Molecular biologists have characterized a class of intracellular proteins, termed transcription factors, which are rapidly synthesized in response to extracellular signals and subsequent changes in intracellular second-messenger systems, and which then serve to alter gene transcription. Transcription factors thus provide the molecular interface between gene and environmentally induced changes in cellular activity. The challenge for understanding the pathways by which maternal care alters gene expression is to describe the relevant extracellular and intracellular signals, including the target transcription factors.
Both postnatal handling, which increases maternal LG (see above), and rearing by a high-LG mothers enhance serotonin (5-hydroxytryptamine [5-HT]) turnover in the hippocampus in day-6 rat pups.73,74
Interestingly, postnatal handling results in specific increases in 5-HT in the hippocampus and prefrontal cortex, where GR expression is increased.74
5-HT levels in the hypothalamus, septum, and amygdala are unaffected; GR levels in these regions are not altered by handling. Thus, the sensory input associated with maternal LG selectively alters 5-HT activity in specific brain regions.
The obvious question is whether the increase in 5-HT might directly influence GR gene expression. This issue is remarkably difficult to address with in vivo studies, in which pharmacological manipulations targeting a specific neurotransmitter system inevitably alter other systems, as well as systems in other brain regions. This issue begs an in vitro approach in which the relevant system, the hippocampal neurons, can be examined in a cell culture system. In vitro, the treatment of primary hippocampal cell cultures with 5-HT increases GR expression and this effect is mediated by 5-HT7
receptor is positively coupled to cyclic adenosine monophosphate (cAMP) and GR expression in cultured hippocampal neurons is also significantly increased after treatment with 8-bromo-cAMP (a stable cAMP analog) or with various doses of the specific 5-HT7
receptor agonists, such as 5-carboxamidotryptamine (5-CT). For all conditions, the effect on GR expression is apparent only after 4 days of treatment, a seemingly obscure fact whose importance will later become evidence. The effect of 5-CT on GR expression is blocked by methiothepin. Likewise, 5-CT produces a significant increase in cAMP levels and the effect is blocked by methiothepin. Pindolol, which binds to the 5-HT1A
, but not the 5-HT7
receptor, has little effect (see also reference 76). These results further implicate the 5-HT7
receptor. The intracellular effects of cAMP are commonly mediated by cyclic nucleotide-dependent protein kinases (PKA) and, predictably, a PKA inhibitor (H8) blocks the effects of 5HT or cAMP on hippocampal GR expression. Over the course of these studies, we found that other serotonergic agonists (quipazine, TFMPP [1-(trifluoromethylphenyl) piperazine], and DOI [(+/-)-2,5-demethoxy-4-iodoamphetamine]) could partially mimic the 5-HT effect on GR levels and, in all studies, the magnitude of the serotonergic effect on cAMP concentrations is highly correlated (r
=0.97) with that on GR expression.78
This observation is consistent with the idea that the effect of 5-HT on GR expression in hippocampal neurons is mediated by a 5-HT7
receptor via activation of cAMP Importantly, both postnatal handling and increased maternal LG increase hippocampal concentrations of both cAMP and PKA in the rat pup. The conclusion of these studies provides the identification of an extracellular signal, 5-HT, and an intracellular, secondary messenger system, cAMP-PKA. Importantly, the in vivo effects of postnatal handling are blocked with compounds that serve as 5-HT7
The in vitro hippocampal cell culture system mimics the in vivo world with surprising authenticity. The increase in GR levels in cultured hippocampal neurons following 5-HT treatment persists following 5-HT removal from the medium; for as long as the cultures can be maintained, there is a sustained increase in GR levels as long as 50 days beyond the removal of 5-HT from the medium. Thus, 5-HT can act directly on hippocampal neurons to increase GR expression, and the effect of 5-HT on GR expression is observed in hippocampal culture cells mimics the long-term effects of early environmental events. These findings provide an in vitro “programming” model. Activation of cAMP pathways can regulate gene transcription through effects on a number of transcription factors, including, of course, the cAMP-response element binding protein (CREB) via an enhanced phosphorylation of CREB. In this instance, the second-messenger system alters the activity of the transcription factor, through enzymatic modification and phosphorylation, rather than production. CREB regulates gene transcription through pathways that involve the transcriptional cofactor, CREB -binding protein (CBP). Primary hippocampal cell cultures treated with 8-bromo-cAMP, 5-CT, or 5-HT show a significant increase in CBP expression.
