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Biomedical subjects

John A Cidlowski

Publications and source records attributed to John A Cidlowski.

At least 19 recordsLinked to original sources
PubMed2020

Glucocorticoids mobilize macrophages by transcriptionally up-regulating the exopeptidase DPP4.

Glucocorticoids are potent endogenous anti-inflammatory molecules, and their cognate receptor, glucocorticoid receptor (GR), is expressed in nearly all immune cells. Macrophages are heterogeneous immune cells having a central role in both tissue homeostasis and inflammation and also play a role in the pathogenesis of some inflammatory diseases. Paradoxically, glucocorticoids have only a limited efficacy in controlling the resolution of these macrophage-related diseases. Here, we report that the transcriptomes of monocyte-like THP-1 cells and macrophage-like THP-1 cells (THP1-MΦ) have largely conserved gene expression patterns. In contrast, the differentiation to THP1-MΦ significantly altered the sensitivity of gene transcription to glucocorticoids. Among glucocorticoid-regulated genes, we identified the exopeptidase dipeptidyl peptidase-4 (DPP4) as a critical glucocorticoid-responsive gene in THP1-MΦ. We found that GR directly induces DPP4 gene expression by binding to two glucocorticoid-responsive elements (GREs) within the DPP4 promoter. Additionally, we show that glucocorticoid-induced DPP4 expression is blocked by the GR antagonist RU-486 and by GR siRNA transfection and that DPP4 enzyme activity is reduced by DPP4 inhibitors. Of note, glucocorticoids highly stimulated macrophage mobility; unexpectedly, DPP4 mediated the glucocorticoid-induced macrophage migration, and siRNA-mediated knockdowns of GR and DPP4 blocked dexamethasone-induced THP1-MΦ migration. Moreover, glucocorticoid-induced DPP4 activation was also observed in proinflammatory M1-polarized murine macrophages, as well as peritoneal macrophages, and was associated with increased macrophage migration. Our results indicate that glucocorticoids directly up-regulate DPP4 expression and thereby induce migration in macrophages, potentially explaining why glucocorticoid therapy is less effective in controlling macrophage-dominated inflammatory disorders.

Animals↗
PubMed2006

Multiple glucocorticoid receptor isoforms and mechanisms of post-translational modification.

Glucocorticoids regulate diverse physiological effects in virtually every organ and tissue in the body. Glucocorticoid actions are mediated through the glucocorticoid receptor (GR), a ligand-dependent transcriptional factor that activates or represses gene transcription. Since, the cloning of the human GR in 1985, research efforts have been focused on describing the mechanism of action exerted by one of the GR isoforms, GRalpha. However, recent studies from our lab and others have suggested that multiple isoforms of hGR are generated from one single gene and one mRNA species by the mechanisms of alternative RNA splicing and alternative translation initiation. These isoforms display diverse cytoplasm-to-nucleus trafficking patterns and distinct transcription activities. In addition, this new information predicts that each hGR protein can be subjected to a variety of post-translational modifications, such as phosphorylation, sumoylation and ubiquitination. The nature and degree of post-translational modification, as well as subcellular localization, may differentially modulate stability and function among the GR isoforms in different tissues providing an additional important mechanism for regulation of GR action. We outline the recent advances made in identifying the processes that generate and modify multiple GR isoforms and the post-translational modifications that contribute to the increasing diversity in the glucocorticoid signaling pathway.

Alternative Splicing↗
PubMed2006

Glucocorticoids regulate tristetraprolin synthesis and posttranscriptionally regulate tumor necrosis factor alpha inflammatory signaling.

Glucocorticoids are used to treat various inflammatory disorders, but the mechanisms underlying these actions are incompletely understood. The zinc finger protein tristetraprolin (TTP) destabilizes several proinflammatory cytokine mRNAs by binding to AU-rich elements within their 3' untranslated regions, targeting them for degradation. Here we report that glucocorticoids induce the synthesis of TTP mRNA and protein in A549 lung epithelial cells and in rat tissues. Dexamethasone treatment leads to a sustained induction of TTP mRNA expression that is abrogated by RU486. Glucocorticoid induction of TTP mRNA is also blocked by actinomycin D but not by cycloheximide, suggesting a transcriptional mechanism which has been confirmed by transcription run-on experiments. The most widely characterized TTP-regulated gene is the AU-rich tumor necrosis factor alpha (TNF-alpha) gene. Dexamethasone represses TNF-alpha mRNA in A549 cells and decreases luciferase expression of a TNF-alpha 3' untranslated region reporter plasmid in an orientation-dependent manner. Small interfering RNAs to TTP significantly prevent this effect, and a cell line stably expressing a short-hairpin RNA to TTP conclusively establishes that TTP is critical for dexamethasone inhibition of TNF-alpha mRNA expression. These studies provide the molecular evidence for glucocorticoid regulation of human TTP and reflect a novel inductive anti-inflammatory signaling pathway for glucocorticoids that acts via posttranscriptional mechanisms.

