Differential gene expression by 1,25(OH)2D3 in an endometriosis stromal cell line

https://doi.org/10.1016/j.jsbmb.2017.01.011Get rights and content

Highlights

  • An endometriosis stromal cell line was treated with 1,25(OH)2D3.

  • 1,25(OH)2D3 strongly up-regulated CYP24A1 in endometriosis stromal cells.

  • Neuroangiogenesis, cellular motility, and invasion pathways were down-regulated.

  • Endometriosis lesions had higher CYP24A1 expression than normal endometrium.

Abstract

Endometriosis is a common female reproductive disease characterized by invasion of endometrial cells into other organs, frequently causing pelvic pain and infertility. Alterations of the vitamin D system have been linked to endometriosis incidence and severity. To shed light on the potential mechanism for these associations, we examined the effects of 1,25(OH)2D3 on gene expression in endometriosis cells. Stromal cell lines derived from endometriosis tissue were treated with 1,25(OH)2D3, and RNA-seq was used to identify genes differentially expressed between treated and untreated cells. Gene ontology and pathway analyses were carried out using Partek Flow and Ingenuity software suites, respectively. We identified 1627 genes that were differentially expressed (886 down-regulated and 741 up-regulated) by 1,25(OH)2D3. Only one gene, CYP24A1, was strongly up-regulated (369-fold). Many genes were strongly down-regulated. 1,25(OH)2D3 treatment down-regulated several genetic pathways related to neuroangiogenesis, cellular motility, and invasion, including pathways for axonal guidance, Rho GDP signaling, and matrix metalloprotease inhibition. These findings support a role for vitamin D in the pathophysiology of endometriosis, and provide new targets for investigation into possible causes and treatments.

Introduction

Endometriosis is one of the most common female health disorders, affecting 10–15% of all women of reproductive age. Frequently, the condition causes chronic pelvic pain and infertility, impacting the patient’s quality of life. It also places a considerable economic burden on society with approximately $20 billion disease-related costs spent in the United States per year [1]. Endometriosis is characterized by growth of endometrial tissue outside of the uterine cavity. Functional epithelial and stromal elements can be differentiated into peritoneal, ovarian and deep infiltrating lesions according to their location [2].

Exact causes and pathogenetic pathways of this heterogenetic disease are not fully understood. In line with the widely accepted implantation theory [3] endometrial cells enter the abdominal cavity and attach, proliferate and invade local structures to form ectopic foci. Estrogen dependency and changes of cellular characteristics, e.g. epithelial-mesenchymal transition, are a prerequisite for the establishment and maintenance of lesions. It is notable that endometriosis shares several characteristics with cancer, including excessive proliferation, invasion into other organs, peripheral metastasis, inflammation, and (for some cancers) estrogen dependence. During their lifetime, up to 30% of endometriosis patients will also suffer from ovarian cancer; the risk to develop low-grade serous or endometrioid cancers is increased approximately 2-fold, and for clear cell subtypes by 3-fold [4].

The Vitamin D system is important for numerous crucial processes in the human reproductive system. The endometrium is not only a target for 1,25 dihydroxyvitamin D3 (1,25(OH)2D3), but also constitutes a site of 1,25(OH)2D3 synthesis. Specifically, stromal endometrial cells were shown to express Vitamin D receptors and 1-alpha hydroxylase, which activates 25OHD [5]. Serum levels of 25(OH)D have been reported to be both higher [6] and lower [7], [8] in endometriosis patients than in healthy individuals. Compared to controls, endometriosis patients have been reported to have higher serum 1,25(OH)2D3 [9] and higher urinary DBP levels [10]. Also, expression of 1-alpha hydroxylase has been reported to be higher in endometriosis tissues compared to normal endometrium [11]. Further, vitamin D has been widely studied as a possible cancer preventive agent. Anti-tumor effects of 1,25(OH)2D3 and its analogues are mediated by multiple pathways in a cell type and tissue specific manner. Mechanisms include inhibition of proliferation, cell motility, invasion, metastasis, and inflammation (reviewed by Ma [12]).

Since findings on endometriosis and the vitamin D system remain inconclusive and this complex relationship needs further investigation, the objectives of this study were (1) to identify genes and genetic pathways that are regulated by vitamin D in endometriosis cells, and (2) to determine whether the vitamin D system is more active in endometrial tissue and ectopic lesions than in normal endometrium. We treated stromal endometriosis cell lines with 1,25(OH)2D3 and examined global gene expression using next-generation sequencing. We also examined expression levels of one gene that is strongly regulated by vitamin D, CYP24A1, in tissue from endometriosis lesions and normal endometrium.

Section snippets

Cell culture

The endometriosis cell line (ESC22B) used in this study was of stromal origin, and derived from peritoneal endometriosis lesions [13], [14]. Cells were cultured for 25 days in media composed of Dulbeco’s Modified Eagle’s Medium/F12 (DMEM/F12), 10% fetal bovine serum (FBS), and 5% l-glutamine with antibiotics Penicillin-Streptomycin in humidified 5% CO2 and 95% air at 37 °C. Cells were passaged twice, divided into 6 tissue culture dishes (3 treatment and 3 control), and allowed to proliferate to

Differential gene expression by 1,25(OH)2D3 in endometriosis stromal cells

There were 1627 genes that were at least two-fold differentially expressed (886 down-regulated and 741 up-regulated) by 1,25(OH)2D3. Only one gene, CYP24A1, was strongly up-regulated (369-fold). The other 740 up-regulated genes had less than 7-fold change in expression. Many genes were strongly down-regulated by 1,25(OH)2D3. Among the 886 down-regulated genes, differential expression was more than 100-fold for 20 genes, more than 50-fold for 49 genes, and more than 10-fold for 240 genes. The

Conclusions

The vitamin D system appears to be activated in both eutopic and ectopic endometriosis tissue relative to normal endometrium, as indicated by increased CYP24A1 expression. Genetic pathways that may be altered by 1,25(OH)2D3 in endometriosis tissues were identified by analysis of global gene expression. Treatment of endometriosis stromal cells with 1,25(OH)2D3 altered expression of genetic pathways involved in neuroangiogenesis, cellular motility and invasion. These results support a role for

Conflict of interest

The authors declare to have no conflict of interest.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Acknowledgements

We are grateful to Prof. Dr. A. Starzinski-Powitz (Goethe University, Frankurt/Maine) for providing the cell line used in this study, to Dr. Daniel Weisenberger for expert advice on RT-PCR, and Dr. Meng Li for bioinformatics assistance. The bioinformatics software and computing resources used in the analysis are funded by the USC Office of Research and the Norris Medical Library.

Cited by (0)

1

L. Wu and B.T. Liu contributed equally to this manuscript.

2

Present address: Institute of Occupational, Social and Environmental Medicine, Goethe University, 60329 Frankfurt/Main, Germany.

View full text