TGFβ1 attenuates expression and release of prolactin in a SMAD-dependent manner.

<p>Incubation with TGFβ1 (10 ng/ml; 72 h) significantly reduced expression of mRNAs encoding IGFBP-1 and PRL (p<0.001 IGFBP1; graph A 1.0 =  absolute value of ΔCT  = 12.2, p<0.05 PRL; graph C 1.0 =  absolute value of ΔCT  = 11) and the amount of each of these proteins recovered from culture media (p<0.001 IGFBP1, graph B, p<0.01 PRL, graph D) after incubation of decidualized ESCs for 72 h. Depletion of SMAD 4 using two independent target-specific siRNAs had no significant impact on the TGFβ1-dependent reduction in IGFBP-1 mRNA or protein (graphs A 1.0 =  absolute value of ΔCT  = 12.2, B) but reversed the reduction in PRL mRNA (p<0.001, graph C 1.0 =  absolute value of ΔCT  = 11.25) and protein (p<0.01, graph D) induced by treatment. No response was observed with cells depleted of MAPK siRNA. Data are mean ± S.E.M; * p<0.05, ** p<0.01, *** p<0.001 vs. TGFβ1 alone. n = 5 endometrial samples (in triplicate).</p

TGFβ1 inhibited expression of decidualization markers when ESC were decidualized in the presence or absence of TGFβ1 for up to 72 h.

<p>A: TGFβ1 reduced expression of mRNA IGFBP-1 in a time dependent manner, 48 h, p<0.01, 72 h, p<0.001. 1.0 =  absolute value of ΔCT  = 12. B: TGFβ1 inhibited release of IGFBP-1 protein release after only 2 h of treatment (p<0.001) and this continued to decline in a time-dependent manner (all time points p<0.001). C: TGFβ1 inhibited expression of PRL mRNA in a time dependent manner, 48 h (p<0.01), 72 h (p<0.001). 1.0 =  absolute value of ΔCT  = 11. D: TGFβ1 inhibited PRL protein release after only 2 h of treatment (p<0.05) and this was sustained for up to 72 h (12 h, p<0.01, 24 h, p<0.05, 48 h and 72 h, p<0.01). E: TGFβ1 inhibited expression of TF mRNA in a time dependent manner; 24 h (p<0.05), 48 h (p<0.001), 72 h (p<0.001). 1.0 =  absolute value of ΔCT  = 14. Data are mean ± S.E.M; * p<0.05, ** p<0.01, *** p<0.001 vs. control. n = 8 endometrial samples (in triplicate).</p

TGFβ1 suppresses expression and release of markers of decidualization by cells from 1^st trimester decidua.

<p>A: TGFβ1 reduced expression of IGFBP-1 mRNA after 72 h treatment. (p<0.05) 1.0 =  absolute value of ΔCT  = 12.2. B: TGFβ1 was without significant effect on protein release of IGFBP-1. C: TGFβ1 reduced expression of PRL mRNA after 72 h treatment (p<0.001). 1.0 =  absolute value of ΔCT  = 11.5. D: TGFβ1 was without significant effect on PRL protein release, although did display a trend toward inhibition. E: TGFβ1 reduced expression of TF mRNA after 72 h treatment (p<0.05). 1.0 =  absolute value of ΔCT  = 13.75. Data are mean ± S.E.M; * p<0.05, *** p<0.001 vs. no TGFβ1 treatment. n = 7 decidual samples (in triplicate).</p

Anti-TGFβ1 antibody neutralizes endogenous TGFβ1 and potentiates the decidualization process.

<p>Cultured ESCs were decidualized <i>in vitro</i> in the prescence and absence of TGFβ1. To confirm the specificity of the TGFβ1 response anti-TGFβ1 antibody (1 µg/ml) or mouse IgG control were added, for 72 h. A: TGFβ1 inhibited expression of decidual PRL (p<0.05), addition of anti-TGFβ1 blocked this reduction. 1.0 =  absolute value of ΔCT  = 11.5. B: TGFβ1 reduced expression of IGFBP-1 mRNA (p<0.001), whilst anti-TGFβ1 antibody increased expression IGFBP-1 (p<0.001) above that of controls. 1.0 =  absolute value of ΔCT  = 12.3. Data are mean ± S.E.M; * p<0.05, *** p<0.001. n = 6 endometrial samples (in triplicate).</p