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Human Molecular Genetics Advance Access originally published online on September 19, 2007
Human Molecular Genetics 2007 16(24):3081-3087; doi:10.1093/hmg/ddm266
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

BNP is a transcriptional target of the short stature homeobox gene SHOX

Antonio Marchini1,{dagger},{ddagger}, Beate Häcker1, Tiina Marttila1, Volker Hesse2, Joyce Emons3,4, Birgit Weiss1, Marcel Karperien3,4 and Gudrun Rappold1,*

1 Institute of Human Genetics, Ruprecht-Karls-University, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany, 2 Department of Pediatrics, Sana Klinikum Lichtenberg, Gotlindestrasse 2-20, 10356 Berlin, Germany, 3 Department of Pediatrics, Leiden University Medical Center, PO Box 9600, 2300 RC, Leiden, The Netherlands and 4 Department of Endocrinology and Metabolic Diseases, Leiden University Medical Center, PO Box 9600, 2300 RC, Leiden, The Netherlands

* To whom correspondence should be addressed at: Institute of Human Genetics, Ruprecht-Karls-University at Heidelberg, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany. Tel: +49 6221565059; Fax: +49 6221565332; Email: gudrun_rappold{at}med.uni-heidelberg.de

Received July 27, 2007; Revised September 6, 2007; Accepted September 16, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Short stature due to SHOX deficiency represents a common congenital form of growth failure and is involved in the aetiology of ‘idiopathic’ short stature and the growth deficits and skeletal anomalies in Leri–Weill, Langer and Turner syndromes. Although much is known on the clinical and molecular aspects of SHOX haploinsufficiency, the integration of SHOX in the signalling pathways regulating bone growth is currently not defined. Here we identify NPPB encoding the natriuretic peptide, BNP, a well-known cardiac and natriuretic peptide hormone, as a transcriptional target of SHOX. The ability of SHOX to transactivate the NPPB endogenous promoter was demonstrated in luciferase reporter assays using serial deletions of the NPPB promotor region. Binding of SHOX to the NPPB promoter was also demonstrated in vivo by chromatin fixation and immunoprecipitation. We also demonstrate the lack of promoter activation in two SHOX mutants from patients with Leri–Weill syndrome. In addition, immunohistochemical analysis of human growth plate sections showed for the first time a co-expression of BNP and SHOX in late proliferative and hypertrophic chondrocytes. Together these data strongly suggest that BNP represents a direct target of SHOX.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Proliferation and maturation of chondrocytes in growth plates and replacement of the hypertrophic cartilage by bone is a highly regulated process (1). Systemic factors such as growth and thyroid hormone regulate longitudinal growth during childhood and puberty, and oestrogen and androgen contribute to this process (2,3). They provide important signals for the regulation of bone growth by modulating expression of locally produced factors. These include the bone morphogenetic proteins, Wnts, fibroblast growth factor receptors, hedgehog proteins and insulin-like growth factors which directly influence chondrocytic differentiation (4–8). Changes in gene expression within the chondrocytic life cycle consisting of proliferation, differentiation, maturation and apoptosis are mediated by various transcription factors including Sox9, Cbfa1 and different members of the homeobox containing gene family (1). Mutations or deregulation of these factors have been shown to lead to disturbed chondrogenesis and skeletal malformation. This was demonstrated for various homeobox genes including HoxA11 (radioulnar synostosis with amegakaryocytic thrombocytopenia, OMIM 605432 [OMIM] ), HoxD11 (synpolydactyly, OMIM 142989 [OMIM] ), Msx2 (craniosynostosis, Boston type, OMIM 604757 [OMIM] ) and SHOX (mesomelic short stature, OMIM 604271 [OMIM] ) (9). SHOX is present in all vertebrate species with the notable exception of rodents, in which this gene is absent (10). Relatively strong expression of SHOX was detected in hypertrophic chondrocytes, suggesting a role for this protein in the life cycle of chondrocytes (11,12).

