Sotos Syndrome Is Associated with Deregulation of the MAPK/ERK-Signaling Pathway Remco Visser1*, Ellie B. M. Landman2, Jelle Goeman3, Jan M. Wit1, Marcel Karperien2 1 Department of Pediatrics, Leiden University Medical Center, Leiden, The Netherlands, 2 Department of Developmental BioEngineering, MIRA Institute for Biomedical Technology and Technical Medicine, Enschede, The Netherlands, 3 Department of Medical Statistics and Bioinformatics, Leiden University, Medical Center, Leiden, The Netherlands Abstract Sotos syndrome (SoS) is characterized by tall stature, characteristic craniofacial features and mental retardation. It is caused by haploinsufficiency of the NSD1 gene. In this study, our objective was to identify downstream effectors of NSD1 and to map these effectors in signaling pathways associated with growth. Genome-wide expression studies were performed on dermal fibroblasts from SoS patients with a confirmed NSD1 abnormality. To substantiate those results, phosphorylation, siRNA and transfection experiments were performed. A significant association was demonstrated with the Mitogen- Activated Protein Kinase (MAPK) pathway. Members of the fibroblast growth factor family such as FGF4 and FGF13 contributed strongly to the differential expression in this pathway. In addition, a diminished activity state of the MAPK/ERK pathway was demonstrated in SoS. The Ras Interacting Protein 1 (RASIP1) was identified to exhibit upregulated expression in SoS. It was shown that RASIP1 dose-dependently potentiated bFGF induced expression of the MAPK responsive SBE reporter providing further support for a link between NSD1 and the MAPK/ERK signaling pathway. Additionally, we demonstrated NSD1 expression in the terminally differentiated hypertrophic chondrocytes of normal human epiphyseal growth plates. In short stature syndromes such as hypochondroplasia and Noonan syndrome, the activation level of the FGF-MAPK/ERK-pathway in epiphyseal growth plates is a determining factor for statural growth. In analogy, we propose that deregulation of the MAPK/ERK pathway in SoS results in altered hypertrophic differentiation of NSD1 expressing chondrocytes and may be a determining factor in statural overgrowth and accelerated skeletal maturation in SoS. Citation: Visser R, Landman EBM, Goeman J, Wit JM, Karperien M (2012) Sotos Syndrome Is Associated with Deregulation of the MAPK/ERK-Signaling Pathway. PLoS ONE 7(11): e49229. doi:10.1371/journal.pone.0049229 Editor: Farid F. Chehab, University of California San Francisco, United States of America Received February 19, 2012; Accepted October 9, 2012; Published November 14, 2012 Copyright: ß 2012 Visser et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Stichting Nationaal Fonds ‘‘het Gehandicapte Kind’’ and The Netherlands Organisation for Health Research and Development (920-03-325 to R.V.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction patients, was demonstrated to regulate transcription of the Hox-A gene locus [8]. In a tissue-specific manner NSD1 exhibits both Sotos syndrome (SoS; MIM 117550) is characterized by tall repressing as well as activating capacities as was exemplified by stature, facial dysmorphism and mental retardation [1]. It is reduced expression of the MEIS1 oncogene in a neuroblastoma caused by haploinsufficiency of the nuclear receptor binding SET model, while increased expression was detected in transfected cells domain protein 1 (NSD1) gene [2]. Originally, mouse Nsd1 was containing the NUP98–NSD1 fusion protein [8,9]. identified in a two-hybrid screen with the retinoic acid receptor Based on its role in regulation of gene transcription, it has been alpha (RARa) as bait [3]. It was shown that Nsd1 interacted with a hypothesized that heterozygous inactivation of NSD1 results in number of nuclear hormone receptors, such as the estrogen loss of repression of growth promoting genes [4]. Consequently, receptor, retinoic acid and thyroid hormone receptors [3]. It was increased activity of these genes would lead to the characteristic postulated that NSD1 could either act as a co-repressor or a co- overgrowth phenotype of SoS. Unfortunately, experimental activator of these nuclear receptors depending on the cellular evidence for this hypothesis is lacking due to the fact that context and the presence or absence of the respective hormones heterozygous knock out mice of Nsd1 do not show a SoS [3]. Since then, several in vitro experiments showed that an phenotype [4], especially with regard to overgrowth and important function of NSD1 is the methyltransferase