| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* INSERM U413, European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, UA CNRS, University of Rouen, Mont-Saint-Aignan, France;
Department of Neurotransmission and Neuroendocrine Secretion, UMR 7168/LC2 CNRS, Institut des Neurosciences Cellulaires et Intégratives, Université Louis Pasteur, Strasbourg, France;
Department of Adaptative Neurosciences and Physiology, Perinatal Stress Unit, University of Lille I, France;
CNRS UMR5203, INSERM U661, Functional Genomics Institute, Department of Endocrinology, University of Montpellier I et II, Montpellier, France;
|| Genome Technology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, USA; and
¶ Institut National de la Recherche Scientifique-Institut Armand-Frappier, University of Quebec, Pointe Claire, Montréal, Canada
2Correspondence: European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U413, UA CNRS, University of Rouen 76821 Mont-St.-Aignan, France. E-mail: youssef.anouar{at}univ-rouen.fr
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key Words: cell differentiation • pituitary adenylate cyclase-activating polypeptide • cAMP • PC12 cells
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. Selenium is selectively incorporated into the rare amino acid selenocysteine (Sec), which is uniquely present in selenoproteins. The genes encoding selenoproteins harbor, at their 3'-untranslated region (UTR), a specific hairpin motif, designated the Sec insertion sequence (SECIS), which is responsible for the recognition and decoding of the inframe UGA stop codon as a signal for the incorporation of the Sec residue (2)
. Evidence supporting a role for selenoproteins in diverse physiological and pathophysiological processes comes from animal knockout models and epidemiologic/genetic studies. Thus, targeted disruption of the gene encoding the Sec transfer RNA provokes early embryonic lethality in mice (3)
, and conditional removal of this gene in breast epithelium (4)
or liver (5)
causes several defects in these tissues. In addition, high rates of disease, notably cancer, have been linked with low selenium status (6
, 7)
or with genetic variations in certain selenoproteins (6
, 8
, 9)
. The selenoproteins the function of which is known, such as thioredoxin reductases, glutathione peroxidases, and iodothyronine deiodinases, are involved in various redox reactions due to the high nucleophilic activity of the Sec residue (2)
. However, the precise function of many selenoproteins identified in eukaryotes remains undetermined (10)
.
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38-amino acid peptide that exerts cAMP-dependent regulatory as well as trophic effects in the central nervous system and in peripheral tissues through two types of G protein-coupled receptors: a PACAP-selective receptor, named PAC1-R, and two PACAP/vasoactive intestinal polypeptide (VIP) receptors, named VPAC1-R and VPAC2-R (11)
. The effects of PACAP have been extensively studied using sympathoadrenal cells, i.e., adrenochromaffin cells and sympathetic neurons, which develop from the neural crest (12)
and which express the peptide and the PAC1-R early during neurogenesis (13
, 14)
. In fact, PACAP is able to induce the differentiation of sympathetic neuroblasts (13)
as well as that of rat pheochromocytoma PC12 cells (15)
, a sympathoadrenal-derived cell model widely used to characterize the actions of growth factors during neuronal differentiation (16)
. PACAP also regulates the biosynthesis and the secretion of catecholamines and neuropeptides in sympathetic neurons of the superior cervical ganglion (17)
and in the adrenal medulla (18
19
20)
. In a previous study, we showed that PACAP elicits a dual neuronal and neuroendocrine differentiation in PC12 cells as evidenced by morphological changes, the development of Na+ and Ca2+ currents, and the regulation of various gene markers (21)
. The global changes in gene expression induced by PACAP during sympathoadrenal cell differentiation have been subsequently determined by microarray and suppressive subtractive hybridization analyses, showing the regulation of at least 150 genes involved in various cellular processes, including proliferation, survival, secretion, and adhesion/motility (22
, 23)
.