receptor is positively coupled to adenylyl cyclase, and thus the activation of cAMP In vivo, both handling and increased maternal LG result in an increased level of hippocampal cAMP concentrations and the activation of PKA over the first week of postnatal life.76
Activation of PKA results in the tissue-specific induction of a number of transcription factors. The day6 offspring of high-LG mothers or pups of the same age exposed to handling show increased hippocampal expression of NGFIA (nerve growth factor-induced clone A, also known as zif-268, krox-24, egr-1, and zenk) (Weaver IGC et al, unpublished results).79
In vitro, 5-HT increases NGFIA expression in cultured hippocampal neurons and the effect of 5-HT on GR expression in hippocampal cultures is completely blocked by concurrent treatment with an oligonucleotide antisense directed at the NGFIA mRNA.80
The antisense is a synthetic strand of nucleotides that hybridizes with the native mRNA and prevents transcription of the NGFIA protein. These studies serve to identify a relevant transcription factor and to link the activation of the transcription factor NGFIA to the activation of GR expression in response to 5-HT.
Maternal LG results in an increased expression of NGFIA, which in turn might then regulate GR expression. Other rodent models examining environmental regulation of hippocampal GR expression also suggest a correspondence between NGFIA levels and GR expression.81,82
In each case, increased levels of NGFIA are associated with enhanced GR expression. However, the critical site for GR regulation remains to be defined.
These findings provide a platform for the study of direct gene-environment interactions. However, the important missing piece is the identification of the relevant DNA target. We assumed that a potential target for regulation is the regulatory region of the GR gene. Regulatory regions contains sequences that alter the activity of the gene, such as promoters or suppressors, and are commonly found in front (or upstream) of the coding region of the gene that actually produces the protein. We identified and characterized several new GR mRNAs cloned from rat hippocampus ()
All mRNAs encode a common protein, but differ in their 5 '-leader sequences presumably as a consequence of alternative splicing of, potentially, several different sequences from the 5' noncoding exon 1 region of the GR gene. In this case, the variation in the mRNAs reflects the different promoters that are spliced onto the coding region during transcription to create diverse GR mRNAs. The promoter, while spliced onto the mRNA, does not alter the translational phase by which mRNA is “translated” into the amino acid sequence that defines the protein product. The alternate exon 1 sequences are unlikely to alter the amino acid sequence of the GR protein; there is an “stop” codon present immediately 5' to the translation initiation site in exon 2, common to all the mRNA variants. Hence, only the coding region is actually translated into protein. Of the alternate exon 1 sequences identified, four correspond to exon 1 sequences previously identified in mouse, exons 11
, and 110
Most alternative exons are located in a 3-kb CpG island upstream of exon 2 that exhibits substantial promoter activity in transfected cells (Figure 2).
Ribonuclease protection assays demonstrate significant levels of six alternative exon 1 sequences in vivo in the rat, with differential expression in the liver, hippocampus, and thymus presumably reflecting tissue-specific differences in promoter activity. The different promoters respond to different signals, which forms the basis for tissue-specific laterations in gene expression. Simply put, it is the process by which environmental or hormonal signals can alter GR expression in one region of the body, without affecting expression in another. Hippocampal RNA contains significant levels of the exon 17
-containing GR mRNA variants expressed at undetectable levels in liver and thymus. These studies thus identify a brain-specific GR promoter, the exon 17
Figure 2. Map of the noncoding exon 1 region of the glucocorticoid receptor (GR) gene cloned from rat hippocampus.83 The sequence of the critical exon 17 region is provided below, highlighting the NGFIA (nerve growth factor-induced clone A) consensus sequence. (more ...)
In transient transfection experiments, a construct encoding the entire regulatory region of the GR gene, including eight of the alternate exon 1 sequences and the splice acceptor site within the intron 5' of exon 2, was fused to a lucif erase reporter gene. The lucif erase gene is activated by the coupled promoters and its activity thus reflects the ability of the regulatory sites to activate gene transcription - hence the term reporter gene.
