FEBS Letters
Volume 583, Issue 14 , Pages 2401-2406, 21 July 2009

Thyroid hormones reverse the UV-induced repression of APP in neuroblastoma cells

Edited by Jesus Avila

Instituto de Investigaciones Biomédicas (C.S.I.C.), Consejo Superior de Investigaciones Científicas, Arturo Duperier, 4, 28029 Madrid, Spain

Received 27 March 2009; received in revised form 2 June 2009; accepted 22 June 2009. published online 29 June 2009.

Article Outline

Abstract 

As a precursor of the neurotoxic amyloid-β peptide, APP plays a central role in Alzheimer’s disease. We have recently reported that the tumor suppressor p53 inhibits APP gene transcription through the same DNA sequences that mediate an inhibitory effect of thyroid hormones. Now, we have analyzed whether the thyroid hormone T3 can modulate the effects of p53 on APP expression. Exposition to UVC radiation leads to a marked decrease of intracellular APP levels that is paradoxically reversed by T3. Repression by UVC and reversion by the hormone are not observed in cells depleted of p53, demonstrating a p53-dependent mechanism. These results suggest the existence of a cross-talk between p53 and T3 that could play an important role in Alzheimer´s disease.

Keywords: Alzheimer’s disease, Amyloid precursor protein, Neuroblastoma cell, p53, Thyroid hormone, Gene expression

 

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1. Introduction 

Alzheimer’s disease is a neurodegenerative disorder of the human central nervous system that causes mental deterioration and progressive dementia. The disease is characterized by a massive loss of neurons that is accompanied by neuropathological lesions, including the formation of senile plaques that are mainly composed of β-amyloid protein, a hydrophobic peptide of 39- to 43-residues amino acid [1], [2]. The β-amyloid protein derives from a set of alternatively spliced β-amyloid precursor proteins (APP), which are encoded by a single gene located in human chromosome 21 [3]. APP plays a central role in Alzheimer’s disease, and it has been suggested that an increase in the production of this protein might actively contribute to the development of this pathology [4], [5], likely by inducing the synthesis and deposit of β-amyloid [5], [6], and the subsequent neurotoxicity [4]. In contrast, it has been also reported that physiological APP levels may elicit neurotrophic effects [7], [8], [9], and protect neuronal cells against the apoptosis induced by insults such as ultraviolet radiation [10], or hypoglycemia [8], thus preventing neurodegeneration. APP is ubiquitously expressed in mammalian tissues, and its expression has been proved to be regulated by a variety of cellular mediators. In particular, we have recently reported that p53, a transcription factor that is increased in Alzheimer’s disease [11], may inhibit the expression of APP in neuroblastoma cells [12]. Transient expression of p53, as well as activation of this factor by camptothecin, a DNA damaging agent, significantly reduce the intracellular levels of APP, an effect that appears to be mediated by sequences of the gene located downstream of the transcriptional start site.

Of interest, we have previously described that thyroid hormones decrease transcriptional activity of APP through sequences located in the same region of the gene [13], and this coincidence together with previous descriptions about physical and functional interactions between p53 and the thyroid hormone receptor (TR) [14], [15] suggest that both transcription factors could cooperate to regulate the APP gene expression. To check this hypothesis we have now analyzed whether or not thyroid hormones may affect the response of APP to p53 activated by UVC irradiation.

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2. Materials and methods 

2.1. Chemicals 

The A-8717 antibody, which recognizes the c-terminal region of APP, the anti α-tubulin (#T6199) and DAPI were purchased from Sigma. Antibodies against phospho-p53 and Caspase-3 antibody were from cell signaling (cat. #9284 and 9661), and the antibody against PARP as well as the goat anti-rabbit IgG-HRP were from Santa-Cruz Biotechnology (cat. #2030). The pCMV-E6 expression vector was kindly provided by Dr. S. Llanos (C.N.I.O., Spain) and the siRNA against p53 was obtained from Dharmacon. An in situ cell death detection kit from Roche was used for TUNEL labelling.

2.2. Cell culture and UV radiation 

Murine N2a-β cells, a subclon of N2a that constitutively expresses the β-isoform of the thyroid hormone receptor (TRβ), were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum as previously described [16]. Previous to the experiments, the culture medium was replaced with a similar medium containing serum depleted of thyroid hormone by treatment with resin AG1X8 as described [17], and the cells were then incubated in this medium for an additional 24-h period before the beginning of experiments. For UV radiation, medium was removed from the plates (p60) and the cells were exposed to different fluencies of UVC (254nm) by using a Stratalinker 2400 UV crosslinker from Stratagene (La Jolla, Ca, USA). Fresh medium was then added and the cells were incubated by different time periods before harvesting. At the indicated times, neurons were collected for proteins determination, or fixed and subjected to DAPI staining and TUNEL labelling to estimate the UVC-induced cell death. Cell death was estimated to be relative to the level of apoptotic nuclei (TUNEL) in relationship to the total number of cells (DAPI).

