Expression of distinct RNAs from 3′ untranslated regions

The 3′ untranslated regions (3′UTRs) of eukaryotic genes regulate mRNA stability, localization and translation. Here, we present evidence that large numbers of 3′UTRs in human, mouse and fly are also expressed separately from the associated protein-coding sequences to which they are normally linked, likely by post-transcriptional cleavage. Analysis of CAGE (capped analysis of gene expression), SAGE (serial analysis of gene expression) and cDNA libraries, as well as microarray expression profiles, demonstrate that the independent expression of 3′UTRs is a regulated and conserved genome-wide phenomenon. We characterize the expression of several 3′UTR-derived RNAs (uaRNAs) in detail in mouse embryos, showing by in situ hybridization that these transcripts are expressed in a cell- and subcellular-specific manner. Our results suggest that 3′UTR sequences can function not only in cis to regulate protein expression, but also intrinsically and independently in trans, likely as noncoding RNAs, a conclusion supported by a number of previous genetic studies. Our findings suggest novel functions for 3′UTRs, as well as caution in the use of 3′UTR sequence probes to analyze gene expression

Control of human endometrial stromal cell motility by PDGF-BB, HB-EGF and trophoblast-secreted factors

Human implantation involves extensive tissue remodeling at the fetal-maternal interface. It is becoming increasingly evident that not only trophoblast, but also decidualizing endometrial stromal cells are inherently motile and invasive, and likely contribute to the highly dynamic processes at the implantation site. The present study was undertaken to further characterize the mechanisms involved in the regulation of endometrial stromal cell motility and to identify trophoblast-derived factors that modulate migration. Among local growth factors known to be present at the time of implantation, heparin-binding epidermal growth factor-like growth factor (HB-EGF) triggered chemotaxis (directed locomotion), whereas platelet-derived growth factor (PDGF)-BB elicited both chemotaxis and chemokinesis (non-directed locomotion) of endometrial stromal cells. Supernatants of the trophoblast cell line AC-1M88 and of first trimester villous explant cultures stimulated chemotaxis but not chemokinesis. Proteome profiling for cytokines and angiogenesis factors revealed neither PDGF-BB nor HB-EGF in conditioned media from trophoblast cells or villous explants, while placental growth factor, vascular endothelial growth factor and PDGF-AA were identified as prominent secretory products. Among these, only PDGF-AA triggered endometrial stromal cell chemotaxis. Neutralization of PDGF-AA in trophoblast conditioned media, however, did not diminish chemoattractant activity, suggesting the presence of additional trophoblast-derived chemotactic factors. Pathway inhibitor studies revealed ERK1/2, PI3 kinase/Akt and p38 signaling as relevant for chemotactic motility, whereas chemokinesis depended primarily on PI3 kinase/Akt activation. Both chemotaxis and chemokinesis were stimulated upon inhibition of Rho-associated, coiled-coil containing protein kinase. The chemotactic response to trophoblast secretions was not blunted by inhibition of isolated signaling cascades, indicating activation of overlapping pathways in trophoblast-endometrial communication. In conclusion, trophoblast signals attract endometrial stromal cells, while PDGF-BB and HB-EGF, although not identified as trophoblast-derived, are local growth factors that may serve to fine-tune directed and non-directed migration at the implantation site

Signaling pathways activated by PDGF-AA, PDGF-BB, HB-EGF, TCM, or VECM.

<p>(<b>A</b>) Decidualized St-T1b were starved in Opti-MEM and then treated for 5 or 30 min with PDGF-BB, HB-EGF, TCM or PDGF-AA. Levels of phosphorylated (p-) or total ERK1/2, Akt, and p38 were determined by Western blotting. (<b>B</b>) Decidualized hESC were starved in Opti-MEM and then treated for 5 min with PDGF-BB, HB-EGF, TCM, 5 individual VECM preparations, villous explant control medium (VECM-Co), or PDGF-AA. Western blotting was performed as above.</p

Chemotactic response of hESCs to trophoblast secretory products identified by proteome profiling.

