Quantitative analysis of modulations in numerical and lateral distribution of intramembrane particles during the cell cycle of neuroblastoma cells.

Modulations in the internal structure of the plasma membrane during the cell cycle of mouse C1300 neuroblastoma cells (clone Neuro-2A) have been studied by freeze-fracture electron microscopy. Both the numerical and lateral distributions of the intramembrane particles (IMP) of the P face of the medium-exposed plasma membrane were determined as a function of the IMP diameter. The lateral IMP-distribution was quantified by a differential density distribution analysis, that could distinguish between random, aggregated, and dispersed distributions of IMP-subpopulations at various levels of spatial organization. Nonrandom lateral IMP-distribution was considered to indicate significant directional constraints on the lateral mobility of the represented molecules. The analysis demonstrated that the density, the size distribution, and the lateral distribution of the IMP are modulated during the cell cycle, such that characteristic structural and dynamic membrane properties can be attributed to the various cell cycle phases (M, G1, S, and G2). The results are interpreted in terms of asynchronous assembly of different membrane components and dynamic reorganizations within the plasma membrane during the cell cycle. Furthermore, they provide a structural manifestation of earlier observed changes in the dynamic properties of membrane proteins and lipids, and functional membrane transport properties in these neuroblastoma cells.

Ionic responses and growth stimulation induced by nerve growth factor and epidermal growth factor in rat pheochromocytoma (PC12) cells.

Rat pheochromocytoma cells (clone PC12) respond to nerve growth factor (NGF) by the acquirement of a phenotype resembling neuronal cells. In an earlier study we showed that NGF causes an increase in Na+,K+ pump activity, as monitored by ouabain-sensitive Rb+ influx. Here we show that addition of epidermal growth factor (EGF) to PC12 cells resulted in a stimulation of Na+,K+ pump activity as well. The increase of Na+,K+ pump activity by NGF or EGF was due to increased Na+ influx. This increased Na+ influx was sensitive to amiloride, an inhibitor of Na+,H+ exchange. Furthermore, no changes in membrane potential were observed upon addition of NGF or EGF. Amiloride-sensitive Na+,H+ exchange in PC12 cells was demonstrated by H+ efflux measurements and the effects of weak acids on Na+ influx. These observations suggest that both NGF and EGF activate an amiloride-sensitive, electroneutral Na+,H+ exchange mechanism in PC12 cells. These findings were surprising in view of the opposite ultimate biological effects of NGF and EGF, e.g., growth arrest vs. growth stimulation. However, within 24 h after addition, NGF was found to stimulate growth of PC12 cells, comparable to EGF. In the presence of amiloride, this stimulated growth by NGF and EGF was abolished. In contrast, amiloride did not affect NGF-induced neurite outgrowth of PC12 cells. From these observations it is concluded that in PC12 cells: (a) NGF has an initial growth stimulating effect; (b) neurite outgrowth is independent of increased amiloride-sensitive Na+ influx; and (c) growth stimulation by NGF and EGF is associated with increased amiloride-sensitive Na+ influx.

Identification of target cells for the genomic effects of estrogens in bone.

Estrogen has bone protective effects, but the exact mechanism behind these effects remains unclear. The aim of the present study was to identify the primary target cells in bone for the classical genomic effects of estrogens in vivo. For this purpose we have used reporter mice with a luciferase gene under the control of three estrogen-responsive elements (EREs), enabling detection of in vivo activation of gene transcription. Three-month-old ovariectomized mice were treated with a single dose (50 mug/kg) 17beta-estradiol (E2). Luciferase activity was analyzed in several tissues and in different bone marrow-derived lymphocyte enriched/depleted preparations using MacsMouse CD19 (for B lymphocytes) or CD90 (for T lymphocytes) MicroBeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Histological characterization of cells with high luciferase content was performed using immunohistochemistry. Both cortical bone and bone marrow displayed a rapid (within 1 h) and pronounced E2-induced increase in luciferase activity. The luciferase activity in total bone marrow and in bone marrow depleted of lymphocytes was increased six to eight times more than in either B-lymphocyte or T-lymphocyte enriched cell fractions 4 h after the E2 injection, demonstrating that mature lymphocytes are not major direct targets for the genomic effect of estrogens in bone. Immunohistochemistry identified clear luciferase staining in hypertrophic growth plate chondrocytes, megakaryocytes, osteoblasts, and lining cells, whereas no staining was seen in proliferative chondrocyte. Although most of the osteocytes did not display any detectable luciferase staining, a subpopulation of osteocytes both in cortical and trabecular bone stained positive for luciferase. In conclusion, hypertrophic growth plate chondrocytes, megakaryocytes, osteoblasts, lining cells, and a subpopulation of osteocytes were identified to respond to estrogen via the classical ERE-mediated genomic pathway in bone. Furthermore, our findings indicate that possible direct estrogenic effects on the majority of osteocytes, not staining positive for luciferase, on proliferative chondrocytes and on mature lymphocytes are mediated by non-ERE actions.