Among the identified genes, we isolated a rat expressed sequence tag (EST) that exhibited a strong sequence homology with human selenoprotein T (SelT), a protein originally identified using a bioinformatics algorithm that allows for the recognition of the SECIS element (24)
. More recently, three orthologous isoforms of SelT have been characterized in the zebrafish (25)
. However, the function of this selenoprotein remains unknown. It has been shown that the putative Sec-containing redox center of the human SelT (C-V-S-U motif where U is the Sec) is similar to those of the selenoproteins Sep15, SelM, and SelW, implying its suitability for the catalysis of redox reactions involving a mechanism typical of thiol/disulfide oxidoreductases (26
, 27)
. Here, we show that the gene encoding the rat SelT is up-regulated by PACAP and cAMP in a Ca2+-dependent manner, and we demonstrate that SelT, which localizes mainly to the endoplasmic reticulum (ER), is implicated in the PACAP-induced increase of intracellular Ca2+ concentration ([Ca2+]i) and subsequent neuroendocrine secretion from differentiated PC12 cells.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. VIP was from American Peptide (Sunnyvale, CA, USA). Nerve growth factor (NGF), chelerythrine, BAPTA-AM, phorbol 12-myristate 13-acetate (PMA), dibutyryl cAMP (dbcAMP), and thapsigargin were obtained from Sigma-Aldrich. H89 was purchased from Coger (Paris, France). Twenty four hours after plating, differentiation of PC12 cells was initiated by adding 100 nM PACAP38. The inhibitors were added 1 h before the onset of PACAP treatment.
Reverse transcription-polymerase chain reaction (PCR), cloning, and sequencing
Total RNA was isolated using the Tri-Reagent (Sigma-Aldrich) and reverse-transcribed using the ImProm-II Reverse Transcription System (Promega, Charbonnières, France). Forward and reverse primers to amplify the full-length cDNA of rat SelT including the SECIS domain were designed according to the sequences of the mouse SelT (accession number AK013022) and the rat EST CB556645 and were terminated at the 5' end by EcoRI and KpnI restriction sites as follows: 5'-GAATTCAGATGCAGTACGCCACGGGGC-3' and 5'-GGTACCGCCAGCATGGAAAACTGTCT-3'. Another forward primer, 5'-GAATTCCGATGAGGCTCTTGCTGCTTCTG-3', was designed and used along with the reverse primer described above to amplify an N-terminally truncated form of rat SelT. PCR was performed in Perkin-Elmer GeneAmp PCR system 9700 (Applied Biosystems, Courtaboeuf, France), and the products were ligated to the pGEMT vector (Promega). Sequencing was performed with a Li-Cor 4200 DNA sequencer (ScienceTec, Les Ulis, France).
Real-time PCR
Total RNA was reverse-transcribed and used to quantify SelT gene expression by real-time PCR with the following forward and reverse primers that were designed using the Primer Express 2 software (Applied Biosystems): 5'-GGTCTAAGCTGGAATCTGGACATC-3' and 5'-TGCACATTGAGTTTCATTTCATTG-3'. PCR was carried out in SYBR Green PCR Master Mix in the presence of 300 nM of primers, using an ABI Prism 7700 (Applied Biosystems). The relative amplification efficiency of SelT and GAPDH (used as an internal control) was assessed and the amounts of SelT mRNA were determined using the comparative CT method (Applied Biosystems).
Construction of expression and RNA interference (RNAi) vectors and cell transfection
The full-length and truncated SelT, cloned in pGEMT, were digested with EcoRI and KpnI and the inserts were subcloned into the expression vector pCMV-Myc (BD Biosciences, Saint-Quentin en Yvelines, France), resulting in the tagging of the two forms of SelT with the Myc epitope. To disrupt the hydrophobic domain of SelT at positions 87–102, a PCR was carried out on the Myc-SelT plasmid with the primers 5'-ACTAGTTGGCAAAGATCCTTTTGC-3' and 5'-GGTACCGCCAGCATGGAAAACTGTCT-3' that contain SpeI and KpnI sites at their 5' extremity, respectively. The PCR product that encodes amino acids 102 to the end of the protein was digested by SpeI/KpnI and inserted in frame to the Myc-SelT plasmid that was cut with the same enzymes to remove the sequences between the naturally occurring SpeI site encoding a part of amino acids 93–95 and a KpnI site at the end of the coding sequence. This construction resulted in the deletion of seven amino acids at positions 95–101 of the hydrophobic domain. To convert the Sec residue to an Ala, we used the QuickChange II Site-Directed Mutagenesis Kit (Stratagene, Amsterdam, The Netherlands) to introduce two nucleotide substitutions in the corresponding codon (TGA to GCA). A PCR was carried out on the Myc-SelT plasmid using the PfuUltra DNA polymerase with the mutagenic primers 5'-CAGATTTGTGTATCCGCAGGGTACAGGCGGGTG-3' and 5'-CACCCGCCTGTACCCTGCGGATACACAAATCTG-3' according to the manufacturer’s protocol.
