John A. Cidlowski

Characterization of mechanisms involved in transrepression of NF-kappa B by activated glucocorticoid receptors.

Glucocorticoids are potent immunosuppressants which work in part by inhibiting cytokine gene transcription. We show here that NF-kappa B, an important regulator of numerous cytokine genes, is functionally inhibited by the synthetic glucocorticoid dexamethasone (DEX). In transfection experiments, DEX treatment in the presence of cotransfected glucocorticoid receptor (GR) inhibits NF-kappa B p65-mediated gene expression and p65 inhibits GR activation of a glucocorticoid response element. Evidence is presented for a direct interaction between GR and the NF-kappa B subunits p65 and p50. In addition, we demonstrate that the ability of p65, p50, and c-rel subunits to bind DNA is inhibited by DEX and GR. In HeLa cells, DEX activation of endogenous GR is sufficient to block tumor necrosis factor alpha or interleukin 1 activation of NF-kappa B at the levels of both DNA binding and transcriptional activation. DEX treatment of HeLa cells also results in a significant loss of nuclear p65 and a slight increase in cytoplasmic p65. These data reveal a second mechanism by which NF-kappa B activity may be regulated by DEX. We also report that RU486 treatment of wild-type GR and DEX treatment of a transactivation mutant of GR each can significantly inhibit p65 activity. In addition, we found that the zinc finger domain of GR is necessary for the inhibition of p65. This domain is also required for GR repression of AP-1. Surprisingly, while both AP-1 and NF-kappa B can be inhibited by activated GR, synergistic NF-kappa B/AP-1 activity is largely unaffected. These data suggest that NF-kappa B, AP-1, and GR interact in a complex regulatory network to modulate gene expression and that cross-coupling of NF-kappa B and GR plays an important role in glucocorticoid-mediated repression of cytokine transcription.

A Primary Role for K+ and Na+ Efflux in the Activation of Apoptosis

Cell shrinkage is a major characteristic of apoptosis, but the mechanism and role of this process in cell death are poorly understood. The primary factor that controls volume regulation in all cells is ions, and thus we have examined the movement of ions at the single cell level in lymphocytes during apoptosis. Activation of the death program with several stimuli that act through independent pathways to stimulate apoptosis results in a synchronous shift of cells from a normal cell size to a shrunken cell size. Only the shrunken cells exhibit DNA fragmentation and an approximate 4-fold elevation of caspase-3-like activity. Analysis of K+ and Na+ ion content of individual cells by flow cytometry revealed that the intracellular ionic strength of apoptotic cells decreased substantially from their non-shrunken counterparts. Additionally, we show apoptosis is enhanced under conditions where the intracellular K+ concentration is diminished and that apoptosis is inhibited when K+ efflux is prevented. These data show that the efflux of ions, primarily potassium, plays a necessary and perhaps a pivotal role in the cell death program.

The Human Glucocorticoid Receptor Isoform EXPRESSION, BIOCHEMICAL PROPERTIES, AND PUTATIVE FUNCTION

Alternative splicing of the human glucocorticoid receptor (hGR) primary transcript produces two receptor isoforms, hGRα and hGRβ, which differ at their carboxyl termini. The hGRα isoform conveys endocrine information to target tissues by altering patterns of gene expression in a hormone-dependent fashion. In contrast to hGRα, very little is known about the hGRβ splice variant. Using hGRα- and hGRβ-specific riboprobes on human multiple tissue Northern blots, we show that the hGRβ message has a widespread tissue distribution. We also prove by reverse transcriptase-polymerase chain reaction that the alternative splicing event underlying the formation of the hGRβ message occurs in these tissues. Because the hGRβ protein differs from hGRα at the extreme COOH terminus, we investigated several of the biochemical properties of hGRβ expressed in transfected cells. hGRβ does not bind the glucocorticoid agonist dexamethasone nor the glucocorticoid antagonist RU38486 in vivo. Moreover, in contrast to hGRα, hGRβ is located primarily in the nucleus of transfected cells independent of hormone administration. Finally, in the absence of hGRα, hGRβ is transcriptionally inactive on a glucocorticoid-responsive enhancer. However, when both isoforms are expressed in the same cell, hGRβ inhibits the hormone-induced, hGRα-mediated stimulation of gene expression. Thus, hGRβ potentially functions as a dominant negative inhibitor of hGRα activity.

The role of DNA fragmentation in apoptosis.

The formation of distinct DNA fragments of oligonucleosomal size (180-200 bp lengths) is a biochemical hallmark of apoptosis in many cells. Recent observations also suggest large DNA fragments and even single-strand cleavage events occur during cell death. These observations have raised many questions. What are the types of DNA cleavage observed during apoptosis? What are the nucleases involved? And what is the role of these nucleolytic events in apoptosis?

Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes

Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.

