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REPRODUCTION-DEVELOPMENT |
Centre de Recherche en Reproduction Animale, Faculté de Médecine Vétérinaire, Université de Montréal, St. Hyacinthe, Québec, Canada J2S 7C6
Address all correspondence and requests for reprints to: Dr. Bruce D. Murphy, Centre de Recherche en Reproduction Animale, Faculté de Médecine Vétérinaire, Université de Montréal, 3200 rue Sicotte, St. Hyacinthe, Québec, Canada J2S 7C6. E-mail: murphyb{at}medvet.umontreal.ca
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Abstract |
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Introduction |
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The deduced amino acid (aa) sequence for the human NPC-1 gene displays a sterol-sensing domain similar to that seen in cholesterol-sensitive proteins such as 3-hydroxy-3-methyl glutaryl-coenzyme A reductase and sterol regulatory binding element cleavage activating protein (11). It also has 13 transmembrane domains and a lysosome-targeting motif (9, 11, 12). The mouse ortholog of human NPC-1 is known, and it displays all of the human domains and an overall identity in excess of 90% to the deduced human aa sequence (13).
NPC-1 functions in cholesterol trafficking and may be essential for normal steroidogenesis in the adrenal and ovary. Two mouse models display spontaneous mutations of the NPC-1 gene (4, 14). In the BALB/c npcnih mouse there is a mutation in the open reading frame such that there is an insertion of intronic sequences and a premature termination sequence (13). The mutation that interferes with NPC-1 function is not currently known in the second model, the C57BL/6Jspm/spm. Homozygotes of both mouse strains are infertile (15) (Gévry, N., and B. D. Murphy, unpublished observations). There is substantial derangement of basal and ligand-induced testicular steroidogenesis in the BALB/cnpcnih mouse (16). A feline model in which mutation of the NPC-1 gene has been demonstrated by complementation studies (17) displays adrenal insufficiency (18). Together, these studies provide evidence for steroidogenic defects associated with NPC-1 mutation.
Steroid synthesis is ligand dependent. Thus, it is expected that cAMP, the second messenger in the linear signaling pathway of ACTH-, FSH-, and LH-induced steroidogenesis, could be expected to modulate the expression of NPC-1. In human granulosa-lutein cells, no effects of cAMP on NPC-1 expression were apparent (19). The purposes of this investigation were to establish the presence of the homologous NPC-1 sequences in a nonhuman, nonmurine model, the pig, to investigate expression of NPC-1 during luteal differentiation, and to determine whether cAMP modulates the abundance of its gene products in primary culture of ovarian cells and in steroidogenic cell lines.
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Materials and Methods |
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) were based first on conserved regions from the interior portion of the mouse (13) and human ( 9) genes. Subsequent to the amplification of an authentic internal fragment, porcine primers and rapid amplification of cDNA ends kits (Roche, Laval, Canada) were employed to amplify the extremities. The RT reaction was performed with SuperScript II (Life Technologies, Inc., Burlington, Canada) containing 5 µg total RNA from pig granulosa cells and 20 pmol deoxythymidine primer. PCRs employed the Expand High Fidelity Kit (Roche) using the method described by the manufacturer. They were performed in a Hybaid Omnigene Thermal Cycler (Intersciences, Markham, Canada) for 35 cycles of 94 C for 15 sec, 60-52 C for 30 sec, and 72 C for 2–4 min, followed by an extension amplification at 72 C at the end of the PCR reaction.
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). The Expand High Fidelity kit served for amplification of sequences in excess of 2 kb. The first PCR amplification was performed with seven cycles of 94 C for 15 sec and 72 C for 4 min and 30 sec, followed by 32 cycles of 94 C for 15 sec, 65 C for 30 sec, and 68 C for 4 min plus 5 sec additional/cycle, with a final step at 68 C for 7 min. The cycling conditions for the nested PCR were five cycles of 94 C for 15 sec and 72 C for 4 min and 30 sec, followed by 24 cycles of 94 C for 15 sec and 68 C for 4 min and 30 sec plus 5 sec additional/cycle and 68 C for 7 min for the final elongation step. The resulting cDNA and 5'-flanking fragments were cloned into pGEM-T Easy (Promega Corp., Nepean, Canada) and sequenced by automated DNA sequencing (Service d’Analyze et de Synthèse d’Acides Nucléiques de Université Laval, Québec, Canada). Sequence analysis was undertaken using MatInspector (Abteilung Genetek, Braunschweig, Germany), Grail (Oak Ridge Laboratories, Oak Ridge, TN), and Clustal (European Bioinformatics Institute, Cambridge, UK) software.