Alterations in SHOX activity are known to result in altered human growth. Trisomy of the SHOX gene in Klinefelter or Triple X patients, for example, causes tall stature (13,14). Conversely, haploinsufficiency of the SHOX gene either leads to mesomelic short stature as seen in patients with Leri–Weill dyschondrosteosis (OMIM 127300 [OMIM] ), Langer mesomelic dysplasia (OMIM 240700) and Turner syndrome or to proportional short stature in patients with isolated short stature (OMIM 604271 [OMIM] ) (9). Besides short stature, patients with SHOX deficiency can present a wide variety of skeletal malformations such as shortening and bowing of the forearms, Madelung deformity, short fourth metacarpals, cubitus valgus and micrognathia ranging from very mild to severe (15,16). Mutations within a single gene such as SHOX can therefore lead to multiple and variable phenotypes affecting different skeletal elements. Elucidation of the signalling cascades that are activated by the transcription factor SHOX within the growth plate is of crucial importance as this may lead to a better understanding of the complex process of bone formation and provide insights into potential therapeutic avenues.

In this study, we report the first identification of a SHOX target gene, NPBB, encoding the brain natriuretic peptide (BNP).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
SHOX expression leads to an upregulation of BNP in osteosarcoma and chondrosarcoma cell lines
To identify putative SHOX target genes, we have used the previously described stably transfected osteosarcoma cell lines U2OS-ST and U2OS-STM and performed transcriptional profiling. Osteosarcoma cells were used, as the SHOX transactivating activity has been previously demonstrated to be cell type specific and to be functional in osteosarcoma cells (17). Upon doxycyclin induction, U2OS-ST expresses the wildtype SHOX protein, whereas U2OS-STM expresses a SHOX C-terminally truncated mutant, STM. This truncation mutant resembles a mutation initially identified in patients with ‘isolated idiopathic’ short stature and patients with Leri–Weill syndrome and lacks 99 amino acids at the end. STM shows identical DNA-binding properties and an identical nuclear localization compared with the wildtype SHOX protein, but lacks its transactivating potential (17). RNA from both cell lines was prepared after 12 and 24 h of induction and hybridized to the Affymetrix Human GeneChip (Hu95A) microarrays.

After 12 h of SHOX induction, the gene most significantly (13.6-fold) upregulated was NPBB which encodes the BNP (18). This increase in NPBB mRNA was specific for the SHOX wildtype protein, as it was not observed in cells expressing the C-terminally truncated mutant. After 24 h of SHOX expression, NPBB was upregulated 17.1-fold (Supplementary Table 1).

SHOX-induced upregulation of NPBB was confirmed by quantitative real-time PCR using RNA from different U2OS cell preparations in time course experiments. In agreement with the microarray data, upregulation of NPBB expression was observed only after the induction of wildtype protein but not upon the expression of the SHOX-STM mutant (Fig. 1A and B). Similar results were obtained using a second osteosarcoma cell line, Saos-SHOX, and a chondrosarcoma cell line, SW1353-SHOX (data not shown). Together, these results indicate that expression of the full-length SHOX leads to an upregulation of NPBB expression in osteosarcoma and chondrosarcoma cell lines.


Figure 1
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  		Figure 1. BNP expression upon the induction of SHOX. (A) U2OS-SHOX (ST or STM) and U2OS parental cell lines were grown in the presence of doxycycline for the time indicated. Cells were collected and total RNA was extracted. Concentration of BNP and SHOX mRNA was determined by quantitative RT–PCR carried out in duplicate using GAPDH as a standard. (B) Comparison of BNP mRNA levels between U2OS-ST (expressing SHOX wild type) and U2OS-STM (expressing SHOX mutant) grown either in the presence (+) or absence (–) of doxycycline. mRNA levels in U2OS-ST (black diamonds) increased significantly over time compared to the uninduced cells (white circles) and to U2OS-STM-induced (black triangles) or uninduced (gray squares) cells.

 
The NPBB regulatory region contains SHOX responsive elements
To confirm a direct regulation of NPBB by SHOX, a reporter plasmid was generated, in which 1940 nucleotides of the 5' flanking region of the NPBB gene were cloned upstream of a firefly luciferase reporter gene in a pGL3-Basic vector (BNP-1940) (Supplementary Fig. 1). U2OS cells were transiently transfected with the BNP-1940 construct and grown for 48 h in the presence (SHOX induction) or absence of doxycyline.

