Adrenocortical carcinomas are rare tumors with poor prognosis and limited treatment options. Although widely used as in vitro models to test novel therapeutic strategies, the adrenocortical carcinoma-derived cell lines NCI-H295R and SW-13 have only partially been described genetically. Our aim was to characterize the mutational landscape of these cells to improve their experimental utility and map them to clinical subtypes of adrenocortical carcinoma. Genomic DNA from NCI-H295R and SW-13 cells was subjected to whole-exome sequencing. Variants were filtered for non-synonymous mutations and curated for validated adrenocortical and pan-cancer driver gene mutations. Genes mutated in the cell lines were mapped using gene ontology and protein pathway tools to determine signaling effects and compared to mutational and clinical characteristics of 92 adrenocortical carcinoma cases from The Cancer Genome Atlas. NCI-H295R and SW-13 cells carried 1325 and 1836 non-synonymous variants, respectively. Of these, 61 and 76 were known cancer driver genes, of which 32 were shared between cell lines. Variant interaction analyses demonstrated dominant TP53 dysregulation in both cell lines complemented by distinct WNT (NCI-H295R) and chromatin remodeling (SW-13) pathway perturbations. Both cell lines genetically resemble more aggressive adrenocortical carcinomas with worse prognosis, for which development of targeted therapies is most critical. Careful incorporation of the genetic landscapes outlined in this study will further the in vitro utility of these cell lines in testing for novel therapeutic approaches for adrenocortical malignancy.
Norman G Nicolson, Reju Korah and Tobias Carling
Kanchan Gupta, Vijay Kumar Sirohi, Suparna Kumari, Vinay Shukla, Murli Manohar, Pooja Popli and Anila Dwivedi
Our earlier studies have demonstrated the cyclic variation and also the altered expression of sorcin in endometrium during early-to-mid-secretory phase transition in women with unexplained infertility. The current study was undertaken to establish the functional role of sorcin in endometrial receptivity in mice. Results indicated that sorcin was highly expressed during the window of implantation in mice and functional blockage of sorcin caused significant reduction in number of implanted blastocyst. The receptivity markers (i.e.Integrin β3, HBEGF, IGFBP1, WNT4 and Cyclin E)) were found to be downregulated in sorcin knocked down uterine horn on day 5 as compared to untreated horn. The reduced attachment and expansion of BeWo spheroids on RL95-2 endometrial cells with sorcin knock down, in in vitro model of endometrium–trophoblast interaction further supported these findings. Uterine sorcin expression pattern during estrous cycle and in delayed implantation mice model suggested the upregulation of sorcin by estrogen. The functional blockade of sorcin induced the intracellular Ca+2 levels in endometrial epithelial cells (EECs), which indicated that altered Ca+2 homeostasis might be responsible for implantation failure. Sorcin silencing led to significant reduction in the expression of angiogenic factor VEGF and its downstream effector molecules i.e. PI3K, Akt and NOS. The migratory and invasive properties of HUVECs were abrogated by anti-VEGF or by adding culture media from sorcin blocked EECs, which indicated that sorcin might mediate angiogenesis during implantation. Taken together, sorcin is involved in the regulation of Ca+2-mediated angiogenesis via VEGF/PI3K/Akt pathway in endometrial cells and plays a crucial role in preparing the endometrium for implantation.
