The nuclear receptor NOR-1 regulates the small muscle protein, X-linked (SMPX) and myotube differentiation

Recent works have highlighted the role of NOR-1 in both smooth and skeletal muscle, and have proposed this nuclear receptor as a nexus that coordinates muscle performance and metabolic capacity. However, no muscle specific genes regulated by NOR-1 have been identified so far. To identify NOR-1 target genes, we over-expressed NOR-1 in human vascular smooth muscle cells (VSMC). These cells subjected to sustained over-expression of supraphysiological levels of NOR-1 experienced marked phenotypic changes and up-regulated the skeletal muscle protein X-linked (SMPX), a protein typically expressed in striated muscle and associated to cell shape. By transcriptional studies and DNA-protein binding assays, we identified a non-consensus NBRE site in human SMPX promoter, critical for NOR-1 responsiveness. The expression of SMPX was higher in human skeletal muscle myoblasts (HSMM) than in human VSMC, and further increased in HSMM differentiated to myotubes. NOR-1 silencing prevented SMPX expression in HSMM, as well as their differentiation to myotubes, but the up-regulation of SMPX was dispensable for HSMM differentiation. Our results indicate that NOR-1 regulate SMPX in human muscle cells and acts as a muscle regulatory factor, but further studies are required to unravel its role in muscle differentiation and hypertrophy.

Scientific RepoRts | 6:25944 | DOI: 10.1038/srep25944 VSMC subjected to sustained over-expression of supraphysiological levels of NOR-1 experienced marked phenotypic changes and up-regulated a gene typically expressed in striated muscle (skeletal muscle protein X-linked, SMPX). We characterized the regulation of SMPX by NOR-1 and uncovered the critical role of this nuclear receptor in the differentiation of human skeletal myoblasts to myotubes, a process which also entails the up-regulation of SMPX.

NOR-1 over-expression in VSMC modifies cell shape and increases SMPX expression. Human
VSMC were transduced to over-express NOR-1 ( Supplementary Fig. S1). VSMC over-expressing NOR-1 exhibited striking differences in cell morphology compared to control cells (Fig. 1a). This phenotypic switch in cells expressing high and sustained levels of NOR-1 was associated to a low proliferation rate ( Supplementary Fig. S2a) and signs of cell hypertrophy such as increased total cellular protein and enhanced cellular surface area ( Supplementary Fig. S2b,c). As a first approach to identify genes regulated by NOR-1, we analyzed the mRNA levels of several genes related to cell shape/morphology encoding for cytoskeleton and cytoskeletal-associated proteins. The over-expression of NOR-1 did not alter the expression of most of these genes, but slightly induced the expression levels of the transcript corresponding to the ubiquitously expressed form of caldesmon (l-CaD; ≈ 1.9-fold). The most striking results, however, was the strong up-regulation of SMPX (a stretch-responsive skeletal muscle protein) (Fig. 1b). SMPX was up-regulated up to 170-fold as compared with control cells (transduced with pLVX or pLVX/EGFP) (Fig. 1c). The regulation of both SMPX and l-CaD was confirmed by Western blot (Fig. 1d). SMPX expression was detected at low levels in human vascular tissues (aorta and coronary arteries) compared to its expression in skeletal muscle ( Supplementary Fig. S3).

