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Retinol up-regulates the receptor for advanced glycation endproducts (RAGE) by increasing intracellular reactive species
Toxicology in Vitro 22 (2008) 1123–1127
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Retinol up-regulates the receptor for advanced glycation endproducts (RAGE) by increasing intracellular reactive species Daniel Pens Gelain *, Matheus Augusto de Bittencourt Pasquali, Fernanda Freitas Caregnato, Alfeu Zanotto-Filho, José Cláudio Fonseca Moreira Centro de Estudos em Estresse Oxidativo, Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Laboratório 32, Rua Ramiro Barcelos 2600 anexo, CEP 90035-003, Porto Alegre, RS, Brazil
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Article history: Received 10 January 2008 Accepted 26 February 2008 Available online 5 March 2008
Keywords: Retinol RAGE AGE Free radicals Oxidative stress
a b s t r a c t Retinol (vitamin A) and other retinoids have been suggested to exert an important antioxidant function in biological systems, besides their more established role as regulators of cell growth and differentiation. On the other hand, many authors have recently observed pro-oxidant activities of vitamin A and other retinoids in vitro and in vivo, resulting in cell death and/or transformation associated to increased oxidative damage. However, the mechanisms by which retinol causes oxidative stress are still not fully understood. Receptors for advanced glycation endproducts (RAGE) have been recently implied as promoters and/or amplifiers of oxidant-mediated cell death induced by diverse agents, and increased RAGE expression is observed in conditions related to unbalanced production of reactive species, such as in atherosclerosis and neurodegeneration. In the present work, we observed that retinol supplementation increases RAGE protein expression in cultured Sertoli cells, and antioxidant co-treatment reversed this effect. Retinolincreased RAGE expression was observed only at concentrations that induce intracellular reactive species production, as assessed by the DCFH assay. These results indicate that retinol is able to increase RAGE expression by an oxidant-dependent mechanism, and suggest that RAGE signaling may be involved in some of the deleterious effects observed in some retinol-supplementation therapies. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Retinol (vitamin A) is a major regulator of cell cycle, controlling apoptosis, cell division and differentiation. On the other hand, the full range of the biological activities of retinol, along with their mechanisms, is still not fully understood. Besides the more established mechanism involving the activation of the retinoid nuclear receptors RAR and RXR (Marill et al., 2003), many authors advocate that some biological properties of retinoids are related to their ability to scavenge toxic forms of oxygen and other free radicals in living systems (Palace et al., 1999). Nonetheless, a growing body of evidence has been suggesting that retinol and other retinoid derivatives have pro-oxidant properties, which might lead to cell oxidative damage, transformation and/or cell death (Polyakov et al., 2001; Mayne et al., 1991). It was observed that retinol may induce DNA oxidative damage in vitro (Murata and Kawanishi, 2000), and that cultured cells incubated with retinol had increased lipid, protein and DNA damage, and enhanced antioxidant enzyme activities, such as catalase, superoxide dismutase and glutathione peroxidase (Dal-Pizzol
* Corresponding author. Tel.: +55 51 3308 5578; fax: +55 51 3308 5535. E-mail address: [email protected] (D.P. Gelain). 0887-2333/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2008.02.016
et al., 2000, 2001). Thus, retinoids may also promote oxidative stress in living systems. Under conditions of unbalanced reactive species production and oxidative stress, advanced glycation end-products (AGEs) are generated by the sequential non-enzymatic glycation of protein amino groups and by oxidation reactions (Brownlee et al., 1988). AGE accumulation in cells alters the intracellular redox balance and triggers a vicious cycle between oxidative stress and further AGE generation (Yan et al., 1994). AGEs also activate different cell surface receptors which alter the function of many proteins, including antioxidant and metabolic enzymes, calcium channels, lipoproteins, and transcriptional and structural proteins (Vasdev et al., 2007). The best characterized AGE receptor is the so-called RAGE (Receptor for Advanced Glycation Endproducts), which belongs to the IgG superfamiliy of cell surface molecules, and most of the deleterious effects caused by AGEs are ascribed to RAGE activation (Neeper et al., 1992). In the present work, we investigated the effect of retinol treatment on RAGE protein immunocontent in cultured Sertoli cells, a well-characterized model to study physiological as well as pro-oxidant actions of vitamin A (Silva et al., 2002; Dalmolin et al., 2007; Klamt et al., 2008). We report here, for the first time, that retinol up-regulates RAGE by a mechanism dependent on the generation
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of intracellular reactive species. This data suggests that RAGE upregulation may exert a role in the deleterious effects observed in some retinol supplementation therapies.
bility measurements, at the end of 24 h of retinol treatment, MTT was added to the wells and the MTT assay was performed as described below. Sertoli cells cultures were estimated to be 90–95% pure, as assessed by the alkaline phosphatase assay.