To inhibit the expression of SelT in PC12 cells, the siRNA Target Finder tool on Ambion Web site (http://www.ambion.com/techlib/misc/siRNA_finder.html) was used to design the oligonucleotides 5'-TCCCACATCATCGATCATAttcaagagaTATGATCGATGATGTGGGAtttttt-3' and 5'-attaaaaaaTCCCACATCATCGATCATAtctcttgaaTATGATCGATGATGTGGGAggcc-3' that were annealed and ligated between the ApaI and EcoRI sites of pSilencer 1.0-U6 plasmid (Ambion, Huntingdon, UK). All constructs were confirmed by sequencing. PC12 cells were transfected with 1.5–3 µg of DNA constructs and 2–4 µl of Lipofectamine 2000 (Invitrogen) for 6 h according to the manufacturer’s protocol.
In situ hybridization
Pregnant female Wistar rats were killed by decapitation, and fetuses at embryonic days E7, E14, and E21 were collected by cesarean section and used for in situ hybridization as described previously (28)
. All manipulations were performed according to the recommendations of the French Ethical Committee and the rules of the European Union Normative (86/609/EEC), under the supervision of authorized investigators. Tissue sections were fixed, prehybridized, and hybridized with [35S]UTP-labeled sense and antisense riboprobes (Riboprobe Combination Systems, Promega) transcribed from the rat SelT cDNA. After several washes (28)
, slides were exposed for 13 days onto β-Max hyperfilm (Amersham Pharmacia, Les Ulis, France) for E14 and E21 fetuses or dipped in photographic emulsion and exposed for 21 days for E7 animals. Counterstaining with cresyl violet was used for tissue identification. Images were acquired using a Biocom 200 image analysis system (Biocom, San Diego, CA, USA).
Immunocytochemistry
An antibody against the rat SelT-derived peptide WSKLESGHLPSMQ was generated in rabbits and used at 1:1000 dilution. Preincubation of this antibody with the synthetic peptide abolished the immunoreaction. Transfected GFP-secretogranin II (SgII) fusion protein was used as a marker of secretory granules (29)
, and a polyclonal antibody directed against the 78 kDa glucose-regulated protein (GRP78) (1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used to label the ER compartment. Mutant forms of SelT tagged with the Myc epitope were immunostained using an anti-Myc monoclonal antibody (1:100; BD Biosciences) for 2 h at room temperature. Secondary antibodies were Alexa 488-conjugated goat anti-mouse, Alexa 568-conjugated donkey anti-goat, and Alexa 594-conjugated goat anti-rabbit gamma globulins (1:300; Invitrogen). Cells were observed on a Leica SP2 upright confocal laser scanning microscope (DMRAX-UV) equipped with the Acousto-Optical Beam Splitter system (Leica Microsystems, Reuil-Malmaison, France). To verify the specificity of the immunoreaction, the primary or secondary antibodies were substituted with PBS.
Western blot
Proteins were prepared from individual cell culture wells as described previously (19)
. Proteins were quantified by the Bradford assay (Bio-Rad, Marnes-la-Coquette, France) and analyzed by SDS-PAGE followed by electrotransfer onto nitrocellulose membranes (Amersham Pharmacia Biotech). The expression of the SelT protein was examined using the polyclonal rabbit antibody directed against SelT at a 1:1000 dilution. The antigen-antibody complexes were visualized by the chemiluminescence ECL Western blotting analysis system (Amersham Pharmacia Biotech) and exposure on Kodak X-OMAT films (Sigma-Aldrich).
Measurement of intracellular Ca2+ levels
PC12 cells were plated on glass coverslips coated with poly-L-lysine in 35 mm culture dishes at a density of 5 x 104 cells/ml. Cells were incubated at 37°C for 45 min with 5 µM of the fluorescent Ca2+ probe indo-1/acetoxymethylester (Invitrogen) in a saline solution containing 125 mM NaCl, 5.5 mM KCl, 2 mM CaCl2, 0.8 mM MgCl2, 10 mM HEPES, 24 mM glucose, and 36.5 mM sucrose, pH 7.3. The cells were washed 2x with the saline solution at 37°C, and [Ca2+]i was monitored by using a dual emission microfluorimeter system, as described previously (30)
.