NOR-1 binds to SMPX promoter through a non-consensus NBRE site.
To further characterize the functionality of the putative NBRE(− 167/− 160) site, we performed electrophoretic mobility shift assays (EMSA) using nuclear extracts from VSMC over-expressing a FLAG-tagged NOR-1. As shown in Fig. 2c, a differential pattern of retarded bands was observed in EMSA using this NBRE site as a probe and nuclear extracts from NOR-1-transduced cells, respect those from control cells (transduced with pLVX/EGFP). NOR-1 over-expression increased the intensity of two of these complexes, that were competed by an excess of unlabeled probe, and one of them (complex I) was supershifted by an anti-FLAG antibody. Conversely, mutation of the NBRE probe abrogated both the enhanced binding found in NOR-1-transduced cells and the supershift. Taken together, these data demonstrate that NOR-1 specifically bind to this NBRE site. To confirm that NOR-1 actually binds to the SMPX promoter in vivo, chromatin immunoprecipitation (ChIP) assays were performed in VSMC over-expressing FLAG-tagged NOR-1. Proteins directly bound to chromatin were immunoprecipitated using an antibody against the FLAG sequence or an unspecific IgG. Conventional and real-time PCR were used to amplify a genomic region (b) Luciferase activity from cells co-transfected with a NOR-1 expression vector (pCMV5/NOR-1; black bars) or the corresponding empty plasmid (pCMV5; white bars) together with different pGL3/SMPX constructs. The activity of constructs mutated in the NBRE site (deleted white circle) is also shown. The core of the NBRE(− 167/− 160) is indicated and changes introduced by mutagenesis on constructs are boxed (n = 6). *P < 0.0001 vs. cells co-transfected with pCMV5; # P < 0.0001 vs. cells co-transfected with pCMV5/NOR-1 and SMPX constructs containing the native NBRE(− 167/− 160) site. (c) Representative autoradiograms of EMSA performed with a SMPX probe containing the NBRE(− 167/− 160) site (NBRE) and nuclear protein extracts from VSMC transduced with lentiviral vectors to express NOR-1-FLAG (pNOR-1F) or EGFP (pGFP). The position of the complexes up-regulated by NOR-1 (I and II) is indicated. Competition assays with a molar excess of unlabeled probe (100-fold; Competitor) and supershift assays with a specific antibody against the FLAG sequence (anti-FLAG Ab) were performed. EMSA carried out with a mutated NBRE probe (mut-NBRE) is also shown. SS: supershifted complex. (d) The relative in vivo association of NOR-1 with SMPX promoter was analyzed by ChIP assays in VSMC transduced with pGFP or pNOR-1F. Immunoprecipitations were performed with an antibody against the FLAG sequence (IP:FLAG) or a non-specific IgG (IP:IgG). The bars graph shows the enrichment of NOR-1 binding quantified by real-time PCR using SMPX promoter specific primers. Data were normalized to the total input DNA (n = 4). *P = 0.0002 vs. pGFP. The panel on right shows the agarose gel electrophoresis of PCR products. Equal input DNA and control IgG immunoprecipitations are shown.

NOR-1 knockdown decreased SMPX mRNA and protein levels in human skeletal muscle myoblasts (HSMM).
To evaluate the impact of NOR-1 knockdown on SMPX expression, a siRNA was used to silence NOR-1; however, the low expression levels of SMPX in primary VSMC cultures preclude this approach. Since SMPX was early identified as a gene preferentially expressed in striated muscle, we analyzed the expression level of SMPX in HSMM cultures. The expression of SMPX was about 800-fold higher in these cells than in primary human VSMC cultures, and further increased in HSMM differentiated to myotubes ( Supplementary Fig. S5). NOR-1 silencing, that was more efficient in non-differentiated ( Fig. 3a) than in differentiated cells (Fig. 3b), was associated with a concomitant reduction of SMPX expression, as determined by real-time PCR and Western blot. The differentiation (in 2% horse serum during five days) transformed HSMM with typical myoblast morphology to elongated and multinuclear myotubes (Fig. 4a). HSMM differentiation increased NOR-1 and SMPX mRNA (≈ 2.4-fold and ≈ 3.5-fold, respectively) and protein levels, as determined by Western blot and immunocytochemistry ( Fig. 4b-d).
The effects of NOR-1 on SMPX and HSMM differentiation are independent events. NOR-1 silencing prevented the phenotypic changes as well as the up-regulation of SMPX (both mRNA and protein levels) associated to HSMM differentiation (Fig. 5). Interestingly, NOR-1 knockdown also prevented the up-regulation of cell differentiation markers such as MyHC and p27 (Fig. 5c,d). In ChIP assays, HSMM differentiation significantly increased the capacity of NOR-1 to bind to the NBRE(− 167/− 160) site in SMPX promoter, while this increase was abolished in cells previously transfected with a siRNA against NOR-1 (Fig. 6). SMPX silencing did not affect HSMM differentiation (Fig. 7). Thus, the NOR-1 mediated increase of SMPX expression associated to HSMM differentiation seems to be dispensable for cell differentiation.