2. Materials and methods
2.3. DCFH-DA assay
2.1. Chemicals and animals
Intracellular reactive species production was determined by the DCFH-DA-based real-time assay using intact living cells, as described by Wang and Joseph (1999). Briefly, Sertoli cells were plated onto 96-well plates and incubated for 1 h with DCFH-DA 100 lM (stock solution in DMSO, 10 mM) in 1% FBS culture medium at 5% CO2 and 37 °C. Then cells were washed and treatments were carried out. During treatment, changes in the fluorescence by the oxidation of DCFH into the fluorogen DCF were monitored in a microplate fluorescence reader (F2000, Hitachi Ltd., Tokyo, Japan) for 1 h at 37 ° C. H2O2 1 mM was used as positive control for intracellular reactive species production. Excitation filter was set at 485 ± 10 nm and the emission filter was set at 530 ± 12.5 nm. Data were recorded every 30 s and plotted in Excel software.
Pregnant Wistar rats were housed individually in Plexiglas cages. Litters were restricted to eight pups each. Animals were maintained on a 12-h light/dark cycle at a constant temperature of 23 °C, with free access to commercial food and water. Male immature rats (15 days old) were killed by cervical dislocation. All-trans retinol alcohol, Trolox, 20 ,70 -dichlorohydrofluorescein diacetate (DCFH-DA), 3-(4,5-dimethyl)-2,5-diphenyl tetrazolium bromide (MTT), Tween-20, and b-mercaptoethanol were from Sigma Chemical Co. (St Louis, MO, USA). Retinol was dissolved in ethanol. Concentrated stocks were prepared immediately before experiments by diluting retinol into ethanol and determining final stock concentration by UV absorption; solution was kept protected from light and temperature during all procedures. Appropriate solvent controls were performed for each condition. Treatments were initiated by adding concentrated solutions to reach final concentrations in the well. The final ethanol concentration did not exceed 0.2% in any experiment. Tissue culture reagents were from Gibco (Invitrogen Corporation, Carlsbad, CA, USA) and were of tissue culture grade. Rabbit polyclonal anti-RAGE and anti-b-actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis (PAGE) reagents were from Bio-Rad Laboratories (Hercules, CA, USA), nitrocellulose membrane (Hybond ECL), enhanced chemiluminescence kit (ECL plus), and anti-rabbit immunoglobulin (horseradish peroxidase-linked whole antibody from donkey) were from Amersham Pharmacia Biotech (Amersham, UK). 2.2. Isolation and culture of Sertoli cells and assays Sertoli cells were isolated as previously described (Dalmolin et al., 2007). Briefly, testes of 15-day-old rats were removed, decapsulated and digested enzymatically with trypsin and deoxyribonuclease for 30 min at 34 °C, and centrifuged at 750g for 5 min. The pellet was mixed soybean trypsin inhibitor, then centrifuged and incubated with collagenase and hyaluronidase for 30 min at 34 °C. After incubation, this fraction was centrifuged (10 min at 40g). The pellet was taken to isolate Sertoli cells and supernatant was discarded. After counting, Sertoli cells were plated in 6x dishes multi-well plates (3 105 cells/cm2) in Medium 199 pH 7.4 1% FBS, and maintained in humidified 5% CO2 atmosphere at 34 °C for 24 h for attachment. The medium was then changed to serum-free medium and cells were maintained for more 24 h. Retinol treatment to Sertoli cell cultures was then initiated at this stage. Medium was replaced by fresh serum-free medium and treatments were immediately initiated by adding concentrated solutions of retinol (dissolved in ethanol) or Trolox (dissolved in water) to reach final concentrations in the well. The final ethanol concentration did not exceed 0.2% in any experiment. Vehicle controls with this concentration of ethanol were performed for each condition, showing no alterations. At the end of 24 h of treatments under the conditions mentioned above, cells were used for assay by the following procedures: for DCFH-DA assay, incubation medium was replaced by the fresh medium containing 1% FBS and DCFH-DA 100 lM and assayed as described below. For immunoblot, retinol incubation was stopped by removal of the incubation medium and addition of Laemmli-sample buffer, followed by the procedures described below at ‘‘immunoblot” subsection. For via-
2.4. Immunoblot To perform immunoblot experiments, Sertoli cells were lysed in Laemmli-sample buffer (62.5 mM Tris–HCl, pH 6.8, 1% (w/v) SDS, 10% (v/v) glycerol) and equal amounts of cell protein (30 lg/well) were fractionated by SDS-PAGE and electro-blotted onto nitrocellulose membranes. Protein loading and electro-blotting efficiency were verified through Ponceau S staining, and the membrane was blocked in Tween–Tris buffered saline (TTBS: 100 mM Tris– HCl, pH 7.5, containing 0.9% NaCl and 0.1% Tween-20) containing 5% albumin. Membranes were incubated overnight at 4 °C with rabbit polyclonal antibody against the amino acids 1–300 of RAGE (dilution range 1:400) in the presence of 5% milk, or anti-b-actin 1:2000, and then washed with TTBS. Anti-rabbit IgG peroxidaselinked secondary antibody was incubated with the membranes for additional 1 h (1:5000 dilution range), washed again and the immunoreactivity was detected by enhanced chemiluminescence using ECL Plus kit. Densitometric analysis of the films was performed with ImageQuant software. Blots were developed to be linear in the range used for densitometry. All results were expressed as a relative ratio between RAGE immunocontent and the b-actin internal control immunocontent. 2.5. MTT assay Following retinol treatment, Sertoli cells viability was assessed by the MTT assay. This method is based on the ability of viable cells to reduce MTT (3-(4,5-dimethyl)-2,5-diphenyl tetrazolium bromide) and form a blue formazan product. MTT solution (sterile stock solution of 5 mg/ml) was added to the incubation medium in the wells at a final concentration of 0.2 mg/ml. The cells were left for 45 min at 37 °C in a humidified 5% CO2 atmosphere. The medium was then removed and plates were shaken with DMSO for 30 min. The optical density of each well was measured at 550 nm (test) and 690 nm (reference). H2O2 1 mM was used as positive control for cell death. An in vitro control experiment was performed with varying concentrations of retinol (1–20 lM) incubated for varying times with MTT (0.2 mg/ml), but no alterations on fluorescence have been observed (not shown).
