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Department of Internal Medicine II (U.D.L., M.D., D.M.S., F.B.), Division of Endocrinology and Metabolism; Department of Internal Medicine I and Center for Clinical Trials (M.D., M.K., T.Z., J.S.S.); and Department of Pharmacology I (J.S.S.), Albert-Ludwigs-University Freiburg, D-79104 Freiburg, Germany; Research Institute for the Biology of Farm Animals (A.H.), Research Unit Genetics and Biometry, D-18196 Dummerstorf, Germany; Department of Internal Medicine (S.H.), Endocrine and Diabetes Unit, University of Wuerzburg, D-97074 Wuerzburg, Germany; Division of Metabolism, Endocrinology, and Diabetes (T.E., G.D.H.), Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan 48109; School of Animal and Microbial Sciences (A.B.B.), University of Reading, Reading RG6 6AH, United Kingdom; Division of Endocrinology and Metabolism (N.R.S.), Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390; Department of Pathology (M.P.K.), University of California San Diego, School of Medicine, La Jolla, California 92093; and Division of Endocrine Research (F.B.), Department of Medicine Innenstadt, Ludwig-Maximilians-University, D-80336 Munich, Germany
Address all correspondence and requests for reprints to: Felix Beuschlein, M.D., Division of Endocrine Research, Department of Medicine Innenstadt, University Hospital Munich, Ziemssenstrasse 1, D-80336 Munich, Germany. E-mail: felix.beuschlein{at}med.uni-muenchen.de.
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Introduction |
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The significance of Pbx1 for steroidogenesis has been established by earlier studies on Pbx1-dependent expression of 17
-hydroxylase/17,20-lyase (Cyp17) (14). Cyp17 is required for cortisol and androgen biosynthesis and therefore plays a key role in biological processes including sexual differentiation. Overexpression of Pbx1 has been demonstrated to enhance the cAMP evoked transcriptional response in an in vitro model, thus indicating a modulation of cAMP-dependent transcription through Pbx1. Additional evidence for Pbx1-dependent effects on adrenal function comes from the phenotypical description of significant adrenal and urogenital differentiation defects including severe adrenal hypoplasia (15) in Pbx1–/– animals associated with minimal expression of steroidogenic factor 1 (Sf-1), an important regulator of adrenocortical cell commitment and steroidogenesis. In accordance with this observation, only recently, Pbx1-dependent initiation of Sf-1 expression during murine adrenal development has been demonstrated in a series of transgenic approaches in vivo (16).
Taken together, Pbx1 has been implicated as an additional key factor for adrenocortical organogenesis and growth. However, the lethal phenotype of Pbx1–/– animals has precluded the elucidation of possible Pbx1-dependent effects on adrenal function in the adult organism. Therefore, we used a viable, haploinsufficient mouse model lacking the critical domain for Pbx1-DNA interaction to further define the contribution of Pbx1 for adrenal growth and steroidogenesis.
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Materials and Methods |
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). The targeted sequence (referred to as target) was composed of Pbx1 exon 6 (corresponding to 1192–1356 bp in PBX1a mRNA, accession no. AF020196) flanked by 700-bp upstream and 280-bp downstream intronic sequences (Fig. 1A). In the course of Pbx1flox1 vector preparation, a HindIII restriction digest site was removed from the genomic sequence upstream of the targeted exon 6 (Fig. 1A). Removal of this restriction site by enzymatic digest and Klenow polymerase fill-in reaction allowed for Southern detection of homologous recombination events with the external Southern probe located upstream of the vector sequences (Fig. 1, A and B).
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). The homologous recombination events, detected by Southern blot, were confirmed by PCR amplification of the 5' and 3' ends of the targeting vector, with one primer based on the sequence inside and one external to the vector sequence (Fig. 1C). Cells were electroporated with 20 µg CMV-Cre plasmid (kindly provided by Dr. Florian Otto) and serially diluted before plating. Colonies were picked and PCR analyzed for the loss of target and selection cassette sequences (data not shown). Four clones of ES cells heterozygous for the Pbx1 knockout were microinjected into blastocysts of C57BL/6 background. A total of 57 microinjected blastocysts were retransferred into pseudopregnant foster mice, and three highly chimeric males were born. One of the male chimeras gave a germline transmission of the Pbx1 knockout allele that was further bred onto the C57BL/6 background. Genotyping was performed by genomic PCR of tail DNA using primers flanking the targeted exon 6 yielding a 600-bp fragment for the knockout allele and a 1.2-kb fragment for the wild-type allele. Because this long fragment was not regularly amplified under the applied PCR conditions, 18s primers resulting in a 151-bp fragment were used as a positive amplification control (Fig. 1D). Timed pregnancies of Pbx1 heterozygote F2 generation mice were set up and d-14.5 embryos obtained. Phenotypically Pbx1–/– embryos were similar to Pbx1–/– mice described previously (10). Among externally visible knockout features, Pbx1–/– pups were distinguished by sc edema, diminished vascularization and consequent pallor, slender thorax and abdomen, hunched posture, and abnormal orientation of both fore and hind limbs (data not shown).