Discussion
NOR-1 exhibits a dissimilar expression pattern in different tissues 4,[20][21][22] and has been shown to regulate diverse and sometimes antithetical processes such as cell survival and apoptosis, and cell proliferation and differentiation [2][3][4][5] . Interestingly, the transient up-regulation of this immediate-early gene by cytokines and growth factors has been associated with VSMC proliferation and migration 4,14-18 , but in skeletal muscle, NOR-1 is induced by muscle contractile activity [28][29][30] and has been related with muscle hypertrophy 23,24 . Since the role of NOR-1 in lipid and (a) HSMM were transfected with a siRNA against NOR-1 (siNOR-1; black bars) or a random siRNA (siRd; white bars). The expression of NOR-1 and that of SMPX in these samples was analyzed by real-time PCR (upper panel) (n = 6). *P < 0.0001 vs. siRd. The down-regulation of SMPX by siNOR-1 was also analyzed by Western blot (lower panel). Levels of β -actin were used as a loading control. (b) HSMM were differentiated to myotubes, and then were transfected with siNOR-1 or siRd. NOR-1 and SMPX mRNA levels (upper panel) and protein levels (lower panel) were analyzed as indicated in (a) (n = 6). *P < 0.0001, and # P = 0.0002 vs. siRd.
glucose metabolism was established, it has been proposed as a nexus that coordinates muscle performance and metabolic capacity 26,27 . Few NOR-1 target genes, however, have been described so far and none specifically related to the muscular tissue. Here we show that SMPX, a gene commonly expressed in striated muscle and identified as a stretch-responsive gene upregulated in skeletal muscle hypertrophy 31 , is a target of NOR-1. Further we assess the role of NOR-1 and SMPX in the differentiation of human myoblasts.
We used lentiviral vectors for stable ectopic expression of NOR-1 in human VSMC. The sustained over-expression of this early gene induced phenotypic changes in VSMC. Morphological and cell phenotypic changes are commonly accompanied by profound alterations in gene expression pattern affecting cytoskeleton and cytoskeletal-associated proteins. Since NOR-1 is a transcription factor and therefore modulates gene expression, as a first approach we analyzed mRNA levels of a set of genes previously associated with changes in cell morphology. Despite the observed reorganization of the actin filaments, neither the expression of β -actin nor that of smooth muscle specific α -SMA encoding genes experienced noticeable changes. Similarly, expression levels of several cytoskeletal associated proteins remained unchanged or experienced minor changes. By far the most striking result was the strong increase of SMPX, a gene typically expressed in both heart and skeletal muscle 32 .
SMPX (also called Chisel, Csl) is a small proline-rich protein (9 kDa) highly conserved among mammalian species 32 , that was firstly identified by reciprocal probing as a muscular protein selectively expressed in human cardiac and skeletal muscle cells 33 . By suppressive subtractive hybridization strategy, SMPX was later identified as a transcriptional target of the cardiac homeodomain factor Nkx2-5 in a mouse line carrying a null allele of Nkx2-5 32 , and as a stretch-responsive gene in mouse skeletal muscle 31 . Low SMPX expression, however, has also been detected in smooth muscle-rich tissues such as stomach 34 , intestine 32,34 and pulmonary veins 32 . In embryos, SMPX is expressed in sub-regions of the developing heart corresponding to the future chamber myocardium and in developing skeletal muscles 35 . In fact, in the heart, SMPX is considered a chamber-specific gene, activated as part of a chamber myocardium-specific program of gene expression, which also includes Natriuretic precursor peptide type A (Nppa), Connexin (Cx) 40 and Cx43 32,36 . Interestingly, the formation of specialized heart structures such as the sinoatrial node (SAN) requires the local repression of these "chamber-specific" genes (including SMPX) [36][37][38] . Surprisingly, SMPX knockout mice showed no obvious phenotype; however, SMPX over-expression in the C2C12 myogenic cell line affect cytoskeletal dynamics and cell shape, and promote cell signaling-dependent myocyte fusion 32,35 . These results suggest that SMPX could play a role in the regulatory network through which muscle cells coordinate their structural and functional states during growth, adaptation, and repair 32 . However, the precise role played by SMPX in the musculature is presently unknown. Mutations affecting SMPX have been associated to x-chromosome linked hearing loss in humans 39,40 , but there is no information regarding SMPX in cardiovascular or muscular human disorders. Therefore, further studies are needed to understand the pathophysiological role of SMPX in human diseases.
As we mentioned above, SMPX is expressed in muscle tissues, predominantly in striated muscle and, in a lesser extent, in several smooth muscle tissues 31,33,34 . We confirmed this expression pattern using a panel of mouse tissues (Supplementary Fig. S6). In accordance with this expression pattern, previous studies of SMPX promoter identified putative cis regulatory elements for muscle-specific factors such as myocyte-specific enhancer factor (MEF2) 31 , a transcription factor involved in myogenesis 41 , MyoD 31 , a factor that triggers cell differentiation into myotubes, and Nkx-2.5 31 , a cardiac homeodomain transcription factor involved in cardiac development 42 . We also show for the first time that SMPX is expressed in the human vasculature (aorta and coronary arteries). Our in silico analysis revealed the presence of several putative NBRE sites potentially involved in the up-regulation of SMPX by NOR-1. We experimentally analyzed the functionality of these elements. Serial promoter deletion and site-directed mutagenesis analysis identified the proximal NBRE(− 167/− 160) site as crucial for the NOR-1-mediated induction of SMPX promoter activity. Accordingly, the direct binding of NOR-1 to this site was demonstrated in vitro and in vivo by EMSA and ChIP assays, respectively. Our results involving NOR-1 in the regulation of SMPX expand the knowledge on the SMPX promoter structure and regulation. The regulation of SMPX by NOR-1 was further confirmed in human skeletal muscle cells. SMPX mRNA levels were high in HSMM, and in the course of the differentiation to myotubes both NOR-1 and SMPX were up-regulated. NOR-1 silencing significantly reduced SMPX expression in both differentiated and non-differentiated HSMM. Interestingly, the differentiation process was dependent on NOR-1 but independent of SMPX. Although in certain contexts NR4A receptors (particularly NOR-1 and Nur77) can be totally or partially redundant 43,44 , which would hinder the observation of phenotypic consequences of deleting a single NR4A receptor, it should be emphasized that the specific silencing of NOR-1 prevented HSMM differentiation. Further studies are needed, however, to unravel the role of NOR-1 in the process of HSMM differentiation.
In summary, our data show that long lasting over-expression of NOR-1 in VSMC led to a non-proliferative, hypertrophic phenotype and to the induction of a protein commonly expressed in striated muscle whose expression further increases in skeletal muscle hypertrophy. Although the role of NOR-1 in hypertrophy remains to be elucidated, these results are coherent with the negative regulation of myostatin (anti-hypertrophic protein) 45 by NOR-1 in the mouse skeletal muscle cell line C2C12 23 and in the muscle of Nur77-transgenic mice 44 . It should be noted, however, that our in silico analysis identify the NBRE responsive for NOR-1 regulation in different mammalian species but not in murine rodents, and our experimental results discard SMPX as a NOR-1 target gene in mouse. Thus, unfortunately further testing on the functional significance of SMPX modulation by NOR-1 by using small animal models is not possible. Previous studies from us and others indicate that there are notable differences between human and murine rodents in both the mechanisms that regulate NOR-1 46 and NOR-1 target