In vivo analyses
Porcine tissues collected from the abattoir included skeletal muscle, brain, liver, lung, kidney, adrenal, and uterus. In addition, ovaries were collected from pigs at intervals of 24 h after the observation of estrus, and these tissues were supplemented with ovaries from the abattoir of the same stages of the estrous cycle, as determined by the macroscopic and microscopic analyses, previously described (20). Testes of adult and neonatal pigs (3–5 d of age) were collected and preserved for the analyses described below.
In vitro investigations
Granulosa cells were aspirated from medium-sized (3–5 mm) follicles of ovaries from prepubertal gilts and cultured as previously described (20). Briefly, 7–9 x 106 viable cells/ml were pooled in MEM (Life Technologies, Inc.) containing 1 mg/liter insulin (Sigma, St. Louis, MO), 0.1 mM nonessential amino acids (Life Technologies, Inc.), 5 x 104 IU/liter penicillin (Life Technologies, Inc.), 50 mg/liter streptomycin (Life Technologies, Inc.), 0.5 mg/liter fungizone (Life Technologies, Inc.), and 10% FCS (Life Technologies, Inc.). Incubation was carried at 37 C. At 48 h after initiation of culture, the cells were washed, and medium was replaced with serum-free medium. Cultures were terminated at 0, 24, 48, 72, and 96 h, and total RNA samples were collected to investigate the sequence of luteinization in vitro. For immunohistochemistry, cells were seeded onto coverslips, which were then fixed in methanol before undergoing the procedure described below. Cells incubated for 96 h were treated with (Bu)2cAMP (300–1000 µM; Sigma) for 24 h in subsequent trials to determine the regulatory mechanisms of NPC-1 expression. To investigate the rate of disappearance of NPC-1 message, cells incubated for 96 h were treated with actinomycin D (ACT-D; 5 µg/ml, Sigma), and cultures were terminated at 0, 6, 12, and 24 h. To provide basic information about the characteristics of NPC-1 mRNA expression, luteinized granulosa cells were treated with the protein synthesis blocker, cycloheximide (CHX; 40 µM; Sigma), and cultures were terminated at 0, 6, 12, and 24 h. Each experiment was repeated three or more times. Total RNA was purified as described below and stored at -80 C until Northern analyses were conducted.
Northern blot analysis
We previously described the methods of Northern analysis for porcine tissues (20, 21). In brief, tissues and cultured cells were homogenized in 4 M guanidine isothiocyanate (Life Technologies, Inc.), 26.5 mM sodium acetate (Sigma), and 0.12 M ß-mercaptoethanol (Sigma) and stored at -70 C until analysis. Total RNA was purified with Easy Spin columns (QIAGEN, Mississauga, Canada). Aliquots of 15 µg total RNA were subjected to electrophoresis on 1% agarose-formaldehyde gels using a 20-mM morpholinopropanesulfonic acid buffer (pH 7.0), transferred overnight to nylon membranes, and cross-linked for 30 sec at 150 mJ in a UV chamber (GS Gene Linker, Bio-Rad Laboratories, Inc., Richmond, CA).
Blots from tissues collected from the abattoir and from cell culture were hybridized with a 1-kb probe from the 5'-region of the porcine NPC-1 open reading frame. In some experiment blots were also hybridized with homologous steroidogenic acute regulatory protein (StAR) cDNA probe, as previously described (21), to serve as positive controls for ligand-regulated steroidogenic genes. To control for variation in pipetting and transfer, blots were then stripped and hybridized with a 1.4-kb fragment from an internal region of the human 28S rRNA gene, provided by Dr. G. Schultz (University of Calgary, Calgary, Canada). The probes were labeled with 32P to specific activity of approximately 1.5–3.0 x 109 dpm/µg by random primer synthesis using a DNA Random Prime kit (Roche). Hybridization was conducted as previously described (21), followed by two high stringency washes at 60 C. Hybridized blots were autoradiographed or subjected to phosphorimaging for visualization and quantitative estimates of the most prominent NPC-1 transcript (5.0 kb), along with the corresponding 28S band. The means of the dimensionless ratio of NPC-1 mRNA to 28S RNA were calculated for graphic representation of the data.