As shown in Figure 2A and B, SHOX expression triggers the activation of the luciferase reporter gene which results in an ~30-fold increase in luciferase activity between induced and non-induced cells. Similar experiments using the U2OS parental cell line, and cells with an R153L SHOX missense mutation, expressing a transcriptionally inactive SHOX mutant (19), did not show activation of the NPBB promoter, indicating that activation is specific for SHOX wildtype expression.


Figure 2
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  		Figure 2. SHOX wildtype activates the BNP promoter. (A) BNP promoter activation after the induction of SHOX.

U2OS-ST cells were co-transfected with the BNP-1940 reporter construct and pRL null vector (Renilla). After 24 h, cells were grown in the presence (SHOX induced) or absence (Not ind.) of doxycyline (0.5 µg/ml) for an additional 48 h. Cells were then lysed and dual-luciferase assays were performed. Firefly luciferase activities were normalized for transfection efficiency with the corresponding Renilla luciferase activity. The graph shows the results of a typical experiment performed in triplicate (average values with their relative standard deviation). (B) Only SHOX wildtype but not R153L and S106A SHOX mutants were able to efficiently transactivate the BNP promoter. Three inducible stable cell lines, the U2OS ST cell line expressing SHOX wildtype (WT) (17), the U2OS-R153L (19) and U2OS-S106A (20) cell lines expressing the SHOX mutants R153L and S106A, respectively, were transiently transfected with the BNP-1940 and pRL vectors. Values represent the ratio between the luciferase activity obtained in induced cells versus non-induced cells (fold induction values). No significant changes in luciferase activity were obtained for the negative control vector, pGL3 empty vector. All experiments were performed at least in triplicate. The bars represent the mean values of at least three independent experiments.

 
Recently, we have demonstrated that SHOX is a phosphoprotein and identified S106 as a SHOX phosphorylation site (20). Substitution of S106 by Ala dramatically reduced the SHOX ability to act as a transcriptional activator, suggesting that phosphorylation modulates the transcriptional activity of the protein. In agreement with previous results, the S106A mutant showed a dramatic reduction in the ability to transactivate the NPBB promoter (Fig. 2B).

To identify putative SHOX responsive elements within the regulatory region of the NPBB gene, sequential deletions of the 1940 bp 5' flanking region were generated and cloned in front of the luciferase gene in a pGL3 basic vector. Transfection of a vector, containing 1030 nucleotides of the NPBB regulative region (Fig. 3), gave similar luciferase activity as cells transfected with full-length BNP-1940, indicating that deletion of the most distal 910 nucleotides of the analysed 5' NPBB flanking region did not affect the capacity of SHOX to transactivate the NPBB promoter. A further deletion of 90 bp (BNP-940 vector) resulted, however, in a dramatic reduction in luciferase activity (drop from ~30 to 5 times), suggesting that the small interval within –1030 and 940 bp contains particularly effective SHOX responsive element(s). The residual luciferase activity obtained with the BNP-940 construct compared with BNP-827 also suggests the presence of another (yet much weaker) SHOX responsive elements between –941 to –827 nucleotides of the NPBB regulatory region.


Figure 3
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  		Figure 3. SHOX responsive elements are contained within the BNP regulatory region. (A) Schematic view of the reporter constructs containing different intervals of the BNP regulatory region, generated for the mapping of the SHOX responsive region. (B) Dual luciferase assays. Indicated constructs were co-transfected together with pRL null vector (Renilla) in U2OS-ST cells. Dual-luciferase assays were performed as described in the legend to Figure 2.