Assay of growth hormone (GH) release from PC12 cells
Expression vectors were introduced into PC12 cells (24-well dishes, 1x105 cells, 0.5 µg/well of each plasmid) using GenePorter (Gene Therapy Systems, San Diego, CA, USA) according to the manufacturer’s instructions. GH release experiments were performed 72 h after transfection. PC12 cells were washed 3x with Locke’s solution containing 140 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 11 mM glucose, and 15 mM HEPES, pH 7.2, and then incubated for 10 min in Ca2+-free Locke’s solution with or without (basal release) 100 nM PACAP. The supernatants were collected, and the cells were harvested by scraping in 10 mM PBS. The amounts of GH secreted into the medium and retained in the cells were measured using an ELISA assay (Roche, Meylan, France). GH secretion is expressed as a percentage of total GH present in the cells before stimulation.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
was the orthologous gene of human SelT. We used oligonucleotides derived from various related ESTs that were initially available in Genbank to isolate a full-length cDNA encoding rat SelT from PC12 cells (Fig. 1
A). Rat SelT mRNA contains a translation start site localized 96 bp upstream of the start codon originally described in the human sequence. This new initiation site is conserved in rat, mouse, and human sequences and generates an open reading frame encoding a rat protein of 195 amino acids (Fig. 1A
). The SECIS element contained in the 3'-UTR would allow the recognition of the inframe UGA triplet present within the coding region of SelT mRNA as a signal for Sec incorporation at position 49 of the protein (Fig. 1A
). Sequence analysis of SelT revealed the occurrence of a 19 amino acid signal peptide and a highly hydrophobic stretch of 16 amino acids at positions 87–102 of the predicted protein, which may represent a transmembrane domain (Fig. 1A, B
).
|
SelT gene expression occurs early during development and is ubiquitous
To characterize SelT gene expression in rat, we analyzed the distribution of its mRNA in different tissues during development and in the adult. Developmental expression of SelT in rat embryos was examined by in situ hybridization. The antisense probe revealed a high expression level of SelT mRNA at E7 both in the embryo and the placenta (Fig. 2
A). At E14, the SelT transcript exhibited a strong and widespread expression in various developing organs (Fig. 2B
). At E21, SelT mRNA was detected in all tissues, with particularly high levels in certain organs such as the brain, salivary glands, thymus, lung, and adrenal, whereas other organs like the heart or the liver showed a less intense signal (Fig. 2C
). As a control, only background labeling could be observed with the sense probe at all embryonic developmental stages studied (Fig. 2D
and data not shown). In adult rat, the expression and abundance of SelT mRNA in different tissues were studied by PCR and real-time PCR. A single band representing the SelT transcript was amplified from all the tissues tested, i.e., brain, pituitary, heart, spleen, kidney, and testis (Fig. 2E
). Real-time PCR showed that the highest concentrations of SelT mRNA occur in the anterior lobe of the pituitary and the testis, whereas moderate levels were found in all other tissues examined except the heart, which exhibited a lower expression level (Fig. 2F
). These data revealed that the SelT gene is widely expressed in rat tissues. Widespread distribution was observed in early as well as late stages of embryogenesis and persisted in the adult, suggesting that SelT may play a basic and general function in various tissues of developing and adult organisms.
|
SelT is localized to the ER through a hydrophobic motif
Next, we studied the subcellular localization of SelT in PC12 cells by confocal immunofluorescence microscopy. Specific antibodies revealed that SelT exhibits a cytoplasmic punctate immunostaining pattern in PC12 cells (Fig. 3
A). Colocalization studies with GFP-SgII showed that SelT is not present in the secretory granules (Fig. 3A
). Using antibodies against the GRP78, a specific marker for the ER, we observed that SelT labeling largely, although not exclusively, colocalizes with the staining of the ER (Fig. 3A
), which suggests that SelT is an ER-resident protein. To determine whether the N terminus and the hydrophobic domain at positions 87–102 of the SelT sequence described in the present study are necessary for the targeting of this protein to the ER, we constructed expression plasmids of SelT tagged with the Myc epitope in which these regions were deleted (Fig. 3B
). SelT
N, which lacked the N terminus exhibited a cytoplasmic localization that did not virtually differ from that of the full-length protein (Fig. 3C
). However, disruption of the hydrophobic domain at positions 87–102 of SelT (SelTHD) resulted in loss of most of the labeling (Fig. 3C
), strongly suggesting that the highly hydrophobic region predicted by sequence analysis is required for the targeting of SelT to intracellular compartments including the ER.