Animal experiments
All experiments involving animals were performed in accordance with institutionally approved and current animal care guidelines. Animals were maintained under standard conditions of temperature (22 C) and lighting (12 h light, 12 h dark) and with food and water given ad libitum. Pbx1+/– haploinsufficient and wild-type mice were obtained by breeding Pbx1+/– mice with wild-type animals (C57BL/6; Jackson Laboratory, Bar Harbor, ME). Generation of Sf-1+/– haploinsufficient animals has been described earlier (18).
For evaluation of adrenal morphology, expression studies and hormonal measurements, 10-wk-old female Pbx1+/– mice and wild-type controls were isolated and kept in a quiet environment overnight. Blood samples for hormonal measurements (n = 6 per genotype) were obtained by retroorbital bleeding after isoflurane anesthesia for restrained stress and ACTH-stimulated experiments and trunk blood collection for baseline measurements. In all instances, blood collection was done within 60 sec after initial mouse handling. Restrained stress was induced by placing the animals in a 50-ml Falcon tube for 30 min. ACTH stimulation was performed after injection of weight-adjusted doses of ACTH and blood collection after 30 min as described earlier (19). After microdissection, adrenals were measured and the tissues were snap frozen for protein/RNA extraction and in situ hybridization or immersed in paraformaldehyde for 3 h for histological analysis.
For compensatory adrenal growth experiments, 9-wk-old male Pbx1+/– or wild-type mice were subjected to left-sided adrenalectomy (n = 6 per genotype) on d 0 following standard procedures as described earlier (20). Mice were euthanized on d 3, and the remaining adrenals were collected. All adrenal weights were measured after careful microdissection.
For real-time expression analysis of Pbx1, 8-wk-old female Sf-1+/– animals and age-matched wild-type controls (n = 3 each) were used with adrenals snap frozen after microdissection.
Adrenal morphology
For morphological evaluation, tissues were dehydrated, embedded in paraffin, sectioned, and stained with hematoxylin and eosin following standard procedures. Hematoxylin- and eosin-stained adrenal sections from wild-type and Pbx1+/– mice were examined with a standard light microscope using x400 magnification. Cell nuclei within the zona fasciculata of three independent sections from three different animals per group were counted under standardized conditions. Cell counts were expressed as cell number per high-power field. Morphometric analysis for quantification of the x-zone was performed on at least three individual mice per genotype as described earlier (20).
Immunohistochemistry
For immunohistochemical analysis, paraffin-embedded sections were rehydrated, blocked with 0.3% H2O2 in methanol for 10 min, and incubated with blocking buffer for 15 min. Primary antibodies [proliferating cell nuclear antigen (PCNA), 1:500 (Santa Cruz Biotechnology, Santa Cruz, CA); SF-1, 1:500 (K. Morohashi); PBX1, 1:100 (Abcam, Cambridge, UK); and 20-hydroxysteroid dehydrogenase (20HSD), 1:5000 (Y. Weinstein)] were incubated overnight at 4 C after dilution in blocking buffer containing 3% BSA (Roche Diagnostics, Mannheim, Germany), 5% goat serum (Jackson ImmunoResearch Laboratories, West Grove, PA), and 0.5% Tween 20. After rinsing for 15 min in PBS, secondary antibody (goat antirabbit biotinylated IgG; Vector Laboratories, Burlingame, CA) was applied for 30 min at room temperature. Bound primary antibody was detected using the Immunocruz Kit (Santa Cruz Biotechnology) according to the manufacturer’s protocol.
Western blot analysis
Adrenals were sonicated on ice until tissue was disrupted in RIPA buffer (50 mM Tris, 150 mM sodium chloride, 1.0 mM EDTA, 1% Nonidet P40, and 0.25% sodium deoxycolate, pH 7.0) that was supplemented with complete protease inhibitor cocktail (Roche, Mannheim, Germany) and phosphatase inhibitor cocktail 1 (Sigma Chemical Co., St. Louis, MO). After incubation by rotating at 4 C for 1 h, followed by a centrifugation step at 10,000 x g at 4 C for 5 min, protein concentration was assessed using a protein assay (Bio-Rad, Munich, Germany). Then, 10 µg of the protein preparation was run on a 10% acrylamide gel at 110 V for 1 h and transferred to nitrocellulose at 120 mA for 1 h. The nitrocellulose was then incubated with 5% nonfat dry milk overnight at 4 C with either rabbit anti-PCNA antibody (Santa Cruz Biotechnology) at a dilution of 1:1000, rabbit anti-SF-1 antibody at 1:1500 (K. Morohashi), or chicken anti-IGF-I receptor antibody at 2 µg/ml (Upstate, Charlottesville, VA), respectively. The washed blots were then incubated with suitable secondary antibodies conjugated to horseradish peroxidase (sheep antimouse, donkey antirabbit, or rabbit antichicken, all from Amersham Biosciences, Little Chalfont, UK) at a dilution of 1:10,000 for 1 h at room temperature. Antibody binding to the membrane was visualized using the KPL Western Luminescence ECL plus (Amersham) chemiluminescent detection system. All immunoblots were performed on at least six different animals per genotype. Bands were quantified using volume quantification (Quantity one, version 4.2.0; Bio-Rad, Hercules, CA), and resulting values were expressed as percent changes compared with wild-type controls.