 
To confirm the binding of SHOX to the NPBB regulatory region in vivo, we carried out chromatin fixation and chromatin immunoprecipitation (ChIP) assays using induced or non-induced U2OS-ST cells. After treatment with formaldehyde to crosslink proteins to DNA, cells were harvested and sonicated and cell lysates incubated with either anti-SHOX antibodies (11) or with pre-immune serum. Isolated DNA was then analysed by RT–PCR using either primers able to amplify the NPBB regulatory region including the SHOX putative binding sites or primers annealing to an intronic region (negative control). An NPBB-related PCR product was obtained only when we used as template chromatin immunoprecipitated by SHOX antibodies in SHOX-expressing cells. In the same pellet, we did not observe an accumulation of the fragment corresponding to the intronic region, proving sequence specificity of the chromatin fixation assay and specific binding of SHOX to the NPBB regulatory region (Fig. 4).


Figure 4
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  		Figure 4. Chromatin immunoprecipitation. U2OS-ST cells were grown for 24 h in the presence (induction of SHOX expression, IND.) or absence (NOT) of doxycycline. ChIPs were performed using either rabbit-pre-immune serum (Pre) or anti-SHOX antibody (a-SHOX ab) as described in the Material and Methods section. PCRs on the immunoprecipitated DNA were carried out using either primers amplifying the BNP regulatory region containing the 90 bp putative SHOX binding sites or an intronic DNA fragment (negative control). Lanes 1 and 2 show the PCR products obtained with one-tenth of the DNA input used for the ChIPs; lanes 3 and 4, PCR products obtained using as template immunoprecipitated DNA from non-induced U2OS-ST cells; lanes 5 and 6, PCR product obtained using as template immunoprecipitated DNA from SHOX-expressing cells.

 
In summary, our results show that the promoter region of NPBB is recognized in vivo by the SHOX protein, suggesting that NPBB represents a direct target for the transcription factor SHOX.

SHOX and BNP are expressed in the chondrocytes of the growth plate
To analyse SHOX and BNP expression in the human growth plate, quantitative RT–PCR of growth plate material was carried out. Both SHOX and BNP were expressed in the growth plate of four different pre-pubertal and pubertal children. The BNP mRNA levels were lower to those of the natriuretic peptide, CNP (data not shown), previously reported to be expressed in this structure (21). In addition, immunohistochemical analysis for SHOX and BNP expression on growth plate sections of tibia (Fig. 5, Suppl. Fig. 2) and hip (data not shown) from two different individuals was carried out. We have previously shown that SHOX localizes strongly to the zone of hypertrophic chondrocytes with lower expression in proliferating and resting chondrocytes (11,12). Similar to SHOX, the BNP protein was specifically detected in both growth plate sections in the late proliferative, prehypertrophic and hypertrophic chondrocytes but not in the resting and early proliferating cells. Assuming a direct activation of BNP through SHOX, we expect SHOX and BNP to localize not only within the same region of the growth plate but also within the very same cells. A co-localization of SHOX and BNP within the same cell was observed in the majority of the chondrocytes of sequential growth plate sections. These findings provide the first evidence for a role of the BNP protein in the human growth plate. Since both SHOX and BNP proteins are found in the late proliferative, prehypertrophic and hypertrophic chondrocytes, BNP, similar to SHOX, may play a role in chondrocyte maturation and the secreted factor BNP as a downstream target of SHOX might act as a mediator of the SHOX actions.


Figure 5
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  		Figure 5. Immunohistochemical staining of a human growth plate. Sections of the tibial growth plate of a 15-year-old boy with severe overgrowth were analysed for SHOX and BNP expression. (A) and (B) depict characteristic staining obtained with anti-SHOX (SHOX) and anti-BNP antibody (BNP), respectively. Specific staining can be observed in the zone of hypertrophic chondrocytes for SHOX as well as for BNP. Controls for the immunohistochemistry analyses are shown in Supplementary Figure S2.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
A major role in the regulation of human height has been attributed to the transcription factor and homeodomain protein SHOX (22). To address which genes are influenced and controlled by the expression of SHOX, we carried out transcriptional profiling. The gene with the most significant and by far strongest upregulation upon SHOX induction was the NPPB gene encoding the peptide BNP (Suppl Table 1). We have demonstrated that SHOX can bind to the NPPB 5' flanking region and activates the NPBB promoter in vivo. SHOX, as other paired-related homeodomain proteins, can also bind to the palindromic sequence TAAT(N)nATTA (17,23). Sequence analysis of the 5' flanking region of the NPBB gene revealed a putative SHOX-binding site, TAATGATAATTA, between –1479 and 1469 nucleotides upstream of the NPBB open-reading frame. However, deletion of this site did not reduce SHOX-induced NPBB promoter activity (data not shown). Instead, serial deletions of the 5' NPBB flanking region identified an interval of 90 bp between –940 and –1090 upstream of the translation initiation site that is mainly responsible for SHOX-induced promoter activation. An imperfect palindromic sequence, TAATGAATTG, at –837 and –827 nucleotides upstream of the start codon may also add to the role in SHOX-induced NPBB promoter activity.