Immunohistochemistry
Porcine ovarian and testicular tissues were fixed in Zamboni’s solution, embedded in paraffin, and sectioned at 7 µm. For antigen recovery, issues were treated with 0.1% trypsin at 37 C for 10 min. All subsequent procedures were conducted at room temperature. Tissues were incubated for 1 h with the NPC-1 antibody (provided by H. Watari and J. F. Strauss III) (19, 22), employed at 1:100 and 1:200 concentrations. Tissues were washed in PBS and subjected to incubation for 30 min with goat antirabbit second antibody conjugated to fluorescein (BioCan, Mississauga, Canada) before microscopic visualization and digitalization of the images. The method was the same for porcine cells derived from primary culture, except no antigen retrieval was performed. Control tissues were subjected to the same procedures, except that NPC-1 antibody was replaced with normal rabbit serum.
Cell culture, transient transfections, and promoter activity assays
Three steroidogenic cell lines were employed. Y1 mouse adrenal tumor cells (CCL-79, American Type Culture Collection, Manassas, VA) were maintained in DMEM/F-12 supplemented with 10% horse serum, 2.5% FBS, and antibiotics. The mouse Leydig tumor cell line (MA-10), a gift from Dr. Mario Ascoli (23), was cultured in Waymouth’s MB/752 medium supplemented with 15% horse serum. SVG40 human granulosa cells (gift from Dr. P. Leung) were cultured in Opti-MEM, supplemented with 5% FBS and antibiotics. The cells were transfected with 100 nM/well of 1.86 kb of the pig NPC-1 promoter (pNPC-Luc) in a firefly luciferase reporter gene in the vector pGL3. Basic (Promega Corp.) using Effectene reagent (QIAGEN) according to the manufacturer’s protocol. Cells were cotransfected with the SV40 Renilla luciferase control vector pRL.SV40 (Promega Corp.) at a ratio of 10:1 for pNPC-LUC/pRL.SV40 to normalize results for transfection efficiency. Some cultures were treated with 1 mM (Bu)2cAMP (Sigma) for 24 h. Luciferase activity was detected by means of the Promega Corp. Dual Luciferase Assay system, and chemiluminescence was measured in a Berthold 9501 luminometer. Control transfections included the inclusion of an equal amount of the promoterless pGL3 basic plasmid (Promega Corp.).
Statistical analysis
One-way ANOVA was performed on mRNA abundance and luciferase activity data. Individual comparisons between means were made by the Tukey-Kramer test. The level of significance was P < 0.05.
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Results |
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provides information about the domain homology between the porcine NPC-1 sequence and the human (9) and mouse ( 13) sequences. Homology with potential posttranslational modification sites in the human protein are present in the pig sequence, including a potential tyrosine phosphorylation site (aa 506) and 17 cysteine residues that are conserved between the human and mouse sequences (9). The putative lumenal loop 3 (10, 11), also known as the cysteine-rich loop, present in the human sequence (24, 25) contains six cysteine residues in the pig. There is a strong tendency for clustering of the 20 potential glycosylation sites in the pig sequence. All but one (aa 314) are within the proposed lumenal loop regions of the human protein (10, 11). Five are in the N-terminal putative loop 1, 7 in loop 2, and seven in loop 3. Computer analysis revealed 20 potential myristoylation sites, of which only 3 were in the putative loop 1 compared with 6 in the human (11); none was in loop 2, and 8 were found in loop 3.
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). Although no consensus TATA box motif was present, an important GC-rich region (CpG island) was found in the promoter (Fig. 1). It is significant to this report that a site (TGACGTGC) showing high homology to the consensus cAMP response element-binding protein (CREB)-binding site (TGACGTCA) (26, 27) was present in the porcine promoter at -312 from the putative transcription initiation site (ATG). A second sequence with lower homology to consensus CREB was present at -152 to -148 from the ATG. The overall homology between the region of the pig and human sequences 400 bp upstream of the translation initiation site was 61%.
