Glucocorticoids are one of the most widely prescribed therapeutics in the world. They are used to treat a wide variety of inflammatory disorders including asthma, rheumatoid arthritis, Crohn disease, and on occasion, sepsis (1–4). Unfortunately, patient response to steroids is inconsistent since some patients fail to respond or even develop a resistance to treatment. Sepsis is a major clinical concern, with 850,000 to 3,000,000 patients in the United States affected yearly; up to 25% of septic patients die (4, 5). The problem is even greater in burn patients, with 50% to 84% of cases listing sepsis as the primary cause of death (6). Despite the widespread administration of glucocorticoid therapy in treating sepsis, the effectiveness of its use is still debated, primarily because of different patients having varying responses to treatment (7, 8). In one study, septic patients were tested for their response to adrenocorticotropic hormone (ACTH). Those patients who failed to respond to ACTH were found to do better with exogenous steroid treatment (9). Unfortunately, subsequent studies failed to support those findings (10). Despite several prospective studies demonstrating no improvement, the Surviving Sepsis Campaign still supports the use of hydrocortisone for a septic patient who is unresponsive to other treatments (4).
Glucocorticoids mediate their function via binding to the human glucocorticoid receptor (hGR). hGR is a member of the steroid nuclear receptor family and its activation induces an anti-inflammatory response in the body. There are several mechanisms for hGR activity but little is known about why some people respond to glucocorticoid therapy and others do not. Different theories have been proposed to explain these variations, such as timing of when glucocorticoid treatment is administered, changes in hGR expression levels, shifts in the hGR isoform ratio, alterations in nuclear translocation, and variations in hGR ligand binding activity (7, 11–13). Our study of a normal population identified numerous previously unreported naturally occurring variations in hGR (14). We have found that these alterations can impact the transactivation potential of hGR, and more importantly, their responsiveness to glucocorticoid treatment in a type and dosage-specific manner (14, 15). This has led us to hypothesize that a major cause of variable response to steroids is due to differences in hGR isoform expression. We also believe that each person's hGR expression profile can change through stress-induced alternative splicing of hGR. mRNA splicing involves processing the pre-mRNA to remove the intron sequences and then joining the remaining exons. Therefore, alternative splicing effectively generates genetic diversity through the inclusion or exclusion of different combinations of exons, thereby creating multiple proteins from one gene. It has been reported that over 80% of human genes are believed to undergo alternative splicing (16). The hGR is known to be made up of nine exons (17). Exon nine can be alternatively spliced into either exon 9α or exon 9ß. Exon 9α creates the reference isoform, hGRα, which binds ligand and is responsible for the well-known anti-inflammatory actions of hGR. Exon 9ß, on the other hand, generates hGRß, which has been found to be a competitive inhibitor of hGRα (17, 18). Although other splice variants have been identified (hGR-S1, hGRγ, hGR-A, and hGR-P), hGRα and hGRß are the most widely studied (15, 19, 20).
Recently, our lab also identified multiple hGR splice isoforms in burn patients whose hGR expression changed throughout their disease courses. Since patients with extensive burns have multiple septic episodes, we tested the hypothesis that lipopolysaccharide (LPS) stress could induce the expression of these multiple hGR variants. This manuscript describes the identification and functional characterization of three novel LPS-responsive hGR splice variants, hGR-B(93), hGR-B(77), and hGR-B(54) (Fig. 1A).