Adenovirus E1A functions as a cofactor for retinoic acid receptor beta (RAR beta) through direct interaction with RAR beta.

Transcription regulation by DNA-bound activators is thought to be mediated by a direct interaction between these proteins and TATA-binding protein (TBP), TFIIB, or TBP-associated factors, although occasionally cofactors or adapters are required. For ligand-induced activation by the retinoic acid receptor-retinoid X receptor (RAR-RXR) heterodimer, the RAR beta 2 promoter is dependent on the presence of E1A or E1A-like activity, since this promoter is activated by retinoic acid only in cells expressing such proteins. The mechanism underlying this E1A requirement is largely unknown. We now show that direct interaction between RAR and E1A is a requirement for retinoic acid-induced RAR beta 2 activation. The activity of the hormone-dependent activation function 2 (AF-2) of RAR beta is upregulated by E1A, and an interaction between this region and E1A was observed, but not with AF-1 or AF-2 of RXR alpha. This interaction is dependent on conserved region III (CRIII), the 13S mRNA-specific region of E1A. Deletion analysis within this region indicated that the complete CRIII is needed for activation. The putative zinc finger region is crucial, probably as a consequence of interaction with TBP. Furthermore, the region surrounding amino acid 178, partially overlapping with the TBP binding region, is involved in both binding to and activation by AF-2. We propose that E1A functions as a cofactor by interacting with both TBP and RAR, thereby stabilizing the preinitiation complex.

Neuroblastoma cells express c-sis and produce a transforming growth factor antigenically related to the platelet-derived growth factor.

Mouse neuroblastoma Neuro-2A cells produce transforming growth factors during exponential growth in a defined hormone-free medium, which, on Bio-Gel columns in 1 M HAc, elute at a molecular size of 15 to 20 kilodaltons (kDa). These neuroblastoma-derived transforming growth factors have strong mitogenic activity, but they do not compete with epidermal growth factor for receptor binding (E. J. J. van Zoelen, D. R. Twardzik, T. M. J. van Oostwaard, P. T. van der Saag, S. W. de Laat, and G. J. Todaro, Proc. Natl. Acad. Sci. U.S.A. 81:4085-4089, 1984). In this study approximately 80% of the mitogenic activity was immunoprecipitated by antibodies raised against platelet-derived growth factor (PDGF). Immunoblotting indicated a true molecular size of 32 kDa for this PDGF-like growth factor. Analysis of poly(A)+ RNA from Neuro-2A cells demonstrated the expression of the c-sis oncogene in this cell line, whereas in vitro translation of the RNA yielded a 20-kDa protein recognized by anti-PDGF antibodies. Separation by reverse-phase high-pressure liquid chromatography demonstrated the presence of two distinct mitogenic activities in neuroblastoma-derived transforming growth factor preparations, one of which is antigenically related to PDGF. Both activities had the ability to induce anchorage-independent growth in normal rat kidney cells, both in the presence and in the absence of epidermal growth factor. It is concluded that Neuro-2A cells express c-sis with concomitant production and secretion of a PDGF-like growth factor, which plays a role in the induction of phenotypic transformation on normal rat kidney cells.

Cell scattering of SK-N-MC neuroepithelioma cells in response to Ret and FGF receptor tyrosine kinase activation is correlated with sustained ERK2 activation.

The c-ret proto-oncogene encodes a receptor tyrosine kinase which plays an important role in kidney and enteric nervous system development. Germline mutations in c-ret are responsible for the dominantly inherited cancer syndromes, multiple endocrine neoplasia types 2A and 2B and familial medullary thyroid carcinoma as well as the developmental disorder Hirschsprung's disease. Using SK-N-MC neuroepithelioma cells stably transfected with an EGFR/Ret chimeric receptor, we have studied cellular consequences and signalling events following activation of exogenous EGFR/Ret and endogenous FGF and PDGF receptor tyrosine kinases in cells of neuroectodermal origin. Here we report that Ret activation led to cell scattering, growth inhibition and loss of anchorage-independent growth. Basic FGF, but not PDGF, evoked similar responses in those cells. Nevertheless, activation of all three receptor tyrosine kinases led to ERK2 activation. Analysis of the kinetics of ERK2 activation and downstream events revealed that Ret and FGF receptor activation led to sustained ERK2 activation and SRE transactivation, while PDGF treatment led to transient ERK2 activation and failed to induce SRE transactivation. Our results suggest that sustained, but not transient ERK2 activation may be involved in cell scattering.