In summary, these data suggest that SHOX and BNP may act on the same pathway in bone development. Supporting this hypothesis, quantitative RT–PCR (data not shown) and immunohistochemical analysis of several human growth plate sections has revealed for the first time a co-expression of SHOX and BNP in late proliferative, prehypertrophic and hypertrophic chondrocytes.

The identification of the peptide hormone BNP (24) as a direct target of SHOX was puzzling at first sight, as this marker had been primarily known for its cardiovascular function, constituting a biomarker for the diagnosis and prognosis of heart failure (25,26). Because of its wide range of vasodilatory, natriuretic and diuretic properties, BNP is also known as a potent peptide hormone drug (‘Nesiritide’, Scios) in the treatment of decompensated heart failure (27). However, growing evidence indicates that natriuretic peptides BNP and CNP as well as the receptors (NPRs) represent important regulators of endochondral bone ossification. Mice which overexpress or lack natriuretic peptides or NPRs exhibit pronounced skeletal growth defects (21,28,29), and CNP has been shown, for example, to enhance longitudinal growth in organ-cultured bones and to prevent shortening of the bones in FGFR3 constitutively active mice with achondroplasia (29,30). Heterozygous defects of the NPR-B receptor have recently been reported in idiopathic short stature, whereas the homozygous loss-of-functional NPR-B causes acromesomelic dysplasia-type Maroteaux (31). Together, these data demonstrate an important role of the natriuretic peptides and its receptors in bone growth and metabolism.

To study the effects of BNP overexpression on the cardiovascular and the diuretic system, Suda et al. (24) developed a BNP transgenic mice. These mice exhibit reduced blood pressure and cardiac weight and an elevated plasma cGMP concentration, confirming the physiological relevance of the natriuretic peptides in the vasculature. They also display a marked skeletal overgrowth characterized by elongated limbs and paws, a kyphosis and a crooked tail. They display alterations and overgrowth of the growth plate cartilage; the hypertrophic and non-hypertrophic zones of the cartilage were enlarged as were the hypertrophic chondrocytes (24). Together, mice overexpressing wildtype BNP present a phenotype with overgrowth, kyphosis and reduced blood pressure and further heart and renal anomalies that may be the consequence of a vasculature defect. Interestingly, it appears that some of these features represent mirror images of the well-studied clinical stigmata in females with Turner syndrome, which have a haploinsufficiency of the SHOX gene. Patients with Turner syndrome are on average 20 cm smaller, often present a scoliosis and have high blood pressure and variable heart and renal abnormalities.