PATIENTS AND METHODS
Collection of burn patient buffy coats
All protocols were approved by the Institutional Review Board (IRB) of the University of California, Davis, and written informed consent was obtained for all participants. One hundred ten patients from Shriners Hospitals for Children Northern California (SHCNC) and the University of California, Davis Medical Center (UCDMC) Burn Units were enrolled in the study. Inclusion criteria were patients greater than 1 year of age with ≥20% total body surface area burn and admitted to the hospital within 96 hours of injury. Excluded were patients with a known adrenal or hypothalamic pre-existing condition or prior administration of exogenous steroids for clinical purposes. The study population was approximately half adult (A) and half children under 18 years of age (C) (A:C, 45%:55%), and predominately male (74% male: 26% female). Blood was collected from patients after admission, every 2 weeks thereafter, and during septic episodes. Buffy coat was isolated from the blood samples for RNA analysis. Sepsis was determined by the attending burn surgeons at SHCNC and UCDMC based on the American Burn Association's Consensus Definitions for sepsis and septic shock (fever >39o C, tachycardia, tachypnea, dropping platelet count, feeding intolerance, diarrhea; along with a suspected source of infection) (21).
Stimulation of PBMCs with LPS
Three Leukopaks (50-year-old female, 53-year-old male, 64-year-old male) were obtained from a local blood bank (BloodSource, Mather, Calif). Leukopaks are a white blood cell concentrate of the donor's whole blood prepared by leukoreduction using a Trima collection device from Caridian (Lakewood, Colo). Peripheral blood mononuclear cells (PBMCs) were then isolated using a Histopaque-1077 gradient (Sigma, St. Louis, Mo) followed by removal of residual red blood cells with ammonium-chloride-potassium lysis buffer. Cells were then resuspended in RPMI-1640 media (ThermoFisher Scientific, Waltham, Mass) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, Ga), counted, plated (9.7 × 106 to 10 × 106 cells/well), and incubated at 37°C with 5% CO2 for 1 h. PBMCs were then treated, in triplicate, with 5 μg/mL LPS from E coli serotype 026:B6 (Sigma), or vehicle control (equivalent dilution of H2O). 5 μg/mL of LPS was found to optimally stimulate the production of tumor necrosis factor α, interleukin (IL)-1ß, and IL-6 cytokines in whole blood and PBMCs by De Groote et al. (22). PBMCs were then allowed incubate for an additional 1, 3, or 13 h at 37°C with 5% CO2 before harvest.
Identification and nomenclature of hGR isoforms
Total RNA was isolated from PBMCs using an RNeasy mini kit (Qiagen, Valencia, Calif) and from buffy coat samples with a modified RNeasy protocol following lysis with TRIzol Reagent (ThermoFisher Scientific). Alternative spice variants were then identified by RT-PCR with a QuantiTect RT kit (Qiagen) using an oligo dT primer, Taq DNA polymerase (Qiagen), and a series of primers that amplified various hGRα exon-to-exon combinations. The exon-to-exon RT-PCR enables a high-resolution screening for hGR variations that may be missed in a single full-length hGRα amplification due to small size differences or lower transcript quantity. This method is able to detect novel splicing events by the appearance bands either above or below the expected band size. hGR-B(93) and hGR-B(77) were identified in exon 2-to-5 amplifications (forward 5’-tggaataggtgccaagg-3’, reverse 5’-cagggtaggggtgagttgtggt-3’), and hGR-B(54) was identified in an exon 2-to-6 amplification (forward 5’-gtcattccaccaattcccgttgg-3’, reverse 5’-cacagcaggtttgcac-3’). Full-length hGRα was amplified using Phusion High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, Mass) or OneTaq DNA Polymerase (New England Biolabs) and a specific primer set (forward 5’-ttcactgatggactccaaagaatcattaac-3’, reverse ‘5- ggtgccatccttctttgactgtg-3’). Amplicons were ligated into the pGEM-T Easy vector (Promega, Madison, Wis) and sequenced at GENEWIZ (South San Francisco, Calif) or Molecular Cloning Laboratories (South San Francisco, Calif). Splice variants were identified by comparison to the National Center for Biotechnology Informatics (NCBI) reference sequences: NM_000176.2 and NG_009062. Subsequently, primers were specifically designed to screen for the new cryptic exons by RT-PCR (54 bp: forward 5’-tcacctcctgccatgattctg-3’, reverse 5’-cttccactgctcttttgaagaa-3’; 93 bp: forward 5’-gtcattccaccaattcccgttgg-3’, reverse 5’-tcatgctggggcgatgtattag-3’; 77 bp: forward 5’-gtcattccaccaattcccgttgg-3’, reverse 5’-tcatgctggggagttctgcatgg-3’). Beta-2-microglobulin (B2M) was used as an expression control (forward 5’-agcagagaatggaaagtcaaa-3’, reverse 5’-tgttgatgttggataagagaa-3’).
