image of Differentially regulated genes by DES in Msx2 uterus
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Homeodomain Transcription Factor Msx-2 Regulates Uterine Progenitor Cell Response to DES

Potential downstream targets of MSX-2, as well as genes whose regulation by DES is modulated through MSX-2

2015 Study Abstract

The fate of mouse uterine epithelial progenitor cells is determined between postnatal days 5 to 7. Around this critical time window, exposure to an endocrine disruptor, diethylstilbestrol (DES), can profoundly alter uterine cytodifferentiation.

We have shown previously that a homeo domain transcription factor MSX-2 plays an important role in DES-responsiveness in the female reproductive tract (FRT). Mutant FRTs exhibited a much more severe phenotype when treated with DES, accompanied by gene expression changes that are dependent on Msx2. To better understand the role that MSX-2 plays in uterine response to DES, we performed global gene expression profiling experiment in mice lacking Msx2 By comparing this result to our previously published microarray data performed on wild-type mice, we extracted common and differentially regulated genes in the two genotypes.

In so doing, we identified potential downstream targets of MSX-2, as well as genes whose regulation by DES is modulated through MSX-2. Discovery of these genes will lead to a better understanding of how DES, and possibly other endocrine disruptors, affects reproductive organ development.

Discussion

Since the discovery of the adverse effects of DES on human health, numerous studies in model animals have been conducted trying to uncover the underlying molecular mechanisms. The neonatal DES mouse model is widely used, because DES-treated animals exhibited a variety of developmental abnormalities and pathologies recapitulating the human syndrome including uterine metaplasia and vaginal adenosis. We have previously conducted global gene profiling studies, and found that neonatal DES treatment in mice caused a series cellular changes including proliferation, apoptosis, metabolic as well as other physiological changes through modulation of uterine gene expression.

We have also discovered that the transcription factor MSX-2, can modulate the responsiveness of uterus and vagina to DES.

To better understand how Msx2 modulates DES response in the uterus, here we performed microarray analyses using RNA extracted from oil- or DES-treated Msx2−/− uteri, and compare this result to that of our previous array data performed on wild-type animals. By comparing the two data sets, we uncovered three groups of genes in general: roup I contains genes that are similarly regulated by DES both in wild type and in Msx2−/− uteri. Thus Msx2 is dispensable for the regulation of this group of 212 genes by DES. Group II consists of 171 genes whose regulation by DES is altered (in either direction) in Msx2 mutant uteri. Thus Msx2 plays important roles in modulating their regulation by DES. Group III consists of 27 genes that are not regulated by DES in wild type uterus, but are now DES targets in Msx2 mutant uteri. These genes are likely not MSX-2 transcription targets but rather exhibit altered DES responsiveness due to secondary changes, such as cell fate change. We further subdivided group II genes into three categories: genes whose regulations by DES are either lost or attenuated in Msx2 mutant uterus; those whose regulations are enhanced by Msx2 mutation; and those oppositely regulated by DES in the two genotypes. RT-PCR was used to validate representative genes from each subgroup. Interestingly, we observed that most DES-induced genes showed a greater response in Msx2−/− uterus, evidenced by the red peaks in the star glyph. On the other hand, DES-repressed genes lost this repressive regulation in Msx2 mutant uterus, evidenced by the blue peaks. These results are in agreement with previous studies suggesting that Msx2 mainly functions as a transcriptional repressor. In this scenario, DES can induce target gene expression to a greater degree due to the removal of a repressor that normally counteracts this induction. On the other hand, repression of certain gene expression by DES may require MSX-2 participation, as its absence leads to failure of repression by DES.

We previously showed that the regulation of Aqp3 by DES was abolished in Msx2 mutant vaginal epithelium. Surprisingly, here we found that Aqp3 was similarly induced in both wild-type and Msx2 mutant uterus. These seemingly contradictory results can be reconciled by the fact that the intermediate cell layers expressing Aqp3 in the developing vagina are missing in Msx2 mutants, and thus failure of induction by DES in Msx2 mutant vagina is secondary to a defective patterning event. In this sense, Aqp3 is likely not a target of Msx2 either in the uterus or in the vagina.

We showed recently that lipid metabolism in the neonatal uterus was dramatically affected by DES exposure. This change is mediated through the upregulation of a transcription factor Pparγ, which in turn led to gene expression changes in an array of genes involved in lipid trafficking and metabolism. By analyzing the expression of those genes in DES-treated Msx2−/− uterus, we found that Msx2 is not involved in the DES-regulation of this process. Pparγ was similarly induced in Msx2−/− as in controls, so were many Pparγ downstream genes. Consistently, lipid droplet accumulation in the uterine epithelium was also observed in the Msx2−/− uterus upon DES exposure. Interestingly, it was previously reported that in myofibroblasts, Msx2 stimulates osteogenesis but suppress adipogenesis by activating Osx transcription and concomitantly suppressing Pparγ expression. Our results that Pparγ showed no change in basal level in the absence of Msx2 indicate that Msx2 is unlikely to be a transcriptional suppressor of Pparγ, at least in the uterus. In addition, DES increases Pparγ, and alters lipid metabolism in the uterus in an Msx2-independent way.

Future investigations on the function of these MSX-2 target candidates are needed to further evaluate their function(s) during normal uterine development as well as during DES-induced pathogenesis of the female reproductive tracts. Nevertheless, identification of these genes may shed light on how Msx2 regulate terminal differentiation of the uterus, and possibly other Msx2-expressing tissues.

Sources and more information
  • Full study (free access) Homeodomain Transcription Factor Msx-2 Regulates Uterine Progenitor Cell Response to Diethylstilbestrol, Journal of stem cell and transplantation biology, NCBI PubMed PMC4596533, 2015 May.
  • Differentially regulated genes by DES in Msx2−/− uterus featured image credit PMC4596533/figure/F1.
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