In situ hybridization
PCR was used to amplify fragments of the appropriate Hox genes from mouse adrenal cDNA. All fragments were chosen within an untranslated sequence that showed no significant homology to other members of the Hox family. Corresponding sequences were as follows: for A9 (accession no. NM 010456), bases 627–999; for B8 (accession no. NM 010461), bases 252–612; and for C8 (accession no. NM 010466), bases 1001–1430. PCR fragments were cloned into pGEM-T Easy (Promega, Madison, WI) and subsequently sequenced to confirm integrity. The plasmids were linearized, and nonradioactive sense and antisense riboprobes labeled with digoxigenin were synthesized using in vitro transcription using either T7 or SP6 RNA polymerase as previously described (21).
Adrenals from wild-type controls were frozen in isopentane cooled in dry ice, and 25-µm sections were cut using a cryostat. In situ hybridization was carried out essentially as previously described (21). For the sense controls, tissue sections were probed with a cocktail of all three probes mixed together in equal quantities. Detection of the probes was via an antidigoxigenin antibody (Roche) coupled with the Renaissance Tyramide Signal Amplification System (NEN Life Science Products, Boston, MA). The cell nuclei were counterstained with TOTO-3 (Molecular Probes, Eugene, OR) and the sections analyzed using confocal microscopy.
Immunoprecipitation
HEK293T cells were transfected with pcDNASf-1 and/or pcDNAPbxHA using the calcium phosphate-mediated transfection method. After 24 h, cells were harvested by scraping them in a buffer containing 50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 5 mM EDTA; 0.5% Nonidet P-40; 0.1 mM phenylmethylsulfonyl fluoride (22), and 100 mg protein was incubated at 4 C with 5 µl anti-Sf-1 (rabbit polyclonal; Upstate) or anti-HA (mouse monoclonal, 6E2; Cell Signaling, Danvers, MA). After 12 h, 25 µl packed protein A (Invitrogen, Carlsbad, CA) or protein G agarose beads (Sigma) were added and incubated at 4 C for another hour. After three consecutive washes with 1 ml IPH buffer, agarose beads were heated to 95 C in loading buffer containing SDS and mercaptoethanol for 5 min. Samples were subjected to 10% SDS-PAGE, transferred to nylon membrane (Bio-Rad) by semidry technique. Protein detection was conducted using anti-SF-1 (rabbit polyclonal) and anti-HA (rabbit polyclonal, Y11; Santa Cruz Biotechnology) and the West Dura extended duration kit (Pierce, Rockford, IL). Alternatively, instead of IPH buffer, experiments with immunoprecipitation were carried out in modified RIPA buffer and 1:1 PBS:modified RIPA buffer obtaining the same results.
RT-PCR
Individual adrenals from each group were used for RNA extraction using the QIAGEN (Valencia, CA) RNA mini kit following the instructions of the manufacturer. cDNA was created using a RT kit (Promega) and 1.0 µg total RNA. Aliquots of the cDNA samples were subjected to the subsequent PCR, which were performed with primer pairs and annealing temperatures as given in supplemental Table 1. For detection of Hox gene products, identical primers as for the generation of the in situ probes were used. Amplification products were separated on 1% agarose gel and stained with ethidium bromide.
Real-time PCR
IGF system, Pcna, and Sf-1 in vivo.