3. Results Sertoli cells were incubated with increasing concentrations of retinol for 24 h and intracellular reactive species production was
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Fig. 1. Intracellular reactive species production and cell viability. Sertoli cells were incubated for 24 h with increasing concentrations of retinol and used for assay. (A) Intracellular reactive species production was assessed by the real-time DCFH-DA assay as depicted in ‘‘Material and Methods”. The rate of DCF oxidation during 24 h was calculated and plotted as fold increase compared to control group (C). (B) Cell viability was measured by the MTT assay. Vehicle (ethanol 0.5%) had no effect in both assays (not shown). *p < 0.05 compared to control, as analyzed by ANOVA with Duncan’s post-hoc test.
Fig. 2. RAGE immunocontent regulation by retinol. (A) Sertoli cells were incubated with ethanol 0.5% (Veh) and retinol 7 lM (Ret) for 24 h, and RAGE immunocontent was determined by western blot. RAGE immunoreactivity was quantified with the ImageQuant 5.1 software and normalized with the immunoreactivity of b-actin. (B) The effect of increasing concentrations of retinol on RAGE was assessed by the same method. (C) Antioxidant co-treatment with Trolox 100 lM (T) inhibits the effect of retinol 7 lM (R). (D) To confirm the antioxidant effect of Trolox, intracellular reactive species in cells treated with 24 h was assessed by the DCFH-DA assay. *p < 0.05, **p < 0.01 compared to control; #p < 0.05 compared to retinol treatment, as analyzed by ANOVA (Duncan’s post-hoc test).
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evaluated by the real-time DCFH assay. Retinol enhanced the rate of intracellular reactive species production in a dose-dependent manner, reaching a maximum effect at 7 lM (Fig. 1). At the end of the 24 h period of incubation, cell viability was assessed by MTT (Fig. 1b). Retinol at the concentration of 14 lM significantly decreased cell viability. Based on these results, we used retinol 7 lM for further experiments. To assess the effect of retinol on RAGE expression, we incubated Sertoli cells for 24 h with retinol 7 lM and performed an immunoblot using a polyclonal antibody against the amino acids 1 and 300 of the RAGE protein. This antibody reacts both the soluble (extracellular) and the membrane-attached domains of the RAGE protein, which are detected as two distinct bands at approximately 50 and 46 kDa (Dattilo et al., 2007). Retinol increased RAGE immunoreactivity by 2.5-fold (Fig. 2A), and this effect had a dose-dependent response (Fig. 2B). To establish the involvement of intracellular reactive species in this effect, we co-incubated Sertoli cells with the antioxidant Trolox, a hydrophilic analogue of a-tocopherol. Trolox 100 lM co-incubation reversed the increase in RAGE immunocontent by retinol (Fig. 2C). The antioxidant treatment with Trolox also inhibited the production of intracellular reactive species by retinol, as assessed by the real-time DCFH assay (Fig. 2D).