Adrenalectomy↗
PubMed2006

Functional analysis of the LXXLL motifs of the human glucocorticoid receptor: association with altered ligand affinity.

Although LXXLL motifs in coactivators mediate binding to liganded nuclear receptors, the roles of comparable motifs within nuclear receptors are less understood. We investigated the role of the LXXLL motifs in the human glucocorticoid receptor both in transcriptional activation and repression of gene expression. The two conserved LXXLL motifs within the ligand binding domain of the receptor, amino acids 532-536 (helix 1) and 718-722 (helix 10), were characterized by evaluating LXXLL mutant receptors as well as comparable mutants in other helices of the ligand binding domain. All mutant receptors were expressed at comparable levels to wild type in COS-1 cells. Mutation of 532-536 LXXLL to LXXAA completely disrupted dexamethasone induced transcription, whereas the 718-722 LXXAA mutant fully activated reporter genes at high ligand concentrations. Ligand binding analysis demonstrated that both LXXLL motif mutations resulted in disruption of ligand binding capacity without altering their association with hsp90. Proteolytic cleavage studies suggested that mutations of the LXXLL motifs introduced changes in the receptor conformation. Interestingly, the 532-536 LXXAA mutant was not able to transrepress NF-kappaB activity, whereas the 718-722 LXXAA mutant did so in the presence of ligand. These data suggest that although LXXLL motifs in helices 1 and 10 appear to lie outside the predicted ligand binding pocket they may contribute to receptor ligand binding affinity.

Amino Acid Motifs↗
PubMed2006

SLCO/OATP-like transport of glutathione in FasL-induced apoptosis: glutathione efflux is coupled to an organic anion exchange and is necessary for the progression of the execution phase of apoptosis.

Apoptosis is characterized by the activation of specific biochemical pathways that lead to the organized demise of cells. Intracellular GSH depletion has been observed during apoptosis; however, neither the mechanisms involved in the reduction of the intracellular GSH concentration, [GSH](i), nor its link to the progression of apoptosis have been elucidated. We have studied this issue using Fas ligand (FasL)-induced apoptosis in Jurkat cells where changes in [GSH](i) can be analyzed biochemically and at the single cell level by flow cytometry. A reduction in the total [GSH](i) in response to FasL occurs in two distinct stages prior to the loss of membrane integrity. Jurkat cells express several members of the multidrug resistance protein (ABCC/MRP), and the organic anion-transporting polypeptide protein (SLCO/OATP) families of GSH efflux pumps at the mRNA level. Glutathione loss and its accumulation in the extracellular medium, induced by FasL, was trans-stimulated by the organic substrates MK571, probenecid, taurocholic acid, estrone sulfate, and bromosulfophthalein and inhibited by high concentrations of extracellular GSH. Single cell analysis demonstrated that intracellular GSH loss was paralleled by the activation of an organic anion uptake process, supporting the role of an anion exchange mechanism (SLCO/OATP-like transport) in GSH efflux induced by FasL. Additionally, high extracellular GSH inhibited the activation of the execution caspases, the cleavage of their substrates poly(ADP-ribose) polymerase (PARP) and alpha-fodrin, and DNA degradation. In contrast, the trans-stimulation of GSH efflux by MK571 increased the cleavage of the execution caspases and their substrates. Together these results suggest that GSH efflux during FasL-induced apoptosis is mediated by a SLCO/OATP-like transport mechanism that modulates the progression of the execution phase of apoptosis.

Apoptosis↗
PubMed2006

Glucocorticoid receptor isoforms generate transcription specificity.

Glucocorticoids are necessary for life and are essential in all aspects of health and disease as they regulate processes from mitosis to apoptosis, from metabolism to growth and development. However, responses to glucocorticoids vary among individuals, cells and tissues. Recent evidence indicates that multiple glucocorticoid receptor (GR) isoforms are generated from one single GR gene by alternative splicing and alternative translation initiation. These isoforms all have unique tissue distribution patterns and transcriptional regulatory profiles. Furthermore, each is subject to various post-translational modifications that affect receptor function. Thus, increasing evidence suggests that unique GR isoform compositions within cells could determine the cell-specific response to glucocorticoids. Here, we discuss a new molecular model potentially underlying tissue-specific glucocorticoid resistance and selectivity.

