Heterodimerization between mineralocorticoid and glucocorticoid receptor: a new principle of glucocorticoid action in the CNS.

In the mammalian central nervous system, responsiveness to glucocorticoids is mediated by both the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). These pharmacologically distinct receptors are believed to bind to common response elements as homodimers. We provide evidence that MR and GR can form a heterodimeric complex with DNA-binding and transactivation properties different from those of the respective homodimers. There was a high degree of cooperativity of MR and GR in binding to a glucocorticoid response element. Transient transfection of a neuroblastoma cell line revealed a transcriptional response pattern of coexpressed MR and GR distinct from that obtained by MR or GR alone. Our findings demonstrate that heterodimerization of MR and GR is a hitherto unrecognized principle for the transcriptional regulation of glucocorticoid-responsive genes in tissue coexpressing these receptors.

Progesterone receptor-mediated effects of neuroactive steroids.

Several 3 alpha-hydroxysteroids accumulate in the brain after local synthesis or after metabolization of steroids that are provided by the adrenals. The 3 alpha-hydroxy ring A-reduced pregnane steroids allopregnanolone and tetrahydrodeoxycorticosterone are believed not to interact with intracellular receptors, but enhance GABA-mediated chloride currents. The present study shows that these neuroactive steroids can regulate gene expression via the progesterone receptor. The induction of DNA binding and transcriptional activation of the progesterone receptor requires intracellular oxidation of the neuroactive steroids into progesterone receptor active 5 alpha-pregnane steroids. Thus, at physiological concentrations, these neuroactive steroids regulate neuronal function through their effects on both transmitter-gated ion channels and steroid receptor-regulated gene expression.

Molecular and functional evidence for in vitro cytokine enhancement of human and murine target cell sensitivity to glucocorticoids. TNF-alpha priming increases glucocorticoid inhibition of TNF-alpha-induced cytotoxicity/apoptosis.

Cytokine-induced glucocorticoid secretion and glucocorticoid inhibition of cytokine synthesis and pleiotropic actions act as important safeguards in preventing cytokine overreaction. We found that TNF-alpha increased glucocorticoid-induced transcriptional activity of the glucocorticoid receptor (GR) via the glucocorticoid response elements (GRE) in L-929 mouse fibroblasts transfected with a glucocorticoid-inducible reporter plasmid. In addition, TNF-alpha also enhanced GR number. The TNF-alpha effect on transcriptional activity was absent in other cell lines that express TNF-alpha receptors but not GRs, and became manifest when a GR expression vector was cotransfected, indicating that TNF-alpha, independent of any effect it may have on GR number, has a stimulatory effect on the glucocorticoid-induced transcriptional activity of the GR. Moreover, TNF-alpha increased GR binding to GRE. As a functional biological correlate of this mechanism, priming of L-929 cells with a low (noncytotoxic) dose of TNF-alpha significantly increased the sensitivity to glucocorticoid inhibition of TNF-alpha-induced cytotoxicity/apoptosis. TNF-alpha and IL-1 beta had the same stimulatory action on glucocorticoid-induced transcriptional activity of the GR via the GRE, in different types of cytokine/glucocorticoid target cells (glioma, pituitary, epithelioid). The phenomenon may therefore reflect a general molecular mechanism whereby cytokines modulate the transcriptional activity of the GR, thus potentiating the counterregulation by glucocorticoids at the level of their target cells.

Heterodimerization between mineralocorticoid and glucocorticoid receptors increases the functional diversity of corticosteroid action.

Gene regulation by steroids is mediated by the binding of the endogenous or pharmacological ligand to the corresponding nuclear receptor. Ligand-activated steroid receptors usually regulate the expression of responsive genes by binding to common response elements on DNA as homodimers. However, recent findings indicate that mineralocorticoid and glucocorticoid receptors are able to interact by forming heterodimers. In tissues coexpressing both of these corticosteroid receptors, heterodimerization between them may be a hitherto unrecognized modality for the transcriptional regulation of corticosteroid-responsive genes. In this review, Thorsten Trapp and Florian Holsboer discuss the potential impact of this heterodimerization on corticosteriod physiology and pharmacology.

Necrotic cell-derived high mobility group box 1 attracts antigen-presenting cells but inhibits hepatocyte growth factor-mediated tropism of mesenchymal stem cells for apoptotic cell death.