4. Discussion Diverse works have observed that vitamin A and other retinoids presented strong antioxidant properties in biological systems (see Hix et al., 2004, for review). Free radicals and other forms of reactive species have been correlated with increased risk and incidence of diverse diseases, such as atherosclerosis, hypertension, inflammation, Parkinson’s and Alzheimer’s diseases, type-2 diabetes and cancer, among others. In some cases, serum retinol deficiency was reported in these diseases (King et al., 1992; Glasø et al., 2004; Sarni et al., 2005), and the supplementation of retinoids in the diet or as pharmacological agents have been proposed as ‘‘antioxidant therapies” for treatment and/or prevention of these conditions. Clinical trials have been carried out based on the potential antioxidant role of retinoids, but in some studies retinoid supplementations had to be discontinued due to the increased mortality related to lung cancer and cardiovascular disease incidence (Omenn et al., 1994; Omenn, 2007). Cell culture as well as other in vitro assays confirmed that retinoids also presented cytotoxic and/or pro-oxidant effects, causing oxidative damage to biomolecules (Murata and Kawanishi, 2000; Penniston and Tanumihardjo, 2006; Dal-Pizzol et al., 2000, 2001; Klamt et al., 2003). Cultured Sertoli cells are considered a good model to study retinol actions at cellular level, as vitamin A physiologically regulates many reproductive-related functions of these cells (Silva et al., 2002). We previously observed that specific concentrations of retinol (above 5 lM) are able to increase intracellular reactive species formation, and this led to DNA damage, pre-neoplasic transformation and increased cell proliferation (Klamt et al., 2003; Dal-Pizzol et al., 2000). In endothelial cells, mononuclear phagocytes and adipocytes, increased RAGE expression and activation elevate intracellular reactive species generation by two main mechanisms: NAPDH oxidase activation and enhanced mitochondrial function (Coughlan et al., 2007). In these conditions, the unbalanced production of reactive species activates redox-sensitive signaling pathways such as ERK1/2, PKC and NF-kB (Nerlich et al., 2007; Fuentes et al., 2007), which may lead to a cyclic increase in RAGE expression and enhancement of deleterious effects arising from its activation, especially in conditions of increased production of RAGE agonists such as amphoterin and AGEs (Stern et al., 2002). Here, we observed that retinol induced an increase in RAGE immunocontent in Sertoli cells by a mechanism dependent on
intracellular reactive species formation. Several evidences presented here indicate that intracellular reactive species formation induced by retinol treatment is causing this effect. First, retinol concentrations up to 5 lM did not induce intracellular reactive species formation (Fig. 1), and also did not affect RAGE expression (Fig. 2). Second, retinol pro-oxidant concentrations (7 lM and above) increased RAGE immunocontent in Sertoli cells after 24 h, and the antioxidant co-treatment with Trolox inhibited this effect. As Trolox abolished intracellular reactive species formation by retinol 7 lM (Fig. 2D), this strongly suggests that the increase in RAGE immunocontent by retinol is a redox-dependent effect. In the concentration range between 2 and 5 lM, retinol is known to induce the expression of several genes in Sertoli cells, which in turn results in increased immunocontent of several proteins, such as Id2 and Id3 (Buzzard et al., 2003). However, concentrations up to 5 lM did not have any effect on RAGE immunocontent in Sertoli cells, reinforcing our suggestion that retinol up-regulates RAGE by a non-classical mechanism, dependent on intracellular reactive species generation. This is the first time that an increase in RAGE expression was observed to be caused by a redox-dependent mechanism. The consequences of RAGE up-regulation by retinol may vary, but most works report that, under conditions of oxidative stress, these consequences are generally deleterious. RAGE activation was reported to induce PKC delta and NADPH oxidase in neurons, leading to neuronal cell death in vitro (Nitti et al., 2007, 2005). RAGE activators accumulate not only in diabetes, as it was first suggested, but also in conditions of enhanced oxidative stress, inflammation and neurodegeneration (Ramasamy et al., 2005; Smith et al., 1995). As we have seen in previous reports, retinoids supplementation protocols have been applied to some clinical therapies, but side-effects related to increased reactive species generation and oxidative damage to biomolecules have been generally neglected. It is possible that many of the deleterious effects observed in some clinical trials with retinoids may be related to the oxidant-dependent RAGE up-regulation observed here. The ATBC and the CARET trials that evaluated the effect of retinol supplementation on the prevention of lung cancer, for instance, had increased mortality related to lung cancer and cardiovascular disease (Omenn et al., 1994; Omenn, 2007), and both tissues express high levels of RAGE (Stern et al., 2002). It is not known whether the deleterious effects observed at clinical trials using vitamin A are related to modifications in the redox state of cells. However, it is well known that retinol supplementation increases dramatically the serum concentrations of vitamin A in the alcohol form (from 0.1 lM to about 3.5–4.0 lM), and also in other forms, such as retinoic acid, retinyl esters and other metabolites (Penniston and Tanumihardjo, 2006). This is reflected in an increase in cellular uptake, storage and metabolism of retinol in vitamin A-storing cells. In hepatic stellate cells, the main site of retinol storage in liver, physiologic concentrations of retinol have been reported to range between 2 and 5 lM (Ross, 1993), and liver retinol content was observed to increase from 3 up to 20-fold (levels considered toxic) in adults taking highly enriched supplementations (Allen and Haskell, 2002). Physiologic concentrations of retinol in Sertoli cells were also reported to range between 2 and 5 lM (Napoli et al., 1993), but there is no information available concerning the vitamin A status in Sertoli cells of animals or humans taking vitamin A supplementation. Here, we compared retinol concentrations considered physiologic (from 2 up to 5 lM) with a range of supra-physiologic concentrations (7 up to 14 lM). It is important to point that concentrations of retinol only slightly above the level considered physiologic (i.e., 7 lM, compared to 5 lM) was able to induce reactive species production and alter RAGE immunocontent, and these concentrations may be readily achievable at some supplementation protocols.
