The 17{beta}-estradiol induced upregulation of the adhesion G-protein coupled receptor (ADGRG7) is modulated by ESR{alpha} and SP1 complex [RESEARCH ARTICLE]

INTRODUCTION

Adolescent idiopathic scoliosis (AIS) is a complex three-dimensional deformity of the spine that mostly occurs during late childhood or puberty (Konieczny et al., 2013). Severe forms of AIS are more common in girls compared to boys (Cheng et al., 2015). The difference between girls and boys, as well as the etiology of AIS, are still unclear. Several studies suggest that AIS could be an endocrinal disease and that various hormones, especially estrogens, have a role in its onset, development and spinal curve progression (Barrios et al., 2011). Lower peak bone mass and osteopenia at puberty have been reported in 27%–38% of AIS patients, suggesting that AIS may be correlated with hormonal disturbances involving estrogen, melatonin and leptin (Ishida et al., 2015). Estrogens (including estradiol) (E2) and estrogen receptors (ERs), including the ERα and ERβ isoforms, are suspected of influencing AIS severity and delayed puberty which has been directly associated with a higher prevalence of AIS in girls than in boys with an incidence ratio of 7.1:1 (Konieczny et al., 2013). Indeed, several ER polymorphisms were found in AIS (Stavrou et al., 2002), but the predisposition to and severity of AIS was not clearly demonstrated (Janusz et al., 2014). E2 participation in puberty, spinal growth and bone metabolism is an important factor to consider in AIS. Until now, it was not clear how E2 could affect the initiation or progression of AIS. However, estrogens interact with many physiopathological factors (including neuroendocrine, neurological, muscular, biochemical and structural) relevant to the etiology of scoliosis, and there is interdependence between the concentration of E2 and development of scoliosis (Leboeuf et al., 2009).

Estradiol is the major hormonal regulator of puberty and bone metabolism and acts through genomic and non-genomic pathways. The genomic effects of E2 are exerted by its binding to the ER in the cytoplasm. This is followed by the translocation to the nucleus and binding of the complex to target genes. In addition to the direct genomic signaling through ER (Dahlman-Wright et al., 2006; Prossnitz et al., 2008), ERs can also mediate their transcriptional potential through tethered interaction with other transcription factors, such as specificity protein 1 (SP1) and activator protein (AP1). In these cases, most estrogen-responsive genes are devoid of estrogen response elements (ERE) (Björnström and Sjöberg, 2005), suggesting enhanced recruitment of ER ligands to target promoters through protein–protein interaction such as SP1 (Safe, 2001). SP1 is a ubiquitously expressed transcription factor that binds and acts through GC-rich elements to regulate gene expression in mammalian cells (Li et al., 2007; Keay and Thornton, 2009).

The adhesion G protein-coupled receptor 7 (ADGRG7), previously known as the G protein-coupled receptor 128 (GPR128), is a membrane-bound protein encoded by ADGRG7 (Fredriksson et al., 2002; Bjarnadóttir et al., 2004; Arac et al., 2012a). In humans and mice, the ADGRG7 gene is on chromosome 3q12.2 and 16; 16 C1.1, respectively (Fredriksson et al., 2002; Bjarnadóttir et al., 2004; Arac et al., 2012a). ADGRG7 is an orphan receptor that belongs to the family of proteins that consists of over 33 homologous proteins (Bjarnadóttir et al., 2004; Yona et al., 2008; Yona and Stacey, 2010). Like most members of ADGRG family, the extracellular region often a N-terminal protein module is extended and linked to a transmembrane (TM) 7 region via the GPCR-autoproteolysis inducing (GAIN) domain (Arac et al., 2012a). ADGRG7, which is phylogenetically related to ADGRG2 and ADGRG1, lacks the conserved N-termini domains present in other GPCRs (Foord et al., 2002; Bjarnadóttir et al., 2004; Huang et al., 2012). ADGRG7 was shown to be expressed in the mucosa of the intestine restricted to the epithelial cells (Badiali et al., 2012; Ni et al., 2014).

The physiological role of ADGRG7 remains mostly unclear. The GPCR family of proteins are mainly involved in cellular adhesion, migration, cell–cell and cell–matrix interactions (Yona et al., 2008). In mice, targeted deletions of the ADGRG7 gene reduced weight gain and increased the frequency of peristaltic contractions of the small intestine, suggesting a role in intestinal absorption of nutrients (Badiali et al., 2012). An important paralog of this gene is ADGRG6, which is suggested as playing a role in musculoskeletal disorders such as AIS and pectus excavatum (PE) (Kou et al., 2013; Karner et al., 2015).

In humans, ADGRG6 gene variants were first associated with AIS in the Japanese population and then a single nucleotide polymorphism (SNP) in ADGRG6 gene (rs657050) was replicated in Han Chinese and European-ancestry AIS population. In zebrafish, the adgrg6 knockdown causes delayed ossification of the developing spine (Kou et al., 2013) and in a mouse model, the loss of Adgrg6 in osteochondroprogenitor cells affects spinal column development and intervertebral disk morphogenesis (Karner et al., 2015).

ADGRG7 was also suggested among the genetic causes or genetic contributors for the pathogenesis of AIS. The ADGRG7 gene maps on the chromosome 3q12.1. Through linkage analysis in multigenerational AIS families with dominant inheritance this locus was reported as one of the two locations containing the gene for AIS (Edery et al., 2011). Our recent study (Patten et al., 2015) identified by exome sequencing two candidate gene variants (SNV) among the novel or rare [minor allele frequency (MAF) 5%] variants: one in ADGRG7 and the other in POC5 (Patten et al., 2015). The ADGRG7 SNV (1274AG) did not perfectly co-segregate with AIS in all the members of this multigenerational AIS family; consequently, the ADGRG7 gene was concluded as a contributory/modifier gene in the pathogenesis of AIS. Based on these findings, and because ADGRG7 is closely related to the ADGRG6 (gene implicated in AIS), we hypothesized that ADGRG7 is regulated by E2 and consequently can contribute to the cellular events in AIS.

To examine how ADGRG7 is regulated at the transcriptional and protein level by E2, we conducted promoter and deletion analysis. We also conducted gene and protein expression study in human osteoblasts, Huh7 and MCF7 cells. Human ADGRG7 gene was cloned and analyzed for functional cis-elements mediating the effects of E2. Deletion analysis of the promoter identified the SP1 site required for both basal activity and hormone-induced activation. Chromatin immunoprecipitation (ChIP) assay confirmed that SP1/ERα binds to ADGRG7 promoter. Our study suggests that the regulation of ADGRG7 expression by E2 is due to the association of ERα and SP1 proteins to ADGRG7 promoter.