Perturbations to placental gene expression may result in the altered metabolic and endocrine function of the placenta and may also affect the placenta's ability to transport water, gas, nutrients and waste products crucial for the proper growth and survival of the fetus.7
Previous findings have reported that DNA methylation marks are stable and accessible for measurement in placenta,24
and that the GR gene is expressed in placenta.25
Taken collectively, our data combined with this previous work suggests that DNA methylation of the GR gene promoter may be important in regulating GR gene expression, and this epigenetic alteration is linked to infant birthweight. Specifically, we have demonstrated that infants considered LGA have significantly higher GR methylation and that birthweight classification as LGA explains a great deal of the variability in methylation of this promoter region. While there is a difference in the percent methylation observed in the placental choriocarcinoma cell lines versus that in tissue, the levels of GR methylation that we observed in tissue are consistent with previous findings in other primary human tissues.18
As birthweight is a multifactorial outcome as well as a predictor of later life health and disease, this finding begins to provide a mechanistic link between the intrauterine environment and health throughout life.
Previous work has shown that altered methylation status of the NGF1-A consensus binding site within the rat exon 1–7 and analogous human exon 1F of the GR gene can lead to reduced NGF1-A binding, and reduced expression.14,15,19,26
Our work is an extension of the previous work focused on the 13 CpG sites in exon 1F,15,18
driven by the hypothesis that the environment may be playing a critical role in determining the DNA methylation status of this gene regulatory region. The NGFI-A binding site occurs at CpG sites 3 and 4. Our results suggest elevated methylation at these sites in LGA compared to SGA and AGA infants, although the differences were not significant. Methylation at these sites in infant cord blood was previously associated with maternal mood and stress.18
Although we could not examine these associations in this population, our results suggest that the environment represented by birthweight may have greater impacts at other CpG sites in the 1F promoter than at the NGF1A sites. This difference may be related to differences in the environment influencing methylation, or may be related to differences in susceptibility of the tissues examined. Specific examinations of potential environmental influences on methylation of this region in placenta and other tissues are warranted to better explain this difference. There are also several alternative first exons of the GR involved in gene expression regulation in a tissue specific manner. Future studies aimed at exploring and more completely characterizing the complex regulatory mechanisms controlling GR gene expression are needed in the placenta and in other primary human tissues.
Our in vitro work in human placenta choriocarcinoma cells suggests that methylation of this region of the gene is also associated with reduced gene expression in this cell type, thereby suggesting a relationship between the methylation of this region in human placentas. Our cell culture data suggests that JEG-3 cells do not express GR unless treated with 5-azaC, and increasing doses of 5-azaC resulted in a dose-dependent increase in GR gene expression. Because 5-azaC acts as a non-specific DNA methyltransferase inhibitor, we cannot exclude the possibility that its effects were not directly related to changes in exon 1F, but these results are consistent with previous work performed in other cell types.26
Future follow-up studies can be performed to more completely characterize the molecular mechanisms underlying control of GR gene expression.
Rich maternal forecasts represented by LGA status can prove to be incorrect if the child is born into a nutrient poor environment. Moreover, high birthweight, especially birthweights that would categorize babies as LGA, is associated with an increased risk for disease in both children and adults. Previous work has shown high birthweight as a risk factor for insulin resistance11
as well as for a number of cancers,13
and it is clear that glucocorticoid signaling can play a role in these pathologic processes. While normal physiological levels of glucocorticoids and GR are essential for metabolic control, altered glucocorticoid action has also been shown to be associated with a variety of metabolic diseases such as obesity and type 2 diabetes.27
Altered methylation of the GR in the placenta and subsequent dysregulated expression of the GR (and thereby altered glucocorticoid signaling) may lead to specific adult phenotypes, such as glucocorticoid resistance, as well as to increased risks of metabolic disorders, such as Type 2 diabetes and obesity.28–30
To date, associations between placental GR methylation and adult metabolic outcomes have yet to be elucidated. Ke and colleagues demonstrated an increase in expression of some forms of GR in the hippocampus of IUGR rats.17
Our data suggest an increase in DNA methylation of the exon 1F of the GR associated with LGA status, which would be consistent with the findings of Ke and colleagues. More research is needed to identify risk factors that may lead to the developmental outcome of high birthweight, as well as further elucidation of the role of the glucocorticoid pathways and other critical molecular mediators leading to this phenotype and the associated later onset disease risk including metabolic disorders.
In summary, our study is one of the first to show an association between the methylation status of the promoter of the GR gene in human placenta and LGA status. This may serve as an early epigenetic marker of maternal exposures and may be involved in the pathway leading to adverse health outcomes associated with increased birthweight. More work is necessary to further elucidate the mechanisms involved in this complex and multifactorial pathway and will be crucial for better understanding the developmental origins of health and disease.