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Journal of Endocrinology (2004) 183, 101-114       DOI: 10.1677/joe.1.05649
© 2004 Society for Endocrinology
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Sex steroid regulation and identification of different transcription units of the SA gene in mouse kidney

Cristina Aresté, M Jesús Melià, Joan Isern, José Luis Tovar1 and Anna Meseguer

Centre d’Investigacions en Bioquímica i Biologia Molecular (CIBBIM), Hospital Universitari Vall d’Hebron, Barcelona, Spain
1 Servei de Nefrologia, Hospital Universitari Vall d’Hebron, Barcelona, Spain

(Requests for offprints should be addressed to A Meseguer; Email: ameseguer{at}vhebron.net)


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although the SA gene was first identified as a putative candidate gene to understand the molecular basis of hypertension in rat and humans, the concept has not been supported in recently generated SA-null mice. We had first identified the mouse SA gene on the basis of its strong androgenic regulation in mouse kidney and further characterized its genomic organization, transcription start site and chromosomal location. Northern blot, RT-PCR and in situ hybridization assays determined mouse strain, tissue distribution, sex-hormone dependence and cell expression of the SA mRNA. Kidney and liver constitute the main expression sites of the SA gene; in particular it is expressed in epithelial proximal tubule cells in the presence of androgens. This androgen-dependent expression is abrogated when estrogens are also present. By using the sensitive RT-PCR technique, minor SA expression sites, corresponding to testes, stomach, heart and lung, have also appeared. Like in kidney, expression of the SA gene in heart and lung is androgen-dependent. Production of rabbit antibodies against SA-synthetic peptides identified the SA protein, a moiety of unknown function, which has been defined as a member of the acyl-CoA synthetase family. We have determined that the SA protein follows the same distribution and regulation as its corresponding mRNA. Transient transfection assays followed by confocal microscopy identified the mitochondria of proximal tubule-derived PCT3 cells as the subcellular location of the SA protein. Different transcriptional units produced by splicing events, occurring before the translation initiation site, have been identified from mouse kidney. This work provides the basis to further understand the molecular mechanisms that control the sex–steroid-dependent expression of the SA gene in mouse kidney, heart and lung, where SA is also expressed in an androgen-dependent manner.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Understanding the processes by which extracellular stimuli modulate the expression of specific genes in a temporal and/or tissue-specific manner is crucial for unravelling the molecular mechanisms underlying cellular growth, homeostasis, differentiation and development. The molecular nature of tissue-specific gene regulation by androgens has not been well defined, partly as a result of the variable expression and incomplete regulation of currently available gene models. To overcome this problem we aimed to establish more informative models by identifying alternative genes whose expression would be tightly and co-ordinately regulated by androgens. By means of the subtractive hybridization techniques of Random arbitrarily printed (RAP)-PCR and representative differential analysis of cDNA (cDNARDA) we isolated differentially expressed genes from kidneys of female C57BL/6 mice dosed with dihydrotestosterone (DHT). In addition to well-characterized androgen-regulated genes (e.g. kidney androgen related protein), we demonstrated the differential expression of other genes previously not known to be under androgen control (Melià et al. 1998). Among them, and because the physiological significance of androgen-inducible gene expression in the kidney is quite unknown, we were particularly interested in the SA gene, since it was initially identified on the basis of its marked overexpression in kidneys of spontaneously hypertensive rats compared with the normotensive Wistar–Kyoto rats (Iwai & Inagami 1991). SA gene markers were subsequently shown to co-segregate with blood pressure (BP) in F2 cohorts of different rat crossings (Iwai and Inagami 1992, Iwai et al. 1992, Harris et al. 1993, Lindpaintner et al. 1993). Other authors not only demonstrated co-segregation of the gene with BP, but also that genotype at the SA locus determined the level of expression of the SA mRNA in kidney (Samani et al.1993, Kaiser et al. 1994). Further evidence of its putative association with BP was provided by mapping the SA locus on to the chromosome 1 linkage group (Lindpaintner et al. 1993), a well-characterized chromosomal region that contains several genes of potential relevance to cardiovascular function that is synthenic to the human chromosome 16, where the SA gene was also mapped (Samani et al. 1994). Although association between a polymorphism at the SA locus and hypertension has remained controversial (Iwai et al. 1994, Harrap et al. 1995), it has recently been reported that different alleles of the SA gene are associated with multiple risk factors including hypertriglyceridemia, hypercholesterolemia, obesity and hypertension (Iwai et al. 2002). A genetic polymorphism in the SA gene has also been related to BP and prognosis of renal function in patients with immunoglobulin A nephropathy (Narita et al. 2002). Finally, the SA locus has been found positively linked with loci regulating water and sodium metabolism and membrane ion transport in essential hypertension (Chu et al. 2002). Different reports on chromosome-transfer studies in congenic strains, to isolate chromosome regions that contain the BP quantitative trait locus (QTL) in the region around the SA gene from different rat strains, have also provided controversial data (St Lezin et al. 1997, Frantz et al. 1998, Iwai et al. 1998, Hübner et al. 1999, Saad et al. 1999, St Lezin et al. 2000, Frantz et al. 2001). In a recent paper, Walsh et al.(2003) have shown direct evidence of the lack of SA involvement in the regulation of either basal or salt-related BP in SA-null mice, demonstrating that the absence of differential BP in these animals is not the consequence of compensatory activation of the renin–angiotensin system.