Alternative Splicing↗
PubMed2006

The physiology of human glucocorticoid receptor beta (hGRbeta) and glucocorticoid resistance.

The development of glucocorticoid (GC) resistance is a serious problem that complicates the treatment of immune-related diseases, such as asthma, ulcerative colitis, and hematologic cancers. hGRalpha and hGRbeta are two isoforms of the human glucocorticoid receptor, which differ in the structural composition of the carboxy-terminal end of the ligand-binding domain and therefore in their ability to bind glucocorticoid ligand and in their physiological function. hGRalpha is the classically functional GR, while hGRbeta seems to act mainly as a dominant negative to the function of hGRalpha. Because of the ability of hGRbeta to antagonize the action of hGRalpha, it has been hypothesized that changes in the expression of hGRbeta may underlie the development of glucocorticoid resistance. In this article we review what is known about the expression and physiological action of hGRbeta in normal cells and tissue as well as in several disease states. Taken together, the evidence suggests that the ratio of hGRalpha:hGRbeta expression is indeed critical to the glucocorticoid responsiveness of various cells. This ratio can be altered by changing the expression level of hGRalpha, hGRbeta, or both receptors simultaneously. Higher ratios correlate with glucocorticoid sensitivity, while lower ratios correlate with glucocorticoid resistance. Thus hGRbeta can be an important modulator of glucocorticoid responsiveness.

Animals↗
PubMed2005

Selective role of intracellular chloride in the regulation of the intrinsic but not extrinsic pathway of apoptosis in Jurkat T-cells.

Apoptosis is a genetic program for the removal of unwanted cells from an organism, which is distinct from necrosis by its characteristic volume loss or apoptotic volume decrease. This cell shrinkage is the result of ion redistribution that is crucial for both the activation and execution of apoptosis. Here we report that UV-C but not Fas ligand treatment results in a significant decrease in intracellular chloride that can be abolished by modulation of chloride flux using either the chloride channel inhibitor SITS or medium with a reduced chloride concentration. Accordingly, downstream events are diminished during UV-C-induced apoptosis following chloride flux modulation, whereas Fas ligand-induced apoptotic characteristics are not affected. Moreover, the activation of the mitogen-activated protein kinase signal transduction pathway early in the apoptotic signaling cascade was affected by chloride flux in Jurkat T-cells. Thus, an alteration of intracellular chloride plays an important role in the activation of signaling molecules upstream of the mitochondria, specifically impairing the intrinsic but not extrinsic apoptotic pathway.

Apoptosis↗
PubMed2005

CD38 expression is insensitive to steroid action in cells treated with tumor necrosis factor-alpha and interferon-gamma by a mechanism involving the up-regulation of the glucocorticoid receptor beta isoform.

Evidence shows that the CD38 molecule, recently involved in the two main features of asthma, bronchial hyper-responsiveness and airway inflammation, could represent a new potential therapeutic target for asthma. In this study, we investigated whether glucocorticoid (GC), the most effective treatment for lung diseases, can affect CD38 expression in human airway smooth muscle (ASM) cells treated with different pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNFalpha) and interferons (IFNs). We found that CD38 expression induced by TNFalpha alone was completely abrogated by fluticasone (100 nM), dexamethasone (1 microM), or budesonide (100 nM). In contrast, the synergistic induction of CD38 by the combination of TNFalpha with IFNgamma or IFNbeta, but not with IL-1beta or IL-13, was completely insensitive to the GC inhibitory effects. We also found that TNFalpha and IFNgamma impaired GC responsiveness by inhibiting steroid induced both 1) GRalpha-DNA binding activity and 2) GC-responsive element-(GRE)-dependent gene transcription. Although levels of the GC receptor (GR) alpha isoform remained unchanged, expression of GRbeta, the dominant-negative GR isoform, was synergistically increased by TNFalpha and IFNgamma with a GRalpha/GRbeta ratio of 1 to 3. More importantly, fluticasone failed to induce GRE-dependent gene transcription and to suppress TNFalpha-induced CD38 expression in ASM cells transfected with constitutively active GRbeta. We conclude that, upon pro-inflammatory cytokine stimulation, CD38 expression becomes insensitive to GC action by a mechanism involving the up-regulation of GRbeta isoform, thus providing a novel in vitro cellular model to dissect GC resistance in primary cells.

ADP-ribosyl Cyclase 1↗
PubMed2005

Estrogens and glucocorticoids have opposing effects on the amount and latent activity of complement proteins in the rat uterus.