hGRα represents the sequence matching to the NCBI reference sequence, NM_000176.2. The new splice variants were named as “hGR-” followed by the intron in which the cryptic exon was located, and then the size of the exon in parentheses. For example, the 93 bp cryptic exon isoform is named hGR-B(93).
Creation of hGR-B(93), hGR-B(77), and hGR-B(54) expression constructs
hGR-B(93) and hGR-B(77) isoforms were identified in hGRα full-length amplicons after ligating into the pGEM-T Easy vector (Promega) and sequence confirmation. The sequence containing the coding region was then cut with restriction enzymes and sub-cloned into a pcDNA4/HisMax expression vector (ThermoFisher Scientific). The 54 bp exon was identified in an exon 2–6 amplicon. hGR-B(54) was created by combining a full-length hGRα clone and the exon 2–6 amplicon clone containing the 54 bp exon into pcDNA4/HisMax using the NEBuilder HiFi DNA Assembly Cloning Kit (New England BioLabs) and Phusion High-Fidelity DNA Polymerase (New England BioLabs).
tsA201 cells (an HEK 293 cell subclone stably transfected with the SV40 large T-antigen) (Sigma-Aldrich, St. Louis, Mo) were seeded at 10,000 cells per well in a 96-well plate in 100 μL of antibiotic free Dulbecco's Modified Eagle Medium (ThermoFisher Scientific) supplemented with 10% fetal bovine serum (Atlanta Biologicals) and incubated at 37°C with 5% CO2 overnight. Cells were then transfected with an hGR isoform (0.5 ng) and a GRE-luciferase reporter plasmid (PathDetect GRE Cis-Reporter Plasmid; Agilent Technologies, La Jolla, Calif) (0.5 ng) using Fugene 6 (Promega) (0.2 μL) per the protocol provided by the manufacturer. At 24 h after transfection, the cells were treated with graded doses of hydrocortisone at 0.1 μM to 10 μM or similarly diluted 0.9% saline (vehicle control) for another 24 h. Pharmaceutical-grade hydrocortisone sodium succinate (Pfizer, New York, NY) was used. The transactivation potential was then determined with a Luciferase Assay Kit (Agilent Technologies) and a luminescence plate reader, the Spectramax i3x (Molecular Devices, Sunnyvale, Calif).
tsA201 cells were seeded at 5 × 105 cells/well in 6-well plates and transfected with Fugene 6 (Promega) per the manufacturer's protocol (1 μg DNA: 3 μL Fugene 6). Whole cell protein lysates were prepared with Cell Lysis Buffer (Agilent) and cOmplete Protease Inhibitor Cocktail (Roche, Indianapolis, Ind). Nuclear and cytoplasmic protein fractions were prepared with a Cell Fractionation Kit (Cell Signaling Technology, Danvers, Mass). Equal amounts of protein were run on 4% to 20% Criterion TGX gels (BioRad, Hercules, Calif) and analyzed with the Glucocorticoid Receptor (D8H2) XP antibody (1:1,000; Cell Signaling Technology) by Western blot. A ß-actin (1:10,000; Abcam, Cambridge, Mass) Western blot was added as a loading control for the whole cell lysates.
All luciferase assays were run in triplicate, except for vehicle controls which were run in duplicate. The experiment was repeated three times to confirm the patterns, and the data shown is combined from the three experiments. Data are presented as means with error bars representing the standard error of the mean. One-way ANOVA followed by a Tukey post hoc test was used to confirm statistical significance.
