Andrew C. B. Cato

Antitumor promotion and antiinflammation: Down-modulation of AP-1 (Fos/Jun) activity by glucocorticoid hormone

Glucocorticoid hormones counteract inflammation and phorbol ester tumor promotion and drastically decrease the expression of several extracellular proteases, including collagenase I. Glucocorticoid hormone inhibits basal and induced transcription of collagenase by interfering with AP-1, the major enhancer factor of the collagenase promoter. The mechanism of interference is novel in that it does not require protein synthesis, it depends on the hormone receptor but not its binding to DNA, it occurs at hormone doses one order of magnitude below those required for gene activation, and it involves down-modulation of the trans-activating function of preexisting unbound and DNA-bound AP-1. Coprecipitation experiments suggest direct AP-1-hormone receptor interaction, which also possibly explains the reverse experiment: overexpression of Fos or Jun inhibits the expression of hormone-dependent genes.

A distinct modulating domain in glucocorticoid receptor monomers in the repression of activity of the transcription factor AP-1.

Steroid receptors activate and repress genes. An important class of genes that they repress is controlled by the transcription factor AP‐1. The activity of AP‐1 is inhibited by the receptor, a mechanism exploited for the therapy of various forms of pathological hyperproliferation in humans. We show here by point mutations in the DNA binding domain and by the choice of steroid ligands that repression of AP‐1 activity and transactivation functions of the glucocorticoid receptor (GR) are separable entities. While DNA binding and activation of glucocorticoid‐regulated promoters require GR dimerization, we present data that suggest that repression is a function of GR monomers.

Glucocorticoids inhibit MAP kinase via increased expression and decreased degradation of MKP-1

Glucocorticoids inhibit the proinflammatory activities of transcription factors such as AP‐1 and NF‐κB as well as that of diverse cellular signaling molecules. One of these signaling molecules is the extracellular signal‐regulated kinase (Erk‐1/2) that controls the release of allergic mediators and the induction of proinflammatory cytokine gene expression in mast cells. The mechanism of inhibition of Erk‐1/2 activity by glucocorticoids is unknown. Here we report a novel dual action of glucocorticoids for this inhibition. Glucocorticoids increase the expression of the MAP kinase phosphatase‐1 (MKP‐1) gene at the promoter level, and attenuate proteasomal degradation of MKP‐1, which we report to be triggered by activation of mast cells. Both induction of MKP‐1 expression and inhibition of its degradation are necessary for glucocorticoid‐mediated inhibition of Erk‐1/2 activation. In NIH‐3T3 fibroblasts, although glucocorticoids up‐regulate the MKP‐1 level, they do not attenuate the proteasomal degradation of this protein and consequently they are unable to inhibit Erk‐1/2 activity. These results identify MKP‐1 as essential for glucocorticoid‐mediated control of Erk‐1/2 activation and unravel a novel regulatory mechanism for this anti‐inflammatory drug.