I J Bujalska, M Quinkler, J W Tomlinson, C T Montague, D M Smith and P M Stewart
Obesity is associated with increased morbidity and mortality from cardiovascular disease, diabetes and cancer. Although obesity is a multi-factorial heterogeneous condition, fat accumulation in visceral depots is most highly associated with these risks. Pathological glucocorticoid excess (i.e. in Cushing’s syndrome) is a recognised, reversible cause of visceral fat accumulation. The aim of this study was to identify depot-specific glucocorticoid-target genes in adipocyte precursor cells (preadipocytes) using Affymetrix microarray technique. Confluent preadipocytes from subcutaneous (SC) and omental (OM) adipose tissue collected from five female patients were treated for 24 h with 100 nM cortisol (F), RNA was pooled and hybridised to the Affymetrix U133 microarray set. We identified 72 upregulated and 30 downregulated genes by F in SC cells. In OM preadipocytes, 56 genes were increased and 19 were decreased. Among the most interesting were transcription factors, markers of adipocyte differentiation and glucose metabolism, cell adhesion and growth arrest protein factors involved in G-coupled and Wnt signalling. The Affymetrix data have been confirmed by quantitative real-time PCR for ten specific genes, including HSD11B1, GR, C/EBPα, C/EBPβ, IL-6, FABP4, APOD, IRS2, AGTR1 and GHR. One of the most upregulated genes in OM but not in SC cells was HSD11B1. The GR was similarly expressed and not regulated by glucocorticoids in SC and OM human preadipocytes. C/EBPα was expressed in SC preadipocytes and upregulated by F, but was below the detection level in OM cells. C/EBPβ was highly expressed both in SC and in OM preadipocytes, but was not regulated by F. Our results provide insight into the genes involved in the regulation of adipocyte differentiation by cortisol, highlighting the depot specifically in human adipose tissue.
Elisabeth Sambroni, Antoine D Rolland, Jean-Jacques Lareyre and Florence Le Gac
The general rules established from mammalian species for the regulation of spermatogenesis by gonadotropins may not be fully relevant in fish. Particularly, Fsh is as potent as Lh to stimulate steroidogenesis and the Fsh receptor is expressed in Leydig cells. In seasonal breeders, Fsh is likely the major gonadotropin involved in spermatogenesis onset and Lh is required to support spermatogenesis progression and gamete release. However, the genes that relay the action of Fsh and Lh have been poorly investigated in fish. The present study was aimed at identifying gonadotropin-dependent genes expressed in the testis during fish puberty. We cultured pubertal trout testicular explants for 96 h, with or without gonadotropin, and analyzed transcriptome variations using microarrays. Fsh and Lh had similar effects on a large group of genes while other genes were preferentially regulated by one or the other gonadotropin. We showed that most of the responsive genes were expressed in somatic cells and exhibited relevant patterns during the seasonal reproductive cycle. Some genes preferentially modulated by Lh could be involved in testicular cell fate (pvrl1 and bty) or sperm maturation (ehmt2 and racgap1) and will deserve further examination. Besides Fsh's effects on the steroidogenic pathway, our study demonstrates that Fsh coordinates relevant stimulatory and inhibitory paracrine factors known to regulate early germ cell proliferation and differentiation. Some of these genes belong to major regulatory pathways including the Igf pathway (igf1b/igf3 and igfbp6), the Tgfb pathway (amh, inha, inhba, and fstl3), the Wnt pathway (wisp1), and pleiotrophin (mdka).
Lekha Jain, Tayaza Fadason, William Schierding, Mark H Vickers, Justin M O’Sullivan and Jo K Perry
Growth hormone (GH) is a peptide hormone predominantly produced by the anterior pituitary and is essential for normal growth and metabolism. The GH locus contains five evolutionarily related genes under the control of an upstream locus control region that coordinates tissue-specific expression of these genes. Compromised GH signalling and genetic variation in these genes has been implicated in various disorders including cancer. We hypothesised that regulatory regions within the GH locus coordinate expression of a gene network that extends the impact of the GH locus control region. We used the CoDeS3D algorithm to analyse 529 common single nucleotide polymorphisms (SNPs) across the GH locus. This algorithm identifies colocalised Hi-C and eQTL associations to determine which SNPs are associated with a change in gene expression at loci that physically interact within the nucleus. One hundred and eighty-one common SNPs were identified that interacted with 292 eGenes across 48 different tissues. One hundred and forty-five eGenes were regulated in trans. eGenes were found to be enriched in GH/GHR-related cellular signalling pathways including MAPK, PI3K-AKT-mTOR, ERBB and insulin signalling, suggesting that these pathways may be co-regulated with GH signalling. Enrichment was also observed in the Wnt and Hippo signalling pathways and in pathways associated with hepatocellular, colorectal, breast and non-small cell lung carcinoma. Thirty-three eQTL SNPs identified in our study were found to be of regulatory importance in a genome-wide Survey of Regulatory Elements reporter screen. Our data suggest that the GH locus functions as a complex regulatory region that coordinates expression of numerous genes in cis and trans, many of which may be involved in modulating GH function in normal and disease states.