The sequence data from this study have been deposited into GeneBank and have been assigned accession numbers: hGR-B(93), MH938595; hGR-B(54), MH938956; hGR-B(77), MH938597.
Identification of cryptic exons
In order to understand how hGR responds to septic stress, an in vitro LPS stimulation model was used. In this study, PBMCs isolated from Leukopaks were stimulated with LPS, followed by a screening for variations in hGR expression. The exon-to-exon screening of the LPS-treated PBMCs revealed the expression of three novel cryptic exons of 93 bp, 77 bp, and 54 bp located between exons two and three of hGRα (Fig. 1A). Cryptic exons are exons that are not annotated in the genomic databases and are not part of the canonical transcript but are occasionally incorporated; what leads to their inclusion is not yet fully understood (23). The 93 bp exon was identified in an exon 2-to-5 screening and 54 bp exon in an exon 2-to-6 screening. Retention of the 93 bp exon resulted in a 416 amino acid putative protein in which the last 21 bases differ from reference hGRα. This isoform was designated as hGR-B(93). hGR-B(54), the isoform retaining the 54 bp exon, produced a slightly smaller putative protein of 402 amino acids, of which the last seven amino acids diverge from hGRα. Lastly, the new 77 bp exon was found in an exon 2-to-5 amplicon from an LPS-stressed Leukopak. This variant was initially identified in buffy coat cells isolated from a burn patient. hGR-B(77), the resultant isoform, had the shortest putative protein at 395 amino acids, in which only the last amino acid differs from hGRα. The hGR-B(93) and hGR-B(77) isoforms were also subsequently identified in full-length hGRα RT-PCR reactions. A survey of the hGR genomic sequence determined that the standard splice donor and acceptor sites are present for all of the new cryptic exons (Fig. 1B).
Stress-induced expression of cryptic exon splicing
The initial hGR screening of the LPS-treated PBMCs detected the presence of the novel splice variants with exon-to-exon RT-PCR reactions. To directly examine the expression of hGR-B(93), hGR-B(77), and hGR-B(54) in response to LPS stimulation, primer sets were designed to specifically amplify the new exons. We found that the expression of all three exons increased as a result of LPS stimulation in the Leukopak PBMCs in a time-dependent manner (Fig. 2). Their observed expression was greatest at 3 h and started to decline by 13 h. The 54 bp exon isoform, however, increased in expression as early as 1 h after LPS exposure. Another interesting phenomenon was observed in the 93 bp exon RT-PCR. A slightly higher band (indicated with arrow) was found to be a combination of the 93 bp and 77 bp exons. The observed expression of this fragment was consistently greatest at 3 h.
Subsequently, we determined if the new exon isoforms were expressed in burn patients. The buffy coat was isolated from blood samples collected from patients and the same primer sets were used to screen for the presence of the new exons. Preliminary results from multiple subjects indicated that the cryptic exon isoforms are expressed in the patients (Fig. 3). However, unlike the LPS-treated Leukopak PMBCs, the results from multiple experiments using the patient samples were variable. The Leukopak PBMCs were subjected to a single stressor, LPS, which is significantly different from the burn patients who experience multiple stressors that dynamically change and may synergistically influence hGR isoform expression. Consequently, it is apparent that there is variability in the expression patterns of hGR-B(93), hGR-B(77), and hGR-B(54), and that one potential factor is LPS. The patients’ samples were tested only to confirm the presence of the three cryptic exon isoforms, not for a correlation with clinical conditions. Understanding what influences the expression of alternatively spliced hGR isoforms is extremely more complex than we thought and will require more studies to clearly decipher the underlying mechanisms.