Rapid Actions of Steroid Receptors in Cellular Signaling Pathways

Steroid hormones regulate cellular processes by binding to intracellular receptors that, in turn, interact with discrete nucleotide sequences to alter gene expression. Because most steroid receptors in target cells are located in the cytoplasm, they need to get into the nucleus to alter gene expression. This process typically takes at least 30 to 60 minutes. In contrast, other regulatory actions of steroid hormones are manifested within seconds to a few minutes. These time periods are far too rapid to be due to changes at the genomic level and are therefore termed nongenomic or rapid actions, to distinguish them from the classical steroid hormone action of regulation of gene expression. The rapid effects of steroid hormones are manifold, ranging from activation of mitogen-activated protein kinases (MAPKs), adenylyl cyclase (AC), protein kinase C (PKC), and heterotrimeric guanosine triphosphate-binding proteins (G proteins). In some cases, these rapid actions of steroids are mediated through the classical steroid receptor that can also function as a ligand-activated transcription factor, whereas in other instances the evidence suggests that these rapid actions do not involve the classical steroid receptors. One candidate target for the nonclassical receptor-mediated effects are G protein-coupled receptors (GPCRs), which activate several signal transduction pathways. One characteristic of responses that are not mediated by the classical steroid receptors is insensitivity to steroid antagonists, which has contributed to the notion that a new class of steroid receptors may be responsible for part of the rapid action of steroids. Evidence suggests that the classical steroid receptors can be localized at the plasma membrane, where they may trigger a chain of reactions previously attributed only to growth factors. Identification of interaction domains on the classical steroid receptors involved in the rapid effects, and separation of this function from the genomic action of these receptors, should pave the way to a better understanding of the rapid action of steroid hormones. Steroid hormones control diverse processes in reproduction and development. In target cells, they diffuse through the plasma membrane and bind to cytoplasmic receptors that mediate their action. Upon hormone binding, the steroid receptors undergo a conformational change and translocate to the nucleus, where they bind discrete nucleotide sequences to enhance the expression of specific genes. Steroid receptors also negatively regulate gene expression by binding to and down-regulating the activity of distinct transcription factors involved in cell proliferation, differentiation, and programmed cell death. Because all these responses require nuclear localization of the receptors, they are termed genomic functions, and they require about 30 to 60 minutes. These regulatory properties of steroid hormones can be distinguished from more rapid actions (<10 minutes) reminiscent of the activities of peptide hormones in the activation of signal transduction cascades. The mechanisms involved in initiating the rapid responses are still unclear, but they range from contributions of unidentified receptors to the involvement of heterotrimeric guanosine triphosphate-binding proteins (G proteins), or even the classical steroid receptors. Considerable advances have been made recently in the elucidation of the rapid responses mediated by steroid receptors. Accumulating evidence shows that a small but significant percentage of the conventional steroid receptors reside at the plasma membrane after ligand binding. From this location, they trigger diverse signaling cascades needed for cell proliferation and other biological processes. The effector molecules activated by the steroid receptors include mitogen-activated protein kinases (MAPKs), adenylyl cyclase (AC), phosphatidylinositol 3-kinase (PI3K), and protein kinase C (PKC). Thus, steroid receptors integrate classical functions as transcription factors in the nucleus with functions as signaling molecules at the plasma membrane. These two properties together make up the entire repertoire of regulatory functions so far attributed to steroid hormones.

Dual specificity phosphatase 1 (DUSP1) regulates a subset of LPS-induced genes and protects mice from lethal endotoxin shock.

Activation of the mitogen-activated protein kinase (MAPK) cascade after Toll-like receptor stimulation enables innate immune cells to rapidly activate cytokine gene expression. A balanced response to signals of infectious danger requires that cellular activation is transient. Here, we identify the MAPK phosphatase dual specificity phosphatase 1 (DUSP1) as an essential endogenous regulator of the inflammatory response to lipopolysaccharide (LPS). DUSP1-deficient (DUSP1−/−) bone marrow–derived macrophages showed selectively prolonged activation of p38 MAPK and increased cytokine production. Intraperitoneal challenge of DUSP1−/− mice with LPS caused increased lethality and overshooting production of interleukin (IL)-6 and tumor necrosis factor α. Transcriptional profiling revealed that DUSP1 controls a significant fraction of LPS-induced genes, which includes IL-6 and IL-10 as well as the chemokines CCL3, CCL4, and CXCL2. In contrast, the expression of the important mediators of endotoxin lethality, interferon γ and IL-12, was not significantly altered by the absence of DUSP1. These data together demonstrate a specific regulatory role of DUSP1 in controlling a subset of LPS-induced genes that determines the outcome of endotoxin shock.

The hormone regulatory element of mouse mammary tumour virus mediates progesterone induction

Sequences within the long terminal repeat region (LTR) of mouse mammary tumour virus (MMTV) confer progestin inducibility to either the tk‐promoter or the MMTV‐promoter in T47D cells, a human mammary tumour cell line which possesses high constitutive levels of progesterone receptor. In a clone of MCF7 cells, another human mammary tumour cell line with a low level of progesterone receptor, as well as in rat fibroblasts, glucocorticoid but not progestin induction is observed. The effect of the progesterone analogue R5020 is much more pronounced than the effect of dexamethasone, and at the concentrations required for maximal induction, R5020 does not significantly compete with binding of dexamethasone to the glucocorticoid receptor. In conjunction with previous results on the DNA binding of the glucocorticoid and progesterone receptors, these data show that two different steroid hormones, acting through their respective receptors, can mediate the induction of gene expression by interacting with the same DNA sequences. Our results suggest that the hormone regulatory element of MMTV may primarily be a progesterone‐responsive element in mammary cells.