The SA-encoded protein is significantly homologous to bovine xenobiotic-metabolizing medium-chain fatty acid-:CoA ligase (Vessey & Kelley 1997) and recent studies have identified the SA protein as a medium-chain acyl-CoA synthetase (MACS; Fujino et al. 2001a, 2001b). Acetyl-CoA synthetase (ACS; also called acetate-CoA ligase) is an enzyme of energy metabolism known to be present in mitochondria and responsible for acetate production accompanied by ATP generation. The SA and ACS genes probably derived from duplication of an ancestral gene, but acquired different functions (Karan et al. 2001) which are not completely understood for the SA gene. It remains important to understand the physiological function of this highly restricted tissue-specific gene to gain insight into the physiological effects of sexual steroids in the kidney. In this report, we further explored the tissue distribution and sex-steroid regulation of the mouse SA gene and compared them with the protein profile obtained using specific antibodies raised against SA-derived synthetic peptides. Moreover, we determined the genomic organization of the mouse SA gene and identified its transcription start units. This work forms the basis for further study of molecular mechanisms that control the androgen-dependent and kidney-restricted expression of the mouse SA gene.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and treatments

C57BL/6, BALB/c and 129/SvJ mice were obtained from IFFA CREDO (L’Arbescle, France) at 6 weeks of age and housed in animal facilities as described elsewhere (Melià et al. 1998). Male mice were castrated at the age of 8 weeks under droperidol and midazolam anesthesia and allowed to recover for 1 week post-surgery. Male and castrated mice were treated for 6 weeks with DHT and 17-ß-estradiol (Sigma) with subcutaneous injections of 120 and 240 µg/day, respectively. Control mice received vehicle alone (95% sesame oil/5% ethanol). After treatment, animals were killed by cervical dislocation. Several tissues were collected and immediately frozen in liquid N2.

RNA extraction and Northern blot analysis

Total RNA was extracted from different tissues using the guanidium thiocyanate/acid phenol method (Chomczynski & Sacchi 1987). Total RNA (15 µg) was electrophoresed in 6.5% formaldehyde/1.4% agarose gel, transferred to ZetaProbe membranes (Bio-Rad) and hybridized at 42 °C overnight with random-primed [{alpha}–32P]dCTP (Amersham Pharmacia Biotech)-labeled cDNA probes, washed following the membrane manufacturer’s instructions and exposed to Hyperfilm (Amersham Pharmacia Biotech). A probe corresponding to cDNA of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal control on each hybridization. Where noted, band intensity was measured by densitometric scanning of the resultant autoradiograph using the Bio-Rad GS700 image densitometer and Molecular Analyst 1.40 program.

RT-PCR and Southern blotting

Total RNA from various tissues of male 129/SvJ mice was isolated using the total RNA preparation kit (Qiagen) and subjected to RT-PCR analysis. A total amount of 500 ng of each tissue was reverse-transcribed using specific primers (see Table 1Go) and the SuperScript One-Step system (Invitrogen) following the manufacturer’s instructions. RT-PCRs were performed under linear conditions with respect to RNA input and the number of amplification cycles. PCRs using SA1 and SA2 primers were determined as linear for 25 cycles and those performed with primers E1', E1, E2, E3 and E4 were determined as linear for 35 cycles. Cyclophilin A was amplified as a control for RNA amount and integrity. Amplification products were separated on 2% agarose gel and transferred to ZetaProbe membranes (Bio-Rad). The blots were probed with specific random primed [{alpha}-32P]dCTP-labeled cDNA. Hybridization, washes and exposure were performed as above. Amplified products were subcloned into the TopoTA cloning pCR2.1 vector (Invitrogen) and sequenced in both directions.


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  		Table 1 Specific primers used in SA fragment amplification
 
Synthesis of riboprobes and in situ hybridization histochemistry

35S-labeled sense and antisense transcripts from a Bluescript plasmid containing 148 bp fragment of the mouse SA cDNA were prepared as previously described (Melià et al. 1998). Preparation of renal sections, hybridization protocol and autoradiographic analysis were all performed as reported (Meseguer & Catterall 1990, 1992).