Tissue damage due to apoptotic or necrotic cell death typically initiates distinct cellular responses, leading either directly to tissue repair and regeneration or to immunological processes first, to clear the site, for example, of potentially damage-inducing agents. Mesenchymal stem cells (MSC) as well as immature dendritic cells (iDC) and monocytes migrate to injured tissues. MSC have regenerative capacity, whereas monocytes and iDC have a critical role in inflammation and induction of immune responses, including autoimmunity after tissue damage. Here, we investigated the influence of apoptotic and necrotic cell death on recruitment of MSC, monocytes and iDC, and identified hepatocyte growth factor (HGF) and the alarmin high mobility group box 1 (HMGB1) as key factors differentially regulating these migratory responses. MSC, but not monocytes or iDC, were attracted by apoptotic cardiomyocytic and neuronal cells, whereas necrosis induced migration of monocytes and iDC, but not of MSC. Only apoptotic cell death resulted in HGF production and HGF-mediated migration of MSC towards the apoptotic targets. In contrast, HMGB1 was predominantly released by the necrotic cells and mediated recruitment of monocytes and iDC via the receptor of advanced glycation end products. Moreover, necrotic cardiomyocytic and neuronal cells caused an HMGB1/toll-like receptor-4-dependent inhibition of MSC migration towards apoptosis or HGF, while recruitment of monocytes and iDC by necrosis or HMGB1 was not affected by apoptotic cells or HGF. Thus, the type of cell death differentially regulates recruitment of either MSC or monocytes and iDC through HGF and HMGB1, respectively, with a dominant, HMGB1-mediated role of necrosis in determining tropism after tissue injury.

Glucocorticoids enhance oxidative stress-induced cell death in hippocampal neurons in vitro.

In patients with Alzheimer's disease, hippocampal cells are among the first neuronal cells of the brain to degenerate. Both rat primary hippocampal neurons and cells of the clonal mouse hippocampal cell line HT22 express endogenous functional glucocorticoid receptors (GRs), as shown by transient transfection of cells with a luciferase reporter plasmid containing GR-responsive elements. The influence of activated GRs on oxidative stress-induced neuronal cell death in vitro was investigated employing these hippocampal model systems. Two oxidative stressors were investigated, the free radical-inducing Alzheimer's disease-associated amyloid beta-protein, which is toxic to hippocampal neurons, and the excitatory amino acid glutamate, which induces oxidative cell death in HT22 cells via an increase in intracellular peroxides. Cellular viability was assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide test and trypan exclusion staining, followed by microscopical cell counting. Glucocorticoids strongly increased the vulnerability of the hippocampal cells to amyloid beta-protein and glutamate. This increase could be blocked by the specific GR antagonist RU486. Our data suggest that changes in hippocampal GR homeostasis and regulation may render hippocampal neurons more vulnerable to oxidative stress-induced neuronal degeneration.

Regulation of rat mineralocorticoid receptor expression in neurons by progesterone.

We have studied the effects of progesterone on the transcription of the mineralocorticoid receptor (MR) gene in neurons in vitro and in vivo. Progesterone treatment caused a 2.5-fold increase in activity of the MR promoter in transiently transfected N2A neuroblastoma cells. Similarly, MR promoter activity in GH3 pituitary cells was increased 2-fold after treatment with the specific progesterone receptor agonist R5020, with an even greater induction after priming with 17 beta-estradiol. Progesterone treatment also produced a dose-dependent increase in MR messenger RNA (mRNA) levels in primary hippocampal neuron cultures. In vivo, chronic administration of progesterone to estrogen-primed adrenalectomized/ovariectomized rats significantly increased MR mRNA levels in all hippocampal subfields, as determined by semiquantitative in situ hybridization histochemistry. Whereas chronic estradiol treatment decreased MR mRNA levels in the hippocampus, progesterone administration in the absence of estradiol priming was without any effect. These results indicate that 1) progesterone increases MR mRNA levels in vitro and in vivo; 2) the stimulatory effects of progesterone are at least partially mediated by induction of MR promoter activity; and 3) estrogen priming is essential for the effect of progesterone upon MR mRNA in vivo. Further, they suggest the possibility of heterologous regulation of corticosteroid receptors in the brain, whereby the responsiveness of the limbic-hypothalamo-pituitary-adrenal system to corticosteroids may be modulated.

