The role of glucocorticoids and progestins in inflammatory, autoimmune, and infectious disease.

A bidirectional communication exists between the CNS and the immune system. The autonomic nervous system, through neurotransmitters and neuropeptides, works in parallel with the hypothalamic-pituitary-adrenal axis through the actions of glucocorticoids to modulate inflammatory events. The immune system, through the action of cytokines and other factors, in turn, activates the CNS to orchestrate negative-feedback mechanisms that keep the immune response in check. Disruption of these interactions has been associated with a number of syndromes including inflammatory, autoimmune, and cardiovascular diseases, metabolic and psychiatric disorders, and the development of shock. The hypothalamic-pituitary-gonadal axis also plays an important part in regulating immunity through the secretion of sex hormones. Although numerous studies have established a role for immunomodulation by estrogen and testosterone, the role of progesterone is less well understood. Progesterone is crucial for reproductive organ development and maintenance of pregnancy, and more recent studies have clearly shown its role as an important immune regulator. The main focus of this review will be about the role of steroid hormones, specifically glucocorticoids and progesterone, in inflammatory responses and infectious diseases and how dysregulation of their actions may contribute to development of autoimmune and inflammatory disease.

The large clostridial toxins from Clostridium sordellii and C. difficile repress glucocorticoid receptor activity.

We have previously shown that Bacillus anthracis lethal toxin represses glucocorticoid receptor (GR) transactivation. We now report that repression of GR activity also occurs with the large clostridial toxins produced by Clostridium sordellii and C. difficile. This was demonstrated using a transient transfection assay system for GR transactivation. We also report that C. sordellii lethal toxin inhibited GR function in an ex vivo assay, where toxin reduced the dexamethasone suppression of the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha). Furthermore, the glucocorticoid antagonist RU-486 in combination with C. sordellii lethal toxin additively prevented glucocorticoid suppression of TNF-alpha. These findings corroborate the fact that GR is a target for the toxin and suggest a physiological role for toxin-associated GR repression in inflammation. Finally, we show that this repression is associated with toxins that inactivate p38 mitogen-activated protein kinase (MAPK).

Survival and invasiveness of astrocytomas promoted by erythropoietin.

OBJECT: The hypoxia-inducible pleiotropic hormone, erythropoietin (EPO), has recently been found to promote the development and survival of neurons and astrocytes. Since hypoxia has been implicated in the malignant progression of some human cancers, the authors investigated whether EPO signaling influenced the malignant properties of human astrocytoma cells. METHODS: Reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemical studies were used to measure EPO and its receptor (EPOR). Cell viability, Matrigel invasion assays, metalloprotease assays, EPO neutralizing antibodies, and EPOR overexpression were used to study the biological actions of EPO. Expression of both EPO and EPOR was observed in the hypoxic regions and invasive margins of glioma specimens obtained at biopsy, and expression of EPOR correlated with the stage of the tumor. The EPOR was also functionally upregulated by hypoxia in cultured glioblastoma multiforme (GBM) cells. Both hypoxia and EPO protected cultured GBM cells from cisplatin cytotoxicity and promoted the invasiveness of GBM cells through Matrigel by potentiating metalloprotease activity. Hypoxia-enhanced cell invasion was attenuated in cells that overexpressed a nonfunctional EPOR. CONCLUSIONS: Hypoxia-inducible autocrine and paracrine EPO signaling participates in the malignant progression of GBMs.

Reversible inactivation of HIF-1 prolyl hydroxylases allows cell metabolism to control basal HIF-1.

Continuous hydroxylation of the HIF-1 transcription factor alpha subunit by oxygen and 2-oxoglutarate-dependent dioxygenases promotes decay of this protein and thus prevents the transcriptional activation of many genes involved in energy metabolism, angiogenesis, cell survival, and matrix modification. Hypoxia blocks HIF-1alpha hydroxylation and thus activates HIF-1alpha-mediated gene expression. Several nonhypoxic stimuli can also activate HIF-1, although the mechanisms involved are not well known. Here we show that the glucose metabolites pyruvate and oxaloacetate inactivate HIF-1alpha decay in a manner selectively reversible by ascorbate, cysteine, histidine, and ferrous iron but not by 2-oxoglutarate or oxygen. Pyruvate and oxaloacetate bind to the 2-oxoglutarate site of HIF-1alpha prolyl hydroxylases, but their effects on HIF-1 are not mimicked by other Krebs cycle intermediates, including succinate and fumarate. We show that inactivation of HIF-1 hydroxylation by glucose-derived 2-oxoacids underlies the prominent basal HIF-1 activity commonly seen in many highly glycolytic cancer cells. Since HIF-1 itself promotes glycolytic metabolism, enhancement of HIF-1 by glucose metabolites may constitute a novel feed-forward signaling mechanism involved in malignant progression.

High-throughput clonal selection of recombinant CHO cells using a dominant selectable and amplifiable metallothionein-GFP fusion protein.

Transfected mammalian cells can be used for the production of fully processed recombinant proteins for medical and industrial purposes. However, the isolation of high-producing clones is traditionally time-consuming. Therefore, we developed a high-throughput screening method to reduce the time and effort required to isolate high-producing cells. This involved the construction of an expression vector containing the amplifiable gene metallothionein (MT), fused in-frame to green fluorescent protein (GFP). The fusion gene (MTGFP) confers metal resistance similar to that of the wild-type metallothionein and expression can be monitored using either flow cytometry or a fluorometer to measure green fluorescence. Expression of MTGFP acted as a dominant selectable marker allowing rapid and more efficient selection of clones at defined metal concentrations than with the antibiotic G418. Cells harboring MTGFP responded to increasing metal concentrations with a corresponding increase in fluorescence. There was also a corresponding increase in recombinant protein production, indicating that MTGFP could be used as a selectable and amplifiable gene for the coexpression of foreign genes. Using our expression vector encoding MTGFP, we demonstrate a high-throughput clonal selection protocol for the rapid isolation of high-producing clones from transfected CHO cells. We were able to isolate cell lines reaching specific productivities of >10 microg hGH/10(6) cells/day within 4 weeks of transfection. The advantage of this method is that it can be easily adapted for automated procedures using robotic handling systems.

Site-directed mutagenesis of platelet glycoprotein Ib alpha demonstrating residues involved in the sulfation of tyrosines 276, 278, and 279.

The interaction between platelet glycoprotein (GP) Ib alpha and von Willebrand factor (VWF) is essential for initiation of hemostasis. The sulfation of the 3 tyrosine residues 276, 278, and 279 in GPIb alpha is an important posttranslational modification that seems to promote the interaction with VWF. The environment where sulfation of tyrosines occurs has been proposed to contain highly acidic residues. This investigation has examined the highly acidic region from Asp249 to Asp287 in the mature GPIb alpha protein. Changes to most of the carboxylic acids in this region resulted in decreased reactivity to VWF. Only 3 mutants (Glu270Gln, Asp283Asn, Asp283Asn/Glu285Gln/Asp287Asn) resulted in the abolition of sulfation. Two novel mutations were also created. First, a deletion of the 7 amino acids from Tyr276 to Glu282 led to a loss of sulfation and totally abolished VWF binding in the presence of botrocetin. This confirms that it is these 3 tyrosines that undergo sulfation and that this region is crucial for botrocetin-mediated VWF binding. The second mutation involves changing the lysine residues at 253, 258, and 262 to alanine. This also led to distinct changes in VWF binding and abolition of sulfation.