|
SelT is rapidly induced by PACAP and cAMP but is not regulated by NGF, VIP, and other factors
As a first approach to understand the role of SelT in neuroendocrine cell differentiation, we studied the time course of the stimulatory effect of PACAP and the action of various factors known to affect sympathoadrenal cell differentiation and function on the expression of this gene in PC12 cells. Treatment with PACAP provoked a rapid increase in SelT mRNA levels in PC12 cells, which was significant after 1 h of treatment. This effect reached 2- to 3-fold above control at 6–24 h and decreased to almost basal value after 72 h of treatment (Fig. 4
A), suggesting that SelT exerts a rapid and long-lasting action during PC12 cell differentiation. In contrast, neither NGF (100 ng/ml), a trophic factor that also differentiates sympathoadrenal-derived cells, nor VIP (100 nM), a neuropeptide structurally related to PACAP, stimulated SelT gene expression (Fig. 4B
). The permeable cAMP analog dbcAMP (10–4 M) provoked a significant increase in the abundance of SelT gene transcripts, mimicking thus the effect of PACAP, whereas cell depolarization by high potassium (50 mM KCl) or activation of protein kinase C (PKC) by PMA (10–7 M) were without effect (Fig. 4B
). Finally, we tested the effect of other peptides such as angiotensin II, urotensin II, and endothelin I, which all activate phospholipase C (PLC) and Ca2+ mobilization, but none had an effect on SelT gene expression (data not shown). Together, these data indicate that the SelT gene is regulated by PACAP but not NGF, VIP, or other peptides in differentiating PC12 cells, through a mechanism that is likely to be dependent on cAMP.
|
PACAP and the cAMP/protein kinase A (PKA) pathway stimulate SelT gene expression through Ca2+ mobilization
To further elucidate the molecular mechanisms responsible for the increase of SelT mRNA levels in response to PACAP and cAMP stimulation, we explored pathways implicating PKA, PKC, and Ca2+ mobilization, which play important roles in the trophic and regulatory effects exerted by the neuropeptide in various tissues and particularly in cells of the sympathoadrenal lineage (11
, 31
32
33)
. The PKA inhibitor H89 (20 µM) significantly reduced the stimulatory effect of PACAP on SelT gene expression (Fig. 5
A). In contrast, the PKC inhibitor chelerythrine (5 µM) did not affect SelT mRNA levels (Fig. 5A
), consistent with the lack of effect of PMA (Fig. 4B
). The cytosolic Ca2+ chelator BAPTA-AM (50 µM) and the nonselective Ca2+ channel blocker nickel (3 mM NiCl2) completely abrogated the stimulatory effect of PACAP (Fig. 5B
), indicating that Ca2+ is crucial for the regulation of SelT gene expression by PACAP in PC12 cells. Furthermore, incubation of PC12 cells with the Ca2+ blockers also impaired dbcAMP-induced stimulation of SelT mRNA levels (Fig. 5C
). These data suggest that Ca2+ signaling plays a pivotal role in the expression of SelT after the stimulation of the cAMP/PKA pathway by PACAP during neuroendocrine cell differentiation.
|
SelT regulates [Ca2+]i in differentiating PC12 cells
To investigate the possible implication of SelT in the modulation of [Ca2+]i, PC12 cells were transfected with the full-length SelT or SelTN described above, and cytosolic Ca2+ levels were measured by microfluorimetry (Fig. 6
). Overexpression of full-length SelT in PC12 cells induced a rise in [Ca2+]i compared with control cells transfected with the empty vector (Fig. 6A
). In contrast, transfection of the N-terminally truncated form of SelT did not affect [Ca2+]i (Fig. 6A
), suggesting that the integrity of the protein sequence characterized in the present study is necessary for the effect of SelT on Ca2+ homeostasis. To assess whether the putative redox center is involved in the effect of SelT on [Ca2+]i, a mutant SelT in which the Sec residue was converted to an Ala was overexpressed in PC12 cells and [Ca2+]i was determined in comparison with cells overexpressing the wild-type protein. Mutation of the Sec residue prevented the effect of SelT on [Ca2+]i (Fig. 6B
), implying that the selenoprotein could regulate Ca2+ homeostasis by a redox mechanism through the Sec-containing motif.
|
To determine whether SelT affects Ca2+ release from internal stores, we tested the effect of thapsigargin, a sarcoendoplasmic reticulum Ca2+ATPase (SERCA) inhibitor, on [Ca2+]i in PC12 cells overexpressing or not SelT. Treatment of control cells with 100 nM thapsigargin induced a rise in [Ca2+]i, consistent with the Ca2+ store-depleting effect of this drug (Fig. 6C
). When SelT-transfected cells were exposed to thapsigargin, a further increase in [Ca2+]i was observed in comparison with untreated SelT-overexpressing cells (Fig. 6C
). However, thapsigargin and SelT did not exert additive effects on [Ca2+]i (Fig. 6C
), indicating that the SERCA inhibitor and SelT may act on similar pools of Ca2+ in ER. In addition, the fact that thapsigargin further increased [Ca2+]i in SelT-overexpressing cells (Fig. 6C
) suggests that SelT does not act by repressing the function of SERCA.