Primer-extension analysis

Two SA-specific primers, located within the 5' region of the cDNA–E4, 4–24 upstream, and E3, 109–89 downstream from the ATG initiating codon–were end-labeled with [{gamma}-32P]ATP. Labeled primer (200 fmol) was annealed to 15 µg mouse kidney total RNA in a 12 µl reaction by heating at 90 °C for 2 min and then cooling to 58 °C at 1 °C/min. The annealing reaction was held at 58 °C for 30 min then snap-chilled on ice. Annealed primers were extended at 42 °C for 2 h by the addition of 200 U of Superscript II reverse transcriptase (Gibco–BRL), 1 µl RNasin, 1 µl 10 mM dNTPs, 4 µl 5x first-strand reaction buffer and nuclease-free water to 20 µl. The reaction was terminated by the addition of 3 µl 0.2 M EDTA (pH 8.0), and the RNA was degraded by the addition of 1 µl RNase A followed by incubation at 37 °C for 30 min. The primer-extension reaction was then ethanol-precipitated and the pellet resuspended in 5 µl loading buffer. Samples were heated at 75 °C for 10 min prior to loading on a sequencing gel. A sequencing reaction for comparison with the primer-extension product was performed with the Sequenase version 2.0 DNA sequencing kit (USB, Cleveland, OH, USA) according to the manufacturer’s protocol. The SeqSa6 low primer was used as a template. Labeled cDNAs were separated through a 6% polyacrylamide/8 M urea gel. Dried gel was exposed to Kodak X-Omat AR film for 48 h at –80 °C.

Intron/exon mapping

A 129/SvJ mouse genomic library Lambda FIX II vector (Stratagene) was screened with a 2.0 kb probe corresponding to mouse SA full-length cDNA. Briefly, approximately 500 000 independent clones were plated and transferred to nitrocellulose membranes (Duralose-UV, Stratagene, Saint Quentin en Yvelines, France). Prehybridization was carried out for a minimum of 2 h at 42 °C in the same hybridization buffer consisting of 50% formamide, 2x Pipes, 0.5% SDS and salmon sperm DNA (100 µg/ml). 32P-random primed labeled probe was added to the hybridization solution (1x106 c.p.m./ml) and incubated overnight at 42 °C. The next day, filters were washed twice in 1xSSC/0.1% SDS for 5 min at room temperature, followed by three high-stringency washes in 0.1xSSC/0.1% SDS for 15 min at 65 °C. Plaque filters were then exposed to autoradiographic films (X-Omat; Kodak) at –70 °C for approximately 20 h. Positive plaques were identified and after four further rounds of purification of phage DNA, five genomic clones–designated {gamma}SA1, {gamma}SA2, {gamma}SA3, {gamma}SA4 and {gamma}SA5–were isolated. Genomic DNA from positive clones was isolated with the QIAGEN Lambda MiniKit and Maxi Kit. Double-stranded DNA was sequenced using ABI Prism Big Dye terminator chemistry (PE Applied Biosystems). Exon sizes were determined by nucleotide sequencing and intron sizes determined by either nucleotide sequencing or estimation from the size of the corresponding PCR-generated DNA fragments using exon-specific primers.

Production of anti- SA polyclonal antibodies

A short peptide, pSA, corresponding to amino acids NH2-CGNFKGMKIKPGSMGK-COOH from position 380 to 395, was selected on the basis of its putative immunogenicity and synthesized in the Servei de Síntesi de Pèptids, Facultat de Química, Universitat de Barcelona, Barcelona, Spain. Two New Zealand male rabbits (261 and 262) were immunized on days 0, 14 and 28 and boosted at days 55 and 84 with 200 µg doses of pSA peptide conjugated with keyhole limpet hemocyanin. Antisera containing the anti-peptide antibody were tested by Western blot.

Western blot analysis

Tissues were homogenized by N2 cavitation in RIPA buffer (0.5% Nadeoxycholate, 1% Nonidet P-40, 0.1% SDS and protease inhibitors in 1xPBS). For Western blot analysis, samples were normalized for protein concentration using the Bradford assay (Bio-Rad), adjusted for equal protein levels, and separated on 10% polyacrylamide gel electrophoresis under denaturing conditions. Proteins were transferred to PVDF (Shleicher & Schuell) membranes and blots blocked overnight at 4 °C in 5% non-fat dried milk in PBS. Primary polyclonal antibodies were tested at different concentrations; the best results were obtained with the 261 antiserum diluted at 1:350 in blocking buffer. Washes were performed following the membrane manufacturer’s instructions and secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit; Dako A/S), diluted 1:5000, incubated for 1 h at room temperature. After washing, bands were detected using the ECL+ chemiluminescence detection method (Amersham Pharmacia Biotech) and exposed to Hyperfilm.

Cell culture and transfection assays

Proximal convoluted tubule cells PKSV-PCT (PCT3 clone) were cultured as described previously (Lacave et al. 1993, Soler et al. 2002). The pFLAG-CMV-5a-SA construct was obtained by cloning the open reading frame of the mouse SA cDNA in the SalI site of the pFLAG-CMV-5a expression vector from Sigma.

Transient transfections were performed using LipofectAMINE PLUS reagent kit (Life Technologies) according to the manufacturer’s instructions. Briefly, cells were seeded at 8x105 cells in a 60 mm dish and transfected 18 h later with 6 µg of the pFLAG-CMV-5a-SA construct. The DNA–liposome mixture was added to cell culture dishes containing an appropriate volume of OPTIMEM I Reduced Serum Medium (Life Technologies). At 3 h of incubation at 37 °C, complete fresh medium was added. Twenty-four hours after transfection, cells were trypsinized and seeded onto glass slides for immunocytochemistry assays.