LJ Williams and AB Abou-Samra
The parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor regulates extracellular calcium concentrations and is therefore important for mineral homeostasis. ROS 17/2.8 cells, a rat osteoblast-like osteosarcoma cell line, express the PTH/PTHrP receptor and provide a good model for examining the transcriptional regulation of its gene. The rat PTH/PTHrP receptor gene has two promoters, U1 and U3, which were shown to be important for its expression. Using extracts from ROS 17/2.8 cells, we have demonstrated two regions (termed FP1 and FP2) of nuclear protein/DNA interaction within promoter sequences previously shown to be important for the activity of the U3 promoter. Nuclear extracts from rat 2 fibroblasts, which do not express the PTH/PTHrP receptor, produced one site of protein/DNA interaction which was found at a position on the promoter identical to the position of FP1 produced by a ROS 17/2.8 nuclear extract. Mutation of these two sites of protein/DNA interaction resulted in reduced U3 promoter activity. Furthermore, we have demonstrated that the transcription factors SP1 and MAZ regulate U3 promoter expression and have shown their functional significance using mutational analysis. These data demonstrate that SP1 and MAZ bind to the PTH/PTHrP receptor promoter and that they are involved in cell-specific expression of its gene product.
Dongxing Zhu, Neil C W Mackenzie, Jose Luis Millan, Colin Farquharson and Vicky E MacRae
The process of vascular calcification shares many similarities with that of skeletal mineralisation and involves the deposition of hydroxyapatite crystals in arteries and cardiac valves. However, the cellular mechanisms responsible have yet to be fully elucidated. In this study, we employed microarray analysis to demonstrate the upregulation of more than >9000 genes during the calcification of murine vascular smooth muscle cells (VSMCs), of which the most significantly, differentially expressed gene was Igf2. Following the validation of increased IGF2 expression by RT-qPCR and immunoblotting in calcifying murine VSMCs, IGF2 expression was further demonstrated in the calcified aorta of the Enpp1 −/− mouse model of medial aortic calcification. Having confirmed that IGF1R and IGF2R were expressed in cultured murine VSMCs, cell-signalling studies in these cells revealed that IGF2 (50 ng/ml) significantly stimulated the phosphorylation of Akt and Erk1/2 (P<0.05). These results potentially indicate that IGF2 may mediate VSMC calcification via the stimulation of Erk1/2 and Akt signalling. This study suggests that the increased IGF2 expression in calcifying VSMCs may reflect the well-established prenatal role of IGF2, particularly as the osteogenic phenotypic transition of VSMCs in a calcified environment recapitulates many of the events occurring during embryonic development. A full understanding of the importance of IGF2 in this pathological process will lead to a better understanding of the aetiology of vascular calcification.