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Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Retinol up-regulates the receptor for advanced glycation endproducts (RAGE) by increasing intracellular reactive species Daniel Pens Gelain *, Matheus Augusto de Bittencourt Pasquali, Fernanda Freitas Caregnato, Alfeu Zanotto-Filho, José Cláudio Fonseca Moreira Centro de Estudos em Estresse Oxidativo, Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Laboratório 32, Rua Ramiro Barcelos 2600 anexo, CEP 90035-003, Porto Alegre, RS, Brazil
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Article history: Received 10 January 2008 Accepted 26 February 2008 Available online 5 March 2008
Keywords: Retinol RAGE AGE Free radicals Oxidative stress
a b s t r a c t Retinol (vitamin A) and other retinoids have been suggested to exert an important antioxidant function in biological systems, besides their more established role as regulators of cell growth and differentiation. On the other hand, many authors have recently observed pro-oxidant activities of vitamin A and other retinoids in vitro and in vivo, resulting in cell death and/or transformation associated to increased oxidative damage. However, the mechanisms by which retinol causes oxidative stress are still not fully understood. Receptors for advanced glycation endproducts (RAGE) have been recently implied as promoters and/or amplifiers of oxidant-mediated cell death induced by diverse agents, and increased RAGE expression is observed in conditions related to unbalanced production of reactive species, such as in atherosclerosis and neurodegeneration. In the present work, we observed that retinol supplementation increases RAGE protein expression in cultured Sertoli cells, and antioxidant co-treatment reversed this effect. Retinolincreased RAGE expression was observed only at concentrations that induce intracellular reactive species production, as assessed by the DCFH assay. These results indicate that retinol is able to increase RAGE expression by an oxidant-dependent mechanism, and suggest that RAGE signaling may be involved in some of the deleterious effects observed in some retinol-supplementation therapies. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Retinol (vitamin A) is a major regulator of cell cycle, controlling apoptosis, cell division and differentiation. On the other hand, the full range of the biological activities of retinol, along with their mechanisms, is still not fully understood. Besides the more established mechanism involving the activation of the retinoid nuclear receptors RAR and RXR (Marill et al., 2003), many authors advocate that some biological properties of retinoids are related to their ability to scavenge toxic forms of oxygen and other free radicals in living systems (Palace et al., 1999). Nonetheless, a growing body of evidence has been suggesting that retinol and other retinoid derivatives have pro-oxidant properties, which might lead to cell oxidative damage, transformation and/or cell death (Polyakov et al., 2001; Mayne et al., 1991). It was observed that retinol may induce DNA oxidative damage in vitro (Murata and Kawanishi, 2000), and that cultured cells incubated with retinol had increased lipid, protein and DNA damage, and enhanced antioxidant enzyme activities, such as catalase, superoxide dismutase and glutathione peroxidase (Dal-Pizzol
* Corresponding author. Tel.: +55 51 3308 5578; fax: +55 51 3308 5535. E-mail address: [email protected] (D.P. Gelain). 0887-2333/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2008.02.016
et al., 2000, 2001). Thus, retinoids may also promote oxidative stress in living systems. Under conditions of unbalanced reactive species production and oxidative stress, advanced glycation end-products (AGEs) are generated by the sequential non-enzymatic glycation of protein amino groups and by oxidation reactions (Brownlee et al., 1988). AGE accumulation in cells alters the intracellular redox balance and triggers a vicious cycle between oxidative stress and further AGE generation (Yan et al., 1994). AGEs also activate different cell surface receptors which alter the function of many proteins, including antioxidant and metabolic enzymes, calcium channels, lipoproteins, and transcriptional and structural proteins (Vasdev et al., 2007). The best characterized AGE receptor is the so-called RAGE (Receptor for Advanced Glycation Endproducts), which belongs to the IgG superfamiliy of cell surface molecules, and most of the deleterious effects caused by AGEs are ascribed to RAGE activation (Neeper et al., 1992). In the present work, we investigated the effect of retinol treatment on RAGE protein immunocontent in cultured Sertoli cells, a well-characterized model to study physiological as well as pro-oxidant actions of vitamin A (Silva et al., 2002; Dalmolin et al., 2007; Klamt et al., 2008). We report here, for the first time, that retinol up-regulates RAGE by a mechanism dependent on the generation
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of intracellular reactive species. This data suggests that RAGE upregulation may exert a role in the deleterious effects observed in some retinol supplementation therapies.
bility measurements, at the end of 24 h of retinol treatment, MTT was added to the wells and the MTT assay was performed as described below. Sertoli cells cultures were estimated to be 90–95% pure, as assessed by the alkaline phosphatase assay.
