Experimental protocols on animals were approved by the Institutional Animal Care and Use Committee of Texas Southwestern Medical Center

Experimental protocols on animals were approved by the Institutional Animal Care and Use Committee of Texas Southwestern Medical Center. Supplementary Material Supplementary FileClick here to view.(636K, pdf) Acknowledgments We acknowledge the helpful assistance of Parkland Hospital for tissue acquisition. early (1 h) and late (24 h) time points. Interestingly, PGE2 controlled its own metabolism by down-regulating the major PGE2 catabolic enzyme 15-PGDH and up-regulating expression of two genes involved in PGE2 synthesis (and and and (Fig. 1and and repressing in a feed-forward regulatory mechanism. Three different EP2-selective antagonists (PF-04418948, TG4-155, and TG8-4) blocked PGE2-mediated gene repression (Fig. 1and 0.05 compared with DMSO treatment. RFE (AU), relative fold expression (arbitrary units). Pathway analysis of the RNA-seq data indicated that the most significantly affected pathway by PGE2 was Ca2+ signaling (Fig. 2and repression was confirmed using a cell-permeable intracellular Ca2+ chelator BAPTA-AM (Fig. 2repression in growth medium containing Ca2+. In Ca2+-free medium, however, BAPTA-AM blocked PGE2-mediated repression (Fig. 2is transduced through EP2 receptors and is Ca2+-dependent in hCSCs. Open in a separate window Fig. 2. PGE2-mediated gene regulation is Ca2+-dependent. (and 0.01 compared with DMSO, ANOVA followed by Tukeys post hoc testing. RFE (AU), relative fold expression (arbitrary units). PGE2 Down-Regulates Gene Expression in Vitro by Increasing HDAC4. Because intracellular Ca2+ results in activation of HDACs (13) and HDAC inhibitors (HDACis) have been shown to induce gene expression in several mammalian cell types (14C16), we hypothesized that PGE2-mediated Ca2+ signaling activates HDACs, which in turn regulate promoter decreased fivefold in response to PGE2 treatment (Fig. 3and gene repression. Open in a separate window Fig. 3. PGE2 results in deacetylation of chromatin associated with the 15-PGDH gene promoter. ( 0.05 compared with DMSO, Students test. = 3. ( 0.05 compared with DMSO, Students test. = 3. (after treatment with HDACi. * 0.05 compared with DMSO (= 3. ( 0.05 compared with corresponding DMSO control. To identify the specific HDAC involved in PGE2-mediated regulation of in hCSCs, we XL184 free base (Cabozantinib) first studied PGE2-mediated changes in expression of various genes using our RNA-seq dataset. Among class I and II HDACs, and mRNA decreased in response to PGE2 (Fig. 3mRNA increased fourfold (Fig. 3in hCSCs (Fig. 4and gene expression (expression (Fig. 4and did not affect gene expression in hCSCs (gene expression (in hCSCs in media Ca2+, pretreated with DMSO or BAPTA-AM (1 M) for 1 h, then with DMSO or PGE2 (25 nM) for 15 h. (and expression levels after siRNA-mediated knockdown with control negative siRNA or siHDAC4 for 48 h followed by treatment with DMSO or PGE2 (100 nM) for 24 h. (and 0.01, ANOVA followed by Dunnetts test using vehicle or DMSO/si-Neg as control. Experiment was repeated in three cell preps with identical results. To determine if Ca2+ is involved in PGE2-mediated gene expression, experiments were conducted as described in Fig. 2gene expression in all conditions (Fig. 4gene expression via EP2 receptors, and and genes are inversely regulated by PGE2 in a Ca2+-dependent manner. Interestingly, PGE2 treatment did not change gene expression in cell types with different EP receptor profiles [MCF7 (17) and MEL5 (18); increased, whereas adenovirus-mediated overexpression decreased basal levels of 15-PGDH mRNA, suggesting that is an HDAC4 target gene (Fig. 4and abrogated PGE2-mediated down-regulation of gene expression (Fig. 4expression levels (Fig. 4knockdown effects on PGE2-mediated down-regulation of (Fig. 4gene expression (Fig. 4did not alter gene expression in these cells, indicating that the effects of LMK-235 are mediated through inhibition of HDAC4, not HDAC5 (together with LMK-235 treatment led to cumulative loss of HDAC4 mRNA and synergistic activation of gene expression (Fig. 4 and in hCSCs. PGE2 Mediates HDAC4 Nuclear Import in Vitro. Immunofluorescence studies show weak HDAC4 immunostaining distributed in both cytoplasmic and nuclear compartments in baseline hCSCs treated with vehicle (Fig. 5 and and and and and and 0.05 compared with DMSO, Students test. (and 