Ana P Irazoqui, Ricardo L Boland and Claudia G Buitrago
Previously, we have reported that 1,25(OH)2-vitamin D3 (1,25D) activates p38 MAPK (p38) in a vitamin D receptor (VDR)-dependent manner in proliferative C2C12 myoblast cells. It was also demonstrated that 1,25D promotes muscle cell proliferation and differentiation. However, we did not study these hormone actions in depth. In this study we have investigated whether the VDR and p38 participate in the signaling mechanism triggered by 1,25D. In C2C12 cells, the VDR was knocked down by a shRNA, and p38 was specifically inhibited using SB-203580. Results from cell cycle studies indicated that hormone stimulation prompts a peak of S-phase followed by an arrest in the G0/G1-phase, events which were dependent on VDR and p38. Moreover, 1,25D increases the expression of cyclin D3 and the cyclin-dependent kinase inhibitors, p21Waf1/Cip1 and p27Kip1, while cyclin D1 protein levels did not change during G0/G1 arrest. In all these events, p38 and VDR were required. At the same time, a 1,25D-dependent acute increase in myogenin expression was observed, indicating that the G0/G1 arrest of cells is a pro-differentiative event. Immunocytochemical assays revealed co-localization of VDR and cyclin D3, promoted by 1,25D in a p38-dependent manner. When cyclin D3 expression was silenced, VDR and myogenin levels were downregulated, indicating that cyclin D3 was required for 1,25D-induced VDR expression and the concomitant entrance into the differentiation process. In conclusion, the VDR and p38 are involved in control of the cellular cycle by 1,25D in skeletal muscle cells, providing key information on the mechanisms underlying hormone regulation of myogenesis.
Caroline M Gorvin
Twenty-five years have elapsed since the calcium-sensing receptor (CaSR) was first identified in bovine parathyroid and the receptor is now recognized as a fundamental contributor to extracellular Ca2+ (Ca2+ e) homeostasis, regulating parathyroid hormone release and urinary calcium excretion. The CaSR is a class C G-protein-coupled receptor (GPCR) that is functionally active as a homodimer and couples to multiple G-protein subtypes to activate intracellular signalling pathways. The importance of the CaSR in the regulation of Ca2+ e has been highlighted by the identification of >400 different germline loss- and gain-of-function CaSR mutations that give rise to disorders of Ca2+ e homeostasis. CaSR-inactivating mutations cause neonatal severe hyperparathyroidism, characterised by marked hypercalcaemia, skeletal demineralisation and failure to thrive in early infancy; and familial hypocalciuric hypercalcaemia, an often asymptomatic disorder associated with mild-moderately elevated serum calcium concentrations. Activating mutations are associated with autosomal dominant hypocalcaemia, which is occasionally associated with a Bartter’s-like phenotype. Recent elucidation of the CaSR extracellular domain structure enabled the locations of CaSR mutations to be mapped and has revealed clustering in locations important for structural integrity, receptor dimerisation and ligand binding. Moreover, the study of disease-causing mutations has demonstrated that CaSR signals in a biased manner and have revealed specific residues important for receptor activation. This review presents the current understanding of the genetic landscape of CaSR mutations by summarising findings from clinical and functional studies of disease-associated mutations. It concludes with reflections on how recently uncovered signalling pathways may expand the understanding of calcium homeostasis disorders.
Lena Espelage, Hadi Al-Hasani and Alexandra Chadt
The two closely related RabGAPs TBC1D1 and TBC1D4 are key signaling factors of skeletal muscle substrate utilization. In mice, deficiency in both RabGAPs leads to reduced skeletal muscle glucose transport in response to insulin and lower GLUT4 abundance. Conversely, Tbc1d1 and Tbc1d4 deficiency results in enhanced lipid use as fuel in skeletal muscle, through yet unknown mechanisms. In humans, variants in TBC1D1 and TBC1D4 are linked to obesity, insulin resistance and type 2 diabetes. While the specific function in metabolism of each of the two RabGAPs remains to be determined, TBC1D1 emerges to be controlling exercise endurance and physical capacity, whereas TBC1D4 may rather be responsible for maintaining muscle insulin sensitivity, muscle contraction, and exercise. There is growing evidence that TBC1D1 also plays an important role in skeletal muscle development, since it has been found to be associated to meat production traits in several livestock species. In addition, TBC1D1 protein abundance in skeletal muscle is regulated by both, insulin receptor and insulin-like growth factor-1 (IGF-1) receptor signaling. This review focuses on the specific roles of the two key signaling factors TBC1D1 and TBC1D4 in skeletal muscle metabolism, development and exercise physiology.