Changes of cell morphology and prolactin secretion induced by 2-Br-alpha-ergocryptine, estradiol, and thyrotropin-releasing hormone in rat anterior pituitary cells in culture.

The secretion of prolactin in cultured pituitary cells was studied in correlation with the cellular changes induced by stimulatory or inhibitory agents. The techniques used in this study were: radioimmunoassay, immunocytochemistry, scanning (SEM) as well as transmission (TEM) electron microscopy. Prolactin secretion was stimulated by 17 beta-estradiol (10 nM) as well as thyrotropin-releasing hormone (TRH) (3 nM) and inhibited by 2-Br-alpha-ergocryptine (CB-154) (1 muM). The total prolactin (release and cell content) increased between 2 and 8 d of estradiol treatment, indicating an increase of both synthesis and release of prolactin. This finding was in agreement with TEM observations because, in estradiol-treated prolactin cells, the Golgi saccules were distended and Golgi elements were increased, thus indicating increased synthetic activity of these cells. The addition of TRH over a 4-h period resulted in a significant degranulation of prolactin cells. In contrast, prolactin secretory granules became accumulated in the cells after CB-154 treatment for a period ranging from 4 to 24 h. In agreement, light microscope immunocytochemistry showed an increased reaction for prolactin after short-term (< 24 h) incubation with CB-154. Because prolactin cells represent approximately 70% of the glandular cell population as revealed by immunocytochemistry, it was then possible to observe the changes of cell surface by SEM. In most cells, estradiol and TRH led to an increase in the number and prominence of microvilli and blebs, whereas CB-154 treatment resulted in a slightly decreased number of microvilli and an increased occurrence of membrane foldings. This report thus provides morphological evidence for the stimulatory effects of estradiol and TRH, and the inhibitory effects of CB-154 on prolactin secretion in pituitary cells in primary culture. These data, moreover, show that acute changes in secretory activity of prolactin-secreting cells are accompanied by marked changes of their morphological characteristics.

Induced expression of the glucocorticoid receptor in the rat intermediate pituitary lobe.

Synthesis and release of pro-opiomelanocortin-derived peptides are under differential regulation in the anterior and intermediate lobes of the pituitary. Glucocorticoids inhibit synthesis of pro-opiomelanocortin-related peptides in the anterior lobe but not in the intermediate lobe. These two lobes are also characterized by differences in neural innervation and blood flow, both of which may represent routes of access for regulatory factors (the intermediate lobe is avascular). Immunoreactive glucocorticoid receptor, which can be demonstrated in many tissues, is absent from the intermediate lobe. Immunocytochemistry was used to demonstrate the presence of immunoreactive glucocorticoid receptor in the intermediate lobe after pituitary stalk transection, neurointermediate lobe grafts to kidney capsule, or monolayer culture of neurointermediate pituitary cells. This appearance of the glucocorticoid receptor is presumably a consequence of removal of intermediate pituitary cells from neural influences that may be responsible for inhibiting their expression under normal conditions in vivo.

Inhibition of transcription factor GATA-4 expression blocks in vitro cardiac muscle differentiation.

Commitment of mesodermal cells to the cardiac lineage is a very early event that occurs during gastrulation, and differentiation of cardiac muscle cells begins in the presomite stage prior to formation of the beating heart tube. However, the molecular events, including gene products that are required for differentiation of cardiac muscle cells, remain essentially unknown. GATA-4 is a recently characterized cardiac muscle-restricted transcription factor whose properties suggest an important regulatory role in heart development. We tested the role of GATA-4 in cardiac differentiation, using the pluripotent P19 embryonal carcinoma cells, which can be differentiated into beating cardiac muscle cells. In this system, GATA-4 transcripts and protein are restricted to cells committed to the cardiac lineage, and induction of GATA-4 precedes expression of cardiac marker genes and appearance of beating cells. Inhibition of GATA-4 expression by antisense transcripts blocks development of beating cardiac muscle cells and interferes with expression of cardiac muscle markers. These data indicate that GATA-4 is necessary for development of cardiac muscle cells and identify for the first time a tissue-specific transcription factor that may be crucial for early steps of mammalian cardiogenesis.