Transcriptional induction of rat mineralocorticoid receptor gene in neurones by corticosteroids.

We investigated the mechanisms by which corticosteroids regulate the expression of the mineralocorticoid receptor (MR) in neurones. Aldosterone and dexamethasone produced a dose-dependent increase of MR and mRNA levels in cultured primary hippocampal neurones. Transient transfection of neuroblastoma cells showed that corticosteroids directly activate the rat MR promoter, indicating that the steroid-induced increase in the MR mRNA concentration is at least partially transcriptional. Progressive 5' deletions of the MR promoter sequence revealed that the promoter induction cannot be assigned to a single element. An oligonucleotide comprising a consensus half-glucocorticoid responsive element located at -319 bp in the MR promoter stimulated the corticosteroid-induced activation of the heterologous promoter. Cloning three of these enhancers in tandem greatly potentiated the responses to glucocorticoids and mineralocorticoids, suggesting that although this element is a weak enhancer it can, in combination with other enhancer elements, induce MR gene expression by both types of corticosteroid receptors.

The oestrogen receptor modulates growth of pituitary tumour cells in the absence of exogenous oestrogen.

The GH3 pituitary cell line has been used to investigate the role of the oestrogen receptor (ER) as a modulator of mitogenic signals in tumour cells in the absence of exogenous oestrogen. Using a chemically defined, serum- and oestrogen-free medium, we have demonstrated that the pure steroidal anti-oestrogens ICI 182780 and ICI 164384 are capable of blocking growth by more than 50% after 5 days of culture. Studies with conditioned medium have indicated that the basal growth is due to the secretion of autocrine growth stimulatory substances. Under serum- and oestrogen-free conditions, insulin and IGF-I increased the growth rate of these cells by twofold over a 5-day treatment period, and this effect was also blocked by the anti-oestrogens ICI 182780 and ICI 164384 (50% of maximum inhibition at 0.6 and 6 nM respectively). To explore the potential mechanism by which the ER apparently facilitates the growth factor effects under oestrogen-free conditions, GH3 cells were transiently transfected with a plasmid reporter containing the vitellogenin oestrogen response element (delta MTV-ERE-LUC). We have shown that as well as oestradiol (OE2), insulin and IGF-I induce luciferase activity by between two- and sevenfold (four experiments), and these effects were completely blocked by ICI 182780. In contrast, growth factors and OE2 were unable to induce luciferase expression when transfections were performed with a plasmid reporter lacking the oestrogen response element. The studies presented here strongly suggest that, in the absence of oestrogen, the ER in these pituitary tumour cells has a role in growth, as peptide factors are able to induce its conversion to a state which is capable of up-regulating the transcription of key growth-promoting genes.

Purification and properties of the 5 alpha-dihydrotestosterone 3 alpha(beta)-hydroxysteroid dehydrogenase from human prostatic cytosol.

5 alpha-Dihydrotestosterone 3 alpha(beta)-hydroxysteroid dehydrogenase [3 alpha(beta)-HSDH] [EC 1.1.1.50/EC 1.1.1.51] which catalyses the conversion of 5 alpha-dihydrotestosterone (5 alpha-DHT) to both 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol was purified to an apparent homogeneous state using cytosol of three human hyperplastic prostates by a 4-step purification procedure. After each purification step 3 alpha-HSDH activity was coincident with 3 beta-HSDH activity. On average, specific 3 alpha-HSDH activity was enriched 856-fold, specific 3 beta-HSDH activity 749-fold compared to human prostatic cytosol using anion exchange, hydrophobic interaction, gel filtration and affinity chromatography. Examination of the purified enzyme by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) revealed a single protein band with silver staining. The molecular weight of the enzyme was estimated as 33 kDa by SDS-polyacrylamide gel electrophoresis and as 28 kDa by Sephacryl S-200 gel filtration indicating that the native 3 alpha(beta)-HSDH is a monomer. In the presence of the preferred co-factor, NADPH, the purified enzyme had a mean apparent Km for 5 alpha-DHT of 3.9 microM and a Vmax of 93.3 nmol (mg protein)-1 h-1 with regard to 3 alpha-HSDH activity, and a Km of 6.3 microM and a Vmax of 20.6 nmol (mg protein)-1 h-1 with regard to 3 beta-HSDH activity.