Immunofluorescense analyses

Trypsinized transfected cells were grown on glass slides for 24 h. After two washes in cold Tris-buffered saline (TBS), cells were fixed in cold acetone/methanol (1:1) for 1 min and washed again in TBS three times. Slides were incubated for 1 h at room temperature with 10 µg/ml anti-FLAG primary antibody (Sigma) in TBS buffer. Upon washing, cells were incubated for 1 h at room temperature with the secondary antibody (FITC-conjugated goat anti-mouse Fab-specific; Sigma) diluted 1:200. Slides were dehydrated and mounted in Aquatex (Merck). Fluorescence labeling was visualized using a Leica DM IRBE confocal microscope.

Mitochondrion-specific staining

For mitochondrial location, living cells grown on glass slides were incubated for 90 min at 37 °C with 500 nM MitoTracker Red CMXRos (Molecular Probes) diluted in complete medium. MitoTracker-loaded cells were fixed in cold acetone/methanol (1:1) for 1 min, washed and visualized under a Leica DM IRBE confocal microscope.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Androgen-dependent expression and cell specificity of the SA gene in different mouse strains

An initial report from our laboratory first described the mouse counterpart of human and rat SA genes (GenBank accession number AF068246 [GenBank] ) on the basis of their profound androgenic regulation, at the mRNA level in mouse kidney (Melià et al. 1998). Since SA gene expression in rat kidney was not completely prevented by castration (MJ Melià & A Meseguer, unpublished observations), we wondered whether the strong androgenic control observed in mice was a general phenomenon in mice or an isolated event occurring in the C57BL/6 strain used. Northern blot assays of kidney RNA from castrated and control C57BL/6, 129/SvJ and Balb/c male mice showed that SA expression is not completely abolished in castrated males but perhaps only undetectable using Northern blot analysis (Fig. 1AGo). Moreover, in situ hybridization of frozen kidney sections using sense- and antisense-specific probes demonstrated that this gene is expressed in epithelial cells of the early (S1 and S2) and late (S3) segments of proximal convoluted tubules (Fig. 1BGo), as determined by periodic acid shift counterstaining (results not shown). SA mRNA was first located in the rat proximal tubule by Samani’s group using in situ hybridization (Patel et al. 1994) and by Yang et al.(1996) using RT-PCR in cDNAs prepared from microdissected nephron segments. While in intact mice the first report on SA mRNA location was made by Takanaka et al.(1998), we demonstrate here that castration prevents expression in all segments of the tubules and DHT replacement restores the expression in castrated mice.



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  		Figure 1 (A) Northern blot analysis of SA mRNA in kidneys of different inbred mouse strains. Total RNA (15 µg) from intact (N) and castrated (Cx) male kidneys of C57BL/6, 129/SvJ and BALB/c strains was electrophoresed and transferred to nylon membranes. Hybridization was performed with mouse SA cDNA under the conditions described in the Materials and Methods section. A mouse cyclophilin A probe was used as an internal control for loading and integrity of RNA. A single 3.0 kb transcript was significantly detected only in intact males in all three mouse strains tested. (B) In situ hybridization analysis of SA mRNA in mouse kidney. Longitudinal kidney sections from castrated males treated with pharmacological doses of DHT (a and b) and castrated males (c and d) were hybridized with strand-specific 35S-labeled RNA probes (antisense, a and c; sense, b and d). After hybridization, sections were exposed to photographic emulsion for 4 weeks. After developing, slides were mounted and examined under a light microscope. The magnification chosen (x25) allows visualization of the three major compartments of the kidney (c, cortex; o, outer medulla; m, inner medulla; see A) and determination of the spatial location of SA mRNA. Kidney sections shown in the panels were each analyzed in a single experiment. Consequently, slides were exposed to the same conditions throughout the procedure, and levels of SA mRNA expression on different slides were comparable. Photomicrographs shown in this figure were selected from similar results obtained in different experiments using animals from different litters.

 
Distribution and androgen regulation of SA mRNA in mouse tissues

Although tissue specificity of SA mRNA has previously been stated (Melià et al. 1998), the more-sensitive RT-PCR/Southern blot technique was used and distribution of SA mRNA determined in a wider panel of tissues including kidney, liver, brain, stomach, prepucial gland, duodenum, spleen, testis, lung and heart. Results in Fig. 2AGo, showing a saturated image on X-ray film after 3 h exposure (left-hand panel), corroborate the concept that kidney and liver tissues are those where the SA gene is expressed preferentially. Moreover, expression in stomach, testis, lung and heart was also detected on overnight exposure of the autoradiographic film (Fig. 2AGo, right-hand panel), and was completely neglected in brain, duodenum and spleen. While Northern blot assays were sensitive enough to detect expression in liver, testes and brain in rat tissues (Iwai and Inagami 1991, Kaiser et al. 1994), no expression in brain was observed in mice by RT-PCR. Although kidney and liver remain the preferential expression sites for the SA gene, our results demonstrate that it exhibits tissue distribution wider than that reported previously in mice (Melià et al. 1998). Some assays performed in castrated male mice indicate that the SA gene appears to be under androgenic control not only in kidney but also in heart and lung. The liver and stomach appear to express the gene in an androgen-independent manner (Fig. 2BGo).



