Gonadotropin releasing hormone analogue (GnRHa) alters the expression and activation of Smad in human endometrial epithelial and stromal cells

Abstract

Gonadotropin releasing hormone analogues (GnRHa) are often used to regress endometriosis implants and prevent premature luteinizing hormone surges in women undergoing controlled ovarian stimulation. In addition to GnRH central action, the expression of GnRH and receptors in the endometrium implies an autocrine/paracrine role for GnRH and an additional site of action for GnRHa. To further examine the direct action of GnRH (Leuprolide acetate) in the endometrium, we determined the effect of GnRH on endometrial stromal (ESC) and endometrial surface epithelial (HES) cells expression and activation of Smads (Smad3, -4 and -7), intracellular signals activated by transforming growth factor beta (TGF-beta), a key cytokine expressed in the endometrium. The results show that GnRH (0.1 microM) increased the expression of inhibitory Smad7 mRNA in HES with a limited effect on ESC, while moderately increasing the common Smad4 and Smad7 protein levels in these cells (P < 0.05). GnRH in a dose--(0.01 to 10 microM) and time--(5 to 30 min) dependent manner decreased the rate of Smad3 activation (phospho-Smad3, pSmad3), and altered Smad3 cellular distribution in both cell types. Pretreatment with Antide (GnRH antagonist) resulted in further suppression of Smad3 induced by GnRH, with Antide inhibition of pSmad3 in ESC. Furthermore, co-treatment of the cells with GnRH + TGF-beta, or pretreatment with TGF-beta type II receptor antisense to block TGF-beta autocrine/paracrine action, in part inhibited TGF-beta activated Smad3. In conclusion, the results indicate that GnRH acts directly on the endometrial cells altering the expression and activation of Smads, a mechanism that could lead to interruption of TGF-beta receptor signaling mediated through this pathway in the endometrium.

Figure 1

Time dependent action of GnRH (leuprolide acetate) on Smad3, Smad4 and Smad7 (arrows) mRNA expression in human endometrial surface epithelial cells (HES) and isolated endometrial stromal cells (ESC). Serum-starved cells were treated with GnRH (0.1 μM) for 2 to 12 hrs and total RNA was isolated from treated and untreated control (Ctrl) cells and subjected to semi-quantitative RT-PCR co-amplifying Smads and G3PDH (lower bands) mRNA shown from a representative experiment. The bar graph show the mean ± SEM of fold change of ratio of Smad7:G3PDH mRNA expression from three independent experiments. b, c, d, and e and c' and are significantly different from a and a', respectively (p < 0.05). M = DNA marker.

Figure 2

Time dependent action of GnRH (leuprolide acetate) on Smad3, Smad4 and Smad7 protein expression in HES and ESC. Serum-starved cells were treated with GnRH (0.1 μM) for 18, 24 and 36 hrs and cell lysates were prepared from treated and untreated control (Ctrl) and analyzed by immunoblotting for Smads and β-actin as loading control, all shown from a representative experiment. Bar graphs show the mean ± SEM of fold change in Smads expression from three independent experiments. The denote d, b' and d' (Smad3), d, b', and c' (Smad4) and b, d, b' and c' (Smad7) are significantly different from a and a', respectively (p < 0.05).

Figure 3

Time- (A) and dose- (B) dependent effects of GnRH (Leuprolide acetate) on the rate of Smad3 activation (phospho-Smad3; pSmad3). HES and ESC were incubated under serum-free condition and treated with GnRH (0.1 μM) for 5, 15 and 30 min, or with GnRH at 0.01 to 10 μM for 15 min. The cell lysates were prepared and analyzed by immunoblotting for pSmad2/3 and β-actin as loading control, shown from a representative experiment. Bar graphs show the mean ± SEM of fold change in the rate of Smad3 activation from three independent experiments. In Fig. A: denotes b, c, d, b', c', and d' and in Fig. B, c, d, e, b', c', d,' and e'are significantly different from a and a', respectively (p < 0.05).

Figure 4

Immunofluorescence localization of Smad3 in HES and ESC. The cells were incubated under serum-free condition for 24 hrs then treated with GnRH (0.1 μM) for 5, 15 and 30 min. Note subcellular localization of Smad3 in untreated control with mostly cytoplasmic and limited nuclear localization, while GnRH-treatment resulted in more cytoplasmic accumulation of Smad3. The figures are shown after 15 min of GnRH treatment with FITC staining used to localize Smad3 and DAPI staining for the nuclei.

Figure 5

The effect of GnRH (0.1 μM) and GnRH antagonist (Antide, 10 μM) on Smad3 activation in HES and ESC. Serum-starved cells were treated with Antide for 2 hrs prior to treatment with GnRH for 15 min. The cells lysates were prepared from treated and untreated control (-) and analyzed by immunoblotting for pSmad2/3 and β-actin as loading control, shown from a representative experiment. Bar graph shows the mean ± SEM of fold change in the rate of Smad3 activation from three independent experiments with denotes b, c, d, b', c' and d'are significantly different from a and a,' respectively (p < 0.05).

Figure 6

The effect of GnRH on TGF-β1-induced Smad3 activation in HES and ESC. Serum-starved HES and ESC were co-treated with TGF-β1 (2.5 ng/ml) and GnRH (0.1 μM) for 15 min and cell lysates from treated (+) and untreated (-) groups were analyzed by immunoblotting for pSmad2/3 and β-actin as loading control, shown from a representative experiment. Bar graph shows the mean ± SEM of fold change in the rate of Smad3 activation from three independent experiments with denotes b, c, b' and c' are significantly different from a and a', respectively (p < 0.05).

Figure 7

The effect of GnRH (0.1 μM) on Smad3 activation in HES and ESC pretreated with TGF-β type II receptor antisense (1 μM) or sense (1 μM) oligonucleotides for 24 hrs. The cell lysates were prepared from GnRH-treated (+) and untreated (-) cells after 5, 15 and 30 min and analyzed by immunoblotting for pSmad2/3 and β-actin as loading control, shown from a representative experiment. Bar graph shows the mean ± SEM of fold change in the rate of Smad3 activation from three independent experiments with denotes ** are significantly different from untreated (*) controls (p < 0.05).