A shift in the ligand responsiveness of thyroid hormone receptor alpha induced by heterodimerization with retinoid X receptor alpha.

Thyroid hormone (T3) receptors (T3Rs) are ligand-modulated transcription factors that bind to thyroid hormone response elements (T3REs) and mediate either positive or negative transcriptional regulation of target genes. In addition, in response to ligand binding, T3Rs can interfere with AP-1 activity and thereby inhibit transcription of AP-1-responsive genes. T3Rs were recently shown to form heterodimers with retinoid X receptors (RXRs), leading to increased binding to T3REs in vitro and potentiation of transcriptional responses in vivo. Here we demonstrate that T3R alpha forms stable heterodimers with RXR alpha in living cells. Most important, we describe a new role for RXR alpha in modulating ligand-dependent T3R alpha activity: heterodimerization with RXR alpha greatly increases transcriptional interference with AP-1 activity, augments T3-dependent transcriptional activation, and potentiates the reversal of ligand-independent activation by T3R alpha. In all three cases, the responses occur at substantially lower T3 concentrations when elicited by T3R alpha plus RXR alpha than by T3R alpha alone. In vitro, the binding of T3 decreases the DNA-binding activity of T3R alpha homodimers but does not affect DNA binding by T3R alpha:RXR alpha heterodimers. We provide evidence that increased activities of T3R alpha at lower T3 concentrations are not due to changes in its T3 binding properties. Instead, the altered response could be mediated by either RXR alpha-induced conformational changes, increased stability of heterodimers over homodimers, especially following T3 binding, or both.

A hormone-encoding gene identifies a pathway for cardiac but not skeletal muscle gene transcription.

In contrast to skeletal muscle, the mechanisms responsible for activation and maintenance of tissue-specific transcription in cardiac muscle remain poorly understood. A family of hormone-encoding genes is expressed in a highly specific manner in cardiac but not skeletal myocytes. This includes the A- and B-type natriuretic peptide (ANP and BNP) genes, which encode peptide hormones with crucial roles in the regulation of blood volume and pressure. Since these genes are markers of cardiac cells, we have used them to probe the mechanisms for cardiac muscle-specific transcription. Cloning and functional analysis of the rat BNP upstream sequences revealed unexpected structural resemblance to erythroid but not to muscle-specific promoters and enhancers, including a requirement for regulatory elements containing GATA motifs. A cDNA clone corresponding to a member of the GATA family of transcription factors was isolated from a cardiomyocyte cDNA library. Transcription of this GATA gene is restricted mostly to the heart and is undetectable in skeletal muscle. Within the heart, GATA transcripts are localized in ANP- and BNP-expressing myocytes, and forced expression of the GATA protein in heterologous cells markedly activates transcription from the natural cardiac muscle-specific ANP and BNP promoters. This GATA-dependent pathway defines the first mechanism for cardiac muscle-specific transcription. Moreover, the present findings reveal striking similarities between the mechanisms controlling gene expression in hematopoietic and cardiac cells and may have important implications for studies of cardiogenesis.

Inhibition of AIDS-Kaposi's sarcoma cell proliferation following retinoic acid receptor activation.

Retinoids, a group of natural and synthetic vitamin A analogues the receptors of which belong to the superfamily of steroid receptors, can exert profound effects on growth and/or differentiation of embryonic and neoplastic cells. Kaposi's sarcoma (KS), previously a rare multicentric neoplasm, has become epidemic with HIV infection, although the etiology of KS remains obscure. In the present study, the effects of two potent retinoids, all-trans-retinoic acid (RA) and 13-cis-RA, on the expression of retinoic acid receptor alpha and the growth of AIDS-related KS (AIDS-KS) cells were examined. The proliferation of AIDS-KS cells was significantly inhibited by RA and 13-cis-RA in a dose-dependent manner with 50% inhibitory concentration of 1.4 x 10(-10) M and 4.7 x 10(-9) M, respectively, which correlate with their potency. Growth inhibition was time dependent with maximal inhibition of 90% after 3 days of treatment with 10(-8) M RA. Growth inhibition by RA was further potentiated by forskolin (1 microM), an intracellular cyclic AMP-inducing agent. Moreover, RA treatment blocked the proliferative effect of oncostatin M and tumor necrosis factor alpha, two major KS autocrine growth factors. The effects of RA were accompanied by a dramatic increase in nuclear staining for retinoic acid receptor alpha and in the relative number of strongly positive retinoic acid receptor alpha nuclei. Finally, RA induced morphological changes as KS cells became more flattened, better spread, and more adhesive to the substrate. These results suggest that retinoids inhibit proliferation of AIDS-KS cells and further support their utility as therapeutic agents in AIDS-KS.