Quantification of mRNA abundance was performed by real-time PCR detection using an ABI PRISM 7700 Sequence Detector (PE Biosystems, Weiterstadt, Germany) and Sybr Green as a double-stranded DNA-specific fluorescent dye. Amplification mixes (25 µl) contained 2.5 µl cDNA solution, 2x Sybr Green PCR Master Mix, 12.5 pM of each primer, and 0.25 IU AmpErase uracil N-glycosylase (PE Biosystems). Amplification primers (Table 1) were designed using the Primer Express software (PE Biosystems). PCR was started with 2 min at 50 C for AmpErase activation and 10 min at 95 C for denaturation. The program continued with 40 cycles of 15 sec at 95 C and 60 sec at 60 C. The amplicons were verified by sequence determination. Each assay included triplicates of cDNA for the gene of interest, no-template control and four dilutions of cDNA pooled from all genetic groups for the gene of interest and for the reference gene ß-actin to calculate the corresponding amplification efficiency (E = 10–(1/b)– 1; b is the regression coefficient). The parameter CT (threshold cycle) is defined as the cycle number at which fluorescence intensity exceeds a fixed threshold. Relative mRNA expression for each gene of interest (I) was calculated using the formula: (1 + E[I])–CT[I]/(1 + E[ß-actin])–CT[ß-actin]. Data were analyzed using the Student’s t test and correlation analysis (Pearson).
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) for 6 sec, and extension at 72 C, at which the time is calculated by the product length in base pairs divided by 25 (Roche, Indianapolis, IN). The melting curve analysis was performed between 65 and 95 C (0.1 C/sec) to determine the melting temperature of the amplified product and to exclude undesired primer dimers. Furthermore, the products were run on a 1% agarose gel to verify the amplified product. Quantification was adjusted using the housekeeping gene ß2-microglobulin. To facilitate overall comparison of individual real-time experiments, expression levels of the particular genes were set as 100% for wild-type animals. For evaluation of endogenous gene expression after forced overexpression of Sf-1 and Pbx1, cells were harvested 48 h after transfection, and real-time analysis was performed as described.
Reporter gene assays
A 2.7-kb fragment of the murine 5' untranslated region of the Pbx1 gene was PCR cloned using primers 5'-PBX1prom-3, TTC CCA CTT GGC GAA AAG AAA TCA GAG CC, and 3'-PBX1prom-3, CCA GAA CCA ATC TGT CCT TCA AAC TGC CTC, from mouse genomic DNA using the Expand Long Template PCR System (Roche, Indianapolis, IN). Cycling conditions and procedure were performed according to the manufacturer’s protocol. The PCR product was first cloned into pCR2.1 vector using TA cloning kit (Invitrogen, Karlsruhe, Germany) and subsequently subcloned into the pGL2 basic vector (Promega).
In addition, a full-length promoter construct of the ACTH receptor (Mc2-r) and a construct with three mutated Sf-1-binding sites that have been described previously (23) as well as an artificial Sf-1-responsive element consisting of multiple core Sf-1-binding sites from the 21-OH genes fused to –36 PRL-LUC (kindly provided by Dr. K. Parker) were used. For stimulation, expression vectors for the full-length murine Pbx1a and Sf-1 were used, respectively.
Mouse adrenocortical Y1 cells were grown to a density of 100,000 per 24-well plate in Ham’s F-10 medium with 7.5% horse serum and 2.5% fetal bovine serum and transiently transfected using ExGen 500 (MBI Fermentas, St. Leon-Rot, Germany) for 5 h according to the manufacturer’s instructions with 250-ng expression vectors (if not stated otherwise) and 500-ng promoter constructs, respectively. Total amount of transfected DNA was kept constant to 1 µg by adding empty pcDNA expression vector. At 48 h after transfection, cells were harvested and luciferase activity was measured (Luciferase Reporter Gene Assay; Roche, Mannheim, Germany). Luciferase values were normalized for total protein content. Each data point represents at least four independent measurements.
Plasma hormone measurements
Plasma corticosterone was measured using a commercially available RIA (MP Biomedicals, Eschwege, Germany). Plasma ACTH was determined with an immunoradiometric assay (Immuno Biological Laboratories, Hamburg, Germany). All assays were performed according to the manufacturers’ protocols. The intra- and interassay coefficients of variation were less than 10% and less than 7%, respectively, for all assays. All hormone measurements were performed on six animals per genotype on single aliquots due to the small amount of plasma available.
Statistical analysis
All results are expressed as mean ± SEM. If not stated otherwise, all statistical comparisons were analyzed by ANOVA and Fisher’s protective least significant difference test. Statistical significance is defined as P < 0.05 and is indicated by asterisks in the figures.
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) as well as by immunohistochemistry (Pbx1, Fig. 2B; Sf-1, Fig. 2C). Moreover, major Pbx1 binding partners such as Hox A9, B8, and C8, which are essential mediators of Pbx1 action (2, 24, 25, 26), were also present in the adrenal cortex by means of in situ hybridization and RT-PCR analysis (Fig. 2D). These results provided the rationale for investigating the effects of Pbx1 haploinsufficiency on the adrenal cortex.