2. Materials and methods
2.3. DCFH-DA assay
2.1. Chemicals and animals
Intracellular reactive species production was determined by the DCFH-DA-based real-time assay using intact living cells, as described by Wang and Joseph (1999). Briefly, Sertoli cells were plated onto 96-well plates and incubated for 1 h with DCFH-DA 100 lM (stock solution in DMSO, 10 mM) in 1% FBS culture medium at 5% CO2 and 37 °C. Then cells were washed and treatments were carried out. During treatment, changes in the fluorescence by the oxidation of DCFH into the fluorogen DCF were monitored in a microplate fluorescence reader (F2000, Hitachi Ltd., Tokyo, Japan) for 1 h at 37 ° C. H2O2 1 mM was used as positive control for intracellular reactive species production. Excitation filter was set at 485 ± 10 nm and the emission filter was set at 530 ± 12.5 nm. Data were recorded every 30 s and plotted in Excel software.
Pregnant Wistar rats were housed individually in Plexiglas cages. Litters were restricted to eight pups each. Animals were maintained on a 12-h light/dark cycle at a constant temperature of 23 °C, with free access to commercial food and water. Male immature rats (15 days old) were killed by cervical dislocation. All-trans retinol alcohol, Trolox, 20 ,70 -dichlorohydrofluorescein diacetate (DCFH-DA), 3-(4,5-dimethyl)-2,5-diphenyl tetrazolium bromide (MTT), Tween-20, and b-mercaptoethanol were from Sigma Chemical Co. (St Louis, MO, USA). Retinol was dissolved in ethanol. Concentrated stocks were prepared immediately before experiments by diluting retinol into ethanol and determining final stock concentration by UV absorption; solution was kept protected from light and temperature during all procedures. Appropriate solvent controls were performed for each condition. Treatments were initiated by adding concentrated solutions to reach final concentrations in the well. The final ethanol concentration did not exceed 0.2% in any experiment. Tissue culture reagents were from Gibco (Invitrogen Corporation, Carlsbad, CA, USA) and were of tissue culture grade. Rabbit polyclonal anti-RAGE and anti-b-actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis (PAGE) reagents were from Bio-Rad Laboratories (Hercules, CA, USA), nitrocellulose membrane (Hybond ECL), enhanced chemiluminescence kit (ECL plus), and anti-rabbit immunoglobulin (horseradish peroxidase-linked whole antibody from donkey) were from Amersham Pharmacia Biotech (Amersham, UK). 2.2. Isolation and culture of Sertoli cells and assays Sertoli cells were isolated as previously described (Dalmolin et al., 2007). Briefly, testes of 15-day-old rats were removed, decapsulated and digested enzymatically with trypsin and deoxyribonuclease for 30 min at 34 °C, and centrifuged at 750g for 5 min. The pellet was mixed soybean trypsin inhibitor, then centrifuged and incubated with collagenase and hyaluronidase for 30 min at 34 °C. After incubation, this fraction was centrifuged (10 min at 40g). The pellet was taken to isolate Sertoli cells and supernatant was discarded. After counting, Sertoli cells were plated in 6x dishes multi-well plates (3 105 cells/cm2) in Medium 199 pH 7.4 1% FBS, and maintained in humidified 5% CO2 atmosphere at 34 °C for 24 h for attachment. The medium was then changed to serum-free medium and cells were maintained for more 24 h. Retinol treatment to Sertoli cell cultures was then initiated at this stage. Medium was replaced by fresh serum-free medium and treatments were immediately initiated by adding concentrated solutions of retinol (dissolved in ethanol) or Trolox (dissolved in water) to reach final concentrations in the well. The final ethanol concentration did not exceed 0.2% in any experiment. Vehicle controls with this concentration of ethanol were performed for each condition, showing no alterations. At the end of 24 h of treatments under the conditions mentioned above, cells were used for assay by the following procedures: for DCFH-DA assay, incubation medium was replaced by the fresh medium containing 1% FBS and DCFH-DA 100 lM and assayed as described below. For immunoblot, retinol incubation was stopped by removal of the incubation medium and addition of Laemmli-sample buffer, followed by the procedures described below at ‘‘immunoblot” subsection. For via-
2.4. Immunoblot To perform immunoblot experiments, Sertoli cells were lysed in Laemmli-sample buffer (62.5 mM Tris–HCl, pH 6.8, 1% (w/v) SDS, 10% (v/v) glycerol) and equal amounts of cell protein (30 lg/well) were fractionated by SDS-PAGE and electro-blotted onto nitrocellulose membranes. Protein loading and electro-blotting efficiency were verified through Ponceau S staining, and the membrane was blocked in Tween–Tris buffered saline (TTBS: 100 mM Tris– HCl, pH 7.5, containing 0.9% NaCl and 0.1% Tween-20) containing 5% albumin. Membranes were incubated overnight at 4 °C with rabbit polyclonal antibody against the amino acids 1–300 of RAGE (dilution range 1:400) in the presence of 5% milk, or anti-b-actin 1:2000, and then washed with TTBS. Anti-rabbit IgG peroxidaselinked secondary antibody was incubated with the membranes for additional 1 h (1:5000 dilution range), washed again and the immunoreactivity was detected by enhanced chemiluminescence using ECL Plus kit. Densitometric analysis of the films was performed with ImageQuant software. Blots were developed to be linear in the range used for densitometry. All results were expressed as a relative ratio between RAGE immunocontent and the b-actin internal control immunocontent. 2.5. MTT assay Following retinol treatment, Sertoli cells viability was assessed by the MTT assay. This method is based on the ability of viable cells to reduce MTT (3-(4,5-dimethyl)-2,5-diphenyl tetrazolium bromide) and form a blue formazan product. MTT solution (sterile stock solution of 5 mg/ml) was added to the incubation medium in the wells at a final concentration of 0.2 mg/ml. The cells were left for 45 min at 37 °C in a humidified 5% CO2 atmosphere. The medium was then removed and plates were shaken with DMSO for 30 min. The optical density of each well was measured at 550 nm (test) and 690 nm (reference). H2O2 1 mM was used as positive control for cell death. An in vitro control experiment was performed with varying concentrations of retinol (1–20 lM) incubated for varying times with MTT (0.2 mg/ml), but no alterations on fluorescence have been observed (not shown).