The mammalian uterus faces unique immunological challenges. It must nurture and protect the semiallogenic fetus from attack by the maternal immune system while guarding against infection by pathogens that compromise fetal and maternal health. Complement has recently been implicated in the etiology of pregnancy loss, but its regulation by steroid hormones and its role in host defense in the uterus are not clearly defined. Here we use biochemical, functional, and physiological assays to elucidate the regulation of complement proteins in the rat uterus. We demonstrate that estrogens (17 beta-estradiol) and glucocorticoids (dexamethasone) have major, but opposing, effects on the amount and latent activity of complement effectors in the uterus. Treatment with 17 beta-estradiol induced vasodilation and an increase in vascular permeability, which resulted in extravasation of plasma and complement into the uterus, rather than de novo complement biosynthesis. In vitro assays revealed that 17 beta-estradiol induced a potent bactericidal activity in uterine luminal fluid and that the antibacterial component was complement. These proinflammatory and immunomodulatory effects were evident within 4 h of treatment and were blocked by coadministration of dexamethasone. We also found that estrogen effects on the vasculature were mediated in part by activation of the contact system and bradykinin B1 receptors. These results indicate that complement plays a central role in innate immunity in the female reproductive tract and suggest that estrogens or glucocorticoids might be used therapeutically to enhance or inhibit complement-dependent processes in the uterus.

Animals↗
PubMed2005

Translational regulatory mechanisms generate N-terminal glucocorticoid receptor isoforms with unique transcriptional target genes.

Glucocorticoids regulate diverse physiological functions ranging from mitosis to apoptosis, although only one glucocorticoid receptor (GR) gene has been discovered. We report here that one single GR mRNA species unexpectedly produces at least eight functional GR N-terminal isoforms via translational mechanisms. These GR isoforms display diverse cytoplasm-to-nucleus trafficking patterns and distinct transcriptional activities. In human osteosarcoma cells, the transcriptional responses to glucocorticoids closely reflect the identity and abundance of the GR isoforms. In addition, each GR isoform regulates both a common and a unique set of genes in the same cell. Interestingly, the levels of these GR isoforms differ significantly among tissues. Based on these observations, we propose that cell-type specific GR isoforms generate specificity in glucocorticoid control of transcription in different tissues.

Alternative Splicing↗
PubMed2005

Ligand-selective targeting of the glucocorticoid receptor to nuclear subdomains is associated with decreased receptor mobility.

The association between nuclear distribution and mobility of the human glucocorticoid receptor was examined in living COS-1 cells using yellow fluorescent protein- and cyan fluorescent protein-tagged receptors. Quantitation of the nuclear distribution induced by an array of glucocorticoid ligands revealed a continuum from a random (cortisone) to a nonrandom (triamcinolone acetonide) receptor distribution. Structure-function analysis revealed that the 9-fluoro and 17-hydroxy groups on the steroid significantly impact nuclear receptor distribution. Using time-lapse microscopy, the triamcinolone acetonide-induced receptor distribution did not change significantly over a period of 15 sec. However, using fluorescence recovery after photobleaching, the individual receptors moved at a much faster rate, indicating rapid exchange of receptors on immobile nuclear subdomains. Receptor mobilities for 13 different steroids, measured by fluorescence recovery after photobleaching, appeared to correlate with receptor distribution. Ligands that induced a nonrandom distribution induced slower receptor mobility and vice versa. Finally, application of 2-photon confocal microscopy revealed differences in receptor mobility between nuclear subdomains. Areas of high receptor concentration showed slower mobility than areas of low receptor concentration. Thus, glucocorticoid receptors can be targeted (depending on the ligand) to relatively immobile nuclear subdomains. The transient association of receptor with these domains decreases the mobility of the receptor.

Animals↗
PubMed2004

Activation of intrinsic and extrinsic pathways in apoptotic signaling during UV-C-induced death of Jurkat cells: the role of caspase inhibition.