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) resulting in a significantly lower adrenal weight (4.4 ± 0.4 mg) in comparison with wild-type controls (7.5 ± 0.3 mg, P < 0.0001; Fig. 3, A and C). Despite the lower adrenal weight, morphometric analyses demonstrated hypertrophy of the zona fasciculata as indicated by a lower cell number per high-power field in Pbx1+/– mice (63.0 ± 3.3%; Fig. 3, E and F) than in wild-type animals (89.3 ± 8.3%, P = 0.014; Fig. 3, D and F).
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HSD (Fig. 3, G and H) suggested a decrease in the size of the x-zone in nulliparous female mice in the context of Pbx1 haploinsufficiency (Fig. 3H) in comparison with wild-type controls (Fig. 3G). These observational findings could be verified by morphometric analysis (ratio x-zone area/whole adrenal cortex area, 100.0 ± 6.3 vs. 57.3 ± 13.9%, P < 0.05; Fig. 3I).
Pbx1 haploinsufficiency is associated with lower adrenal cellular proliferation, decreased compensatory adrenal growth, and changes of expression in the IGF system
Because the observed lower adrenal weight in Pbx1+/– animals could have been explained by a proliferation defect, detailed expression analysis of the proliferation marker Pcna was performed. Indeed, Pbx1 haploinsufficient mice displayed a marked reduction in adrenal expression levels of Pcna, which was observed in real-time PCR (52.3 ± 12.5 vs. 100 ± 7.6%, P = 0.01; Fig. 4A), Western blot analysis (Fig. 4B), and immunohistochemistry (Fig. 4C).
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Because the IGF system is a major determinant of adrenocortical growth in adrenal physiology and tumorigenesis, this system was investigated thoroughly by real-time analysis. Although IGF-I and IGF-II expression levels were not different between the two genotypes, IGF-I receptor was significantly down-regulated (46 ± 6 vs. 100 ± 10%, P = 0.007). Furthermore, IGF-binding protein 2 (IGFBP-2) was up-regulated about 10-fold (1033.0 ± 408.5 vs. 100 ± 8.3%, P = 0.04), whereas IGFBP-3 and IGFBP-5 expression levels remained unaltered by Pbx1 haploinsufficiency (Fig. 4E). In accordance with the lower IGF-I receptor mRNA expression levels, adrenals from Pbx1+/– also displayed lower IGF-I receptor protein levels by means of Western blot analysis (67.3 ± 4.1 vs. 100.0 ± 7.8%; P = 0.03; Fig. 4E, inset).
Taken together, these findings indicate that a full complement of Pbx1 is indispensable for proper growth and proliferation of the adrenal cortex. In addition, the transcriptional modulation of the intraadrenal IGF system associated with Pbx1 haploinsufficiency might contribute to the observed adrenal growth defect.
Pbx1 haploinsufficient mice have a blunted steroid response after stimulation
On a functional level, baseline corticosterone levels were similar in Pbx1+/– (18.8 ± 6.2 ng/ml) and wild-type (16.7 ± 4.7 ng/ml, P = 0.8) animals. In contrast, corticosterone stimulation was significantly blunted in Pbx1+/– mice after ACTH administration (184.4 ± 57.6 ng/ml vs. wild-type 358.8 ± 28.3 ng/ml, P = 0.03), whereas no statistical difference could be observed after restrained stress experiments (375.0 ± 36.3 ng/ml vs. wild-type 495.9 ± 51.2 ng/ml, P = 0.14; Fig. 5). Accordingly, basal ACTH levels were similar between the two groups (259.0 ± 25.2 pg/ml vs. wild-type 296.0 ± 37.0 pg/ml, P = 0.60) but significantly higher in Pbx1+/– mice after restrained stress (1177.5 ± 57.8 pg/ml vs. wild-type 797.3 ± 112.8 pg/ml, P = 0.02; Fig. 5). Collectively, these data correspond with a compensated primary adrenal insufficiency in Pbx1 haploinsufficient mice.
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). Therefore, we further evaluated possible direct interaction of Pbx1 on the transcriptional regulation of the Mc2-r. In addition to the three well-characterized Sf-1-binding sites within the Mc2-r promoter (28), sequence analysis predicted two potential Pbx1/Hox and two potential Pbx1/Meis binding sites, displaying the consensus TGATNNAT (29) and TGACAG (30) at positions –63 to –56, –326 to –319, –686 to –681, and –915 to –910 bp, respectively. Accordingly, promoter studies using a full-length Mc2-r promoter construct revealed a significant increase in promoter activation by Pbx1 (342.0 ± 81.2% vs. pcDNA 100 ± 4.7%, P = 0.007) which was of similar magnitude as that induced by Sf-1 (520.8 ± 121.3 vs. 100 ± 4.7%, P = 0.002). In addition, overexpression of Pbx1 together with Sf-1 resulted in an additional significant increase in Mc2-r promoter activity over Sf-1 alone (1627.4 ± 408.2 vs. 520.8 ± 121.3%, P < 0.001; Fig. 6B).
