Fig 1.
Expression of cellular retinoic acid binding proteins in decidualizing HESCs.
(a) CRABP2 and FABP5 transcript levels in undifferentiated or decidualized cells treated with 8-bromo-cAMP, P4, and E in combination with or without 10−8 M atRA or 10−8 M atRald for 8 days. The results show fold-change (mean ± SEM) relative to vehicle control (dotted lines). CRABP2 mRNA was measured in 8 primary HESC cultures and FABP5 mRNA in 9 cultures. (b) Representative Western blot analysis of retinoic acid binding proteins in whole cell lysates from undifferentiated or cells decidualized in the absence or presence of 10−8 M atRA or 10−8 M atRald. β-actin served as a loading control. E, cortisone; RA, retinoic acid; Rald, retinaldehyde. * indicates P < 0.05; ** P < 0.01.
Fig 2.
Expression of RA nuclear receptors in decidualizing HESCs.
(a) Transcript levels of RARα, RXRα and PPARβ/δ in undifferentiated or cells in the presence or absence of 10−8 M atRA or 10−8 M atRald for 8 days (n = 9–11). Transcripts levels were normalized to expression in vehicle-treated control cells (dotted line). (b) Representative Western blot analysis of a parallel culture. β-actin served as a loading control. * P < 0.05; ** P < 0.01.
Fig 3.
Induction of RA metabolic enzymes in decidualizing cells.
(a) Expression of DHRS3, RDH12, RBP4 and CYP26A1 transcripts in undifferentiated or cells decidualized with 8-bromo-cAMP, P4, and E in the presence or absence of 10−8 M atRA or 10−8 M atRald for 8 days (n = 9–11). The results show fold-change (mean ± SEM) in mRNA levels relative to expression levels in vehicle-treated undifferentiated HESCs (dotted lines). (b) Representative Western blot analysis of culture treated in parallel. β-actin served as a loading control. * P < 0.05; ** P < 0.01.
Fig 4.
Impaired decidualization in responses to exogenous atRA and atRald.
(a) RTQ-PCR analysis of PRL, IGFBP1 and HSD11B1 transcript levels in decidualizing HESCs treated with or without 10−8–10−11 M atRA or 10−8 M atRald for 8 days in 6 primary HESC cultures. The results show fold-change (mean ± SEM) relative to transcript levels in vehicle-treated control cells (dotted lines). (b) Western blot analysis of 11βHSD1 expression in protein lysates from primary undifferentiated cells and cultures decidualized for 4, 8 or 12 days in the presence of increasing concentrations of atRA or 10-8M at Rald. β-actin served as a loading control.
Fig 5.
Cytoxicity of atRA and atRald in decidualizing HESCs.
(a) Cell viability of primary HESCs, decidualized in the presence or absence of 10−8 M atRA or 10−8 M atRald, was analyzed by XTT assay. The data show % of viable cells (± SEM) relative to matched undifferentiated HESCs (dotted line) in 3 independent primary cultures. * P < 0.05; ** P < 0.01. (b) Parallel cultures were analysed over 60 hours using xCELLigence Real-Time Cell Analyzer (RTCA). Cell contact is monitored as an arbitrary “cell index” value, which is an indication of confluency and adherence. Different letters above the error bars indicate that those groups are significantly different from each other at P < 0.05. The results show mean ± SEM of 3 biological repeat experiments.
Fig 6.
Schematic overview of the RA pathway.
Retinol, hydrolyzed from retinyl ester, is converted to retinaldehyde (Rald) and generates retinoic acid (RA) following two-step oxidation. Intracellular RA binds to CRABP2 and activates heterodimer, RAR and RXR, leading to apoptotic machinery. By contrast, RA-dependent activation of nuclear receptor, PPAR β/δ is mediated by FABP5.