activity of its epiphyseal growth plate changes (prof. Losson, personal commu- Su(var)3–9, Enhancer-of-zeste and Trithorax (SET) domain [4–6]. nication). This domain specifically methylates lysine 36 at histone H3 (H3– Our group previously studied the expression of members of the K36), lysine 20 at histone H4 (H4–K20) and other non-histone Growth Hormone (GH)/Insulin like growth factor 1 (IGF1) axis in substrates [4–7], resulting overall in the regulation of chromatin SoS patients [10]. Modestly increased plasma levels of IGFBP-2 transcription. For example, NSD1 was shown to regulate activity and IGFBP-6 and reduced levels of IGF-I, IGF-II, IGFBP-3 and of the transcription factor NF-kB [7]. Furthermore, depletion of IGFBP-4 were detected. Reduced levels of IGF-I and IGF-II in NSD1 reduced the expression of the bone morphogenetic protein particular are, however, more reminiscent of short rather than tall gene 4 (BMP4) [5]. In addition, a fusion protein NUP98–NSD1 stature such as observed in SoS. The relationship between NSD1 which is found in about 5% of human acute myeloid leukemia and the GH/IGF1 axis remains elusive. Thus, although a number PLOS ONE | www.plosone.org 1 November 2012 | Volume 7 | Issue 11 | e49229 Sotos Syndrome and the MAPK/ERK-Signaling Pathway of functions of NSD1 have been identified, the molecular A correction for multiple testing was performed using the mechanisms leading to phenotypic features of SoS remain largely Benjamin-Hochberg method. unclear. The Global Test package (version 4.10.0) was used for the The aim of the current study was therefore to identify pathway analysis [15]. Details about the pathways can be found in downstream effectors of NSD1 and to map these effectors in Table S3. The permutation method was employed and correction signaling pathways associated with growth. Genome-wide mRNA for multiple testing was performed. In all statistical analysis, results expression profiles of dermal fibroblasts from SoS patients with a with an adjusted p.value,0.05 were considered statistically confirmed NSD1 abnormality were compared to expression significant. patterns in age and sex-matched controls. Real-Time Quantitative PCR Materials and Methods Real-Time quantitative PCR (qPCR) using QuantiTect primers (Qiagen, Venlo, The Netherlands) was performed to validate Ethics Statement differentially expressed genes identified in the microarray analysis. This study was approved by the Medical Ethical Committee of The following genes were selected: RASIP1, RBM47, COMP, and the Leiden University Medical Center and written informed FGF13. Fold changes were adjusted for the expression of the consent was given by the patients and/or their parents or legal housekeeping gene b2 microglobulin following the 22DDCt method guardians. [16]. Subjects NSD1 siRNA Study The study included cell lines of skin fibroblasts which were HEK293t cells (American Type Culture Collection CRL- obtained from 9 SoS patients with a confirmed NSD1 abnormality 11268) were cultured for 48 hours after which cells were and 9 sex- and age matched normal donors as described previously transfected with siRNA (Qiagen) for NSD1 or non-targeting by De Boer et al. [10]. Details are presented in Tables S1 [11] and siRNA and incubated for another 48 hours. After RNA isolation, S2. expression levels were determined with qPCR. Cell Culture RASIP1 Transfection Study Skin fibroblasts were cultured in Dulbecco’s modified Eagle’s HEK293t cells (American Type Culture Collection CRL- Medium supplemented with glutamax (Gibco, Breda, The 11268) were co-transfected with the MAPK-responsive SBE Netherlands) and containing 10% fetal calf serum (Lonza, promoter reporter construct in combination with increasing Verviers, Belgium). The cell cultures were maintained at 37uC amounts of a RASIP1 expression vector and pRL-CMV Renilla in a humidified atmosphere of 5% CO2 and 95% O2. In all control, using Fugene HD (Roche Applied Science, Almere, The experiments cell passages were between 2 and 10. With regard to Netherlands). pUC19 DNA was used to adjust DNA concentra- the NSD1 interaction with RARa [3], we hypothesized that tions in the transfection experiment. Twenty-four hours after differences in gene expression between fibroblasts of SoS patients transfection, cells were stimulated with 10 ng/ml bFGF for and controls would be more pronounced in the presence of all 24 hours. Luciferase values were normalized for Renilla and trans-retinoic acid (RA). At reaching 80–90% monolayer conflu- expressed as fold induction compared to control which