Characterization of 42K+ and 86Rb+ transport and electrical membrane properties in exponentially growing neuroblastoma cells.

For measuring K+ efflux from exponentially growing neuroblastoma cells (clone Neuro-2A), two methods were used, a sampling method and a washing method. Both methods indicated that K+ efflux kinetics were as from a two-compartment system, but the two compartments could only be resolved completely using the washing method. A fast compartment, containing 143 +/- 16 nmol K+/10(6) cells, was found to be associated to the cell surface, and a slow compartment, containing 151 +/- 7 nmol K+/10(6) cells, was found to represent the intracellular K+. The rate constant of the slow compartment was 0.0164 +/-0.0005 min-1, and the K+ efflux rate was 2.46 +/- 0.14 nmol K+/10(6) cells per min. Using the appropriate conditions to measure K+ influx, the kinetics of influx were equal to the kinetics of efflux, indicating steady-state conditions. In addition a comparison was made between 42K+ and 86Rb+ as radioactive tracers for K+ flux. It was found that 86Rb+ was specifically bound on both the inside and the outside of the cells, and for this reason was not a suitable tracer for studying K+ flux kinetics in neuro-2A cells. A membrane potential of -42.9 +/- 1.3 mV and intracellular K+ activity of 108.1 +/- 3.0 mM were measured using conventional and ion-selective microelectrodes. A correlation was made between the K+ flux and electrophysiological data, using the equations of electrodiffusion theory. Thus, the permeabilities of K+ and Na+ were calculated as (3.9 +/- 0.4) . 10(-8) cm/s and (0.6 and 0.1) . 10(-8) cm/s respectively, together with K+ conductance of (2.8 +/- 0.3) . 10(-6) omega-1/cm2.

Lateral diffusion of membrane lipids and proteins is increased specifically in neurites of differentiating neuroblastoma cells.

Lateral diffusion of membrane lipids and proteins was determined in differentiating C1300 mouse neuroblastoma cells by fluorescence photo-bleaching recovery measurements. It is demonstrated that upon differentiation the lateral diffusion of membrane lipids and proteins is increased specifically in the extending neurites. This indicates the appearance of a topographical heterogeneity in the cell membrane, whereby more fluid domains become located in the membrane of the neurites.

Microviscosity changes during differentiation of neuroblastoma cells.

Microviscosity (eta) of the plasma-membrane lipid matrix was measured in exponentially growing and differentiating C1300 mouse neuroblastoma cells, attached to a glass substratum, by fluorescence polarisation of 1,6-diphenyl-1,3,5-hexatriene. Upon differentiation eta decreases progressively, reaching values below those observed in the growth phase. Treatment of the cells with dipalmitoyl phosphatidylcholine vesicles reversibly inhibits morphological differentiation. The results show that a high membrane fluidity is a prerequisite for differentiation.

Effect of fatty acids on plasma membrane lipid dynamics and cation permeability in neuroblastoma cells.

In this study the effects of experimental modifications of plasma membrane lipid lateral mobility on the electrical membrane properties and cation transport of mouse neuroblastoma cells, clone Neuro-2A, have been studied. Short-term supplementation of a chemically defined growth medium with oleic acid or linoleic acid resulted in an increase in the lateral mobility of lipids as inferred from fluorescence recovery after photobleaching of the lipid probe 3,3'-dioctadecylindocarbocyanide iodide. These changes were accompanied by a marked depolarization of the membrane potential from -51 mV to -36 mV, 1.5 h after addition, followed by a slow repolarization. Tracer flux studies, using 86Rb+ as a radioactive tracer for K+, demonstrated that the depolarization was not caused by changes in (Na+ + K+)-ATPase-mediated K+ influx or in the transmembrane K+ gradient. The permeability ratio (PNa/PK), determined from electrophysiological measurements, however, increased from 0.10 to 0.27 upon supplementation with oleic acid or linoleic acid. This transient rise of PNa/PK was shown by 24Na+ and 86Rb+ flux measurements to be due to both an increase of the Na+ permeability and a decrease of the K+ permeability. None of these effects occurred upon supplementation of the growth medium with stearic acid.