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Using an artificial Sf-1 promoter construct, which consisted of multiple isolated Sf-1-binding sites, only Sf-1-dependent effects on promoter activation were evident (Sf-1 alone, 433.9 ± 13.8 vs. 100 ± 8.2%, P < 0.001; Pbx1 plus Sf-1, 370.4 ± 16.5 vs. 100 ± 8.2%, P < 0.001), whereas Pbx1 was not able to significantly increase promoter activity over baseline (94.6 ± 8.8 vs. 100 ± 8.2%, P = 0.76) or over Sf-1-dependent activation alone (370.4 ± 16.5 vs. 433.9 ± 13.8%, P = 0.07), respectively (Fig. 6D).
Collectively, these data demonstrate that Pbx1 and Sf-1 have additive effects on Mc2-r promoter activity. Moreover, Sf-1 seems not to be required for Pbx1 to evoke its activating effects on the Mc2-r promoter. Conversely, Pbx1 fails to increase Sf-1-dependent effects on promoter activation in the absence of Pbx1-binding sites.
Because a functional interaction between Pbx1 and Sf-1 could require direct protein-protein binding, we performed immunoprecipitation experiments under various conditions (Fig. 6E and data not shown). However, overexpression of Sf-1 was not able to coprecipitate Pbx1, suggesting that direct interaction between the two transcription factors seems not to play a major role for the observed functional effects on promoter activation.
Pbx1 does not affect Sf-1 expression levels in adrenocortical tumor cell lines in vitro and in adult adrenal glands in vivo
Expression of Fgf15, which represents a direct downstream target of Pbx1 action (31), was up-regulated by overexpression of Pbx1 in Y1 cells in a dose-dependent manner (247.4 ± 29.8% for 1000 ng Pbx1 vs. 100 ± 1.7% for pcDNA, P = 0.03; Fig. 7A). In contrast, endogenous Sf-1 expression levels remained unchanged (119.4 ± 2.4% for 1000 ng Pbx1 vs. 100 ± 6.6%, P = 0.12; Fig. 7C) under the same experimental conditions. Concomitantly, Sf-1 expression levels in adrenal glands of wild-type and heterozygous Pbx1 mice were assessed by real-time PCR and Western blot analysis. In accordance with the in vitro data, Sf-1 expression in adult adrenals on mRNA (115.3 ± 17.0 vs. 100 ± 6.6%, P = 0.39; Fig. 7D) or protein level (102.5 ± 7.3 vs. 100 ± 14.5%, P = 0.90; Fig. 7E) was not significantly different in Pbx1+/– mice in comparison with wild-type controls.
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) as well as endogenous Pbx1 (165.65 ± 19.4% vs. 100 ± 11.6%, P = 0.01; Fig. 7F) were significantly up-regulated in the Sf-1-transfected cells, indicating Sf-1-dependent activation of endogenous Pbx1 transcription.
In accordance with these in vitro findings in Y1 cells, sequence analysis of the Pbx1 promoter predicted four potential Sf-1-binding sites represented by a CAAGG consensus sequence (positions –11 to –15 and –2465 to –2454) and AGGTCA consensus sequence (32) (positions –2465 to –2460 and –2312 to –2307). Notably, four consensus binding sites for Pbx1 were also evident within the Pbx1 promoter region (–1942 to –1933, –982 to –975, –639 to –632, and –535 to –542) by sequence analysis. Functional significance of these findings was verified by Pbx1 promoter stimulation studies. In accordance with the sequence analysis, overexpression of Sf-1 resulted in a dose-dependent increase in Pbx1 promoter activation (125 ng Sf-1, 157.7 ± 6.9%, P = 0.017; 250 ng Sf-1, 188.4 ± 8.9%, P = 0.014; 500 ng Sf-1, 272.2 ± 17.0%, P < 0.001 vs. pcDNA, 100 ± 4.7%; Fig. 7G). Furthermore, Pbx1 promoter activity was also significantly induced by Pbx1 overexpression (331.1 ± 24.0 vs. 100 ± 4.7%, P < 0.001; Fig. 7G). In vivo, adrenal glands of Sf-1 heterozygous animals showed significantly lower Pbx1 levels on the mRNA level (43.3 ± 15.3 vs. 100 ± 12.7%, P = 0.047; Fig. 7H). Taken together, these data are in agreement with the concept of an Sf-1-dependent promoter activation of Pbx1.