0.05 compared with DMSO, Students test. = 3. (and 0.05 compared with DMSO, Students test. = 3. (gene repression. Phosphorylation of class II HDACs by serine/threonine kinases.ANOVA followed by Tukeys multiple comparisons test. pregnancy in vivo and thus can be targeted by novel pharmacologic agents to induce cervical ripening and labor. Results PGE2 Regulates the Transcriptome of hCSCs in Vitro Through EP2-Mediated Increases in Intracellular Ca2+. RNA-sequencing (RNA-seq) data analysis and validation experiments identified PGE2-mediated changes in the transcriptome of CSCs at both early (1 h) and late (24 h) time points. Interestingly, PGE2 controlled its own metabolism by down-regulating the major PGE2 catabolic enzyme 15-PGDH and up-regulating expression of two genes involved in PGE2 synthesis (and and and (Fig. 1and and repressing in a feed-forward regulatory mechanism. Three different EP2-selective antagonists (PF-04418948, TG4-155, and TG8-4) blocked PGE2-mediated gene repression (Fig. 1and 0.05 compared with DMSO treatment. RFE (AU), relative fold expression (arbitrary units). Pathway analysis of the RNA-seq data indicated that the most significantly affected pathway by PGE2 was Ca2+ signaling (Fig. 2and repression was confirmed using a cell-permeable intracellular Ca2+ chelator BAPTA-AM (Fig. 2repression in growth medium containing Ca2+. In Ca2+-free medium, however, BAPTA-AM blocked PGE2-mediated repression (Fig. 2is transduced through EP2 receptors and is Ca2+-dependent in hCSCs. Open in a separate window Fig. 2. PGE2-mediated gene regulation is Ca2+-dependent. (and 0.01 compared with DMSO, ANOVA followed by Tukeys post hoc testing. RFE (AU), relative fold expression (arbitrary units). PGE2 Down-Regulates Gene Expression in Vitro by Increasing HDAC4. Because intracellular Ca2+ results in activation of HDACs (13) and HDAC inhibitors (HDACis) have been shown to induce gene expression in several mammalian cell types (14C16), we hypothesized that PGE2-mediated Ca2+ signaling activates HDACs, which in turn regulate promoter decreased fivefold in response to PGE2 treatment (Fig. 3and gene Rabbit Polyclonal to KR2_VZVD repression. Open in a separate window Fig. 3. PGE2 results in deacetylation of chromatin associated with the 15-PGDH gene promoter. ( 0.05 compared with DMSO, Students test. = 3. ( 0.05 compared with DMSO, Students test. = 3. (after treatment with HDACi. * 0.05 compared with DMSO (= 3. ( 0.05 compared with corresponding DMSO control. To identify the XL184 free base (Cabozantinib) specific HDAC involved in PGE2-mediated regulation of in hCSCs, we first studied PGE2-mediated changes in expression of various genes using our RNA-seq dataset. Among class I and II HDACs, and mRNA decreased in response to PGE2 (Fig. 3mRNA increased fourfold (Fig. 3in hCSCs (Fig. 4and gene expression (expression (Fig. 4and did not affect gene expression in hCSCs (gene expression (in hCSCs in media Ca2+, pretreated with DMSO or BAPTA-AM (1 M) for 1 h, then with DMSO or PGE2 (25 nM) for 15 h. (and expression levels after siRNA-mediated knockdown with control negative siRNA or siHDAC4 for 48 h followed by XL184 free base (Cabozantinib) treatment with DMSO or PGE2 (100 nM) for 24 h. (and 0.01, ANOVA followed by Dunnetts test using vehicle or DMSO/si-Neg as control. Experiment was repeated in three cell preps with identical results. To determine if Ca2+ is involved in PGE2-mediated gene expression, experiments were conducted as described in Fig. 2gene XL184 free base (Cabozantinib) expression in all conditions (Fig. 4gene expression via EP2 receptors, and and genes are inversely regulated by PGE2 in a Ca2+-dependent manner. Interestingly, PGE2 treatment did not change gene expression in cell types with different EP receptor profiles [MCF7 (17) and MEL5 (18); increased, whereas adenovirus-mediated overexpression decreased basal levels of 15-PGDH mRNA, suggesting that is an HDAC4 target gene (Fig. 4and abrogated PGE2-mediated down-regulation of gene expression (Fig. 4expression levels (Fig. 4knockdown effects on PGE2-mediated down-regulation of (Fig. 4gene expression (Fig. 4did not alter gene expression in these cells, indicating that the effects of LMK-235 are mediated through inhibition of HDAC4, not HDAC5 (together with LMK-235 treatment led to cumulative loss of HDAC4 mRNA and synergistic activation of gene expression (Fig. 4 and in hCSCs. PGE2 Mediates HDAC4 Nuclear Import in Vitro..