Effect of external ATP on the plasma membrane permeability and (Na+ +K+)-ATPase activity of mouse neuroblastoma cells.

1. Addition of 3.5 mM ATP to mouse neuroblastoma Neuro-2A cells results in a selective enhancement of the plasma membrane permeability for Na+ relative to K+, as measured by cation flux measurements and electro-physiological techniques. 2. Addition of 3.5 mM ATP to Neuro-2A cells results in a 70% stimulation of the rate of active K+ -uptake by these cells, partly because of the enhanced plasma membrane permeability for Na+. Under these conditions the pumping activity of the Neuro-2A (Na+ +K+)-ATPase is optimally stimulated with respect to its various substrate ions. 3. External ATP significantly enhances the affinity of the Neuro-2A (Na+ +K+)-ATPase for ouabain, as measured by direct [3H]ouabain-binding studies and by inhibition studies of active K+ uptake. In the presence of 3.5 mM ATP and the absence of external K+ both techniques indicate an apparent dissociation constant for ouabain of 2 X 10(-6)M. Neuro-2A cells contain (3.5 +/- 0.7) X 10(5) ouabain-binding sites per cell, giving rise to an optimal pumping activity of (1.7 +/- 0.4) X 10(-20) mol K+/min per copy of (Na+ +K+)-ATPase at room temperature.

Dominant negative retinoic acid receptor beta.

Induction of the retinoic acid receptor beta 2 (RAR beta 2) gene by retinoic acid (RA) is mediated by a RA response element (RARE), which represents a high affinity binding site for RAR/RXR heterodimers acting at this site as RA-inducible transcription activators. In RA resistant RAC65 cells, RAR beta 2 induction is blocked due to expression of a truncated RAR alpha acting as a dominant negative repressor. Here we show that exogenous expression of RAR but not RXR can restore RA-dependent RAR beta 2 promoter activation in RAC65 cells. Structure-function analysis of hRAR beta 2 mutants in RAC65 cells shows, that the transactivation function required to restore RAR beta 2 promoter activation is dependent on the DNA binding, dimerization and RA-dependent transactivation properties of hRAR beta 2, which are retained in a mutant (beta delta 409) lacking the F domain. By contrast, dominant repression of RA-dependent mRAR beta 2 promoter activation by hRAR beta 2 mutants is independent of the DNA binding or RA-dependent transactivation function but requires a region (residues 204-384) in hRAR beta 2 involved in heterodimerization with RXR. These data extend previous observations on structure-function of RARs and provides tools for studying the role of retinoids and RARs during vertebrate development.

Aggregation and cell cycle dependent retinoic acid receptor mRNA expression in P19 embryonal carcinoma cells.

Differentiation of P19 EC cells along different pathways into derivatives resembling cells of the three embryonic germ layers is accompanied by characteristic differences in modulation of expression of each of the three retinoic acid receptor genes, RAR alpha, -beta and -gamma. Differentiation induced by addition of RA to P19 EC cells cultured in monolayer is accompanied by a rapid increase in expression of both RAR alpha and -beta. Induction of RAR beta occurs in a characteristic biphasic manner, suggesting that multiple factors and/or different mechanisms are involved in controlling its expression. RAR beta mRNA is induced to a far higher level during early aggregation in the presence of RA than during early differentiation in monolayer, suggesting that the direction of differentiation depends on the number and/or ratio of alpha and beta type of RA receptors. Aggregation of P19 EC cells in the presence of RA, but not DMSO, is accompanied by repression of RAR gamma, suggesting that the expression of RAR beta and RAR gamma during neuroectodermal differentiation is mutually exclusive. The effects of RA on RAR expression are significantly greater in G1 than in S-phase of the cell cycle. These results extend previous observations that commitment to differentiation is cell cycle dependent and indicates that critical target gene regulation in response to RA has to take place in G1 for differentiation to occur.

Transcriptional regulation of retinoic acid receptor beta in retinoic acid-sensitive and -resistant P19 embryocarcinoma cells.