<p>(<b>A</b>) Decidualized hESCs were analyzed in transwell migration assay in response to PDGF-BB, HB-EGF, TCM, PDGF-AA, PLGF-1 or VEGF-165. Motility indices are shown as means±SD (n = 3), and were analyzed by ANOVA and Dunnett test. ***, <i>P</i><0.001 compared to the control without chemoattractant. (<b>B, C</b>) Effect of neutralization of PDGF activity. Decidualized hESCs were subjected to transwell migration assay with two different doses of PDGF-AA (<b>B</b>) or with TCM and two individual VECM preparations (<b>C</b>) in the absence or presence of a neutralizing antibody to PDGF-AA/-AB/-BB (pan). Motility indices are shown as means±SD (n = 3) and were analyzed by ANOVA and Dunnett or Tukey test. ***, <i>P</i><0.001; **, <i>P</i><0.01 in the absence vs. presence of antibody. <i>a</i>, <i>P</i><0.001; <i>b</i>, <i>P</i><0.01; <i>c</i>, <i>P</i><0.05 compared to the respective control without stimulation or antibody (white or light grey columns).</p

Proteome profiling for cytokines and angiogenesis factors in conditioned medium from AC-1M88 human trophoblast cells, two individual first trimester villous explant cultures, and the St-T1b human endometrial stromal cell line after decidualizing treatment.

<p>Proteome profiling for cytokines and angiogenesis factors in conditioned medium from AC-1M88 human trophoblast cells, two individual first trimester villous explant cultures, and the St-T1b human endometrial stromal cell line after decidualizing treatment.</p

Effect of pathway inhibitors on chemokinetic motility of St-T1b cells.

<p>(<b>A</b>) Decidualized St-T1b cells were seeded in Oris migration plates and preincubated with inhibitors for 1 h: PD98059 (50 µM), Y27632 (100 µM), NSC23766 (50 µM), SB202190 (10 µM) or Wortmannin (200 nM). Then PDGF-BB was added to the indicated final concentration, or the equivalent volume of control medium, and cells allowed to migrate into the detection zone for 18 h. Numbers of migrated cells were normalized to the control in the absence of stimulus and inhibitor (left white column). Results represent the means±SEM of n = 3 independent experiments. Data were analyzed by ANOVA and Dunnett test within each group (unstimulated or treated with PDGF-BB) and revealed significant differences after inhibitor treatment compared to the respective controls without inhibitor: *, <i>P</i><0.05; **, <i>P</i><0.01; *** <i>P</i><0.001. (<b>B</b>) Representative images of detection zones for the treatments evaluated in panel (<b>A</b>), showing cells that had migrated into the cell-free zone within 18 h. Cells were stained with Diff-Quik. Images of each detection zone were reassembled from 4 microphotographs taken with a 4× objective and graphically overlaid with the black mask.</p

Effect of pathway inhibitors on chemotactic migration of hESCs.

<p>Decidualized hESCs in transwell migration inserts were preincubated with PD98059 (50 µM), Y27632 (100 µM), NSC23766 (50 µM), SB202190 (10 µM) or Wortmannin (200 nM) before the addition of TCM to the lower reservoir. Controls received MM1-10% instead of chemoattractant. Motility indices are means±SD (n = 3) and were analyzed by ANOVA and Dunnett test. **, <i>P</i><0.01; ***, <i>P</i><0.001 compared to cells without inhibitor in the group without chemoattractant (white column). No significant differences were seen within the group receiving TCM. Exemplary images of migrated cells are shown in Figure S2.</p

Chemotactic response of St-T1b cells or primary hESCs to PDGF-BB, HB-EGF and trophoblast conditioned medium.

<p>St-T1b cells (<i>upper panel</i>) or hESC (<i>lower panel</i>), non-decidualized (ND) or decidualized (D) by 5 d treatment with 8-Br-cAMP/MPA, were subjected to transwell migration assay. The bottom reservoir contained MM1-10% (controls), PDGF-BB, HB-EGF or conditioned medium (TCM) from the trophoblast cell line AC-1M88 undiluted (100%) or diluted to 80% or 20%. The motility index designates the percentage of migrated cells relative to the total cell number (non-migrated plus migrated) at the end of the 18 h migration period. Shown are results representative of 3 similar experiments (means± SD, n = 3). Data were analyzed by ANOVA and Dunnett <i>post-hoc</i> test. *, <i>P</i><0.05; **, <i>P</i><0.01; *** <i>P</i><0.001 compared to the respective control within the ND or D groups.</p