3. Results Sertoli cells were incubated with increasing concentrations of retinol for 24 h and intracellular reactive species production was
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Fig. 1. Intracellular reactive species production and cell viability. Sertoli cells were incubated for 24 h with increasing concentrations of retinol and used for assay. (A) Intracellular reactive species production was assessed by the real-time DCFH-DA assay as depicted in ‘‘Material and Methods”. The rate of DCF oxidation during 24 h was calculated and plotted as fold increase compared to control group (C). (B) Cell viability was measured by the MTT assay. Vehicle (ethanol 0.5%) had no effect in both assays (not shown). *p < 0.05 compared to control, as analyzed by ANOVA with Duncan’s post-hoc test.
Fig. 2. RAGE immunocontent regulation by retinol. (A) Sertoli cells were incubated with ethanol 0.5% (Veh) and retinol 7 lM (Ret) for 24 h, and RAGE immunocontent was determined by western blot. RAGE immunoreactivity was quantified with the ImageQuant 5.1 software and normalized with the immunoreactivity of b-actin. (B) The effect of increasing concentrations of retinol on RAGE was assessed by the same method. (C) Antioxidant co-treatment with Trolox 100 lM (T) inhibits the effect of retinol 7 lM (R). (D) To confirm the antioxidant effect of Trolox, intracellular reactive species in cells treated with 24 h was assessed by the DCFH-DA assay. *p < 0.05, **p < 0.01 compared to control; #p < 0.05 compared to retinol treatment, as analyzed by ANOVA (Duncan’s post-hoc test).
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evaluated by the real-time DCFH assay. Retinol enhanced the rate of intracellular reactive species production in a dose-dependent manner, reaching a maximum effect at 7 lM (Fig. 1). At the end of the 24 h period of incubation, cell viability was assessed by MTT (Fig. 1b). Retinol at the concentration of 14 lM significantly decreased cell viability. Based on these results, we used retinol 7 lM for further experiments. To assess the effect of retinol on RAGE expression, we incubated Sertoli cells for 24 h with retinol 7 lM and performed an immunoblot using a polyclonal antibody against the amino acids 1 and 300 of the RAGE protein. This antibody reacts both the soluble (extracellular) and the membrane-attached domains of the RAGE protein, which are detected as two distinct bands at approximately 50 and 46 kDa (Dattilo et al., 2007). Retinol increased RAGE immunoreactivity by 2.5-fold (Fig. 2A), and this effect had a dose-dependent response (Fig. 2B). To establish the involvement of intracellular reactive species in this effect, we co-incubated Sertoli cells with the antioxidant Trolox, a hydrophilic analogue of a-tocopherol. Trolox 100 lM co-incubation reversed the increase in RAGE immunocontent by retinol (Fig. 2C). The antioxidant treatment with Trolox also inhibited the production of intracellular reactive species by retinol, as assessed by the real-time DCFH assay (Fig. 2D).