As in other embryocarcinoma (EC) cell lines retinoic acid (RA) rapidly induces expression of the nuclear retinoic acid receptor (RAR) beta in murine P19 EC cells, while RAR alpha is expressed constitutively. In the RA-resistant P19 EC-derived RAC65 cells, however, there is no such induction and an aberrant (smaller) RAR alpha transcript is expressed. RAR gamma 1 is expressed at low levels in both cell lines. To study the regulation of the RAR beta gene and the possible involvement of RAR alpha protein in transcriptional activation of the RAR beta gene we transfected these cells with a construct containing a 1.6 kb promoter fragment of the human RAR beta gene fused to the CAT gene. Upon transient assays in P19 EC cells CAT activity is enhanced rapidly by RA, to more than 100-fold in a concentration-dependent fashion. On the contrary no activity can be observed in the RA-resistant RAC65 cells; however, co-transfection of hRAR alpha, hRAR beta or hRAR gamma 1 restores the RA-dependent induction of CAT activity. These results clearly show that RAR alpha and RAR gamma 1 can transactivate the RAR beta gene; that RAR beta can stimulate its own expression and that resistance to RA in RAC65 cells is probably due to the altered RAR alpha transcript present in these cells.

Expression of retinoic acid 4-hydroxylase (CYP26) during mouse and Xenopus laevis embryogenesis.

Retinoids are important signal molecules during vertebrate embryonic development and their synthesis as well as catabolism should therefore be strictly regulated. The retinoic acid (RA) 4-hydroxylase, belonging to the cytochrome P450 family CYP26, is an enzyme catalyzing the 4-hydroxylation of RA, thereby regulating RA homeostasis. Here we describe the temporal and spatial expression patterns of mouse (mCYP26) and Xenopus laevis (xCYP26) homologues. In mouse, expression is detected in uterine crypt, around differentiating cartilage, several regions of the head, regions of the pharynx, the neural retina, and several regions of the trunk. In Xenopus, Northern blot analysis shows presence of xCYP26 transcripts before the MBT and an increased expression level during gastrulation. Whole-mount in situ hybridization shows a specific expression pattern arising at onset of gastrulation, with a ring around the blastopore. By mid gastrulation there is an anterior and a posterior expression domain, each of which gets more complex later in development. There are some important similarities and differences in expression pattern between Xenopus and mouse.

Expression of estrogen receptor alpha and beta during mouse embryogenesis.

In adult mammals numerous target tissues and organs for estrogens exist. Little is known about possible target organs during embryogenesis other than the reproductive tract and the gonads. This is the first report on the expression of estrogen receptor beta (ERbeta) in comparison with ERalpha mRNA during mouse embryogenesis. We found expression of estrogen receptor mRNA in the reproductive tract, but also in the atrial wall, brain, kidney, urethra, bladder neck, mammary gland primordium, midgut, cartilage primordia and perichondria.

Nuclear factor-kappa B repression in antiinflammation and immunosuppression by glucocorticoids.

The transcription factor nuclear factor-kappaB (NF-kappaB) directs transcription of a large number of key molecules in immunological and inflammatory responses. The recently discovered inhibition of transcriptional activity of this factor by the activated glucocorticoid receptor (GR) provides a molecular basis for the potent antiinflammatory and immunosuppressive properties of glucocorticoids. This repressive activity of the GR can function independently of transcriptional activity. Because it has been shown that certain steroid receptor ligands can differentially address transactivation and transrepression functions, it may be possible to develop ligands that specifically suppress NF-kappaB activity and, as a result, are more efficient in treatment of a variety of important chronic inflammatory diseases with less severe side effects than those of currently available drugs.

Intercellular adhesion molecule-1.