Differential transactivation potential of splice variants in response to exogenous hydrocortisone
Expression constructs were created to evaluate the activity of the hGR-B(93), hGR-B(77), and hGR-B(54) novel splice variants. Their transactivation potential was then measured in comparison with hGRα via a luciferase assay with graded doses of hydrocortisone in tsA201 cells. Our previous studies have shown that hGRα has a dose-dependent response to hydrocortisone treatment that peaks at ∼10–2 μM; however, the optimal dosage range for the splice variant isoforms was shifted higher (0.1 μM, 1 μM, and 10 μM), so it only overlapped the tail end of the hGRα dosage curve (Fig. 4) (15). We found that two of the splice variant isoforms, hGR-B(77) and hGR-B(54), had negligible responsiveness to hydrocortisone in comparison with hGRα with only ∼35% of the activity of hGRα at their peak (10 μM). However, hGR-B(93), which lacks the ligand binding domain, had an augmented response to hydrocortisone stimulation. The activity of hGR-B(93) was significantly greater than hGR-B(77) and hGR-B(54) at all concentrations of hydrocortisone tested and was greatest at 10 μM. At its peak, the activity of hGR-B(93) was ∼1.2 times greater than hGRα.
Differential protein expression patterns of splice variant isoforms
We also examined the protein expression and subcellular localization of all three splice variant constructs by Western blot. The initial Western blot confirmed their expression and found that all three splice variants express proteins around 55 kd (Fig. 5A). hGR-B(77) had the greatest amount of protein expression, while hGR-B(93) had the least, despite being the hyperactive isoform. In addition to alternative splicing, hGR has been found to have alternative translation initiation start sites producing the hGR variants hGR-B (∼91 kd), hGR-C1, C2, C3 (∼84–84 kd), and hGR-D1, D2, D3 (∼53–56 kd) (24). hGR-B(77) also expressed two lower bands at approximately 37 kd and 16 kd which could potentially be isoforms resulting from the alternative translation initiation start sites, C1–3 and D1–3, respectively in conjunction with early terminations (Fig. 5A). Additional bands, however, were not present in the hGR-B(54) and hGR-B(93) samples. Subsequently, cytoplasmic and nuclear fractionated protein extracts were prepared to determine the localization of the isoforms (Fig. 5B). We found that the three splice variants had a similar expression pattern in both the cytoplasmic and nuclear compartments with hGR-B(77) having the greatest expression and hGR-B(93) having the least. However, in the nuclear fraction, the ∼16 kd lower band from hGR-B(77) was not present.
Since the coding sequence for hGR was first identified by Hollenberg et al. (25) in 1985, it has been known that hGR undergoes alternative splicing; however, the function of these splice variants is less understood. One variant, hGRß, has been linked to inflammatory diseases such as glucocorticoid resistant asthma and rheumatoid arthritis (26, 27). hGRß lacks a functional ligand binding domain; however, it can still dimerize with hGRα, the major reference isoform, and thereby impede its function (17, 18, 28). Investigations into hGRß function have found that it is able to regulate the transcription of certain genes independent of the classic glucocorticoid response element (GRE) pathway but its exact method of action is still unknown (29). The hGR-A and hGR-P variants were first identified in multiple myeloma cells lines (19). Exons 5–7 are spliced out from hGR-A and hGR-P retains intron G between exons 7 and 8. Both splice variants result in truncated hGR proteins. hGR-P has since been found in patients with hematological malignancies and other tumors at varying levels of expression and was able to enhance the activity of hGRα in some cell lines (30). Another splice variant that was previously identified by our lab, hGR-S1, retains all of intron H between exons 8 and 9, and was significantly less responsive to glucocorticoids compared to hGRα (15).