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  		Figure 2 Tissue distribution of SA mRNA expression. (A) Expression profile of SA in intact male mouse tissues analyzed by RT-PCR. Total RNA (500 ng) from various murine tissues (indicated at the top) was reverse-transcribed and PCR-amplified using specific SA primers (SA1 and SA2; see Table 1Go). As an internal control, mouse cyclophilin A (CypA) primers were used in (B). Expression profile of SA in castrated male mouse tissues analyzed by RT-PCR, under the same experimental conditions described in (A).

 
Estrogenic effects on SA expression in kidney and liver

We aimed to determine whether estrogens could also exert an effect on SA gene expression. To do so, we performed Northern blot assays using total RNA from kidneys and livers of mice treated with DHT, estrogens or both hormones simultaneously and compared SA expression levels with those obtained in untreated control animals. Results were normalized with the endogenous control GAPDH gene and densitometric analysis performed in non-saturated X-ray films. The SA/GAPDH ratios expressed in arbitrary units are depicted at the bottom of Fig. 3AGo. The same treatments and assays were also made in castrated male mice (Fig. 3AGo). Results from these experiments revealed that pharmacological doses of DHT can induce further expression of the SA gene in untreated control male mice and that levels in castrated males are restored upon treatment, which indicates that the gene responds to androgens in a dose-dependent manner. Estrogenic treatment of control males or DHT-induced intact male mice resulted in a very drastic down-regulating effect on SA mRNA expression, even in the presence of pharmacological doses of androgens (Fig. 3AGo). Expression of SA mRNA in liver was completely independent of steroid hormones, as neither castration nor induction with pharmacological doses of DHT and/or estrogens modified the levels attained by control male mice (Fig. 3BGo). Since estrogens exerted a powerful negative effect on SA kidney expression, we wondered whether an estrogenic-dependent repression was responsible for the lack of expression in female kidney (Melià et al. 1998).



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  		Figure 3 Effects of sex steroid hormones on SA mRNA expression in mouse kidney and liver total RNA (15 µg) from kidneys (A) and livers (B) of intact non-treated males (NT), or males treated with pharmacological doses of DHT (DHT), estrogens (E2) and both hormones together (DHT+E2; left-hand panel), and similar samples obtained from castrated male mice under the same hormonal treatments (right-hand panel) were electrophoresed and transferred to nylon membranes. Hybridization was performed with mouse SA cDNA under the conditions described in the Materials and Methods section. A mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe was used as an internal control for loading and integrity of RNA. Non-saturated autoradiographs were analyzed by densitometry and the ratios between SA and the GAPDH internal control gene were calculated as shown under each lane. Samples from kidneys of intact non-treated (NT) and ovariectomized (Ovx) (C) or intact non-treated (NT) and DHT-treated females (DHT) (D) were analyzed using the same experimental procedure described for (A) and (B).

 
Ovariectomized females failed to express the SA gene in kidney (Fig. 3CGo) and respond to DHT stimulation (Fig. 3DGo), indicating that although estrogens can repress the gene, its kidney expression is fully androgen-dependent. Previous data from our laboratory demonstrated that the effects are mediated by the androgen receptor since flutamide-treated mice do not express the gene (Melià et al. 1998). These results indicate that there are tissue-specific mechanisms underlying SA expression and, therefore, that this gene constitutes an excellent model for understanding the basis of androgen regulation of specific gene expression in kidney.

Genomic organization of the mouse SA gene and identification of different SA transcriptional units

As an initial approach to elucidating the mechanisms regulating SA gene expression, we identified genomic clones through screening a mouse genomic library, using its full-length cDNA as a probe and characterized transcription units of the mouse SA gene. Several positive clones were isolated, cloned until homogeneity and sequenced with specific primers derived from the cDNA sequence of the gene. The genomic structure and intron/exon organization of the mouse SA gene are shown in Fig. 4Go and Table 2Go, respectively. The gene spans approximately 23 kb and consists of 16 exons and 15 introns (Fig. 4Go) and has been annotated at mouse chromosome 7 (ENSMUSG00000030935) at the Ensemble Genome Browser. The translation initiation site is present in exon 4. Exon sizes range from 69 to 274 bp, with the exception of exon 16, which is 616 bp and contains the TAG stop codon and 3' untranslated region, including the polyadenylation signal. The size of the introns was determined by either direct DNA sequencing or long-distance PCR with exon-specific primers; in some cases, alignment with mouse genomic traces from the mouse genome sequencing database was also used to verify and determine the length of some introns. All exon/intron boundaries conform to canonical splice donor and acceptor consensus AG–GT sequences (Mount 1982). The transcription initiation site of the SA gene was mapped by primer extension and 5'-RACE (rapid amplification of cDNA ends). For primer extension, a pair of reverse primers was tested (E3 and E4; see Table 1Go), complementary to the third and fourth exons, respectively. Primer E3, situated 89 nucleotides from the translation initiation ATG codon, rendered three products of 132, 167 and 259 nt which indicated the existence of three transcription start sites, with the smallest being the most prominent, mainly in the 129/SvJ strain (Fig. 5AGo). Results were confirmed using primer E4, which gave a single product of 240 bp corresponding to the 132 site obtained with E3. Results were the same in both mouse strains. From these experiments we located three major potential transcription start sites at 221, 256 and 348 bp upstream from the translation initiation ATG codon. The right-hand panel of Fig. 5AGo depicts the transcriptional units obtained by primer extension that were further confirmed by 5'-RACE. Sequencing of the products revealed that the previously cloned cDNA (Accession number AF068246 [GenBank] ) corresponds to the 167 product; the 259 band includes 35 bp from the 5' site of exon 1 (also present in the 167 and named for convenience 1') and an additional 92 bp situated further down from 1' that complete the entire exon 1. This new form was also deposited in the GenBank database under accession number AY064696 [GenBank] .