2.3. DNA transfection 

For transient transfection of p53 siRNA or the E6 protein-coding expression vector, the cells were plated on the previous day and transfected with Lipofectamine 2000 following the instructions of the manufacturer (Invitrogen, Carlsbad, CA). The transfected cells were incubated for an additional period of 24h, and then irradiated and collected for immunoblotting determination 48h later. Each experiment was repeated at least 2–3 times with similar relative differences in regulated expression.

2.4. Western blot analysis 

Cellular proteins were extracted by lysis with a buffer (150mM NaCl, 50mM Tris pH 8, 2mM EDTA, 1% Triton, 0.1% SDS) containing the protease inhibitors PMSF (1mM) and leupeptin (10μg/ml). The protein content of cells was determined by using the BCA assay (Pierce, IL), following the manufacturer’s instructions, and equal amounts, 40μg, of cell extracts were then electrophoresed in an 8% SDS–polyacrylamide gel, transferred to an immobilon polyvinylidine difluoride membrane. Non-specific binding was blocked with 5% non-fat dried milk in TBS-T (Tris buffered saline, 0.1% Tween 20) for 2–3h at room temperature and the cellular APP and levels of intracellular APP and phospho-p53 analyzed with specific antibodies. After 1h incubation at room temperature, the membrane was washed and incubated with a second biotinylated anti-rabbit antibody for one additional hour, washed again and finally incubated for 1h with 1:2000 peroxidase-conjugated streptavidine. All incubations took place at room temperature, and detection by enhanced chemiluminiscence (ECL, Amersham International plc, England) was carried out according to the manufacturer’s indications. Dilutions of antibodies used against APP, phospho-p53, and the loading control α-tubulin were, respectively, 1:4000, 1:1000 and 1:2000. Dilution of the second biotynilated anti-rabbit antibody was 1:5000.

The apparent molecular mass (kDa) of the detected bands was always determined by using a wide range protein standard (Mark 12 from Novex.-S, Diego, CA).

2.5. Statistical analysis 

Unless indicated otherwise, all the data are the mean of two independent experiments performed in duplicate. Significance of the differences was calculated with the Student’s t-test and is indicated in the figures with an asterisk (∗P<0.05).

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3. Results 

3.1. UVC irradiation decreases cell content of APP in N2aβ neuroblastoma cells by a p53-dependent mechanism 

We have previously demonstrated that exposition of cells to the DNA damaging agent camptothecin leads to a significant decrease in the cellular content of APP, an effect that appears to be mediated by the previous activation of p53 [12]. To further explore the effect of p53 on APP, cells were exposed to different fluencies of UVC irradiation by using a 254nm emitting lamp, a source that is appropriate to induce DNA damage, and therefore to activate the endogenous p53. To verify and exclude doses of UVC that might results lethal to the cells, we first analyzed the effects induced by a wide range of UVC fluences. Cells were irradiated with UVC doses ranging from 20 to 600J/m2, and the induced apoptosis and cell viability were assessed 24h after by immunodetection of apoptotic markers such as activated caspase-3 or PARP or by using DAPI staining and TUNEL labelling. As shown in Fig. 1, all the UV doses used, in a dose-dependent manner caused apoptosis, as assessed by the appearance of cleaved caspase-3 and the specific Mr 85000 cleavage product of PARP. However, as shown in the lower panel of the figure only exposition of cells to UV fluencies higher than 100J/m2 was accompanied of a significant increase in cell death, as determined by the increasing percentage of TUNEL positive cells in relation with the total number of DAPI stained cells, whereas the changes induced by doses of UV irradiation lower than 40J/m2 only minimally affected the cell viability at the studied time. Nevertheless, after 72–96h, even doses of irradiation lower than 100J/m2 were effective in causing cell death (not illustrated).

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  • Fig. 1. 

    Effects of the UVC irradiation on cell viability. N2aβ cells were exposed to different doses of UVC light and further incubated for 24h. Whole cells were then photographed and collected for (A) Western blot detection of activated caspase-3, the 85kDa cleaved fragment of PARP and α-tubulin as a loading control, or (B) cell death analysis by fluorescence microscopy after DAPI staining and TUNEL labelling.