Although the concept of alternative splicing is not new, the prevalence of this phenomenon is only beginning to be understood. With the advent of RNA sequencing (RNA-seq), numerous non-annotated splicing events have been identified in the human genome (23). Here, we identified three novel splice variants that incorporated cryptic exons and their expression consistently increased as a result of LPS stress in vitro. These isoforms also have variable expression in vivo in burn patents. It has been previously reported by Molina et al. that LPS can increase the expression of hGRα in PBMCs, and that LPS can differentially induce the expression of hGRα and hGRß isoforms in a cell type specific manner (31). It has been found that mouse GRß, arising from alternative splicing of intron 8, has similar properties to hGRß (32). In a study examining changes in glucocorticoid receptor expression in response to sepsis in mice, Abraham et al. (33) also found that cecal ligation and puncture in mice predominantly results in a decrease in GRα and an increase in GRß expression in the tissues they examined. They also found a significant increase in hGRß expression in tissues from recently deceased septic patients (33). To our knowledge, LPS induction of hGR cryptic splice variants has not been reported, but it is consistent with these reports that show that LPS stress can induce alternative splicing in hGR. LPS is a major cause of inflammatory responses so it would not be surprising that receptors involved in stress response have developed mechanisms for adapting to its presence. Unfortunately, most hGR splicing studies focus on hGRα and hGRß, thus bypassing other possible splicing events such as the ones reported here. Nonetheless, the inclusion of cryptic exons as a result of stress is not a novel phenomenon. The retention of a 31 bp cryptic exon between exons 4a and 4b of neurofibromatosis type 1 was found to be induced by cold shock (34). And in mouse BALB/c 3T3 fibroblast and teratocarcinoma F9 cells, heat shock induces the alternative splicing of HSP47 to include a 169 bp fragment in the 5’ untranslated region which increased its translation efficiency at high temperatures (35).
The inclusion of cryptic exons into coding sequences often results in premature termination codons (PTCs), which can trigger nonsense mediated decay (NMD) (23). NMD is the process whereby cells degrade potentially deleterious transcripts containing PTCs responsible for truncated proteins (36). Interestingly, it has been found that cellular stress, such as hypoxia, can inhibit NMD (37). It is possible that LPS stress can also inhibit NMD, but that has yet to be determined. Although the 54 bp and 93 bp exons are in-frame insertions, they both have in-frame stop codons which result in truncated proteins. The 77 bp exon is not in-frame and thus causes a shift which results in early termination which is due to a PTC. As such, the increase in their transcription may be a signal of cellular stress. Furthermore, the increase in the transactivation potential of hGR-B(93) at higher concentrations of hydrocortisone indicates that this isoform may play a role in the stress response. hGR-B(77) and hGR-B(54) may also contribute to the stress response through unknown interactions with hGRα or other proteins. We previously reported another truncated hGR splice variant that unexpectedly had an elevated response to hydrocortisone at high concentrations, hGR-S1(-349A), which in addition to retaining intron H also had a deletion at position 349 (15). Because hGR-B(93) and hGR-S1(-349A) lack the hGR DNA and ligand binding domains, it is likely that their effects are mediated by an, as yet, unidentified alternative pathway.
Here, we have identified three novel alternative splice variants of hGR which incorporate cryptic exons. The exact cause and impact of the induction of these splice variants has yet to be determined. However, preliminary results have revealed the expression of all three splice variants in our burn patient population at various time points in their clinical course. A significantly larger patient population and additional data analysis is needed to determine what factors influence their expression, and if any of the splice variants correlate with specific patient conditions, especially sepsis. If the expression of any of these isoforms can be correlated with sepsis pathophysiology, especially hGR-B(54) because of its early induction by LPS, it would be a significant contribution to patient monitoring and early intervention in sepsis treatment. It also needs to be determined whether signaling through pathogen response receptors, such as toll-like receptor-4, is involved in inducing splice variants. In future studies, we will investigate how LPS induces these hGR splice variants and if LPS signaling pathways are involved.
This study was made possible by contributions from the Burn Division Clinical Research Group at Shriners Hospitals for Children Northern California and the UC Davis Medical Center: Mary Beth Lawless, Terese Curri, Katrina Falwell, Carol Kinkennon, Cassie Conover, Angela Mix, Isabel Reyes, and Lynda Painting, who enrolled patients, collected patient samples, maintained patient data, and managed IRB compliance.
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