Figure 5. NOR-1 knockdown prevents HSMM differentiation and down-regulates SMPX expression.
HSMM were transfected with a siRNA against NOR-1 (siNOR-1) or a control random siRNA (siRd), and then were exposed to differentiation conditions during five days.  . NOR-1 knockdown prevents the binding of NOR-1 to SMPX promoter. HSMM cells were transfected or not with a siRNA against NOR-1 (siNOR-1) or a control random siRNA (siRd), and then were exposed to differentiation conditions during five days (black bars). Non-differentiated HSMM were used as a control (white bars). The relative in vivo binding of NOR-1 to the human SMPX promoter was analyzed by ChIP assays. Immunoprecipitations were performed with an antibody against NOR-1 (IP:NOR-1) or a nonspecific IgG (IP:IgG). The bars graph shows the levels enrichment of NOR-1 quantified by real-time PCR using SMPX promoter specific primers. Data were normalized to the total input DNA (n = 6). *P < 0.0001 vs. non-differentiated HSMM; # P < 0.0001 vs. differentiated HSMM transfected or not with siRd. The bottom panel shows the agarose gel electrophoresis of PCR products. Equal input DNA and control IgG immunoprecipitations are shown. genes 47 . Consequently, before extrapolate biomedical consequences from NOR-1 biology in mouse models, it is mandatory to corroborate the data in humans. Therefore, NOR-1 seems to complement other muscle regulatory factors in the regulation of proteins and processes in the musculature 26 , although further studies are required to establish the relevance of this nuclear receptor in the skeletal muscle biology in humans.