SelT is involved in PACAP-induced Ca2+ mobilization and hormone release
It has been shown that PACAP induces a long-lasting increase in [Ca2+]i in chromaffin cells (34
, 35)
. To determine whether SelT is involved in the elevation of Ca2+ levels by PACAP in these cells, SelT gene expression was inhibited through RNA interference gene silencing. We used a vector (pSilencer) that directs the transcription of short hairpin RNA (shRNA), which generates small interfering RNA (siRNA) in mammalian cells. After transfection with the pSilencer encoding SelT shRNA, real-time PCR revealed a significant decrease in SelT expression in PC12 cells in comparison with cells transfected with the empty vector (Fig. 7
A). Furthermore, SelT mRNA levels in PACAP-treated PC12 cells after siRNA action were similar to those in control unstimulated cells (Fig. 7A
). Similarly, PACAP also increased SelT protein levels in PC12 cells, and this stimulatory effect was abrogated in the presence of SelT siRNA (Fig. 7B, C
). In contrast, the action of the neuropeptide was unaffected in cells transfected with the empty vector (Fig. 7B, C
).
|
We first showed that PACAP (100 nM) induces a significant increase in [Ca2+]i in PC12 cells (Fig. 7D
). The specific knockdown of SelT abrogated the effect of PACAP on [Ca2+]i since shRNA-treated cells showed Ca2+ levels that were comparable to control cells untreated by PACAP (Fig. 7D
). This finding demonstrates that SelT plays a critical role in PACAP-induced increase of [Ca2+]i during PC12 cell differentiation.
Because PACAP activates the cAMP/PKA/Ca2+ pathway to increase SelT gene expression and to stimulate catecholamine release (17
, 18
, 31
, 36)
and because the peptide regulates intracellular Ca2+ through SelT expression (this study), we investigated the possibility that SelT could be involved in PACAP-induced secretion from PC12 cells. To test this hypothesis, we used a transfected GH as a secretory reporter (37)
in cells treated or not with SelT shRNA (Fig. 8
). These experiments revealed that SelT knockdown does not alter the effect of PACAP on GH release in undifferentiated cells (Fig. 8A
). However, when PC12 cells were differentiated by PACAP overnight, prior reduction of endogenous SelT expression through shRNA treatment provoked a significant decrease (35.3±2%; n=3) in the net release of GH evoked by a short stimulation with PACAP (Fig. 8B
), indicating that SelT is involved in the acquisition and regulation of the secretory phenotype in differentiating PC12 cells.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
39
40)
, the molecular mechanisms involved in this process are not fully understood. In the present study, we identified the selenium-containing protein SelT as an important actor for the increase of [Ca2+]i induced by PACAP in PC12 cells, thus linking cAMP to selenoproteins and Ca2+ regulation during neuroendocrine cell differentiation. Our findings underline the usefulness of gene expression profiling studies to identify novel genes such as SelT, with unsuspected functions in processes of utmost biological relevance such as cell differentiation.
Characterization and distribution of SelT
Cloning of rat SelT cDNA revealed the presence of a new putative start codon located 96 nucleotides upstream of the beginning of the previously proposed coding region of the human mRNA (24)
. The additional sequence is highly conserved in the human and mouse SelT proteins predicted from sequencing projects and could give rise to a 32-amino acid N-terminally extended protein. The sequence of this new N terminus encompasses a signal peptide that is important for the function of SelT since its deletion abolished the biological activity of the protein.
Since the spatiotemporal localization of SelT gene expression was completely unknown in mammalian species, we undertook a large tissue-distribution study in the rat to gain insight into the potential role of this selenoprotein. In situ hybridization revealed that SelT mRNA is abundantly expressed very early during embryogenesis and is found in all developing tissues at E7, E14, and E21. However, while a diffuse, intense expression pattern was observed at E7-E14, when developmental events are initiated, several organs including the brain, the pituitary, the adrenal, or the thymus exhibited a strongest labeling with the SelT probe at E21, when the organization and specification of embryonic tissues are almost completed. In the adult rat, SelT gene expression was detected in all the tissues examined, with highest mRNA levels in certain tissues such as the anterior lobe of the pituitary and the testis. The widespread expression of the SelT gene and the elevated mRNA levels in tissues like the brain, pituitary, thymus, or testis, in which PACAP exerts important effects (11)
, suggested that this selenoprotein could be involved in a fundamental process regulated by the neuropeptide in different tissues, notably in nerve, endocrine, immune, or reproductive organs. These observations are consistent with a role of SelT in intracellular Ca2+ mobilization as reported in the present study, which is regulated by PACAP in various tissues (11)
.