We have examined UV irradiation-induced cell death in Jurkat cells and evaluated the relationships that exist between inhibition of caspase activity and the signaling mechanisms and pathways of apoptosis. Jurkat cells were irradiated with UV-C light, either with or without pretreatment with the pan-caspase inhibitor, z-VAD-fmk (ZVAD), or the more selective caspase inhibitors z-IETD-fmk (IETD), z-LEHD-fmk (LEHD), and z-DEVD-fmk (DEVD). Flow cytometry was used to examine alterations in viability, cell size, plasma membrane potential (PMP), mitochondrial membrane potential (DeltaPsi(mito)), intracellular Na(+) and K(+) concentrations, and DNA degradation. Processing of pro-caspases 3, 8, and 9 and the pro-apoptotic protein Bid was determined by Western blotting. UV-C irradiation of Jurkat cells resulted in characteristic apoptosis within 6 h after treatment and pretreatment of cells with ZVAD blocked these features. In contrast, pretreatment of the cells with the more selective caspase inhibitors under conditions that effectively blocked DNA degradation and inhibited caspase 3 and 8 processing as well as Bid cleavage had little protective effect on the other apoptotic characteristics examined. Thus, both intrinsic and extrinsic pathways are activated during UV-induced apoptosis in Jurkat cells and this redundancy appears to assure cell death during selective caspase inhibition.

Apoptosis↗
PubMed2004

A single amino acid change in the first zinc finger of the DNA binding domain of the glucocorticoid receptor regulates differential promoter selectivity.

Mammalian species are well known to differ in their sensitivity to glucocorticoids, but the molecular basis for this difference remains largely uncharacterized. To address this issue, the transcriptional activity of the mouse and human glucocorticoid receptor (GR) was analyzed on two model glucocorticoid-responsive promoters. Mouse GR (mGR) displayed unique promoter discrimination in response to a range of glucocorticoids, with enhanced activity on a simple glucocorticoid response element (GRE)-based promoter and diminished activity on the complex mouse mammary tumor virus promoter compared with human GR (hGR). Promoter discrimination between mGR and hGR was mapped to a single amino acid change at residue 437 (glycine to valine) of mGR and to sequence differences within individual GREs of the different promoters. Mouse GR displayed higher activation on GREs with a guanine rather than a thymine at the -6 position. Binding studies indicated mGR (mGR437V) displayed a weaker affinity for GREs containing a thymine at the -6 position than a mGR mutant containing a glycine at residue 437 (mGR437G). Despite distinct transcriptional activities, both receptors had similar affinities for response elements that contain a guanine at the -6 position. Our findings support a model by which the presence of a valine residue at position 437 of mGR induces a conformational change that leads to alterations in affinity and/or transcriptional activation in a promoter-dependent context.

Amino Acid Sequence↗
PubMed2004

The role of apoptotic volume decrease and ionic homeostasis in the activation and repression of apoptosis.

The mechanism of activation and repression of apoptosis has been a central focus of many studies examining the role of programmed cell death in both normal and pathological conditions. Despite intensive research efforts, the precise cellular and molecular mechanisms that trigger and/or prevent apoptosis remain undefined. A universal characteristic of apoptosis is the loss of cell volume or cell shrinkage, recently termed apoptotic volume decrease. While cell shrinkage has traditionally been viewed as a passive event during apoptosis, recent work from several laboratories has shown that the loss of cell volume, or more specifically the flux of ions associated with the change in cell size, play a critical role in the regulation of the cell death machinery. On going studies continue to support the hypothesis that the change in intracellular ions can alter a cells decision to die by apoptosis.

Animals↗
PubMed2004

Glucocorticoids and tumor necrosis factor alpha cooperatively regulate toll-like receptor 2 gene expression.

Tumor necrosis factor alpha (TNF-alpha) and glucocorticoids are widely recognized as mutually antagonistic regulators of adaptive immunity and inflammation. Surprisingly, we show here that they cooperatively regulate components of innate immunity. The Toll-like receptor 2 (TLR2) gene encodes a transmembrane receptor critical for triggering innate immunity. Although TLR2 mRNA and protein are induced by inflammatory molecules such as TNF-alpha, we show that TLR2 is also induced by the anti-inflammatory glucocorticoids in cells where they also regulate MKP-1 mRNA and protein levels. TNF-alpha and glucocorticoids cooperate to regulate the TLR2 promoter, through the involvement of a 3' NF-kappaB site, a STAT-binding element, and a 3' glucocorticoid response element (GRE). Molecular studies show that the IkappaBalpha superrepressor or a STAT dominant negative element prevented TNF-alpha and dexamethasone stimulation of TLR2 promoter. Similarly, an AF-1 deletion mutant of glucocorticoid receptor or ablation of a putative GRE notably reduced the cooperative regulation of TLR2. Using chromatin immunoprecipitation assays, we demonstrate that all three transcription factors interact with both endogenous and transfected TLR2 promoters after stimulation by TNF-alpha and dexamethasone. Together, these studies define novel signaling mechanism for these three transcription factors, with a profound impact on discrimination of innate and adaptive immune responses.

Binding Sites↗