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). Transcripts were detected in all of the tissues investigated (Fig. 2) and were in highest abundance in tissues that synthesize steroids. In the 15 µg total RNA samples examined, the Leydig cell-rich, immature pig testis had NPC-1 mRNA in 2.6-fold greater abundance relative to the same quantity of total liver RNA, corrected for loading with 28S. Adult testis, in which the spermatogenic elements are most abundant and the relative Leydig cell number is reduced, had a lower amount of NPC-1 transcript. Ovarian granulosa cells had 5.3-fold, corpora lutea (CL) 5.8-fold, and adrenal 8.5-fold more abundant expression of NPC-1.
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), and this was significantly different from the postovulatory (stage I) CL (P < 0.05). The message was barely detectable in CL showing morphological regression (P < 0.01 relative to all other stages). Granulosa cells cultured for 96 h displayed the morphological characteristics of in vitro luteinization, including confluence and a cobblestone, epitheloid aspect (20). NPC-1 transcripts were detectable in freshly isolated cells and had increased through 96 h (P < 0.05), concomitant with luteinization (Fig. 3B).
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). The protein was present in a punctate form distributed throughout the cytoplasm. In CL recovered at approximately 24 h after ovulation, a strong signal was present in the thecal-vascular elements that invade the granulosa compartment at this time (Fig. 4B). Incipient punctate expression was observed in the cytoplasm of granulosa cells (Fig. 4C). In midcycle corpora lutea, NPC-1 was highly expressed in large and small luteal cells (Fig. 4D). The protein was also detected in the Leydig cells of the immature pig testis (Fig. 4E). Figure 4F represents the control in which NPC-1 antibody was replaced by normal rabbit serum and no evident signal.
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). As these cells luteinized in culture, the abundance of the punctate NPC-1 protein progressively increased and became associated with larger organelles, presumably the lysosomes. Cultures terminated at 48, 72, and 96 h after harvest, and mRNA abundance was significantly different from that in freshly isolated cells (P < 0.05).
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, A and C). P450scc mRNA disappeared more slowly than StAR, but much more rapidly than NPC-1 (Fig. 6C). Incubation with the translational blocker, CHX, resulted in dramatic increases in NPC transcript abundance (2-fold at 6 h and 5.6-fold at 24 h; Fig. 6, A and B).
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). Subsequent hybridization was conducted with the homologous StAR probe, as indicated in the expected increases in transcript ranging from 8- to 12-fold in these experiments (Fig. 7A).
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; P < 0.05). |
Discussion |
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Regulation of NPC-1 expression by cAMP
The prevailing view is that NPC-1 is a housekeeping gene based on the ubiquity of its expression and evidence for posttranscriptional regulation of message stability (33). In contrast, we contend that, at least in the pig, NPC-1 is a cAMP-regulated gene, and we present three lines of evidence in support of this assertion. First, there are increases in NPC-1 transcript levels in luteinized pig granulosa cells treated with (Bu)2cAMP. Although these are less robust than seen with acute regulation of StAR (21), they are nonetheless consistent and of the same magnitude observed with steroidogenic enzyme transcripts in the same cell system (34). Secondly, analysis of the 5'-flanking region of the porcine NPC-1 gene revealed a consensus elements for a cAMP response, including the sequence for binding CREB and another CREB family member, activating transcription factor-1 (26, 27). Third, we demonstrated that porcine NPC-1 promoter-reporter constructs transfected into three steroidogenic cell lines, human granulosa cells, MA-10 Leydig tumor cells, and Y-1 adrenal cells, displayed consistent increases in promoter activity over constitutive levels after (Bu)2cAMP stimulation. Similar experiments in primary cultures of human granulosa-lutein cells indicated that the 8-bromo analog of cAMP did not increase NPC-1 mRNA abundance (19). This led those researchers to suggest that our preliminary finding (32) of (Bu)2cAMP induction of pig NPC-1 mRNA was due to an effect of progesterone on blockade of intracellular cholesterol transport. We believe that the presence of a cAMP response element in the pig promoter and evidence for cAMP stimulation of NPC-1 promoter-reporter expression argue for a direct, rather than indirect, effect of cAMP on this gene.
Translational and transcriptional inhibition
Estimates of transcript half-life can be made after treatment with ACT-D, nonetheless bearing in mind the inherent uncertainties related to blockade of synthesis of all proteins. We provide an estimate for NPC-1 in luteinized porcine granulosa cells on the order of 20 h. This compares with an estimate of 12 h for the transcript in human granulosa-lutein cells (22). The same authors indicated that exogenous progesterone increased the persistence of the human NPC-1 transcript. Human luteal cells produce relatively little progesterone (19) relative to pig cells (30), and the inherent production of this steroid may account for the long half-life of NPC-1 RNA in the present investigation.