Fusion to the socalled reporter gene permits a measure of the degree to which individual sequences can potentially influence gene expression. This alteration in activity results from various sequences originating at any point within the regulatory region and, we presume, represents the sum of the activity of individual promoters on the genomic DNA fragment. In subsequent studies examining the potency of the individual promoters, we found that the relative activity of the individual exon 1 sequences is similar, with one notable exception, the exon 17
promoter sequence. The fused exon 17
has the highest transcriptional activity of any single promoter construct. More recent studies confirm the transactivational effect of NGFIA at the exon 17
sequence. We used a cotransfection model with human embryonic kidney (HEK) cells (intentionally aiming as far from the neural target as possible) with an NGFIA expression vector and an exon 17
-luciferase construct. Cotransfection of the NGFIA vector and the exon 17
-luciferase construct resulted in a robust increase in luciferase activity, reflecting NGFIA-induced activation of transcription through the exon 17
promoter. These later studies reveal not only the ability of the exon 17 promoter to drive gene expressions, but that it does so in response to an increased NGFIA signal. Recall that an NGFIA antisense completely blocks the effects of 5-HT on GR expression in hippocampal cell cultures.80
Interestingly, the activity of the exon 17
promoter is altered by postnatal handling, which increases GR expression in the hippocampus. Handling selectively elevated GR mRNA containing exon 17
; there is, for example, no effect on exon 110
Predictably, maternal care also affected the expression of GR splice variants: variants containing the exon 17
sequence were also significantly increased in the adult offspring of high-LG mothers (Weaver IGC et al, unpublished results). Thus, transcriptional activation of the GR gene in the hippocampus during adulthood is altered by maternal care over the first week of life.
The exon 17
promoter sequence of the GR gene contains guanine-cytosine nucleotides, so-called GC boxes (GCGGGGGCG), which form the core consensus site (ie, a DNA binding site) for NGFIA (Figure 2)
Thus, increases in NGFIA induced by maternal LG could increase transcription from the exon 17
promoter leading to increased GR mRNA. We previously found that handling increased the binding of NGFIA to a promoter sequence for the human GR promoter containing an NGFIA consensus sequence. Since neonatal handling increases maternal LG, these finding suggest that naturally occurring variations in maternal behavior might regulate GR expression in neonatal offspring through a 5HT-induced increase in NGFIA expression, and the subsequent binding of NGFIA to the exon 17
promoter. Recent findings support this idea, including studies using chromatin immunoprecipitation (ChIP) assay in which the in vivo formation of protein-DNA complexes are examined using cross-linking with paraformaldehyde perfusion and subsequent precipitation from soluble hippocampal samples using specific antibodies. Protein binding, defined by the specificity of the antibody, to specific DNA sequences is then quantified following polymerase chain reaction (PCR) amplification with targeted primers and Southern blotting. PCR allows for identification of precise DNA sequences and Southern blotting permits quantification of those same sequences. The experiment provides information on the amount of a specific DNA sequence bound to a specific protein. The charm of this approach is the ability to directly examine the interaction of specific proteins with specific DNA sequences at the time the biological sample is obtained. ChIP analysis of hippocampal samples from postnatal day-6 pups reveals dramatically increased NGFIA binding to the exon 17
promoter in the offspring of high-LG compared with low-LG mothers.67
These findings confirm that maternal care regulates the binding of NGFIA to the exon 17
promoter sequence in pups.
These findings suggest that maternal LG in the neonate increases NGFIA expression in the hippocampus and NGFIA binding to the exon 17 promoter. NGFIA might then increase GR expression in hippocampal neurons, and these findings might then provide a mechanism for the effect of maternal care over the first week of life. However, while there are striking differences in NGFIA expression in the offspring of high- and low-LG mothers at day 6 of postnatal life, hippocampal NGFIA expression in adulthood is unaffected by maternal care: there is no difference in hippocampal NGFIA expression in the adult offspring of high- and low-LG dams. We are thus left with the defining question of early experience studies: how are the effects of early life events sustained into adulthood?