Immunocytochemical localization of the glucocorticoid receptor in steroid-sensitive and -resistant human leukemic cells.

Because of their lympholytic action, glucocorticoids are included in most therapeutic regimens for the treatment of lymphomas and leukemias. The presence of functional glucocorticoid receptor may be predictive of the response to hormonal therapy in these diseases. We have developed an immunocytochemical procedure for human glucocorticoid receptor (GR) in order to assess its level and subcellular distribution in a well-studied system of childhood lymphoblastic leukemia cells (CEM), where sensitive and resistant subclones have been established. Several fixation and cell permeabilization protocols were compared. The most sensitive and reproducible one for light microscopy was prefixation in Bouin's solution followed by cytocentrifugation. Using various polyclonal and monoclonal antibodies, the GR was consistently localized predominantly in the cell cytoplasm in the absence of steroid. We compared localization of GR following glucocorticoid treatment in the glucocorticoid-sensitive clone CEM C7 with resistant subclones (4R4, 3R43, ICR27). Upon incubation with glucocorticoid, an increase in nuclear staining was clearly observed in the steroid-sensitive C7 cells. Although the resistant cell lines contain immunoreactive GR, they failed to show nuclear translocation following glucocorticoid treatment. This outlines the importance of the immunocytochemical procedure to distinguish between sensitive and resistant leukemic cells. Whether this test could be used prospectively to help select glucocorticoid therapy in human leukemias and lymphomas can now be examined.

Demonstration of the intracellular localization and up-regulation of glucocorticoid receptor by in situ hybridization and immunocytochemistry.

Glucocorticoid response in target cells closely correlates with glucocorticoid receptor (GR) level. We have compared the localization of GR (by immunocytochemistry) and GR mRNA levels (by in situ hybridization) in steroid-sensitive and steroid-resistant subclones derived from the human CEM lymphoid cell line. In addition, GR was localized in lymphoid cells from patients with various hematological malignancies. In the absence of preincubation with the steroid, GR was localized at the light and electron microscopic level predominantly in the cytoplasm and to a weaker extent in the nucleus. In the presence of steroid, the GR was shown to translocate to the nucleus in the steroid-sensitive but not in the steroid-resistant cell lines. Furthermore, GR and GR mRNA levels were increased following glucocorticoid treatment of the sensitive cell line but not the resistant one. These data support a role for receptor regulation and translocation in the overall mechanism of glucocorticoid hormone action.

Immunocytochemical localization of glucocorticoid receptor in target cells.

Antiserum prepared against highly purified glucocorticoid receptor was used for immunocytochemical studies. Rat liver and pituitary were chemically fixed, and histological sections were examined for immunoreactivity by an indirect immunoperoxidase procedure. In liver, immunoperoxidase staining was observed in the nuclei and cytoplasm of most hepatocytes. Kupffer, endothelial, and bile duct cells were not significantly stained. Control sections treated with preimmune serum remained unstained; preadsorption of antisera with purified liver glucocorticoid receptor resulted in a significant decrease in immunocytochemical staining. In adrenalectomized rats, nuclear staining was markedly reduced, whereas in adrenalectomized rats treated with cortisol acetate, the density of nuclear staining was comparable to and often slightly higher than that in intact animals. In the anterior pituitary, numerous cells were immunoreactive; their nuclei, cytoplasm, or both were stained. Alternate histological sections to those stained with antireceptor antibodies were processed for the localization of ACTH. It was found that the number of anterior pituitary cells that stained with the antireceptor antibodies exceeded the number of corticotrophs. Cells of the intermediate lobe pituitary were devoid of staining, whereas cells in the posterior lobe were stained. The presence of immunoreactive glucocorticoid receptors in pituitary cytosolic fractions was biochemically confirmed by immunoadsorption studies with [3H]triamcinolone acetonide-receptor complexes. This morphological localization of glucocorticoid receptor in liver and pituitary tissues demonstrated that immunocytochemistry can be successfully used to study localization of the glucocorticoid receptor. The known lack of response of intermediate pituitary cells to glucocorticoid may be secondary to a very small number or even an absence of glucocorticoid receptors.