Rapid signalling by androgen receptor in prostate cancer cells

Androgens are important growth regulators in prostate cancer. Their known mode of action in target cells requires binding to a cytoplasmic androgen receptor followed by a nuclear translocation event and modulation of the expression of specific genes. Here, we report another mode of action of this receptor. Treatment of androgen responsive prostate cancer cells with dihydrotestosterone leads to a rapid and reversible activation of mitogen-activated protein kinases MAPKs (also called extracellular signal-regulated kinases or Erks). Transient transfection assays demonstrated that the androgen receptor-mediated activation of MAP kinase results in enhanced activity of the transcription factor Elk-1. This action of the androgen receptor differs from its known transcriptional activity since it is rapid and insensitive to androgen antagonists such as hydroxyflutamide or casodex. Biochemical studies as well as analyses with dominant negative mutants showed the involvement of kinases such as MAPK/Erk kinase, phosphatidyl-inositol 3-kinase and protein kinase C in the androgen receptor-mediated activation of MAP kinase. These results demonstrate a novel regulatory action of the androgen receptor and prove that in addition to its known transcriptional effects, it also uses non-conventional means to modulate several cellular signalling processes.

The hormone response element of the mouse mammary tumour virus DNA mediates the progestin and androgen induction of transcription in the proviral long terminal repeat region.

Mouse mammary tumour virus (MMTV) gene expression has been shown to be regulated by glucocorticoids. A hormone response element (HRE) located between ‐202 and ‐59 upstream of the start of transcription in the long terminal repeat (LTR) region of the proviral DNA is required for this induction. We have investigated the role played by the HRE in the induction of MMTV LTR transcription by other classes of steroid hormones. Chimaeric constructs containing the HRE and the authentic LTR promoter linked to an indicator gene or the HRE linked to an otherwise hormone insensitive promoter directing the transcription of an indicator gene, were transfected into the human mammary tumour cell line T47D. Transcription at the MMTV LTR promoter or at the previously hormone‐insensitive promoter was induced by progestins and androgens but not by oestradiol in transfected cells that contained functional receptors for these hormones. These results identify the HRE as the cis‐acting element that mediates the progestin and androgen induction of MMTV LTR transcription. The HRE is therefore a DNA element that is required not just for glucocorticoid but also for progesterone and androgen induction of MMTV LTR transcription.

IκBα-independent downregulation of NF-κB activity by glucocorticoid receptor

IκBα is an inhibitor protein that prevents nuclear transport and activation of the transcription factor NF‐κB. In acute inflammation, NF‐κB is activated and increases the expression of several pro‐inflammatory cytokine and chemokine genes. Glucocorticoids counteract this process. It has been proposed that the glucocorticoid‐dependent inhibition of NF‐κB activity is mediated by increased synthesis of IκBα which should then sequester NF‐κB in an inactive cytoplasmic form. Here, we show by the use of a mutant glucocorticoid receptor and steroidal ligands that hormone‐induced IκBα synthesis and inhibition of NF‐κB activity are separable biochemical processes. A dimerization‐defective glucocorticoid receptor mutant that does not enhance the IκBα level is still able to repress NF‐κB activity. Conversely, glucocorticoid analogues competent in enhancing IκBα synthesis do not repress NF‐κB activity. These results demonstrate that increased synthesis of IκBα is neither required nor sufficient for the hormone‐mediated downmodulation of NF‐κB activity.

Glucocorticoids synergistically enhance nontypeable Haemophilus influenzae-induced Toll-like receptor 2 expression via a negative cross-talk with p38 MAP kinase.

The recognition of invading microbes followed by the induction of effective innate immune response is crucial for host survival. Human surface epithelial cells are situated at host-environment boundaries and thus act as the first line of host defense against invading microbes. They recognize the microbial ligands via Toll-like receptors (TLRs) expressed on the surface of epithelial cells. TLR2 has gained importance as a major receptor for a variety of microbial ligands. In contrast to its high expression in lymphoid tissues, TLR2 is expressed at low level in epithelial cells. Thus, it remains unclear whether the low amount of TLR2 expressed in epithelial cells is sufficient for mediating bacteria-induced host defense and immune response and whether TLR2 expression can be up-regulated by bacteria during infection. Here, we show that TLR2, although expressed at very low level in unstimulated human epithelial cells, is greatly up-regulated by nontypeableHemophilus influenzae (NTHi), an important human bacterial pathogen causing otitis media and chronic obstructive pulmonary diseases. Activation of an IKKβ-IκBα-dependent NF-κB pathway is required for TLR2 induction, whereas inhibition of the MKK3/6-p38α/β pathway leads to enhancement of NTHi-induced TLR2 up-regulation. Surprisingly, glucocorticoids, well known potent anti-inflammatory agents, synergistically enhance NTHi-induced TLR2 up-regulation likely via a negative cross-talk with the p38 MAP kinase pathway. These studies may bring new insights into the role of bacteria and glucocorticoids in regulating host defense and immune response and lead to novel therapeutic strategies for modulating innate immune and inflammatory responses for otitis media and chronic obstructive pulmonary diseases.