4. Discussion Diverse works have observed that vitamin A and other retinoids presented strong antioxidant properties in biological systems (see Hix et al., 2004, for review). Free radicals and other forms of reactive species have been correlated with increased risk and incidence of diverse diseases, such as atherosclerosis, hypertension, inflammation, Parkinson’s and Alzheimer’s diseases, type-2 diabetes and cancer, among others. In some cases, serum retinol deficiency was reported in these diseases (King et al., 1992; Glasø et al., 2004; Sarni et al., 2005), and the supplementation of retinoids in the diet or as pharmacological agents have been proposed as ‘‘antioxidant therapies” for treatment and/or prevention of these conditions. Clinical trials have been carried out based on the potential antioxidant role of retinoids, but in some studies retinoid supplementations had to be discontinued due to the increased mortality related to lung cancer and cardiovascular disease incidence (Omenn et al., 1994; Omenn, 2007). Cell culture as well as other in vitro assays confirmed that retinoids also presented cytotoxic and/or pro-oxidant effects, causing oxidative damage to biomolecules (Murata and Kawanishi, 2000; Penniston and Tanumihardjo, 2006; Dal-Pizzol et al., 2000, 2001; Klamt et al., 2003). Cultured Sertoli cells are considered a good model to study retinol actions at cellular level, as vitamin A physiologically regulates many reproductive-related functions of these cells (Silva et al., 2002). We previously observed that specific concentrations of retinol (above 5 lM) are able to increase intracellular reactive species formation, and this led to DNA damage, pre-neoplasic transformation and increased cell proliferation (Klamt et al., 2003; Dal-Pizzol et al., 2000). In endothelial cells, mononuclear phagocytes and adipocytes, increased RAGE expression and activation elevate intracellular reactive species generation by two main mechanisms: NAPDH oxidase activation and enhanced mitochondrial function (Coughlan et al., 2007). In these conditions, the unbalanced production of reactive species activates redox-sensitive signaling pathways such as ERK1/2, PKC and NF-kB (Nerlich et al., 2007; Fuentes et al., 2007), which may lead to a cyclic increase in RAGE expression and enhancement of deleterious effects arising from its activation, especially in conditions of increased production of RAGE agonists such as amphoterin and AGEs (Stern et al., 2002). Here, we observed that retinol induced an increase in RAGE immunocontent in Sertoli cells by a mechanism dependent on
intracellular reactive species formation. Several evidences presented here indicate that intracellular reactive species formation induced by retinol treatment is causing this effect. First, retinol concentrations up to 5 lM did not induce intracellular reactive species formation (Fig. 1), and also did not affect RAGE expression (Fig. 2). Second, retinol pro-oxidant concentrations (7 lM and above) increased RAGE immunocontent in Sertoli cells after 24 h, and the antioxidant co-treatment with Trolox inhibited this effect. As Trolox abolished intracellular reactive species formation by retinol 7 lM (Fig. 2D), this strongly suggests that the increase in RAGE immunocontent by retinol is a redox-dependent effect. In the concentration range between 2 and 5 lM, retinol is known to induce the expression of several genes in Sertoli cells, which in turn results in increased immunocontent of several proteins, such as Id2 and Id3 (Buzzard et al., 2003). However, concentrations up to 5 lM did not have any effect on RAGE immunocontent in Sertoli cells, reinforcing our suggestion that retinol up-regulates RAGE by a non-classical mechanism, dependent on intracellular reactive species generation. This is the first time that an increase in RAGE expression was observed to be caused by a redox-dependent mechanism. The consequences of RAGE up-regulation by retinol may vary, but most works report that, under conditions of oxidative stress, these consequences are generally deleterious. RAGE activation was reported to induce PKC delta and NADPH oxidase in neurons, leading to neuronal cell death in vitro (Nitti et al., 2007, 2005). RAGE activators accumulate not only in diabetes, as it was first suggested, but also in conditions of enhanced oxidative stress, inflammation and neurodegeneration (Ramasamy et al., 2005; Smith et al., 1995). As we have seen in previous reports, retinoids supplementation protocols have been applied to some clinical therapies, but side-effects related to increased reactive species generation and oxidative damage to biomolecules have been generally neglected. It is possible that many of the deleterious effects observed in some clinical trials with retinoids may be related to the oxidant-dependent RAGE up-regulation observed here. The ATBC and the CARET trials that evaluated the effect of retinol supplementation on the prevention of lung cancer, for instance, had increased mortality related to lung cancer and cardiovascular disease (Omenn et al., 1994; Omenn, 2007), and both tissues express high levels of RAGE (Stern et al., 2002). It is not known whether the deleterious effects observed at clinical trials using vitamin A are related to modifications in the redox state of cells. However, it is well known that retinol supplementation increases dramatically the serum concentrations of vitamin A in the alcohol form (from 0.1 lM to about 3.5–4.0 lM), and also in other forms, such as retinoic acid, retinyl esters and other metabolites (Penniston and Tanumihardjo, 2006). This is reflected in an increase in cellular uptake, storage and metabolism of retinol in vitamin A-storing cells. In hepatic stellate cells, the main site of retinol storage in liver, physiologic concentrations of retinol have been reported to range between 2 and 5 lM (Ross, 1993), and liver retinol content was observed to increase from 3 up to 20-fold (levels considered toxic) in adults taking highly enriched supplementations (Allen and Haskell, 2002). Physiologic concentrations of retinol in Sertoli cells were also reported to range between 2 and 5 lM (Napoli et al., 1993), but there is no information available concerning the vitamin A status in Sertoli cells of animals or humans taking vitamin A supplementation. Here, we compared retinol concentrations considered physiologic (from 2 up to 5 lM) with a range of supra-physiologic concentrations (7 up to 14 lM). It is important to point that concentrations of retinol only slightly above the level considered physiologic (i.e., 7 lM, compared to 5 lM) was able to induce reactive species production and alter RAGE immunocontent, and these concentrations may be readily achievable at some supplementation protocols.
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