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  		Figure 4 Genomic structure of the murine SA gene. Schematic representation of the SA gene. Exons (numbered 1–16) are represented by boxes and oblique-horizontal lines indicate intronic regions (numbered I–XV). White and black boxes represent untranslated and protein-coding regions, respectively.

 

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  		Table 2 Genomic organization of murine SA gene
 


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  		Figure 5 Identification of the transcriptional start sites of the mouse SA gene. (A) The nucleotide sequence flanking the transcription initiation sites is illustrated on the left, with transcription initiation sites obtained by primer extension (arrowheads). The arrows represent the major transcription start sites common to C57BL/6 and 129/SvJ mouse strains as mapped by primer extension, obtained using primers E3 and E4 and mouse kidney total RNA as a template. The sizes of the extended bands are indicated. For further details see the Materials and Methods section. The right-hand side of the figure shows the three major forms and the nature of the exons included. (B) RT-PCR amplification of the mRNAs of mouse kidney SA. PCR products were obtained using six sets of primers, E1'–E2, E1'–E3, E1'–E4, E1–E2, E1–E3 and E1–E4. Representative agarose gels of PCR products obtained with the six sets of primers are shown at the top of the figure. M indicates the molecular DNA markers used (M is 100 bp and #M is 1 kb ladder markers from Invitrogen). A schematic representation of the possible origin of PCR products appears at the bottom. Arrows indicate the annealing regions of the PCR primers, lines above indicate the sizes for the different moieties obtained upon cloning and sequencing of the PCR products. (C) Schematic representation of the splicing cryptic sites found in exons 1 and 2 of the SA gene.

 
Apart from the transcripts obtained in our laboratory, other authors identified a new unit of SA cDNA, which included 208 bp of exon 2 and no sequences from exon 1 (accession number BC015248 [GenBank] ). At that point, we aimed to determine whether other SA mRNA products could also exist in our kidney mRNA samples. By performing RT-PCR assays using different sets of primers, indicated at the top of each gel (Fig. 5BGo, upper panel), we obtained a variety of products that upon cloning and sequencing revealed an even more complex organization of the 5' untranslated region of the SA gene. There are two forms on the lower panel of Fig. 5BGo that correspond to the 167 and 259 fragments of Fig. 5AGo (marked with an asterisk). Both forms have skipped exon 2 and contain either the full exon 1 (1'-1), or exon 1' Apart from these forms, we obtained four new products which show different combinations of exons 1, 2, 1'and 2' (the latter corresponds to the 63 bp from the most 3' part of exon 2), followed by exons 3 and 4. These splicing variants are generated from canonical splice donor and acceptor consensus AG–GT sequences located in exons 1 and 2 of the SA gene that define the boundaries between exon 1'-1 and exon 2–2' (Fig. 5CGo). By comparing the genomic structure and intron/exon organization of the mouse SA gene (see Fig. 4Go and Table 2Go) with rat SA genomic sequences (GenBank accession number AY456695 [GenBank] ) we have observed as the main difference that the rat gene is lacking mouse exon 2. It contains, therefore, 15 exons instead of 16, which share 86–98% homology with the mouse sequence. The translation initiation site and the stop codon are located at exons 3 and 15, respectively. Moreover, rat exon 1 lacks what we have named exon 1' in mouse; the remaining 65 bp of rat exon 1 share 95% homology with the 3' end of mouse exon 1. The main differences between the rat and mouse SA genes correspond to the 5' region, before the translation initiation site, which indicates that both proteins will be very similar.

Location and hormonal control of the SA protein

Polyclonal antibodies raised against SA-specific peptides revealed the appearance of four different molecular species by Western blot assays (Fig. 6Go). From them, the 62 and 118 kDa products disappeared in castrated males and appeared in DHT-treated castrated mice, indicating that their expression is androgen-dependent (Fig. 6AGo, left–hand panel). Assays performed in the presence of specific SA-blocking peptide showed that the 62 kDa protein disappears, while the other three remain under this condition (Fig. 6AGo, right-hand panel). We postulate that the protein of apparent molecular mass 62 kDa corresponds to SA because (i) 62 kDa is close to the expected size of the deduced SA protein, taking the ATG codon in exon 4 as the translation initiation codon, (ii) it disappears in the presence of the specific blocking peptide, (iii) its expression is androgen-dependent and (iv) the anti-SA antibody recognizes the same moiety as the anti-FLAG antibody in cells transiently expressing the SA-FLAG fusion protein (results not shown).