To prove the effects of the UVC irradiation on p53 activation and APP expression, cells were exposed to UVC fluences between 10 and 40J/m2, which as shown in Fig. 1, minimally affect cell survival. Activated (phosphorylated) p53 and APP isoforms were detected from cell lysates by Western blot using the specific antibodies described under Experimental Procedures. Results are illustrated in panel A of Fig. 2. As expected, p53 was activated in all irradiated cells, and this activation was evident even 48h after irradiation. In addition, the UVC irradiation caused a significant decrease of APP levels, an effect that appeared to be dose-dependent and that was more evident in those cells harvested 48h after irradiation. Moreover, these results suggested a correlation between p53 activation and APP decrease, and to further evaluate whether UV-induced repression of APP is actually mediated by p53, we next analyzed the effects of UV radiation in cells transiently transfected with the human papillomavirus E6 gene, which encodes a small protein that binds to and inactivates p53 [18]. Cells were irradiated at 20J/m2 and harvested 48h after as described above. As shown in panel B of the figure, transfection of pCMV-E6 abolished p53 activation and significantly reversed the UV-induced repression of APP, thus confirming a p53-dependent mechanism.

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  • Fig. 2. 

    UVC irradiation decreases intracellular content of APP. (A) Phospho-p53 (p-p53) and APP were determined in cell extracts obtained from N2aβ cells irradiated with UVC doses ranging between 10 and 40J/m2 and incubated for the indicated time periods. (B) Analysis of phospho-p53 and APP levels in E6-expressing cells exposed to 20J/m2 and incubated for 24h. α-Tubulin was used as a loading control in both cases. A relative quantification of those levels is also included at the right of the panel (∗P<0.05).

3.2. Triiodothyronine reverses the UV-dependent reduction of APP 

As shown in the previous figure, activation of endogenous p53 by UVC radiation leads to an evident reduction of intracellular APP, an effect that as previously demonstrated could by exerted at transcriptional level [12]. Because thyroid hormone T3 may also inhibit the APP gene promoter activity through the same DNA sequences [13], [19], we next analyzed whether T3 could affect the APP response to UV. Cells were irradiated at 20J/m2 UVC and incubated for an additional period of 24 or 48h in the presence or in the absence of 200nM T3. Cells were then harvested, the cellular protein extracted and the intracellular levels of activated p53 and APP determined by Western blots. As illustrated in Fig. 3, the UV irradiation caused a significant activation (phosphorylation) of the endogenous p53 that is followed by an evident reduction of APP levels in all the irradiated cells. Of interest, the UVC-induced reduction of APP was partially reversed by the thyroid hormone T3, which would be then playing a significant role in protecting the cells against the effects induced by DNA damaging agents. In addition, activation of p53 by UV was attenuated in T3-treated cells, being this effect more apparent at 48h.

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  • Fig. 3. 

    Triiodothyronine reverses the UV-associated decrease of APP levels. Cells were exposed to UVC irradiation (20J/m2) and phospho-p53 (p-p53) and APP levels were determined in lysates obtained from cells incubated for 24–48h in the presence or in the absence of 200nM T3. A relative quantification of the levels is also included at the bottom of the figure.

In contrast with the results obtained in TR-expressing N2aβ cells, T3 was not able to affect the reduction of APP levels caused by p53 in parental N2a cells, which express very low receptor levels, indicating that this is a thyroid hormone nuclear receptor-dependent action. In addition, the thyroid hormone thyroxine (T4) that binds nuclear TRs with low affinity, but is more effective than T3 on binding to a putative membrane receptor [20] that mediates non-genomic actions of the hormone, was also uneffective (data not shown).