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The observed decrease in the cell number per high-power field in Pbx1+/– mice indicating cellular hypertrophy of the zona fasciculata clearly does not explain the concomitant reduction in adrenal weight. However, similar phenotypic changes have been demonstrated in Sf-1 haploinsufficient mice (19, 20, 33) and have been interpreted as the consequence of latent primary adrenal insufficiency with consecutively elevated levels of ACTH and other proopiomelanocortin-derived peptides together with intracellular compensatory mechanisms including up-regulation of the orphan nuclear receptor nerve growth factor-induced B (NGFI-B) (34). Likewise, although a smaller x-zone might contribute to the observed decrease in adrenal weight in virgin female animals, it does not explain the lower adrenal weight in postpubertal male mice with already regressed x-zone. As such, cellular hyperplasia of the zona fasciculata or differences in zonation seem not to be major determinants of the observed adrenal growth defect.
The adrenal cortex is a dynamic organ with constant cellular replacement from the proposed subcapsular stem cell zone and inward migration of newly differentiated daughter cells (35, 36). As such, growth and adaptation of the adult adrenal cortex is dependent upon the proliferative potential of this stem cell population. Thus, the observed lower levels of the proliferation marker PCNA as detected by RT-PCR, Western blot analysis, and immunohistochemistry suggest a crucial role of Pbx1 for the regulation of adrenocortical stem cell proliferation. In keeping with these findings, severe adrenal hypoplasia has been described in Pbx1–/– embryos (15), which parallels the defects in proliferation of progenitor cells previously reported in other organ systems (7, 10). The lack of compensatory growth in Pbx1+/– animals resembles that of Sf-1 heterozygous mice (20) and provides additional evidence that Pbx1 is indispensable for the maintenance of the adrenal cortex in the adult animal.
IGF-II is considered one of the most potent growth factors for the adrenal cortex in the context of both adrenal development (37) and adrenal tumorigenesis (38). Genetic studies on adrenocortical carcinomas have demonstrated a variety of genetic changes resulting in IGF-II overexpression. Therefore, we hypothesized that alteration in the local IGF system might play a role in the observed lower proliferation at baseline and upon stimulation. Indeed, although no changes in expression levels of IGF-I or IGF-II are evident in adrenals from Pbx1+/– animals, adrenal IGF-I receptor expression is significantly down-regulated, whereas IGFBP-2 expression levels are considerably higher in Pbx1+/– mice. These descriptive data on expression levels in a complex and dynamic in vivo system have to be interpreted with caution. However, because the main effects of IGF-I in the adrenal cortex are mediated by the IGF-I receptor and IGFBP-2 has been demonstrated to modulate IGF-I-dependent action on the adrenal cortex (39), both changes in expression levels could contribute to the evident proliferation defect.
Development of the adrenal cortex and functional maintenance in the adult organism is dependent upon an intact hypothalamus-pituitary-adrenal axis, in that a lack of pituitary proopiomelanocortin causes severe adrenal hypoplasia (40). The Mc2-r is a central part of the hypothalamus-pituitary-adrenal axis by mediating the adrenal steroid output in response to pituitary ACTH (41). As such, the observed lower adrenal expression levels of the Mc2-r are in line with the blunted adrenal responsiveness to ACTH in vivo in Pbx1 haploinsufficient animals.
In addition to its well described binding sites for Sf-1 (23, 28), the Mc2-r promoter contains four binding sites for Pbx1/Hox and Pbx1/Meis heterodimers, respectively. Interestingly, the proximal promoter fragment from position –12 to –109 contains one Pbx1/Hox-binding site that is flanked by two Sf-1-binding sites in close proximity. This promoter sequence is highly conserved between Homo sapiens and Mus musculus, displaying about 80% sequence similarity. Because these findings gave indirect evidence for the functional relevance of these transcription factors for Mc2-r regulation, we used the Mc2-r promoter as a model for Pbx1- and Sf-1-dependent promoter activation. In fact, Pbx1 results in an increase in baseline as well as Sf-1-stimulated promoter activity of the Mc2-r promoter, establishing Mc2-r as a direct target of Pbx1-dependent action. Although the proximity of binding sites for the two transcription factors implies direct protein-protein interaction of Pbx1 and Sf-1, immunoprecipitation provides no evidence for direct physical binding of the two transcription factors under the chosen experimental conditions. In addition, promoter experiments using a Mc2-r construct with site-directed mutated Sf-1-binding sites argue for Pbx1-induced promoter activation independent from Sf-1. Conversely, an artificial promoter construct consisting of multiple Sf-1-binding sites is not activated by Pbx1. Taken together, these findings suggest a parallel albeit independent promoter activation of the Mc2-r promoter by Pbx1 and Sf-1. However, Pbx1 has been demonstrated to penetrate inactive chromatin structures to facilitate the access of other transcription factors to appropriate promoter regions (3). In this regard, we cannot exclude the possibility that in vivo Pbx1 could also facilitate Sf-1 binding to DNA, thus boosting its transcriptional activity.
