IgG antibody (1:1000) (Sigma-Aldrich Srl).

In vitro incubation of cell lysate with PKA

Cells were plated in 6 cm diameter tissue culture dishes at a density of 1 × 106 cells/well and left to grow overnight. The following day, cell membranes were disrupted by putting cells twice in liquid nitrogen and centrifuged at 10 000g for 10 min at 4°C. PKA kinase reaction was performed on the cell lysate following a method previously described (Yano and Yano, 2002). Briefly, the reaction was initiated by the addition of PKA catalytic subunit (200 mU) (Sigma-Aldrich Srl) in 100 μl of the reaction mixture consisting of 50 mM MES, pH 6.9, 10 mM MgCl2, 0.5 mM EDTA, 1 mM DTT and 150 μM ATP. An aliquot removed at the indicated time point (30 min to 6 h) was diluted with distilled water by 100-fold, and the catalase activity in the aliquot was assayed, as described above.

Small interfering RNA-mediated downregulation of MITF

Cells were plated in a 6-well plate at a density of 1.3 × 105 cells/well and left to grow overnight. The following day, cells were transfected with 50 nM of small interfering RNAs (siRNA) dimers and 8 μl of LipofectamineTM reagent (Invitrogen, Milan, Italy). siGENOME SMART pool reagent against mouse MITF (Dharmacon Inc, Lafayette, CO, USA) was used to interfere with MITF expression. A non-specific siRNA was used as a negative control. After 24 h, transfected cells were treated with 10−7 M α-MSH for 24 h and then analysed for protein expression by immuno-blotting, as described above.

Total RNA isolation

Cells were plated in 6 cm diameter tissue culture dishes at a density of 1 × 106 cells/well and left to grow overnight. The following day, cells were treated with 10−7 M α-MSH for different time points (2–4–6 and 24 h). After treatment, cells were washed with PBS and harvested for RT-PCR analysis. The total RNA was isolated using the TRIzol method (Invitrogen) according to the manufacturer’s procedure. Briefly, cells were homogenized in 1 ml TRIzol reagent and then extracted with 0.2 ml CHCl3. Isopropanol (0.5 ml) was added to the aqueous phase. The RNA pellets were washed with 75% ethanol and dissolved in RNase-free water. Total RNA quantity, purity and the absence of ribonuclease digestion were assessed by OD260/280 absorbance measurements and by agarose gel electrophoresis. Total RNA samples were stored at −80°C until use.

Reverse transcriptase-PCR

The oligonucleotide primers for PCR were synthetized by MWG-Biotech AG (Italy) and were as follows: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sense: 5′-GCACCACCAACTGCTTAGC-3′ and antisense: 5′-TGCTCAGTGTAGCCCAGG-3′; catalase sense: 5′-TACCTGTGAACTGTCCCTACCG-3′ and antisense: 5′-GAATTGCGTTCTTAGGCTTCTC-3′. Reverse transcriptase-PCR was carried out using 1 μg of total RNA. After denaturation in diethylpyrocarbonate-treated water (Promega Corporation, Madison, WI, USA) at 70°C for 10 min, RNA was reverse transcribed into cDNA using ImProm-IITM Reverse Transcriptase (1 μl per reaction; Promega) and 0.5 μg of oligo(dT) primers at 42°C for 60 min in a total volume of 20 μl ImProm-IITM 5× reaction buffer (Promega) containing 1.5 mM MgCl2, 0.5 mM dNTP, and 20 U RNase inhibitor. Reverse transcriptase was inactivated at 70°C for 15 min and the RNA template was digested by RNase H at 37°C for 30 min. In each case, samples containing no reverse transcriptase (negative control) were included to exclude amplification from contaminating DNA. PCR reaction was carried out in the PCR Master Mix buffer (Promega) (25 μl total volume containing 1 μl cDNA, 20 pmol of oligonucleotide primers, 1 U Taq DNA polymerase, 1.5 mM MgCl2 and 0.2 mM dNTP). PCR amplification was performed with an iCycler thermal cycler (Bio-Rad, Hercules, CA, USA). Each cycle consisted of 1 min at 94°C, 1 min at 56°C for annealing and 1 min at 72°C for a total of 32 cycles. The final product was extended for 5 min at 72°C. In every case, samples containing no reverse transcriptase (negative control) were included to exclude amplification from contaminating DNA. PCR products were visualized by staining with ethidium bromide after separation on 1.5% tris-acetate-ethylenediamine tretraacetic acid (TAE)-agarose gel. To semiquantitate the relative amounts of catalase mRNA, the band intensities were related densitometrically to the respective GAPDH PCR product of the same sample. Densitometric evaluation was performed using UVIDocMw software (Bio-Rad).

Immunofluorescence and confocal microscopy

Cells grown on coverslips previously coated with 2% gelatin onto 24-well plates and treated with α-MSH 10−7 M (for 2, 4, 6 and 24 h) and forskolin 1 μM (for 24 h), were fixed in cold methanol for 4 min at −20°C. For single immunolabelling experiments, cells were incubated for 1 h at 25°C with anti-catalase monoclonal antibody (1:200 in PBS) (Sigma-Aldrich Srl). The primary antibody was then visualized with goat anti-mouse IgG-FITC antibody (1:200 in PBS) (Chemicon International, Temecula, CA, USA) after appropriate washing with PBS. For double immunolabelling experiments, cells were incubated with the following primary antibodies: anti-tyrosinase polyclonal antibody (1:50 in PBS) (C-19, Santa Cruz Biotechnology Inc) and anti-catalase monoclonal antibody (1:200 in PBS) (Sigma-Aldrich Srl) followed by the secondary antibodies chicken anti-goat IgG-Alexa Fluor 488 (1:1000 in PBS) (Molecular Probes-Invitrogen Life Sciences, Milan, Italy) and rabbit anti-mouse IgG-Texas Red (1:200 in PBS; Jackson ImmunoResearch Laboratories Inc., West Grover, PA, USA) after appropriate washing with PBS. Fluorescence signals were analysed by confocal vertical (x-z) sections (interval: 0.5 μm) obtained with a Zeiss Confocal Laser Scan Microscope (Zeiss, Oberkochen, Germany).

Statistical analysis

Data are presented as means ± SD. Student’s t test was used to analyse differences. Values of P < 0.05 were considered significant.

Acknowledgements

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Materials and methods
  8. Acknowledgements
  9. References

This work has been partially supported by onc_ord 32/07 Grant from ‘Ministero della Salute’, Italy.

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  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Materials and methods
  8. Acknowledgements
  9. References
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