3.3. T3 reverses the UVC-induced repression of APP by a p53-dependent mechanism 

As shown in previous figures, the exposition of cells to UVC irradiation leads to a significant reduction of intracellular APP, an effect that at least in part requires activation of p53, and that is partially reversed by thyroid hormones. To further analyze whether or not this protective effect of T3 is also mediated by a p53-dependent mechanism we next analyzed the intracellular levels of APP in cells depleted of p53 by E6 transient transfection or by siRNA-mediated p53 knock-down. As shown in panel A of Fig. 4, T3 was effective in reversing the UV effects in control cells. However, it was unable to reverse the residual decrease of APP induced by UV in the E6-expressing cells. As an alternative approach, cells were also depleted of p53 by RNA silencing. Cells grown to 70% confluence were irradiated after transfection with control or p53 specific siRNAs, incubated for 48h in the presence or in the absence of 5nM T3 and then harvested for APP and phospho-p53 determination. The results obtained, illustrated in the panel B of the figure, confirm those obtained with the E6-expressing cells. As expected, the UV irradiation caused p53 activation and decreased intracellular APP levels in cells transfected with the control siRNA. Of interest, it should be noted that a dose of T3 that minimally reduces the APP expression levels, was very effective in reversing the UV-associated reduction of APP. In contrast, in the p53 siRNA-transfected cells the inhibitory effect of the UV irradiation on APP was strongly reduced even though only partial depletion of p53 was achieved. In addition, the effect that T3 exerts on the UV-induced repression of APP was essentially abolished in siRNA-transfected cells. These results strongly suggest that both, the inhibitory effect of irradiation on APP as well as the protective activity of T3, would be mediated by a p53-dependent mechanism.

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  • Fig. 4. 

    p53 mediates the suppressive effect of thyroid hormone. Depletion of p53 abolishes the UV-dependent decrease of APP and the T3-induced reversion. (A) Intracellular levels of APP and phospho-p53 (p-p53) were determined in wild type and E6-expressing N2aβ cells exposed to UVC at a dose of 20J/m2 and incubated for 48h in the presence or in the absence of 5nM T3. Two graphs at the right illustrate the densitometric quantification of the intracellular APP bands. Data are the average±S.D. from two separate experiments performed in duplicate, and are expressed relative to the total APP detected in non-irradiated cells. (B) APP and p-p53 levels were analyzed in cells transfected with control (left) or p53 specific (right) siRNA after UVC irradiation and 48h of incubation in the presence or in the absence of T3. A densitometric quantification of data obtained in two independent experiments performed in duplicate is shown at the bottom.

3.4. T3-induced reversion of p53-dependent repression of APP is not mediated at a transcriptional level 

Since T3, as well as p53, have been described to decrease the transcriptional activity of APP through DNA sequences located in the same region (b.p. +55 to +102) of the promoter [12], [13], we next analyzed the existence of a possible crosstalk between p53 and the thyroid hormone receptor (TR) on regulation of the APP promoter. To prove whether or not the effect is actually mediated at a transcriptional level, we analyzed APP promoter activity in transient transfection experiments in the presence of p53 and T3. For this purpose CAT activity was determined in N2a-β cells cotransfected with reporter plasmids containing different fragments (−1099/+101, −307/+101, −15/+101 and +55/+101) of the APP gene and an expression vector for p53 (or a control empty vector). The results obtained in cells incubated for 48 hours in the presence and in the absence of T3 are illustrated in Fig. 5. As expected, promoter activity was clearly reduced by p53 or T3, and the extent of repression was very similar in both cases. However, in contrast with that observed with APP protein levels, repression of APP promoter by p53 was not reversed in cells exposed to both factors simultaneously, thus suggesting that reversion of the UV-induced repression of APP by T3 is not mediated at a transcriptional level.

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  • Fig. 5. 

    Regulation of the APP promoter activity by p53 and T3. CAT activity was determined in N2aβ cells cotransfected with reporter plasmids that contain different fragments (−1099/+101, −307/+101, −15/+101, and +55/+101) of the APP gene, and a p53-expressing vector as indicated in the figure. CAT was determined after a 48-h period of incubation in the presence or absence of 200nM T3. Data are expressed as mean±S.D. of CAT activities obtained from two independent experiments performed in duplicate.

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4. Discussion 

The β-amyloid precursor protein plays a central role in Alzheimer’s disease and its regulation appears to be essential since both, increased as well as decreased levels of this protein could be considered as risk factors for this pathology. High levels of APP may cause a higher production and deposit of β-amyloid, the principal component of senile plaques, and low levels would be accompanied by a significant lost of the APP-dependent neurotrophic effects [8], [21].

A number of factors have been described to regulate APP expression. Among others, we have previously described that p53 [12], as well as the thyroid hormone T3 [19], can regulate APP gene transcription, and inhibit the transcriptional activity of the APP promoter. Of interest, expression of both thyroid hormones and p53 transcription factor has been described to be altered in the brain of Alzheimer patients [22], [23].

Thyroid hormones control growth, development, and metabolism in virtually all mammalian tissues. In particular, they play a key role in the development and maintenance of basal forebrain cholinergic neurons typically involved in Alzheimer’s disease [24], and it has been reported that brain hypothyroidism may occur during the progress of this pathology [23]. Furthermore, we have also demonstrated that thyroid hormone T3 may downregulate APP gene expression in neuroblastoma cells [19], [25].