Neurosteroids: molecular mechanisms of action and psychopharmacological significance.

In addition to the well-known genomic effects of steroid molecules via intracellular steroid receptors, certain steroids rapidly alter neuronal excitability through binding sites on neurotransmitter-gated ion channels. Several of these steroids accumulate in the brain after local synthesis or after metabolization of adrenal steroids. The 3 alpha-hydroxy ring A-reduced pregnane steroids allopregnanolone and tetrahydrodeoxycorticosterone have been thought not to interact with intracellular receptors but enhance gamma-aminobutyric acid (GABA)-medicated chloride currents. When administered systematically in the rat, these neurosteroids display anxiolytic and hypnotic activities that suggest pronounced systemic effects as well as neuropsychopharmacological potential for modulation of sleep and anxiety. We demonstrated that these neurosteroids can regulate gene expression via the progesterone receptor. The induction of DNA-binding and transcriptional activation of the progesterone receptor requires intracellular oxidation of the neurosteroids into progesterone receptor-active 5 alpha-pregnane steroids. Thus, in physiological concentrations these neurosteroids regulate neuronal function through their concurrent influence on transmitter-gated ion channels and gene expression. These findings extend the concept of a "cross-talk" between membrane and nuclear hormone effects and provide a new role for the therapeutic application of these steroids in neurology and psychiatry.

The unliganded estrogen receptor (ER) transduces growth factor signals.

In the absence of serum and estrogen, we show that the growth of the prolactin secreting pituitary tumour cell line, GH3 is stimulated by insulin and insulin-like growth factor-1 (IGF-1) and this response is blocked by the steroidal antiestrogens, ICI 164384 and ICI 182780. From conditioned medium (CM) experiments, growth of low density cells (10k/cm2) is increased by the addition of CM from high density cells (100k/cm2) and this growth effect is also blocked by antiestrogen. Transfection studies with a delta MTV-ERE-LUC reporter plasmid show that in the absence of estrogen and serum, both insulin and IGF-1 induce luciferase expression and this is blocked by the pure antiestrogens. No effect of these treatments was apparent when parallel experiments were conducted with a plasmid construct lacking the vitellogenin estrogen response element. From these and other data discussed in this report, we conclude that for GH3 cells, in the absence of estrogen and serum, the ER is transcriptionally activated by intracellular peptide factor pathways and by this means, acts as the key nuclear factor inducing mitogenesis in response to autocrine and exogenously added growth factors.

Statins potentiate caspase-3 activity in immortalized murine neurons.

Statins are lipid-lowering drugs that have been shown to reduce atherosclerotic cardiovascular morbidity and mortality. However, there is growing evidence from epidemiological studies that long-term treatment with statins has unwanted effects on extrahepatic tissue and increases the risk for neuropathy. To investigate underlying molecular mechanisms we analyzed whether statins influence the activity of caspase-3 in immortalized neurons. Lovastatin and mevastatin are not able to activate caspase-3 but they strongly potentiate its activity when apoptotic signal transduction is initiated by staurosporine. The increase in caspase-3 activity after coincubation with statins and staurosporine was paralleled by an increase in the protein level of the pro-apoptotic GTPase RhoB. Our data provide evidence that statins enhance neuronal apoptosis and therefore give reasons for a careful evaluation when patients with neurological diseases are treated with these drugs.

Photoelectric measurement of laryngeal paralyses correlated with videostroboscopy.

Photoglottography and electroglottography are relatively noninvasive techniques that provide detailed information about vocal fold vibration. However, few significant clinical applications have been made by correlating photoelectric waveforms to specific pathologic changes in laryngeal vibration. Videostroboscopy has recently been used to document vibratory patterns of laryngeal paralyses in a canine model of phonation. A study of PGG and EGG waveforms correlated with videostroboscopy in an in-vivo canine model of phonation with simulated unilateral recurrent or superior laryngeal nerve paralysis is presented. The shift quotient--a new glottographic parameter which identifies flaccid laryngeal paralyses--is presented.

The treatment of T3 glottic carcinoma with vertical partial laryngectomy.