Materials and Methods
Human tissue sampling and preservation. The study was approved by the Hospital de la Santa Creu i Sant Pau (HSCSP) Ethics Committee and was conducted according to the Declaration of Helsinki. Written informed consent was obtained from each patient. Coronary arteries and aorta were collected from patients undergoing heart transplant and coronary artery at the HSCSP, and skeletal muscle samples were obtained from patients undergoing lower limb amputation at the same hospital. Specimens were snap-frozen in liquid nitrogen and stored at − 80 °C until the preparation of protein and RNA extracts.
Cell cultures. VSMC were obtained from human non-atherosclerotic arteries of hearts removed in transplant surgeries at the HSCSP by a modification of the explants technique 48 . All the procedures were approved by the Reviewer Institutional Committee on Human Research of the HSCSP and conform to the Declaration of Helsinki. Written informed consent was obtained from each patient. VSMC were cultured as described 14 . HSMM (Lonza) were cultured following the supplier's recommendations. For HSMM differentiation, cells were cultured during five days in DMEM/F-12 supplemented with 2% horse serum, 2.5 mM L-glutamine, 15 mM HEPES, and 1 mM sodium pyruvate. The HEK293T and HeLa cell lines were cultured in DMEM supplemented with 10% FCS, 2 mM L-glutamine and antibiotics.

Generation of lentiviral particles and transduction.
The human NOR-1 cDNA was obtained from a pBlueScript-NOR1 construct, kindly provided by Dr. N. Ohkura (National Cancer Center Research Institute, Tokyo, Japan) 20 , and the mouse NOR-1 cDNA linked to a FLAG sequence was obtained from the plasmid pCMV5/NOR-1-FLAG, kindly provided by Dr. J. Hastie (University of Dundee, Scotland) 49 . The pLVX/NOR-1, pLVX/NOR-1-FLAG and pLVX/EGFP were obtained as described 10 . These constructs and the empty pLVX-Puro vector (pLVX) were transfected in HEK293T cells according to Lenti-X TM Lentiviral Expression System Kit (Clontech). After 48 h, supernatants containing viral particles were harvested and titrated. Lentiviral transductions of VSMC were performed for 48 h at a multiplicity of infection of 15 in presence of polybrene (8 μ g/mL), and transduced cell populations were enriched by puromycin selection (1.2 μ g/mL). pLVX and pLVX/EGFP vectors were used as controls obtaining similar results.

Analysis of cell proliferation and hypertrophy.
The proliferation rate of lentiviral transduced and non-transduced VSMC was determined by cell counting. Cells were harvested by trypsinization, resuspended in growth medium and cell number was determined by using a Neubauer chamber. Data are expressed as the ratio of cell number/initial cell number. To determine total cell protein, trypsinized cells were washed with PBS, resuspended in lysis buffer (10 mM Tris/HCl pH 7.4, 1 mM Na 3 VO 4 , 0.5% SDS) and protein concentration was determined by the BCA Protein Assay ™ (Pierce). The ratio of total protein/cell number was referred to that of non-transduced cells. Cell surface area was determined in subconfluent cell monolayers. Images were taken from five random fields and cell surface area was measured by using ImageJ 1.49 software (National Institute Health, USA). Data (arbitrary units) are expressed relative to those of non-transduced cells.
Transfections with small interfering RNA (siRNA). Silencer Select Pre-designed siRNA (Ambion) were used for NOR-1 (s15541) and SMPX (s22597) knockdown. The Silencer Select Negative Control #1 was used as a control. Briefly, one day before transfection, HSMM were seeded in complete growth medium. Then, the medium was replaced with antibiotic-free medium and cells were transfected for 8 hours with 20 nM siRNA, using 7.5 μ L of Lipofectamine RNAiMAX Reagent (Invitrogen). After transfection, the medium was replaced with fresh antibiotic-free medium and cells were incubated for 48 h. Transfections of differentiated HSMM were performed in the same conditions, but cells were previously cultured in differentiation medium during five days and, after transfection, medium was replaced by fresh differentiation medium.
Gene expression: real-time PCR. Total RNA was extracted from cell cultures using TRIsure TM (Bioline) according to the manufacturers' protocols. Total RNA (1 μ g) was reverse-transcribed with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Levels of mRNA were assessed by real-time PCR using TaqMan TM gene expression assays-on-demand (Applied Biosystems). TATA-binding protein (TBP) were used as endogenous controls 19 .