The subcellular localization of SelT could also provide important clues to determine the function of this protein. In PC12 cells, SelT exhibited a cytoplasmic immunostaining that largely colocalized with an ER marker. Interestingly, other selenoproteins such as type 2 iodothyronine deiodinase, which is responsible for the activation of thyroid hormone (41)
; methionine-sulfoxide reductase 3A, which repairs oxidized methionine residues in proteins (42)
; and the 15-kDa selenoprotein (26)
are also localized to the ER. Disruption of a highly hydrophobic domain between positions 87 and 102 of the SelT protein resulted in loss of the immunostaining, indicating that this region is important for the localization in the ER. It is likely that this hydrophobic region may represent a transmembrane domain responsible for the integration and maintenance of SelT in ER membranes. In support of this hypothesis, SelT has been classified among transmembrane proteins in a large-scale study designed to identify secreted and transmembrane proteins using bioinformatics algorithms (43)
. Consequently, this domain may anchor SelT to the ER membrane to allow the putative C-V-S-U redox center to form reversible disulfide bonds with redox-sensitive proteins present in this compartment. Thus, like type 2 iodothyronine deiodinase and methionine-sulfoxide reductase 3A, which exert their effects in the ER through a redox activity (26
, 41
, 42)
, SelT could also be involved in redox regulatory mechanisms in the ER. In support of this notion, mutagenesis of the Sec residue in the putative redox center of SelT abolished its action on [Ca2+]i. Note that selenoprotein N, which is mutated in several pathologies such as congenital muscular dystrophy with spinal rigidity and restrictive respiratory syndrome and multiminicore disease (44
, 45)
, is also an ER-resident protein for which a role in regulation of Ca2+ homeostasis via a redox activity has been suggested (46)
.
SelT as a new regulator of Ca2+ homeostasis and hormone release
Several observations indicated that SelT could be involved in regulation of Ca2+ homeostasis: 1) the localization of SelT in the ER, which represents a major source of intracellular Ca2+; 2) the importance of Ca2+ in the regulation of the expression of the SelT gene in PC12 cells in response to PACAP and cAMP stimulation; 3) the implication of Ca2+ in the transduction pathways mediating the various effects of PACAP; and 4) the early and widespread expression of the SelT gene during embryogenesis and in adulthood in rat. Overexpression of SelT and treatment of cells by the Ca2+-ATPase inhibitor thapsigargin showed that SelT could indeed mediate the release of Ca2+ from intracellular stores. Consistent with this finding, specific knockdown of SelT expression using shRNAi abolished the PACAP-induced increase in [Ca2+]i observed in differentiating PC12 cells. These results demonstrate that the increase in SelT expression is a necessary and sufficient condition for the stimulation of [Ca2+]i that is associated with PACAP-induced PC12 cell differentiation. Thus, PACAP stimulates SelT gene expression through rapid Ca2+ efflux from extracellular milieu and Ca2+ mobilization from intracellular stores, resulting in increased SelT protein concentration that is required for long-lasting release of intracellular Ca2+ during PACAP-induced cell differentiation.
Although NGF is also able to differentiate sympathoadrenal-derived cells such as PC12 cells and to stimulate intracellular Ca2+ (22)
, NGF was unable to increase SelT mRNA levels, indicating that the transduction pathways specifically stimulated by PACAP, i.e., elevation of both cAMP and Ca2+, and activation of PKA (11
, 20)
are critical to induce SelT gene transcription and Ca2+ release from internal stores. Consistently, VIP, which mainly activates cAMP production (11)
, and several other peptides, which directly elevate Ca2+ in different cells (data not shown), were ineffective in stimulating SelT gene expression. Collectively, these data suggest that SelT could be specifically involved in PACAP function.