Intracellular K+ Suppresses the Activation of Apoptosis in Lymphocytes

Little is known about the mechanisms of suppression of apoptosis. We have addressed the novel possibility that the level of intracellular K+ regulates the apoptotic process by controlling the activity of death enzymes. We show that K+, at normal intracellular levels, inhibits both apoptotic DNA fragmentation and caspase-3(CPP32)-like protease activation, suggesting that intracellular K+ loss must occur early during apoptosis. Direct measurement of K+ by inductively coupled plasma/mass spectrometry and flow cytometry indicates a major decrease in intracellular K+ concentration in the apoptotic cell. Flow cytometric analysis revealed that caspase and nuclease activity were restricted to the subpopulation of cells with reduced K+. Disruption of the natural K+electrochemical gradient suppressed the activity of both caspase and nuclease independent of the mode of activation of the apoptotic inducing agent, demonstrating that a decrease in intracellular K+ concentration is a necessary, early event in programmed cell death.

The Dominant Negative Activity of the Human Glucocorticoid Receptor β Isoform SPECIFICITY AND MECHANISMS OF ACTION

Alternative splicing of the human glucocorticoid receptor gene generates a nonhormone binding splice variant (hGRβ) that differs from the wild-type receptor (hGRα) only at the carboxyl terminus. Previously we have shown that hGRβ inhibits the transcriptional activity of hGRα, which is consistent with reports of elevated hGRβ expression in patients with generalized and tissue-specific glucocorticoid resistance. The potential role of hGRβ in the regulation of target cell sensitivity to glucocorticoids prompted us to further evaluate its dominant negative activity in other model systems and to investigate its mode of action. We demonstrate in multiple cell types that hGRβ inhibits hGRα-mediated activation of the mouse mammary tumor virus promoter. In contrast, the ability of the progesterone and androgen receptors to activate this promoter is only weakly affected by hGRβ. hGRβ also inhibits hGRα-mediated repression of an NF-κB-responsive promoter but does not interfere with homologous down-regulation of hGRα. We show that hGRβ can associate with the heat shock protein hsp90 although with lower affinity than hGRα. In addition, hGRβ binds GRE-containing DNA with a greater capacity than hGRα in the absence of glucocorticoids. Glucocorticoid treatment enhances hGRα, but not hGRβ, binding to DNA. Moreover, we demonstrate that hGRα and hGRβ can physically associate with each other in a heterodimer. Finally, we show that the dominant negative activity of hGRβ resides within its unique carboxyl-terminal 15 amino acids. Taken together, our results suggest that formation of transcriptionally impaired hGRα-hGRβ heterodimers is an important component of the mechanism responsible for the dominant negative activity of hGRβ.

Apoptosis and glutathione: beyond an antioxidant.

Apoptosis is a conserved homeostatic process critical for organ and tissue morphogenesis, development, and senescence. This form of programmed cell death also participates in the etiology of several human diseases including cancer, neurodegenerative, and autoimmune disorders. Although the signaling pathways leading to the progression of apoptosis have been extensively characterized, recent studies highlight the regulatory role of changes in the intracellular milieu (permissive apoptotic environment) in the efficient activation of the cell death machinery. In particular, glutathione (GSH) depletion is a common feature of apoptotic cell death triggered by a wide variety of stimuli including activation of death receptors, stress, environmental agents, and cytotoxic drugs. Although initial studies suggested that GSH depletion was only a byproduct of oxidative stress generated during cell death, recent discoveries suggest that GSH depletion and post-translational modifications of proteins through glutathionylation are critical regulators of apoptosis. Here, we reformulate these emerging paradigms into our current understanding of cell death mechanisms.

Proinflammatory cytokines regulate human glucocorticoid receptor gene expression and lead to the accumulation of the dominant negative beta isoform: a mechanism for the generation of glucocorticoid resistance.

Inflammatory responses in many cell types are coordinately regulated by the opposing actions of NF-κB and the glucocorticoid receptor (GR). The human glucocorticoid receptor (hGR) gene encodes two protein isoforms: a cytoplasmic alpha form (GRα), which binds hormone, translocates to the nucleus, and regulates gene transcription, and a nuclear localized beta isoform (GRβ), which does not bind known ligands and attenuates GRα action. We report here the identification of a tumor necrosis factor (TNF)-responsive NF-κB DNA binding site 5′ to the hGR promoter that leads to a 1.5-fold increase in GRα mRNA and a 2.0-fold increase in GRβ mRNA in HeLaS3 cells, which endogenously express both GR isoforms. However, TNF-α treatment disproportionately increased the steady-state levels of the GRβ protein isoform over GRα, making GRβ the predominant endogenous receptor isoform. Similar results were observed following treatment of human CEMC7 lymphoid cells with TNF-α or IL-1. The increase in GRβ protein expression correlated with the development of glucocorticoid resistance.

Molecular mechanisms of glucocorticoid action and resistance

The actions of glucocorticoid hormones are mediated by an intracellular receptor, the glucocorticoid receptor (GR). The mechanism of action of this ligand-inducible transcription factor is discussed, focusing on mechanisms of glucocorticoid resistance. Three mechanisms are highlighted: ligand-induced down-regulation of the receptor, the dominant-negative inhibition by the beta-isoform of the receptor, and repression by the transcription factor NF-kappa B. It has been shown that these mechanisms can significantly inhibit glucocorticoid signaling, and could therefore seriously decrease the efficacy of glucocorticoids used clinically.