Silencing of core transcription factors in human EC cells highlights the importance of autocrine FGF signaling for self-renewal

Abstract

Background: Despite their distinct origins, human embryonic stem (hES) and embryonic carcinoma (hEC) cells share a number of similarities such as surface antigen expression, growth characteristics, the ability to either self-renew or differentiate, and control of the undifferentiated state by the same core transcription factors. To obtain further insights into the regulation of self-renewal, we have silenced hES/hEC cell-specific genes in NCCIT hEC cells and analysed the downstream effects by means of microarrays.

Results: RNAi-mediated silencing of OCT4 and SOX2 induced differentiation with mesodermal characteristics. Markers of trophoblast induction were only transiently up-regulated in the OCT4 knock-down. Independent knock-downs of NANOG produced a proliferation rather than a differentiation phenotype, which may be due to high NANOG expression levels in the cell line used. Published ChIP-chip data from hES cells were used to identify putative direct targets. RNAi-mediated differentiation was accompanied by direct down-regulation of known hES/hEC cell markers. This included all three core transcription factors in the case of the OCT4 and SOX2 knock-downs, confirming previous findings of reciprocal activation in ES cells. Furthermore, large numbers of histone genes as well as epigenetic regulators were differentially expressed, pointing at chromatin remodeling as an additional regulatory level in the differentiation process. Moreover, loss of self-renewal was accompanied by the down-regulation of genes involved in FGF signaling. FGF receptor inhibition for short and prolonged periods of time revealed that the ERK/MAPK cascade is activated by endogenously expressed fibroblast growth factors and that FGF signaling is cruicial for maintaining the undifferentiated state of hEC cells, like in hES cells.

Conclusion: Control of self-renewal appears to be very similar in hEC and hES cells. This is supported by large numbers of common transcription factor targets and the requirement for autocrine FGF signaling.

Figure 1

Silencing of OCT4, NANOG, and SOX2 in hEC cells. (A): Initial RNAi screen on hESC marker genes in hEC cells. esiRNA-treated samples were evaluated on the basis of cell morphology and changes in OCT4, NANOG, and SOX2 expression levels (by real-time PCR). Numbers at the bottom are array-based expression ratios of hESCs vs. universal reference RNA. The values for GDF3 and OTX2 are from an in-house platform (our unpublished data) and [34], respectively. Knock-down efficiencies were between 60 and > 90% throughout (not shown). (B): RNAi phenotypes in the OCT4, NANOG, and SOX2 knock-downs. Pictures were taken 2.5 days after esiRNA transfection. The morphology of unmanipulated or mock-treated cells was dependent on the seeding density. When plated at low density as required for esiRNA transfections the cells grew as 3D-shaped colonies rather than in monolayers. Bottom left: Growth curves of NANOG vs. GAPDH esiRNA-transfected cells. (C): Immunostaining of OCT4 protein in samples prepared as in (B). Note that the NANOG RNAi cells are OCT4 positive. (D): Western blot on day 3 RNAi and mock control samples using OCT4, NANOG, and SOX2 antibodies. GAPDH served as a loading control.

Figure 2

Expression profiling of OCT4, NANOG, and SOX2 RNAi samples. (A): Overlaps of differentially expressed genes. The areas of the squares are proportional to the numbers of genes which they represent [77]. O = OCT4 knock-down, N = NANOG k.d., S = SOX2 k.d. (B): Correlation-based dendogram of RNAi samples and control. Note the high similarity between the OCT4 and SOX2 knock-downs. r = linear correlation coefficient. (C): Scatter plots of independent NANOG knock-downs and mock controls. esiRNA 1 and 2 were derived from non-overlapping parts of the NANOG mRNA. The dashed line indicates the expression threshold based upon negative control beads. Only significantly expressed genes were considered in the correlation analysis. (D): Array and real-time PCR-based mRNA expression measurements of NANOG downstream targets using the two independent NANOG esiRNA pools. Note the high degree of concordance between the independent knock-downs and methods of mRNA quantification. Values are means of two biological replicates ± SE.

Figure 3

Autocrine FGF signaling is cruicial for hEC cell self-renewal. (A): Expression changes of genes involved in FGF signaling. Samples were assayed 2 and 4 days after transfection. Transcripts of FGF19, FGF2, and FGFR1 (at 96 h) were undetectable in the OCT4 and SOX2 RNAi samples. (B): Immunostain of OCT4 protein in NCCIT hEC cells 5 days after SU5402 treatment indicating loss of self-renewal. (C): Cellular morphology after 5 days of SU5402 treatment vs. DMSO controls using three different hEC cell lines. (D): Monitoring of OCT4, NANOG, and SOX2 expression levels by real-time PCR in samples from (C). Error bars indicate technical variation (DMSO control: means between cell lines). (E): Short-term effect of SU5402 treatment on MAPK phosphorylation in NCCIT hEC cells. Total MAPK protein (bottom panel) served as loading control. U0126 is a specific inhibitor of MEK (MAPKK) which directly phosphorylates ERK (MAPK). This sample served as positive control for the assay.