Prior studies from our laboratory have demonstrated that an increase in PBX expression in P19 cells is required for the RA-dependent reduction in the expression of several pluripotency genes (OCT-4, SF-1 and DAX-1) and differentiation of P19 cells to endodermal cells (Qin et al, 2004b
; Teets et al., 2012
). Furthermore, the PBX-dependent reduction in SF-1 but not DAX-1 expression is necessary for differentiation of P19 cells to endodermal cells (Teets et al., 2012
). Here we show that the expression of two differentiation associated genes (COUP-TFI and COUP-TFII variant 1) is elevated upon RA treatment of P19 cells and that the increases in their mRNA levels are also PBX-dependent during endodermal differentiation. The overall goal of this work was to determine if expression of COUP-TFI can overcome the loss of PBX induction during RA-dependent differentiation of AS P19 cells. We found that in a dose-dependent fashion COUP-TFI expression leads to growth inhibition, modest cell cycle disruption and early apoptosis. Furthermore, AS cells can overcome their blockage in RA-dependent differentiation leading to endodermal cells when either pharmacological levels of COUP-TFI are expressed or with a combination of physiological levels of COUP-TFI and RA treatment. Finally, COUP-TFI is involved in the regulatory network governing the expression of at least two primary RA response target genes, CYP26A1 and HoxA1. Taken together, our studies demonstrate that elevation of COUP-TFI expression is a critical step in the RA-dependent differentiation cascade/pathway of P19 cells to endodermal cells.
Consistent with the findings of Ben-Shushan et al. (1995)
, COUP-TFI and COUP-TFII mRNA levels inP19 cells were found to be highly elevated upon RA treatment with the maximal increase after 72 hrs. In addition, only variant 1 of COUP-TFII was found to display this RA-dependent increase in mRNA levels. Furthermore, the increase in COUP-TFI and COUP-TFII variant 1 mRNA levels is not a direct response to RA but rather requires the RA-dependent increase in PBX expression. Although our studies did not address whether PBX directly or indirectly regulates the mRNA levels of COUP-TFI and/or COUP-TFII variant 1, it is clear that these genes are downstream of PBX in the cascade of events initiated by RA leading to endodermal differentiation of P19 cells.
The hallmark of AS cells is their failure to induce PBX expression and their subsequent inability to differentiate upon RA treatment (Qin et al., 2004b
). Our data strongly suggest a functional role for COUP-TFI during the differentiation cascade/pathway of P19 cells to endodermal cells. Immunofluorescence analysis of SSEA-1, OCT-4 and TROMA-I expression along with RT-QPCR measurement of several pluripotency genes mRNA levels demonstrates that AS cells expressing a pharmacological level of COUP-TFI for 4 days differentiate to endodermal cells irrespective of RA treatment. On the other hand, RA is needed to induce differentiation when COUP-TFI is expressed at a physiological level. This suggests that AS cells expressing a physiological level of COUP-TFI still require a RA-regulated event(s) distinct from those associated with the elevation of PBX for differentiation. In contrast, cells expressing a pharmacological level of COUP-TFI either do not require this RA-regulated event (s) or high levels of COUP-TFI can replace the need for RA. Since active cell proliferation and cell differentiation are known to be opposing processes, it is possible that the more dramatic growth inhibition observed in cells expressing a pharmacological level of COUP-TFI contributes to the differentiation of these cells to endodermal cells in the absence of RA.
Consistent with the analysis of differentiation marker gene expression, the overall trend from the study of pluripotency gene expression (OCT-4, SF-1 and DAX-1) in AS cells expressing either physiological levels of COUP-TFI along with RA treatment and AS cells expressing only pharmacological levels of COUP-TFI is a reduction in their mRNA levels. These are not surprising findings since prior studies have demonstrated that (i) COUP-TFI can bind to regulatory regions of the SF-1 promoter and repress its transcriptional activity (Xing et al., 2002
; Shibata et al., 2001
), (ii) COUP-TFI can directly repress OCT-4 transcriptional activity (Ben Sushan et al., 1995
), and (iii) COUP-TFI can repress the transcription of DAX-1 (Yu et al., 1998
). Therefore, COUP-TFI is likely to participate as a transcriptional repressor of genes associated with pluripotency thereby facilitating differentiation by promoting escape from pluripotency.
Interestingly, only a very modest increase in COUP-TFII mRNA levels was observed in AS cells expressing COUP-TFI that differentiated to endodermal cells (approximately 50-fold compared with 1000-fold in wild type cells treated with RA). Since COUP-TFI and COUP-TFII genes show a remarkably high degree of homology and their expression pattern in tissues often overlaps (Qiu et al., 1994
), it is likely that COUP-TFI can compensate at least in part for COUP-TFII during differentiation of P19 cells to primitive endoderm. Consistent with this is a recent study (Tang et al., 2010
) that demonstrated using conditional knockout mice that COUP-TFI and COUP-TFII are functionally redundant in the developing eye.