The tumor suppressor p53, which acts as a transcription factor to regulate a variety of cellular functions including gene transcription, DNA repair, cell cycle or cell death [26], has been reported to be involved in many neurodegenerative diseases [27]. In Alzheimer’s disease, p53 levels are increased in damaged neurons [11], and an interesting correlation between the β-amyloid protein and p53 has been reported. The β-amyloid protein may activate p53 in selective neurons [28], [29], and the precursor APP may protect neuronal cells against apoptosis by controlling p53 activation [10].

p53 is constitutively expressed in most types of cells, including neurons, and although is present at a low concentration in normal cells, it can be upregulated and activated in response to a variety of cellular stresses and DNA damage. In this work we have first analyzed the APP expression in cells irradiated with different doses of UVC (254nm), which causes DNA damage and the subsequent activation of p53. As expected, activation (phosphorylation) of p53 was significantly increased in the irradiated cells, and we observed that the intracellular content of APP was reduced in a dose-dependent fashion by UV fluences that minimally affect the cell viability at the studied times. Moreover, the UV-induced reduction of APP was clearly reversed in cells expressing the oncoprotein E6, a small polypeptide that interacts with p53 [30] and promotes its rapid degradation [31], [32], [33], thus suggesting that UV effects are mainly mediated by a p53-dependent mechanism.

We have also analyzed whether or not the thyroid hormone T3 could modulate the inhibitory effect of UVC on APP levels. Surprisingly, the inhibitory effect caused by the UV irradiation was considerably reversed in cells incubated in the presence of T3, which appears to protect the cells against the UV-induced damage. Results obtained in cells rendered deficient in p53 by transient expression of the oncoprotein E6 or by siRNA-mediated p53 knock-down demonstrated that modulation of the APP response to UVC by T3 also requires a functional p53. In the absence of p53, UV irradiation was unable to significantly reduce APP levels, and T3 did not reverse the residual decrease induced by UV, thus confirming that p53 mediates not only the inhibitory effect of the UV, but also the T3-induced reversion of that reduction.

On the other hand, although T3, as well as p53, can transcriptionally inhibit APP expression, the reversion of p53-dependent repression of APP by T3 does not appear to be directly mediated by a transcriptional-dependent mechanism at the APP promoter level. The hormonal effect might be mediated by other mechanism/s that could involve activation of cell signaling pathways that modulate the intracellular content of APP. Among others, the Ras/MAPK or PI3K/Akt signaling pathways that stimulate APP promoter activity [34], can be activated by thyroid hormones through non-genomic mechanisms [35], [36], and may interact with p53 [37]. In particular, the effects of T3 on p53-dependent APP repression could have been mediated by cyclooxygenase-2 (Cox-2), a protein that is increased in the brain of patients with Alzheimer’s disease and enhances the β-amyloid deposition through APP induction [38]. Of interest, it has been previously reported in glioma cells [36], that thyroid hormones may interfere with the nuclear interaction of activated ERK1/2 and COX-2 protein, which is essential to activation of p53, and the subsequent p53-dependent apoptosis. However, COX-2 is not regulated by thyroid hormones in N2aβ cells or in the parental N2a cell line, which does not express the thyroid hormone nuclear receptor. Furthermore, we have analyzed the effects of the thyroid hormones T3 and T4 on the UV-dependent decrease of APP, and have obtained data that appear to discard a non-genomic effect mediated by a membrane receptor and COX-2.

Although new experiments should be performed to definitely clarify the mechanisms that mediate the crosstalk between p53 and thyroid hormones, it is clear that it requires binding of the hormone to the nuclear receptor, since it is not present in cells lacking TRs, and that very likely involves the activation of signals that might regulate APP expression and/or the enzymatic processing of this protein. Moreover, according to the present results the thyroid hormones could have a previously unrecognized role, protecting the brain against p53-mediated neurodegeneration processes, and therefore it would be essential to analyze the effect of p53 and thyroid hormone on APP in vivo, and also confirm whether thyroid hormones can be actually useful in the prevention of this pathology.

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Acknowledgments 

This work was supported by a Grant from the Comisión Interministerial de Ciencia y Tecnología (SAF2006-05577). Fellowship of Ascensión Cuesta was supported by funding of the Spanish Ministerio de Educación y Ciencia.

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PII: S0014-5793(09)00497-9

doi:10.1016/j.febslet.2009.06.040

FEBS Letters
Volume 583, Issue 14 , Pages 2401-2406, 21 July 2009

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