One may speculate that BNP as a downstream effector of SHOX may also open up new potential avenues for the treatment of short stature. BNP in the systemic circulation is likely to reach growth plate chondrocytes. It may either directly influence NPR-B signalling or indirectly increase local CNP levels by saturating the competing receptor NPR-C. As demonstrated in the mouse, increased levels of BNP in the serum increase skeletal growth (24), maybe analogous to the use of recombinant growth hormone and insulin-like growth factor. A systemic application of BNP, possibly in combination with the administration of hGH, may therefore open up new prospects for the treatment of growth impairment in patients with SHOX haploinsufficiency.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Cells and media
Generation and culture conditions of the human T-Rex U2OS osteosarcoma cell lines (R712-07; Invitrogen) and the human embryonic kidney T-Rex HEK293 inducible cell line expressing either wildtype protein (SHOX) or an STM were described previously (17). The S106A SHOX cell line expressing a SHOX mutant containing a substitution of Serine 106 to Alanine was described in Marchini et al. (20), and the R153L SHOX cell line expressing a SHOX mutant in which Argine 153 is mutated in Leucine was described in Schneider et al. (19). Furthermore, another human osteosarcoma cell line T-Rex Saos-SHOX and a human chondrosarcoma cell line SW1353-SHOX were generated expressing wildtype SHOX. Generation of these cell lines was done according to the manufacturer's instruction manual (Invitrogen). Both were cultured in Dulbeccos modified Eagle medium supplemented with 4.5 g/l glucose, penicillin/streptomycin 10 U/10 µg/ml and 10% fetal bovine serum, with the addition of 200 µg/ml Zeocine and 10 µg/ml Blasticidine. In all cell lines, SHOX expression was achieved by adding the tetracycline analogous doxycycline (4 µg/ml).

Affymetrix Hu95A GeneChip hybridization
The Hu95A GeneChip (Santa Clara, CA, USA) contains more than 12 000 probe sets corresponding to 8900 specific human genes (UniGene Build 139). Total RNA for the hybridization was isolated from human U2OS cells expressing either wildtype SHOX or STM. Cells were grown in 145 mM tissue culture dishes and gene expression was induced at 30% confluence, for 12 or 24 h or later. As a negative control, uninduced parental cells were also harvested at corresponding time points. After RNA preparation using the Qiagen RNeasy® Midi Kit, the RNA was quantified and validated for integrity by gel electrophoresis. Induction of the SHOX/STM gene expression was verified by first-strand cDNA synthesis using the SuperscriptTM First-Strand Synthesis System for RT–PCR from GibcoBRL® according to the manufacturer’s protocol and by semi-quantitative RT–PCR. cRNA synthesis, hybridization of the samples to the Hu95A GeneChips, post-hybridization staining, scanning of the stained chips and processing of the data files using the Microsoft Array Suite (MAS) 4.0 and MAS 5.0 software were performed at the Resource Center and Primary Database (RZPD) in Berlin.

Real-time PCR
For quantitative real-time RT–PCR analysis, RNA was extracted from cells described earlier and reverse-transcribed as described earlier. The resulting first-strand cDNA was used as a template in PCR reactions. The following primer pairs were used: GAPDH: 5'-ACC ACA GTC CAT GCC ATC AC-3', 5'-TCC ACC ACC CTG TTG CTG TA-3'; SHOX: 5'-ATG GAA GAG CTC ACG GCT TTT GTA TCC-3', 5'-CGA AGA GTC GCT CGA GCT CGT TC-3'; BNP: 5'-TTC TTG CAT CTG GCT TTC CT-3', 5'-ACC GTG GAA ATT TTG TGC TC-3'. Quantitative PCRs were performed with the LightCycler FastStart DNA Master SYBR green I Kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the following conditions: denaturation of DNA and activation of the polymerase at 94°C for 10 min; 45 cycles of a touchdown PCR with 15 s denaturation at 94°C, 10 s annealing at 65°C with a decrease of 1°C per cycle to a final annealing temperature of 60°C, elongation at 72°C for 30 s. Expression of genes was analysed on the LightCycler from Roche Molecular Systems with the LightCycler Software 3.5. For normalization, we used the expression of the housekeeping gene GAPDH.

Reporter constructs and dual luciferase assays
A DNA fragment containing 1940 bases of the regulatory region of NPBB was PCR-amplified using the primers BNP-1940 5'-AAA GTC GAC AAG CTT GCT TTT TGT AGA AA-3' and BNP Rev 5'-AAA CCA TGG GTC TCT GGA GGG ACT GCG-3', and cloned XhoI/NcoI into pGL3 basic vector (Promega). In the same way, the other DNA fragments containing different portions of the NPBB regulative region were generated. PCRs were performed using the BNP Rev primer in combination with BNP-1130 5'-AAA GTC GAC TTC ACC GTG GTC TCG ATC T-3'; BNP-1080 5'-AAA GTC GAC TCA AAG TGC TGG GAT TAC A-3'; BNP-1030 5'-AAA GTC GAC TGT TTT CCA TCC TGT GTT G-3'; BNP-940 5'-AAA GTC GAC TCA CTG GAC CCT ATC TCT CA-3'; BNP-827 5'-AAA GTC GAC ACT GGG GAA TCA GCA TCC C-3'; BNP-500 5'-AAA GTC GAC TCG CTG TGA GAT CAC CCC GTG-3'.

