Transforming Growth Factor-beta 1 Involved in the Pathogenesis of Endometriosis through Regulating Expression of Vascular Endothelial Growth Factor under Hypoxia

Abstract

Background: Endometriosis (EMs) is a common gynecological disorder characterized by endometrial-like tissue outside the uterus. Hypoxia induces the expression of many important downstream genes to regulate the implantation, survival, and maintenance of ectopic endometriotic lesions. Transforming growth factor-beta 1 (TGF-β1) plays a major role in the etiology of EMs. We aimed to determine whether TGF-β1 affects EMs development and progression and its related mechanisms in hypoxic conditions.

Methods: Endometrial tissue was obtained from women with or without EMs undergoing surgery from October, 2015 to October, 2016. Endometrial cells were cultured and then exposed to hypoxia and TGF-β1 or TGF-β1 inhibitors. The messenger RNA (mRNA) and protein expression levels of TGF-β1, vascular endothelial growth factor (VEGF), and hypoxia-inducible factor-1α (HIF-1α) were measured. A Dual-Luciferase Reporter Assay was used to examine the effect of TGF-β1 and hypoxia on a VEGF promoter construct. Student's t-test was performed for comparison among groups (one-sided or two-sided) and a value of P < 0.05 was considered statistically significant.

Results: TGF-β1, VEGF, HIF-1α mRNA, and protein expression were significantly higher in EMs tissue than that in normal endometrial tissue (t = 2.16, P = 0.042). EMs primary cultured cells exposed to hypoxia expressed 43.8% higher VEGF mRNA and protein (t = 6.84, P = 0.023). VEGF mRNA levels increased 12.5% in response to TGF-β, whereas the combined treatment of hypoxia/TGF-β1 resulted in a much higher production (87.5% increases) of VEGF. The luciferase activity of the VEGF promoter construct was increased in the presence of either TGF-β1 (2.6-fold, t = 6.08, P = 0.032) or hypoxia (11.2-fold, t = 32.70, P < 0.001), whereas the simultaneous presence of both stimuli resulted in a significant cooperative effect (18.5-fold, t = 33.50, P < 0.001).

Conclusions: The data support the hypothesis that TGF-β1 is involved in the pathogenesis of EMs through regulating VEGF expression. An additive effect of TGF-β1 and hypoxia is taking place at the transcriptional level.

Figure 1

Expression of transforming growth factor-beta 1 (TGF-β1), vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) in endometrial tissues (n = 40). (a) Relative messenger RNA expression of the three genes; (b) Relative protein density of the three proteins. (c) Western blotting of the three proteins *P < 0.05. EMs: Endometriosis.

Figure 2

Expression of TGF-β1, VEGF and HIF-1α in primary culture of endometrial cells with different stimuli (n = 40). (a) Relative mRNA expression of the three genes. The VEGF mRNA levels increased in hypoxia and response to TGF-β1 compared with the untreated cells under normoxic conditions. There was a much higher production of VEGF in the combined treatment group with hypoxia and TGF-β1; (b) Relative protein density of the three proteins. The VEGF expression levels increased in hypoxia and response to TGF-β1 compared with the untreated cells under normoxic conditions. There was a much higher expression of VEGF in the combined treatment group with hypoxia and TGF-β1. *P < 0.05; ^†P < 0.01. HIF-1α: Hypoxia-inducible factor-1α VEGF: Vascular endothelial growth factor; TGF-β1: Transforming growth factor-beta 1; mRNA: Messenger RNA.

Figure 3

Relative luciferase activities of the VEGF promoter construct with different stimuli. Induction of VEGF promoter activity was shown in either TGF-β1 or hypoxia, whereas both stimuli resulted in a significant cooperative effect (n = 40). In the presence of the TGF-β1 signal pathway and circumstance of hypoxia, VEGF promoter activity was increased. These results suggest that the collaboration between TGF-β1 and hypoxia occurs at the transcriptional level. *P < 0.05; ^†P < 0.01; ^‡P < 0.001. VEGF: Vascular endothelial growth factor; TGF-β1: Transforming growth factor-beta 1.