Because Sf-1 expression in adrenals from Pbx1-deficient embryos has been reported to be reduced (15), these findings indicate a Pbx1-dependent transcriptional activation of Sf-1 and would place Pbx1 before Sf-1 in the temporal expression of transcription factors during urogenital development. In fact, in a recently published paper, Zubair and co-workers (16) provide ample evidence for a Pbx1-dependent initiation of Sf-1 transcription during early adrenal development that is further maintained by a positive auto-feedback loop by Sf-1 itself at later developmental stages. These findings indicate that Pbx1-dependent transcriptional activation of Sf-1 is dependent on the physiological and cellular context. In fact, as shown herein, Sf-1 expression levels in adrenals from Pbx1 haploinsufficient animals are not diminished in comparison with wild-type controls, and overexpression of Pbx1 in adrenocortical Y1 cells does not affect endogenous Sf-1 expression. Although we cannot exclude the possibility that one remaining allele of Pbx1 is sufficient to drive full expression of Sf-1 in adrenals of Pbx1+/– animals, it is also possible that transcriptional regulation of Sf-1 in the adult organism differs from that during early organogenesis.
Conversely, we present evidence suggesting that Sf-1 induces up-regulation of Pbx1 transcription. First, up-regulation of endogenous Pbx1 can be induced by overexpression of Sf-1 in adrenocortical Y1 cells. Second, sequence analysis of the Pbx1 promoter reveals the presence of four Sf-1-binding sites containing the consensus sequence CAAGG (positions –11 to –15 and –2465 to –2454) and AGGTCA (positions –2465 to –2460 and –2312 to –2307) (32). Third, cloning of the 5' region of the Pbx1 gene and subsequent luciferase experiments demonstrate promoter activation of the Pbx1 promoter fragment by Sf-1 in a dose-dependent manner. Fourth, adrenal Pbx1 levels are down-regulated in Sf-1 haploinsufficient animals. Although differences in the spatiotemporal expression patterns between Pbx1 and Sf-1 with organ-restricted expression for Sf-1 and with ubiquitous and early expression for Pbx1 clearly indicate that Sf-1 is not mandatory for Pbx1 expression in vivo, down-regulation of Pbx1 in Sf-1+/– mice suggests modulation of Pbx1 transcription by Sf-1 most likely in concert with other transcription factors. In keeping with this notion, as we can demonstrate in promoter studies (data not shown), WT-1 is also able to induce transcriptional activation of Pbx1, which in itself might be of functional significance for urogenital organogenesis. In addition, overexpression of Pbx1 itself results in up-regulation of its own promoter, forming a positive feedback loop system that might contribute to a more stable expression of Pbx1 in its target cells. Overall, it is tempting to speculate that the adrenal phenotype in homozygous and heterozygous Sf-1 knockout animals might be mediated in part by effects of Pbx1 down-regulation resulting in the phenotypical similarities between Sf-1- and Pbx1-deficient mouse models.
In conclusion, we demonstrate that Pbx1, in addition to its involvement in urogenital development and adrenocortical cell commitment, is also required for the growth maintenance of the adult adrenal gland. Furthermore, we present data suggesting that the growth defects observed in Pbx1 haploinsufficient mice are mainly caused by a defect in adrenal cell proliferation under baseline and stimulated conditions. In accordance with the overall similarity in the phenotypes of mouse models with haploinsufficiency for Pbx1 and Sf-1, we provide additional evidence for cooperative interaction between the two transcription factors relying on transcriptional regulation of Pbx1 by Sf-1 as well as synergistic up-regulation of mutual target genes such as the Mc2-r.
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Acknowledgments |
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hsd antibody, respectively. |
Footnotes |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online November 2, 2006
Abbreviations: Cyp17, 17-Hydroxylase/17,20-lyase; ES, embryonic stem; 20hsd, 20-hydroxysteroid dehydrogenase; IGFBP, IGF-binding protein; Mc2-r, melanocortin 2 receptor; Pbx1, pre-B-cell transcription factor 1; Pcna, proliferating cell nuclear antigen; SF-1, steroidogenic factor 1.
Received May 19, 2006.
Accepted for publication October 23, 2006.
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Pbx1+/– ES cell clones. B, Southern blot analysis of HindIII-digested DNA from Pbx1 clones after first homologous recombination before Cre treatment indicated a shift of the wild-type (wt) 8-kb band to 16 kb due to the loss of a HindIII site in the homology arm. C, Southern blot analysis was confirmed by long-range PCR with primers within and outside of the targeted sequence yielding 8- and 2-kb fragments of the 5' and 3' homology arms of the vector. D, Genotyping of wild-type (+/+) and heterozygous Pbx1 knockout animals (+/–) was performed by PCR (see text for details). B, BamHI; Bg, BglII; E, EcoRI; H, HindIII; N, NotI; S, SalI.
