Debora Lim

Enhanced steroid response of a human glucocorticoid receptor splice variant.

ABSTRACT Glucocorticoids remain a recommended therapy in advanced septic shock despite the often unpredictable response, and our understanding of the mechanisms regulating the steroid and stress response remains limited. Since the initial sequencing of the human glucocorticoid receptor &agr; and &bgr; gene (hGR&agr; and hGR&bgr;), only three additional splice variants have been identified—all of which have been postulated to contribute to steroid resistance. During a survey of 97 healthy humans’ blood, we identified two novel hGR splice isoforms (hGR-S1 and hGR-S1(−349A) retaining intron H between exons 8 and 9. Human GR-S1(−349A) contained a base deletion causing an early termination and a truncated protein of 118 amino acids, whereas hGR-S1 had an early termination occurring within intron H and resulted in a 745–amino acid protein. Both isoforms had decreased transactivation potentials compared with hGR&agr; when tested in the absence of exogenous steroids. However, after treating with exogenous steroids, dose-response studies showed hGR-S1(−349A) had a substantial augmentation in activity at higher concentrations of hydrocortisone and methylprednisolone when compared with hGR&agr;, whereas hGR-S1 did not. Removal of the 3′ untranslated region (3′UTR) of the hGR-S1(−349A) mRNA sequence resulted in a loss of the augmented response. The isoform hGR-S1(−349A) augments the response to steroids, and this significant response appears to be critically regulated by the 3′UTR. The identification and evaluation of these unique hGR isoforms helps further the understanding of the complex genetic regulation of the stress and steroid response.

Single nucleotide polymorphisms and type of steroid impact the functional response of the human glucocorticoid receptor

BACKGROUND Clinical trials evaluating the use of steroids in septic shock have shown variable outcomes. Our previous studies have implicated human glucocorticoid receptor (hGR) polymorphisms as a possible cause of altered steroid response. To further evaluate this variability, we hypothesized that hGR polymorphisms along with type of steroid influence the functional response. METHODS Total RNA was isolated from healthy human blood samples and surveyed for the hGR gene. The National Center for Biotechnology Information hGRα sequence was used as a reference, and two unique single nucleotide polymorphisms (SNPs) (A214G and T962C) were selected for evaluation. Functional response was measured using a luciferase reporting assay after transfecting hGR isoforms into tsA201 cells and stimulation with graded concentrations of hydrocortisone (HYD), methylprednisolone (MPS), and dexamethasone (DEX). RESULTS Each isoform had a unique dose-response curve with the optimal activity depending on concentration and type of steroid. The presence of either SNP A214G or T962C resulted in a decreased response when compared with hGRα when stimulated with HYD (P < 0.01). The same decreased response occurred for the SNPs with DEX stimulation, but at a much lower concentration range than HYD (P < 0.01). However, in the presence of MPS, SNP A214G resulted in greater activity when compared with hGRα (P < 0.01), whereas the presence of T962C resulted in activity equivalent to hGRα. CONCLUSIONS SNPs, type of steroid, and concentration range impact the functional response of the hGR. A greater understanding of hGR polymorphisms and steroid response may further elucidate mechanisms explaining the variable response seen with patient treatment.

Hyperactive Human Glucocorticoid Receptor Isoforms and Their Implications for the Stress Response

ABSTRACT Glucocorticoids are indispensable therapeutic agents in diseases of inflammation, but their effectiveness in treating advanced septic shock has been inconsistent. Our understanding of the mechanisms causing this variability to steroid therapy remains limited. Previous studies in our laboratory have implicated human glucocorticoid receptor (hGR) polymorphisms as one of the likely reasons for this variability. We examined the effect of two single-nucleotide polymorphisms (SNPs) on the transactivation potential of the hGR in the absence and presence of exogenous steroids. An isoform containing a novel naturally occurring human SNP, T1463C, was found to have a hyperactive response with treatment of all three steroids examined while maintaining low activity in the absence of steroids, relative to reference hGR. In comparison, another hGR isoform with the A2297G SNP, previously identified in our laboratory, demonstrated hyperactive transactivational response in the absence of steroids; however, it had a significant increase in activity after treatment with only one of the glucocorticoids (hydrocortisone) tested. These results offer a possible explanation for the clinical variability seen among individuals in response to stress or shock.

Temporal and spatial rearrangements of a repetitive element array on C57BL/6J mouse genome.

Repetitive elements (REs) make up the vast majority of the mammalian genomes. We identified species-specific genomic libraries of RE arrays. The non-random configurations of RE arrays suggest their functions. We tested whether RE arrays undergo age- and tissue/cell-specific rearrangements. An RE array of C57BL/6J mice, containing tandem repeats of a mosaic of transposable REs, was selected to examine rearrangements in different ages and tissues. There were marked changes in the array configuration in the genomes of the skin and brain in all mice of six weeks and older, whereas the heart and liver had alterations at 29weeks. The temporal variations were confirmed by identifying putative rearrangement junctions. Temporal and spatial rearrangements of certain RE arrays may contribute to the acquired characteristics of the genome information system.