Total laryngectomy has traditionally been considered the optimal treatment for patients with advanced glottic carcinoma who present with a fixed true vocal cord. However, using whole-organ sectioning techniques, it has been demonstrated that vertical partial laryngectomy is a sound oncologic procedure for selected fixed vocal cord lesions. During the period 1969 to 1984, 27 patients who presented at UCLA with T3 glottic carcinoma were treated using vertical partial laryngectomy. Follow-up for these patients averaged 4.0 years. The absolute two-year disease-free survival rate for this group was 85% (23 of 27 patients), and the local cancer recurrence rate during a two-year postoperative interval was 11% (three of 27 patients). These encouraging results support the continued use of partial laryngeal surgery for a subgroup of patients with T3 glottic cancer. Successful patient selection requires a careful analysis of disease extent based on data obtained from physical examination, magnetic resonance imaging or computed tomographic scanning, and direct laryngoscopy.

Melanoma of the nasal and paranasal sinus mucosa.

Melanoma involving the nasal and paranasal sinus mucosa is a rare disease that is difficult to treat and generally has a poor prognosis. Data on 17 patients treated at the UCLA Medical Center during the period 1970 to 1985 were reviewed in a retrospective manner. The five-year disease-free survival was 25% (3/12). Surgery, with or without radiation therapy, is the mode of treatment to control disease in most patients. Treatment failures, which include both local recurrence and distant metastases, may occur many years after initial therapy. We found a correlation between the thickness of tumor and the clinical outcome.

Nonsalivary sinonasal adenocarcinoma.

Thirteen cases of primary non-salivary gland adenocarcinoma of the nasal cavity and paranasal sinuses were studied at UCLA over 20 years. All pathologic specimens were reviewed and those tumors that were histologically distinct from the more common salivary gland-derived tumors were included in the study. Three classifications were identified: well, moderately, and poorly differentiated adenocarcinoma. A distinct variant of sinonasal adenocarcinoma was the intestinal type. The clinical behavior of the latter resembled the well or moderately differentiated types, with behavior mainly predicted by the extent of the disease. These groups have prognostic significance, with the poorly differentiated group having the most virulent course. Nine of 13 tumors occurred in the ethmoid sinuses and all were aggressive locally. Only one case had distant metastases (nodal neck disease in a terminal case). Of five long-term survivors (median five-year follow-up), all had extensive surgical resections and three had full-course radiotherapy. The single most important factor in the treatment of these lesions is adequacy of surgical margins. Four of six patients with confirmed negative margins were cured despite extensive tumors. Three survivors had the cribriform plate taken and one required a combined intracranial/extracranial approach for tumor resection. There were no survivors in four patients treated with primary irradiation.

An accurate method of Teflon injection using functional phonosurgery.

A technique for Teflon injection is described that allows the laryngologist to assess vocal fold vibration during general anesthesia. A tracheostomy tube fitted with a rostral air line allows translaryngeal airflow. During endoscopy with a bivalved laryngoscope, the cords are approximated manually. Vocal fold vibration is produced with the cords adducted. The precise site of defects in glottic closure is clearly seen and corrected with Teflon injection. In cases where standard Teflon injection has failed, utilization of this method has allowed substantial voice improvement. The ability to assess vibratory function of the vocal folds during direct suspension laryngoscopy enhances the precision of vocal fold augmentation techniques in difficult rehabilitation cases.

Office-based system for voice analysis.

There has been recent growing interest in the analysis of various electronically recorded signals as potential tools for objective assessment of vocal dysfunction. In the past, analysis of such signals required an expensive multitrack FM recorder, mainframe computer system, customized software, and significant time commitment. This report describes an adaptation of commercially available components that allow digital recording of multiple electronic signals, storage of data, and subsequent signal analysis using an inexpensive personal microcomputer system. Commercially available software for manipulation and examination signals is discussed as adapted for examination of glottographic and acoustic signals. The relatively inexpensive availability of similar computer systems will, hopefully, encourage assessment of the clinical applications of objective techniques of voice quality.

The effect of air flow and medial adductory compression on vocal efficiency and glottal vibration.

This study used an in vivo canine model to investigate the effects of varying vocal fold resistance by electrically stimulating the recurrent laryngeal nerve while monitoring medial adductory compression of the vocal folds, glottal airflow, and vocal intensity. The effects of increasing airflow on glottal vibration were also examined stroboscopically and by measurement of open quotient. The results indicated that increasing intensity by medial adductory compression was more efficient than by increasing airflow. Increasing airflow produced a significantly greater open quotient and vocal fold vibratory excursion.