Strikingly, the time required for the differentiation of AS cells expressing COUP-TFI appears to be unusually short. Differentiation as indicated by loss of SSEA-1 expression, gain of TROMA-1 expression, and morphological changes of cells was readily apparent after 4 days of COUP-TFI expression. However, in wild type P19 cells treated with RA, an increase in COUP-TFI expression is detected after 48 hrs and differentiation is apparent only after 5 to 7 days. This shortened COUP-TFI dependent timeframe for the differentiation of AS cells is consistent with the concept that there is an RA triggered cascade of events where both the primary RA response genes and PBX induction can be bypassed and this cascade can be entered at some intermediary point that is regulated by COUP-TFI.
In light of the observation that COUP-TFI expression in AS cells resulted in differentiation similar to that seen upon RA mediated differentiation of wild type P19 cells, we next determined if primary RA response gene expression was affected by COUP-TFI expression. Surprisingly we found that the expression of two RA-responsive genes (HoxA1 and CYP26A1) was altered with either an induced expression in the absence of RA treatment (HoxA1) or lack of RA-dependent increase (CYP26A1) in AS cells expressing COUP-TFI. These data suggest that COUP-TFI is able to either directly or indirectly regulate the expression of both HoxA1 and CYP26A1.
HoxA1 contains an RARE in its 3′ enhancer region which has been demonstrated to be controlled by RA-dependent transcriptional regulation through RARγ (Boylan et al., 1993
). Additionally, HoxA1 has been implicated in the antiproliferative effect of RA on ES cells. Hoxa1−/−
cells are more resistant to the growth inhibitory effects of RA than Hoxa1+/−
which are more resistant than Hoxa1+/+
suggesting that the Hoxa1 protein plays a role in the RA-induced inhibition of growth in ES cells in a dose dependent manner (Martinez-Ceballos et al., 2005
). On the other hand, expression of COUP-TFI in mouse ES cells caused the cells to be more resistant to the growth inhibitory effects of RA however the authors did not examine if HoxA1 levels were elevated in these cells (Zhuang et al., 2007). Strikingly, we found that expressing COUP-TFI at either physiological or pharmacological levels resulted in increased expression of HoxA1, with or without RA-treatment, and resulted in a subsequent reduction in the growth of the P19 cells. Although there was an apparent increase in growth inhibition upon expression of a pharmacologic level of COUP-TFI, there was no subsequent increase in HoxA1 expression. This is likely to be explained by the increased amount of cells expressing pharmacological levels of COUP-TFI that entered early apoptosis. Taken together this suggests that the COUP-TFI induced HoxA1 expression may contribute to the growth inhibition observed in these cells and in wild type P19 cells induced to differentiate by RA treatment.
COUP-TFI is well known to serve as a potent transcriptional repressor of genes and to efficiently antagonize transcriptional activation mediated by a number of nuclear receptors including RAR, RXR, PPAR, and ER (Cooney et al., 1993
; Burbach et al., 1994
; Klinge et al., 1997
; Nakshatri and Bhat-Nakshatri, 1998
) by competition for target response elements. Analysis of the promoter region of both mouse and human CYP26A1 using NUBISCAN (www.nubiscan.unibas.ch/
) (Podvinec et al., 2002
), demonstrates one fully conserved DR1 sequence which could potentially bind COUP-TFI. This DR1 lies about 25 base pairs downstream of the R2 RARE located approximately 2 kilobases upstream from the start site of transcription. Both the R2 RARE and the R1 RARE located within the proximal promoter of the CYP26A1 gene have been shown to work synergistically to direct a large increase in transcription upon RA treatment (Loudig et al., 2005
). Deletion of either R1 or R2 results in a dramatic reduction in the level of RA dependent transcription however R2 RARE is more sensitive to low concentrations of RA. It is possible that COUP-TFI binds to this DR1 sequence adjacent to the R2 RARE and interferes with the functioning of the R2 RARE sequence resulting in a loss of RA-dependent increase in transcription.
In summary, we have demonstrated that the RA-dependent increase in PBX protein levels is required for the increase in COUP-TFI and COUP-TFII variant 1 expression in P19 cells induced to differentiate to endodermal cells. An increase in the level of expression of COUP-TFI appears critical for the RA-dependent differentiation of P19 cells to endodermal cells. Here, we report for the first time that physiological levels of COUP-TFI in cells lacking PBX cells combined with RA treatment induces their differentiation to endodermal cells while pharmacologic expression levels circumvents the need for RA treatment. In addition, two additional genes have been found to be regulated by COUP-TF, Hoxa1 expression was increased while the RA-dependent increase in Cyp26a1 was repressed.