NPBB promoter activity studies were carried out in U2OS-ST (SHOX inducible) cell line. Cells were grown in 10 cm dishes and transfected with FUGENE (Roche Diagnostics GmbH) at 30–40% confluence. Transfections were performed using 1 µg of BNP reporter construct in combination with 1 µg of pRL-TK (Promega) used to normalize the transfection efficiency. Cells were then transferred in six-well plates and grown in the presence or absence of DOX for additional 36 h. Luciferase activity was assayed using a Dual-Luciferase Assay Kit (Promega) according to the manufacturer’s protocol. Assays were performed at least in triplicate and transcriptional activation was calculated from at least three independent experiments.

Chromatin immunoprecipitation
Cells were grown on 10 cm plates and were either induced with doxycycline (0.5 µg/ml) for the expression of SHOX WT or not induced and grown for an additional 24 h. ChIP assays were performed using the ChIP Assay Kit (Upstate Biotechnology) according to the manufacturer’s instructions. Sonication of the cells (2 x 106) was conducted four times for 10 s each time, to obtain DNA fragments of 200–1000 bp in length. Pre-immune serum or rabbit anti-SHOX ß-antibody was used for immunoprecipitations. Precipitated DNA samples were analysed by quantitative RT–PCR in the LightCycler as described earlier, using the BNP-ChiP primers 5'-CAT TCC AGC CAT CCT TTT GT-3' (from –1396 to –1368 bases upstream of the BNP translation start site) and 5'-CTC TCA CAA TCC GCC ATC TT-3' (position from –799 to –779). To control specificity of DNA co-immunoprecipitation, the following primers were used—5'-TAC GTG GTA GGG AGG AGG ATT GG-3' and 5'-CAT CCA AGC AAT TGT CAG GGA TGG-3'—that allow the amplification of an intronic DNA fragment residing at 72934–73772 on the X chromosome.

Immunohistochemistry
Sections of growth plates were obtained of a 15-year-old male with a body height of 1.98 m who underwent surgical removal of the tibial growth plates to stop further growth and of a 13-year-old female subjected to surgery due to hip reconstitution after informed parental consent. The use of the material was approved by the medical ethical committee. Immunohistochemistry was performed according to Marchini et al. (11). Antibodies with the relative dilutions were anti-BNP (Immundiagnostic) 1:25; anti-SHOX 1:50, SHOX pre-immuneserum 1:500 and anti-polyHistidine (Sigma) 1:100.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
FUNDING
 REFERENCES
 
Supplementary Material is available at HMG Online.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
REFERENCES
 
This work was supported by a research grant of Eli Lilly and the Deutsche Forschungsgemeinschaft.


   ACKNOWLEDGEMENTS
 
We would like to thank Helene Schmidt and Ralph Roth for technical assistance, Rüdiger Blaschke and Jan-Maarten Wit for discussion, Claire Bacon for reading the manuscript and the RZPD (Deutsches Ressourcenzentrum für Genomforschung) in Berlin for performing the hybridization of the Affymetrix Hu95A filter.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Back

{ddagger} Present address: German Cancer Research Center, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany. Back

The sequence of the BNP gene is available under the accession number BC025785 [GenBank] . Information on the genomic sequence of the BNP promoter region can be obtained under the accession number D16641 [GenBank] .


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 

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B. E. Hjerrild, K. H. Mortensen, and C. H. Gravholt
Turner syndrome and clinical treatment
Br. Med. Bull., June 1, 2008; 86(1): 77 - 93.
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