Similar to Estrogen, Relaxin-Induced Vascular Endothelial Growth Factor (VEGF) Gene Expression in the Rat Uterus Involves Activation of the PI3K/AKT Pathway, Estrogen Receptor α (ERα), and Hypoxia-Inducible Factor 1α (HIF-1α).

Abstract

Uterine edema is a well-known, rapid estrogen (17β-estradiol, E2)-induced physiological effect that occurs as a result of increased expression of vascular endothelial growth factor (VEGF), a potent inducer of increased microvascular permeability. It is essential for both normal uterine growth, including epithelial cell proliferation, and successful implantation. Previous work by this laboratory has shown that the peptide hormone relaxin (Rlx), like E2, also induces edema in the rat uterus and that this effect is also estrogen receptor (ER)-dependent. More recent work has further explored the signaling mechanism(s) involved in E2s uterotrophic effects. It has shown that E2-induced VEGF gene expression and uterine edema involve stimulation of the PI3K/Akt pathway, most likely via membrane ERs, and subsequent recruitment of both nuclear ERα and transcription factor hypoxia-inducible factor 1 (HIF-1) to the VEGF gene promoter. We hypothesized, therefore, that Rlx exerts its effects on the uterus via a similar mechanism. To test this hypothesis, immature female rats (21 days old) were treated with either vehicle (DMSO), the ER antagonist ICI 182,780 (to confirm the role of ERα), or the PI3K inhibitor wortmannin (Wort), to determine the importance of the PI3K/Akt pathway, 1 to 1.5 h prior to treatment with either vehicle (PBS) or Rlx (0.85 μg/g BW). Uteri were collected 1 and 4 h later and processed for RT-PCR, western blot, and chromatin immunoprecipitation (ChIP) analysis. Like E2, Rlx significantly increased 1) uterine wet weight (65% at 4 h), an indicator of edema; 2) VEGF mRNA expression (2.4-fold by 4 h); 3) activation of the PI3K/Akt (>3-fold) and MAPK (>2-fold) pathways at both 1 and 4 h, as indicated by western blot analysis of phospho-AktSer473 and phospho-ERK1/2Thr202/Tyr204; 4) HIF-1α protein expression by 4 h ( >1.5-fold); and 5) binding of both ERα and HIF-1α to the rat VEGF promoter at 1 h, as indicated by ChIP. Pre-treatment with ICI blocked these effects of Rlx, confirming that they were ER-dependent. Wort, which has been previously shown to block E2s rapid uterotrophic effects, also inhibited Rlx-induced activation of the PI3K/Akt pathway, VEGF mRNA expression, and uterine edema, indicating the involvement of the PI3K/Akt pathway. Rlxs effects on the rat uterus were not identical, however, to those of E2. First, while E2 has been shown to increase VEGF mRNA expression by 1 h, Rlxs effect on VEGF expression was somewhat slower, not reaching a significant level until 4 h after treatment. Furthermore, while the recruitment of ERα and HIF-1α were maximal by 1 h, binding of both had returned to baseline levels by 4 h, when VEGF mRNA levels were maximal. It is possible that Rlx also affects the half-life of VEGF mRNA. Overall, these results suggest that 1) Rlx elicits its effects on VEGF expression and edema in the uterus via a signal transduction pathway that is very similar, but not identical, to that of E2; and 2) that the PI3K/Akt pathway and HIF-1 play essential roles in the induction of uterine edema whether in response to E2 or Rlx. [This work was supported by NICHD SCCPRR U54 grant HD36207, R21 ES013061, and R01 HD047275].