Interactions between Glucocorticoid Treatment and Cis-Regulatory Polymorphisms Contribute to Cellular Response Phenotypes

Glucocorticoids (GCs) mediate physiological responses to environmental stress and are commonly used as pharmaceuticals. GCs act primarily through the GC receptor (GR, a transcription factor). Despite their clear biomedical importance, little is known about the genetic architecture of variation in GC response. Here we provide an initial assessment of variability in the cellular response to GC treatment by profiling gene expression and protein secretion in 114 EBV-transformed B lymphocytes of African and European ancestry. We found that genetic variation affects the response of nearby genes and exhibits distinctive patterns of genotype-treatment interactions, with genotypic effects evident in either only GC-treated or only control-treated conditions. Using a novel statistical framework, we identified interactions that influence the expression of 26 genes known to play central roles in GC-related pathways (e.g. NQO1, AIRE, and SGK1) and that influence the secretion of IL6

Comparison of Cellular and Transcriptional Responses to 1,25-Dihydroxyvitamin D3 and Glucocorticoids in Peripheral Blood Mononuclear Cells

Glucocorticoids (GC) and 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3) are steroid hormones with anti-inflammatory properties with enhanced effects when combined. We previously showed that transcriptional response to GCs was correlated with inter-individual and inter-ethnic cellular response. Here, we profiled cellular and transcriptional responses to 1,25(OH)2 D3 from the same donors. We studied cellular response to combined treatment with GCs and 1,25(OH)2 D3 in a subset of individuals least responsive to GCs. We found that combination treatment had significantly greater inhibition of proliferation than with either steroid hormone alone. Overlapping differentially expressed (DE) genes between the two hormones were enriched for adaptive and innate immune processes. Non-overlapping differentially expressed genes with 1,25(OH)2 D3 treatment were enriched for pathways involving the electron transport chain, while with GC treatment, non-overlapping genes were enriched for RNA-related processes. These results suggest that 1,25(OH)2 D3 enhances GC anti-inflammatory properties through a number of shared and non-shared transcriptionally-mediated pathways.</p

Comparison of Cellular and Transcriptional Responses to 1,25-Dihydroxyvitamin D3 and Glucocorticoids in Peripheral Blood Mononuclear Cells

<div><p>Glucocorticoids (GC) and 1,25-dihydroxyvitamin D3 (1,25(OH)₂ D3) are steroid hormones with anti-inflammatory properties with enhanced effects when combined. We previously showed that transcriptional response to GCs was correlated with inter-individual and inter-ethnic cellular response. Here, we profiled cellular and transcriptional responses to 1,25(OH)₂ D3 from the same donors. We studied cellular response to combined treatment with GCs and 1,25(OH)₂ D3 in a subset of individuals least responsive to GCs. We found that combination treatment had significantly greater inhibition of proliferation than with either steroid hormone alone. Overlapping differentially expressed (DE) genes between the two hormones were enriched for adaptive and innate immune processes. Non-overlapping differentially expressed genes with 1,25(OH)₂ D3 treatment were enriched for pathways involving the electron transport chain, while with GC treatment, non-overlapping genes were enriched for RNA-related processes. These results suggest that 1,25(OH)₂ D3 enhances GC anti-inflammatory properties through a number of shared and non-shared transcriptionally-mediated pathways.</p> </div

Interactions between Glucocorticoid Treatment and Cis-Regulatory Polymorphisms Contribute to Cellular Response Phenotypes

Glucocorticoids (GCs) mediate physiological responses to environmental stress and are commonly used as pharmaceuticals. GCs act primarily through the GC receptor (GR, a transcription factor). Despite their clear biomedical importance, little is known about the genetic architecture of variation in GC response. Here we provide an initial assessment of variability in the cellular response to GC treatment by profiling gene expression and protein secretion in 114 EBV-transformed B lymphocytes of African and European ancestry. We found that genetic variation affects the response of nearby genes and exhibits distinctive patterns of genotype-treatment interactions, with genotypic effects evident in either only GC-treated or only control-treated conditions. Using a novel statistical framework, we identified interactions that influence the expression of 26 genes known to play central roles in GC-related pathways (e.g. NQO1, AIRE, and SGK1) and that influence the secretion of IL6

Scatterplot showing log₂ fold change of differentially expressed genes in response to vitamin D (VD) and dexamethasone (dex) on the x- and y-axes respectively after 24 hours.