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  		Figure 6 Western blot analysis of crude homogenates from mouse kidney. (A) Crude kidney homogenates (100 µg/lane) from intact non-treated males (NT), castrated (Cx) or castrated males treated with pharmacological doses of DHT (Cx+DHT) were analyzed by Western blot using rabbit antiserum raised against SA synthetic peptide (left-hand panel). Preabsorbing the antibody with an excess of the antigenic peptide proved the specificity of the reactions (right-hand panel). Arrow points to the 62 kDa apparent-molecular-mass product that corresponds to the SA protein. (B) In this experiment, crude kidney homogenates from intact or castrated males treated with estrogens (E2) or with estrogens and DHT (DHT+E2) were included. Non-treated female kidney homogenates were also studied using the experimental approach described for (A).

 
To further explore whether expression of the protein parallels that of the mRNA, we studied the effects of estrogen treatment on SA protein levels. As shown in Fig. 6BGo, estrogens inhibit expression at physiological and pharmacological doses of androgens, i.e. in control males and in DHT-induced castrated males. In conclusion, the protein follows the same sex steroid-dependent expression pattern as its corresponding mRNA. The nature of the 118 kDa product which follows an androgen-dependent but estrogen-independent pattern of expression is unknown and might not necessarily be related to the SA protein. The mouse SA protein presents high homology with the human and rat SA, 86 and 94%, respectively. Alignment of the three sequences (Fig. 7Go) reveals several functional motifs that include 29 amino acids on the N-terminal region, which corresponds to a mitochondrial translocation signal (see boxed sequence in Fig. 7Go) and an AMP-binding domain, according to the PROSITE program (see underlined sequence in Fig. 7Go). The differences between the 65.5 kDa predicted molecular mass of the mouse SA protein and the estimated 62 kDa size found in Western blot assays suggested that the predicted mitochondrial targeting signal is cleared during transportation of the enzyme into the mitochondrial matrix. SA-FLAG expression vectors were transfected into PCT3 cells and location of the fusion protein was determined by immunocytochemistry using anti-FLAG antibodies. By performing double-labeling confocal fluorescence imaging, using the mitochondrial marker dye MitoTracker Red in transfected SA-FLAG cells, we were able to co-locate both fluorescence signals, demonstrating the location of the SA protein in the mitochondria (Fig. 8Go).



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  		Figure 7 Amino acid sequences of human, rat and mouse SA. The amino acid sequences of mouse (mSA), human (hSA) and rat (rSA) deduced from cDNAs were compared (GenBank accession numbers: mSA, AF068246 [GenBank] ; hSA, D16350 [GenBank] ; rSA, AY456695 [GenBank] ). Amino acids are numbered on the left and identical amino acids are highlighted in solid black. The potential mitochondrion-targeting sequence found at the N-terminus of mouse, human and rat SA is denoted with open boxes. A conserved AMP-binding domain is indicated by a solid underline.

 


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  		Figure 8 Subcellular location of the recombinant SA protein. Transfected SA-FLAG fusion protein in PCT3 cells was labeled with anti-FLAG antibodies and recombinant protein visualized with FITC-conjugated goat anti-mouse secondary antibody (SA-Flag). MitoTracker Red CMXRos was used to stain PCT3 mitochondria (MitoTracker) and analyzed using a confocal microscope. Green fluorescence signals for the recombinant SA protein were overlapped with the red fluorescence signals for mitochondria (overlay).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The mouse SA gene was first described in our laboratory as a kidney-restricted androgen-dependent gene (Melià et al. 1998). By using the subtractive technique of representative differential analysis of cDNA, we aimed to find informative models for studying the nature of tissue-specific gene regulation in kidney. Furthermore, we were also hoping that new target genes might provide insight into the largely unknown physiological significance of androgen-inducible gene expression in the kidney. Apart from SA, other coordinately expressed genes belonging to the organic-anion-transporting (OATP) and cytochrome (Cyp4) families were also identified and characterized (Isern et al. 2001, Isern & Meseguer 2003). As mentioned above, the SA gene has been characterized as a proposed candidate gene for essential hypertension in rat and humans (Iwai & Inagami 1991, Harris et al. 1993, Lindpaintner et al. 1993, Iwai et al. 1994, Harrap et al. 1995, Nabika et al. 1995). Its highly restricted and abundant expression in kidney, a key organ in BP regulation (Guyton et al. 1990, Retting et al. 1993) and its dependence on androgens for expression in mouse kidney, suggested that the SA gene might be involved in a metabolic pathway with regulatory effects on renal vascular resistance, impaired renal hemodynamics and hypertension. A large subset of human hypertension is sexually dimorphic, i.e. more severe in males than in females, with the differences being minimized after menopause (Mantzoros et al. 1995, Chen 1996, August 1999, Garbers & Dubois 1999, Reckelhoff & Granger 1999). In the spontaneously hypertensive rat model, a sexual dimorphism in BP has also been observed (Chen & Meng 1991, Turner et al. 1991, Phillips et al. 1997, Reckelhoff et al. 1999). Despite the scarce information on sex-related BP differences in mice, a recent report from Holla et al.(2001) described lower BPs in female than in age-matched male mice in controls and in the hypertensive phenotype of Cyp4a14-knockout mice, which these authors have developed (Walsh et al. 2003). Despite this evidence Samani’s group have clearly shown that SA is not involved in the regulation of either basal or salt–related BP using SA-null mouse.