It is known that treatment of PC12 cells with PACAP elicits various biological processes, including growth arrest, neuritogenesis, cell survival, and enhanced secretory activity, that characterize the differentiated phenotype (21
, 22
, 36)
. These cellular responses have been associated with the activation of specific transduction pathways involving messengers and protein kinases that are both common and distinct for the induction of the different aspects of cell differentiation (11
, 34
, 36
, 47)
. Activation of the cAMP/PKA pathway and the associated Ca2+ transients induced by PACAP in PC12 cells (31
, 35)
have been shown to mediate the immediate as well as the long-lasting effects of the peptide on catecholamine secretion from sympathetic neurons and chromaffin cells (17
, 18
, 31
, 36)
. In accordance, we found that inhibition of SelT expression reduces PACAP-evoked GH release, thus demonstrating that SelT is involved in the signaling pathways activated by PACAP, most likely through Ca2+ regulation, to trigger secretion from differentiated PC12 cells. In contrast, SelT had no effect on secretion in undifferentiated cells, further indicating that SelT-stimulated expression evoked by PACAP during differentiation is required for enhanced action of the neuropeptide on [Ca2+]i and catecholaminergic cell secretion. Thus, SelT would act by facilitating a "feed-forward" mechanism to potentiate the response to PACAP during PC12 cell differentiation. Because transfected GH is packaged in secretory granules with catecholamines (37)
and since PACAP stimulates catecholamine release from secretory granules of normal and tumoral chromaffin cells (17
, 18
, 36)
, it is reasonable to assume that SelT could be involved in PACAP-induced catecholamine secretion.
SelT as an integrator of cAMP, inositol 1,4,5-trisphosphate (IP3), and Ca2+ signaling through a redox mechanism
Impairment of the effect of SelT on [Ca2+]i by site-directed mutagenesis of the Sec residue implies that PACAP stimulates the expression of SelT and its targeting to the ER to control Ca2+ release through a Sec-mediated redox mechanism. It has recently been shown that the activity of IP3 receptor (IP3R) and SERCA pump, which are responsible for the efflux and the reuptake of Ca2+ in the ER, respectively, is highly dependent on the redox state of critical sulfhydryl residues present in these proteins and located on the lumenal side of the ER. These moieties are modified, through a redox mechanism, by two ER lumenal proteins of the thioredoxin family, ERp44 and ERp57, which act as sensors of the redox potential to inhibit the function of IP3R1 and SERCA 2b, respectively (48
, 49)
. By analogy, the redox motif of SelT could also affect thiol groups of intracellular Ca2+ channels and pumps to modulate their activity, leading to an increase of [Ca2+]i. The similar effects of thapsigargin and SelT on [Ca2+]i in PC12 cells suggest that the selenoprotein may rather stimulate the activity of Ca2+ release channels. Consequently, the selenoprotein SelT and thioredoxins would have opposite functions to fine-tune Ca2+ release and [Ca2+]i in a concerted, redox-dependent manner. In addition, SelT could represent a molecular link that coordinates the effects of cAMP and Ca2+ in response to cues that raise the intracellular concentrations of the two second messengers. Thus, PACAP-induced cAMP production may enhance the concentration of SelT to elicit a sustained Ca2+ efflux from ER via IP3R, which is simultaneously activated by PACAP through stimulation of IP3 biosynthesis (Fig. 9
). In line with our findings, it has recently been shown that selenium affects Ca2+ signaling in endothelial cells (50)
, suggesting that a selenoprotein like SelT, which is ubiquitously expressed, could also be involved in Ca2+ regulation in other cell types with important physiological and pathophysiological consequences.
|
In conclusion, we identified SelT expression as a key step for the increase of [Ca2+]i during PACAP and cAMP/PKA-induced neuroendocrine differentiation. More generally, SelT may act as a regulated coordinator of the effects of the second messengers cAMP, IP3, and Ca2+ through an original redox mechanism, to integrate different signaling pathways (Fig. 9)
during cell differentiation in various tissues, in response to PACAP, during development and in adulthood. This study is the first report on the involvement of a selenoprotein in the control of Ca2+ homeostasis and secretory activity and thus provides new insights to better understand the implication of selenium and selenoproteins in human diseases such as muscular dystrophy and cancer.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Received for publication October 30, 2007. Accepted for publication December 21, 2007.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  D. Vaudry, A. Falluel-Morel, S. Bourgault, M. Basille, D. Burel, O. Wurtz, A. Fournier, B. K. C. Chow, H. Hashimoto, L. Galas, et al. Pituitary Adenylate Cyclase-Activating Polypeptide and Its Receptors: 20 Years after the Discovery Pharmacol. Rev., September 1, 2009; 61(3): 283 - 357. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
20–30 cells were averaged. ***P < 0.001 compared with the control Myc plasmid; ###P < 0.001 compared with untreated cells transfected with the SelT plasmid (Student’s t test).