<p>Each circle represents an individual gene. Black circles indicate genes that are differentially expressed in response to <i>both</i> 1,25 vitamin D and dexamethasone treatment. Red circles are genes that are differentially expressed only in response to 1,25 vitamin D treatment; while, green circles are genes that are differentially expressed only in response to dexamethasone treatment. Names of select differentially expressed genes are indicated that show marked differences in response to 1,25 vitamin D and dexamethasone at 24 hours including fructose 1,6 <i>bisphosphonate</i> (log₂ fold change 3.79 vs. -0.61 in vitamin D vs. dexamethasone, respectively), <i>oncostatin </i><i>M</i> (log₂ fold change 2.71 vs. -1.07 respectively), <i>adenosine </i><i>A3</i> (log₂ fold change -0.66 vs. 4.31 respectively), and <i>zinc </i><i>finger, CCHC </i><i>domain </i><i>containing 12</i> (log₂ fold change 0.27 vs. 3.51 respectively).</p

Intersection and significant gene set enrichment analysis of differentially expressed <i>down-regulated</i> genes in response to dexamethasone and vitamin D at 24 hours.

<p>Overlapping down-regulated genes were enriched for terms including “immune response” and “defense response”. Non-overlapping down-regulated genes unique to dexamethasone treatment included terms involved in RNA processing and function.</p

Intersection and significant gene set enrichment analysis of differentially expressed <i>up-regulated</i> genes in response to dexamethasone and vitamin D at 24 hours.

<p>Non-overlapping up-regulated genes unique to vitamin D treatment were significantly enriched for terms involved in metabolism and cellular respiration. </p

Scatterplot of inhibition of cell proliferation by individual and treatment.

<p>All individuals were treated with dexamethasone (diamond) and 1,25 vitamin D (square). Eight individuals who were least responsive to dexamethasone and for whom sufficient cells were available were treated with both dexamethasone and 1,25 vitamin D (triangle). Individuals treated with both hormones had higher % inhibition compared to either treatment alone.</p

Transcriptional response to vitamin D overall and for specific vitamin D responsive genes.

<p>(<b>a</b>) Volcano plot of transcriptional response to vitamin D at 8 hours and 24 hours respectively summarizing mean log₂ fold change (vitamin D/vehicle) and corresponding evidence of differential expression (-log₁₀ p-value) for each gene shown as a single point. The red line corresponds to an FDR of 1%. (<b>b</b>) Boxplots showing examples of two established vitamin D responsive genes at 24 hours including <i>CYP24A1</i> and <i>CAMP</i>. <i>CYP24A1</i> encodes 1,25-dihydroxyvitamin D 24-hydroxylase, a member of the cytochrome P450 superfamily, that initiates degradation of 1,25 vitamin D and had the largest response to vitamin D treatment (log₂ fold change 4.54, p-value=2.40 x 10^-9). <i>CAMP</i>, cathelicidin anti-microbial peptide, is a known target of the vitamin D receptor and also showed a large response to treatment (log₂ fold change 2.62, p-value=1.64 x 10^-7).</p

Mapping Variation in Cellular and Transcriptional Response to 1,25-Dihydroxyvitamin D3 in Peripheral Blood Mononuclear Cells

The active hormonal form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is an important modulator of the immune system, inhibiting cellular proliferation and regulating transcription of immune response genes. In order to characterize the genetic basis of variation in the immunomodulatory effects of 1,25D, we mapped quantitative traits of 1,25D response at both the cellular and the transcriptional level. We carried out a genome-wide association scan of percent inhibition of cell proliferation (Imax) induced by 1,25D treatment of peripheral blood mononuclear cells from 88 healthy African-American individuals. Two genome-wide significant variants were identified: rs1893662 in a gene desert on chromosome 18 (p = 2.32 x 10−8) and rs6451692 on chromosome 5 (p = 2.55 x 10−8), which may influence the anti-proliferative activity of 1,25D by regulating the expression of nearby genes such as the chemokine gene, CCL28, and the translation initiation gene, PAIP1. We also identified 8 expression quantitative trait loci at a FDRmax and the response eQTL mapping enabled identification of putative Imax-associated candidate genes such as PAIP1 and the transcriptional repressor gene ZNF649. Overall, the variants identified in this study are strong candidates for immune traits and diseases linked to vitamin D, such as multiple sclerosis