Predictions based on its amino acid sequence similarity included the SA protein in the acyl/acetyl-CoA synthetase family (Karan et al. 2001). Later studies confirmed a medium-chain acyl-CoA synthetase nature for the SA protein by means of enzymatic assays using a purified recombinant mouse SA protein heterologously expressed in COS cells. Two reports demonstrated that the SA protein plays a role in the degradation of medium-chain fatty acids for the production of energy. While Fujino et al. (2001b) concluded that isobutyrate constitutes a specific substrate for SA , Iwai et al.(2002) described octanoate as the preferred substrate for CO2 and ATP production. While these reports address a putative function for the SA protein, it remains to be determined what the real substrates and function of this protein are in vivo.

In this report we confirm previous data referring to kidney as the main site for SA mRNA synthesis followed by liver, but also other sites for minor SA production including stomach, testis, lung and heart. Of these, kidney, heart and lung express SA mRNA in an androgen-dependent fashion, indicating that SA constitutes a specific male enzyme for most of the tissues in which it is expressed and that its function must necessarily be important for males. Interestingly, we found a profound negative effect of estrogens on SA kidney mRNA levels since they block the action of androgens at physiological and pharmacological doses. Since ovariectomy in females does not permit SA expression in kidney, and DHT-induction triggers the SA gene in females, we conclude that it is a truly androgen-dependent gene. In order to explain the inhibitory role of estrogens in SA expression, we might speculate as to the presence of a common co-activator for sex steroid receptors, in proximal tubule cells, which becomes unavailable to the androgen receptor in our experimental conditions; alternatively, a newly synthesized estrogen-dependent repressor might be interfering with the mechanisms triggered by androgens, precluding expression. In any event, isolation and functional assays of the proximal promoter of the SA gene will provide insight into the elements and mechanisms governing the sex steroid-controlled expression of the SA gene in kidney and those that permit constitutive expression of the same gene in liver. To this end, we first determined the transcription initiation site by primer-extension analysis, 5'-RACE and RT-PCR, and found multiple forms of SA mRNAs which upon cloning and sequencing appeared to be the result of complex alternative-splicing events which included usage of 19 cryptic internal sites in exons 1 and 2. Although we cannot rule out trans-splicing events, there is no exon repetition that could indicate that this phenomenon is occurring in the mouse SA gene, as has been described for its rat orthologue (Frantz et al. 1999) and the rat carnitine octanoyltransferase gene (Caudevilla et al. 1998). As for the rat SA gene, Frantz et al.(1999) reported exon 2 and exon 2–4 repetition in kidney mRNA from Wistar–Kyoto rats not present in the spontaneously hypertensive strain, which was shown not to correspond to duplications of these specific exons or to the entire gene in the Wistar–Kyoto germ line. Exon 2 is located upstream of the putative translation start site and therefore the presence of the duplication would not be expected to alter the protein product. However, the exon 2–4 duplication would alter the reading frame, resulting in a truncated, altered product of 157 amino acids. Although the physiological significance of these modified transcripts has not been established, the transcripts have also been detected in Milan hypertensive and Dahl salt-sensitive rat strains (Frantz et al. 1999). In mouse, we found no alternative transcript of the SA gene compromising the putative translation initiation site which has been predicted to be in exon 4; Western blot analyses of mouse kidney extracts show that the single moiety that disappears upon blocking the antibody with the specific peptide corresponds to a product with the expected SA protein size. This result indicates that the splicing events occurring further up exon 4 have no impact on the correct synthesis of the SA protein. The biological role, if any, of our findings is unknown but might relate to the use of alternative promoters which might be located on the intronic sequences before exon 4 and upwards, which in turn could contribute to the differential regulation of the gene in kidney and liver. Studies currently being conducted in our laboratory using different reporter gene constructs in transient transfection assays may aid understanding of the complexity of SA gene expression in mouse tissues.


   Acknowledgements
 
We deeply thank Dr A. Vandewalle for providing us with the parental PKSV-PCT and PKSV-PR cells, Dr Juan Carlos López-Talavera for helpful discussions and Miss Christine O’Hara for English revision of the manuscript.


  Funding

This work was supported by grant no. SAF2000-0158 from Ministerio de Ciencia y Tecnología, Plan Nacional de I+D. C A and J I are recipients of predoctoral fellowships from ‘Institut Fundació per a la Recerca Biomè dica i la Docència de la Ciutat Sanitaria i Universitaria Vall d’Hebron’ and from ‘Universitat Autònoma de Barcelona’, respectively.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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Received 22 June 2004
Accepted 2 July 2004



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