The intercellular adhesion molecule (ICAM) 1 is an Ig-like cell adhesion molecule expressed by several cell types, including leukocytes and endothelial cells. It can be induced in a cell-specific manner by several cytokines, for example, tumor necrosis factor-alpha, interleukin-1, and interferon-gamma, and inhibited by glucocorticoids. Its ligands are the membrane-bound integrin receptors LFA-1 and Mac-1 on leukocytes, CD43, the soluble molecule fibrinogen, the matrix factor hyaluronan, rhinoviruses, and Plasmodium falciparum malaria-infected erythrocytes. ICAM-1 expression is predominantly transcriptionally regulated. The ICAM-1 promoter contains several enhancer elements, among them a novel kappa B element which mediates effects of 12-O-tetradecanoylphorbol-13-acetate, interleukin-1, lipopolysaccharide, tumor necrosis factor-alpha, and glucocorticoids. Expression regulation is cell specific and depends on the availability of cytokine/hormone receptors, signal transduction pathways, transcription factors, and posttranscriptional modification. ICAM-1 plays a role in inflammatory processes and in the T-cell mediated host defense system. It functions as a costimulatory molecule on antigen-presenting cells to activate MHC class II restricted T-cells, and on other cell types in association with MHC class I to activate cytotoxic T-cells. ICAM-1 on endothelium plays an important role in migration of (activated) leukocytes to sites of inflammation. ICAM-1 is shed by the cell and detected in plasma as sICAM-1. Regulation and significance of sICAM-1 are as yet unclear, but sICAM-1 is increased in many pathological conditions. ICAM-1 may play a pathogenetic role in rhinovirus infections. Derangement of ICAM-1 expression probably contributes to the clinical manifestations of a variety of diseases, predominantly by interfering with normal immune function. Among these are malignancies (e.g., melanoma and lymphomas), many inflammatory disorders (e.g., asthma and autoimmune disorders), atherosclerosis, ischemia, certain neurological disorders, and allogeneic organ transplantation. Interference with ICAM-1 leukocyte interaction using mAbs, soluble ICAM-1, antisense ICAM-1 RNA, and in the case of melanoma mAb-coupled immunotoxin, may offer therapeutic possibilities in the future. Integration of knowledge concerning membrane-bound and soluble ICAM-1 into a single functional system is likely to contribute to elucidating the immunoregulatory function of ICAM-1 and its pathophysiological significance in various disease entities.

The retinoic acid receptor-beta 2 contains two separate cell-specific transactivation domains, at the N-terminus and in the ligand-binding domain.

In contrast to other members of the steroid/thyroid hormone superfamily, not much is known about the regions involved in transactivation of the receptors for retinoic acid. To determine the transactivation function of RARs, fusion proteins between the DNA-binding domain of the yeast transcription factor GAL4 and retinoic acid receptor-alpha (RAR alpha) or RAR beta were made. Transfection of these constructs resulted in RA-induced activation of a GAL4-responsive element-containing promoter. Deletion analysis revealed that RAR beta-2 has two transcription activation functions (TAFs). TAF-1 activates transcription constitutively and was mapped to the first 32 amino acids of the A-region. TAF-2 is located in the ligand-binding domain between amino acids 137 and 410 and activated transcription only in the presence of RA. The presence of two TAFs was confirmed by cotransfection of RAR beta deletion constructs with the human RAR beta-2 promoter as reporter, showing that the absence of RAR beta TAF-1 causes a decrease in transactivation, whereas truncation of TAF-2 completely blocks this function. Internal deletions in the ligand-binding domain in both GAL-RAR beta and RAR beta expression constructs resulted in a nonfunctional receptor, indicating that the complete ligand-binding domain is required for its transactivation function. Furthermore, we have shown that the contribution of the two TAFs in transcription activation varies among different cell lines, suggesting that they act in a cell-specific manner.

A dual mechanism mediates repression of NF-kappaB activity by glucocorticoids.

Repression of nuclear factor (NF)-kappaB-dependent gene expression is one of the key characteristics by which glucocorticoids exert their antiinflammatory and immunosuppressive effects. In vitro studies have shown protein-protein interactions between NF-kappaB and the glucocorticoid receptor, possibly explaining their mutual repression of transcriptional activity. Furthermore, glucocorticoid-induced transcription of IkappaBalpha was presented as a mechanism in mediation of immunosuppression by glucocorticoids. At present, the relative contribution of each mechanism has not been investigated. We show that dexamethasone induced IkappaBalpha gene transcription in human pulmonary epithelial A549 cells. However, this enhanced IkappaBalpha synthesis did not cause repression of NF-kappaB DNA-binding activity. In addition, dexamethasone was still able to inhibit the expression of NF-kappaB target genes (cyclooxygenase-2, intercellular adhesion molecule-1) in the absence of protein synthesis. Furthermore, we show that the antihormone RU486 did not induce IkappaBalpha expression. However, RU486 was still able to induce, albeit less efficiently, both glucocorticoid- and progesterone receptor-mediated repression of endogenous NF-kappaB target gene expression in A549 cells and the breast cancer cell line T47D, respectively. Taken together, these results indicate that induced IkappaBalpha expression accounts for only part of the repression of NF-kappaB activity by glucocorticoids and progestins. In addition, protein-protein interactions between NF-kappaB and the glucocorticoid or progesterone receptor, resulting in repression of NF-kappaB activity, seem also to be involved. We therefore conclude that NF-kappaB activity is repressed via a dual mechanism involving both protein-protein interactions and induction of IkappaBalpha.