A Tri-Nucleotide Pattern in a 3' Utr Segment Affects the Activity of a Human Glucocorticoid Receptor Isoform.

ABSTRACT We previously identified a truncated human glucocorticoid receptor (hGR) isoform of 118 amino acids, hGR-S1(-349A), that despite lacking the major functional domains, was more hyperactive after glucocorticoid treatment than the full-length receptor. Furthermore, its 3’ untranslated region (UTR) was required. To dissect the underlying mechanisms for hyperactivity, a series of hGR isoforms with consecutive deletions in the 3’ UTR were created to test their transactivation potential using reporter assays. The hGR-S1(-349A) isoform retaining 1303 bp of 3’ UTR displayed unusually high activity with or without glucocorticoid stimulation. Unexpectedly, a complete loss of significant activity was observed with isoforms retaining 1293 bp or 1263 bp of 3’ UTR. Analysis of the 20 bp region neighboring the 1293 bp site showed a pattern: 3’UTR termination at every third base pair in this region resulted in a loss of transactivation potential while the other sites retained hyperactivity with or without glucocorticoid stimulation. Variations in the activity of an hGR isoform, due to changes in the 3’ UTR sequence configuration, may provide an important link in explaining inconsistent responses to glucocorticoid treatment in individuals and ultimately enable tailored, patient-specific care. Furthermore, understanding the mechanisms underlying the cyclic hyperactivity/loss of activity phenomenon may be a step toward identifying a novel mechanism of gene regulation.

Injury-elicited stressors alter endogenous retrovirus expression in lymphocytes depending on cell type and source lymphoid organ

BackgroundMurine leukemia virus-type endogenous retroviruses (MuLV-ERVs) constitute ~10% of the mouse genome and are associated with various pathophysiologic processes. In this study, we examined whether MuLV-ERVs’ response to burn-elicited stressors is specific for certain lymphocyte populations and/or locations of lymphoid organ.ResultsB- and T-cells, which were sorted from nine lymphoid organs of C57BL/6J mice after burn, were subjected to MuLV-ERV expression analyses. Overall, the post-burn MuLV-ERV expression pattern was dependent on lymphocyte type, time after injury, location of lymphoid organ, and MuLV-ERV type. For instance, the MuLV-ERV expression in T-cells from the thymus and three cervical lymph nodes decreased at 3 hours post-burn while the expression of some MuLV-ERVs was augmented in B-cells derived from the mesenteric lymph node. The MuLV-ERV U3 sequences population of the burn-24 hours group was less diverse in comparison to the no burn and burn-3 hours groups. In addition, it was apparent that at the 24 hours time point, the U3 populations of B-cells from both no burn and burn groups were less heterogeneous than the T-cells’ U3 populations. Using the U3 sequences, some of which were isolated only from specific experimental groups (B- vs. T-cells; no burn vs. burn), as probes, 51 putative MuLV-ERVs, including 16 full-length proviruses, were mapped followed by characterization of their biologic properties.ConclusionMuLV-ERVs’ response to burn-elicited stressors may be differentially controlled depending on lymphocyte type, location of lymphoid organ, MuLV-ERV type, and stress duration.

A novel human glucocorticoid receptor SNP results in increased transactivation potential

Glucocorticoids are one of the most widely used therapeutics in the treatment of a variety of inflammatory disorders. However, it is known that there are variable patient responses to glucocorticoid treatment; there are responders and non-responders, or those that need higher dosages. Polymorphisms in the glucocorticoid receptor (GR) have been implicated in this variability. In this study, ninety-seven volunteers were surveyed for polymorphisms in the human GR-alpha (hGRα), the accepted biologically active reference isoform. One isoform identified in our survey, named hGR DL-2, had four single nucleotide polymorphisms (SNPs), one synonymous and three non-synonymous, and a four base pair deletion resulting in a frame shift and early termination to produce a 743 amino acid putative protein. hGR DL-2 had a decrease in transactivation potential of more than 90%. Upon further analysis of the individual SNPs and deletion, one SNP, A829G, which results in a lysine to glutamic acid amino acid change at position 277, was found to increase the transactivation potential of hGR more than eight times the full-length reference. Furthermore, the hGRα-A829G isoform had a differential hyperactive response to various exogenous steroids. Increasing our knowledge as to how various SNPs affect hGR activity may help in understanding the unpredictable patient response to steroid treatment, and is a step towards personalizing patient care.