CHX blockade of translation resulted in increased NPC-1 transcript abundance. A similar result was obtained for human NPC-1, again attributed to an increase in message stability (22). In support of this view is the finding that NPC-1 expression is not superinduced by the combination of CHX and cAMP in steroidogenic cell lines (Gévry, N., and B. D. Murphy, unpublished results).
NPC-1 sequence homology
The data presented herein indicate a high degree of overall homology in the coding sequences of the porcine homolog of the NPC-1 gene and the human and mouse sequences (9, 13). The deduced aa sequence indicates the presence of some variation among the signal sequences, with the pig and human sequences displaying a similar number of hydrophobic residues (9). There is remarkable 100% identity between the pig and human sequences in what has come to be known as the NPC-1 domain (9). This region contains the leucine zipper motif, believed to be involved in protein-protein interactions. The sterol-sensing domain, which has been speculated to be a site for the direct binding of cholesterol (35), displayed 92% homology between the pig and human sequences. The potential glycosylation sites are likewise well conserved and clustered within the putative lumenal loops of the molecule (10, 11), which contain the NPC-1 domain and the N-glycosylation sites obligatory for function (25). Loop 1 ( 10) has an overall homology in excess of 98% between the human and pig sequences. The cysteine-rich loop, also known as loop 3 (10), is mutated in a cluster of NPC-1 disease phenotypes (24). It has recently been shown to be necessary for NPC-1 function by experimental mutagenesis studies (22). It shows some divergence in the pig, with 88% similarity to the human sequence and only 81% to the mouse. Given the importance of the lysosomal targeting sequence (LLNF) to NPC-1 action (25), it is not unexpected that it is conserved in the mouse, human, and pig sequences.
Herein we make the second report on the 5'-flanking region of the NPC-1 gene, and it is therefore possible to make some comparisons with the human sequence (22, 36). The transcription initiation site(s) is not known for either species, but in both there is a substantial 5'-untranslated region amplified by RT-PCR, indicating initiation upstream of the ATG. Both sequences demonstrate the presence of a CpG island, in which 80% or more of the nucleotides are GC. In both cases this is found near the transcription start site, characteristic of constitutively expressed genes, and approximately half of the genes are expressed in a tissue-specific manner (37). The CpG island overlaps putative transcription factor-binding sites, including the CREB/activating transcription factor sequence, consistent with the view that the island is coincident with the region for binding of transcription factors (37). A SP-1 site is also present in the CpG island, and SP-1 has been linked to maintaining the island methylation-free, thereby preventing silencing of the gene (38). This localization notwithstanding, mutation of the human SP-1 site had no effect on the function of the human NPC-1 promoter-reporter constructs (22). There are numerous AP-1 sites on the promoter, and these may be involved in cAMP induction of transcription, as has been seen in rat granulosa cells (39).
In summary, we have analyzed the sequence of the porcine homolog of the human NPC-1 gene. The open reading frame displays high homology to the human and mouse sequences, whereas the promoter region shows greater variation. Expression of NPC-1 is greater in steroidogenic tissue relative to other somatic tissues and increases with the state of differentiation and steroidogenic potential of the CL. Porcine NPC-1 displays both constitutive and cAMP-regulated expression.
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Acknowledgments |
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Footnotes |
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Abbreviations: aa, Amino acids; ACT-D, actinomycin D; AP, activating protein; CHX, cycloheximide; CL, corpus luteum; CREB, cAMP response element-binding protein; NPC-1, Niemann-Pick C-1; nt, nucleotides; P450scc, cytochrome P450 side-chain cleavage enzyme; SP-1, specificity protein-1; StAR, steroidogenic acute regulatory protein.
Received September 4, 2001.
Accepted for publication October 22, 2001.
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and luteal secretion of progesterone in response to oxytocin during the porcine estrous cycle. Biol Reprod 45:950–954[Abstract]
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, Controls; 
, CHX-treated. The effects of ACT-D on the abundance of transcripts for NPC-1 (
), StAR (
), and P450scc (