Tissue-specific dopaminergic regulation of the glucocorticoid receptor in the rat pituitary.

The rat intermediate pituitary lobe is one of the rare tissues that is not a known glucocorticoid target and is devoid of immunoreactive glucocorticoid receptor. The intermediate lobe is poorly vascularized and receives a dopaminergic and serotonergic innervation from the hypothalamus. In previous studies we demonstrated that removal of this hypothalamic input results in the appearance of immunoreactive glucocorticoid receptor in the intermediate lobe, as demonstrated with the use of in vitro intermediate pituitary cultures and two in vivo experimental situations. We now show that this appearance of the glucocorticoid receptor is presumably due to removal of hypothalamic dopamine from the intermediate lobe cells, since in this study dopamine (or its potent agonist bromocriptine) inhibits expression of the glucocorticoid receptor in intermediate pituitary cells in primary culture, as demonstrated by [3H] dexamethasone binding and immunocytochemistry. The dopamine antagonist haloperidol blocks the inhibitory effects of the dopamine agonist. In contrast to the intermediate pituitary cells, bromocriptine does not affect glucocorticoid receptor expression in anterior pituitary cells in culture. The differential dopaminergic regulation of glucocorticoid receptor expression in the pituitary gland raises questions about possible effects of dopamine on glucocorticoid receptor levels and glucocorticoid response in other dopamine target tissues, especially in the brain.

Low molecular weight components but not dimeric HCG inhibit growth and down-regulate AP-1 transcription factor in Kaposi's sarcoma cells.

Kaposi's sarcoma, a sexually dimorphic disease inflicting high mortality in AIDS, remains at present without effective treatment. A recent report (Nature 375:64, 1995) showed that the placental glycoprotein hormone, human chorionic gonadotropin (HCG), and surprisingly its beta subunit, inhibit tumorigenicity and metastasis of Kaposi's sarcoma cells in mice xenografts. The anti-KS efficacy of a commercial HCG was subsequently demonstrated in clinical trials. Experimental data presented herein confirm that commercial HCG preparations (known to be about 25% pure) display significant inhibitory action in a dose-dependent manner. However, pure and biologically active HCG has no effect on Kaposi's sarcoma growth in culture. In fact, incubation of Kaposi's sarcoma cells with either one of four different well characterized preparations of pure HCG dimer or any of its two subunits did not alter cellular proliferation suggesting that a contaminant (or degradation product) may be the active agent. Commercial HCG preparations were subfractionated based on molecular size and each fraction was tested with respect to inhibition of KS cell growth, HCG radioreceptor binding and steroidogenic bioactivity. Results demonstrate that the anti-KS activity resides among low molecular weight components, and not in bona fide (macromolecular) HCG. Our study indicates that HCG activity and anti-KS action are separable. Interestingly, the active components in the crude HCG markedly down-regulate AP-1, a complex of transcription factors of the immediate-early response genes associated with cell growth. We conclude that, as yet unidentified molecules, present in the commercial HCG preparations, are responsible for the growth inhibitory effects presumably via the AP-1 signalling pathway.

Cell-specific expression of the glucocorticoid receptor within granular convoluted tubules of the rat submaxillary gland.

The submaxillary gland, a heterogeneous tissue composed essentially of two functionally distinct cell types (tubular epithelial and acinar), offers an interesting system in which to study the mechanisms of steroid-dependent growth and differentiation. One cell type, the granular convoluted tubular (GCT) cell, secretes a large number of physiologically important polypeptides, including epidermal and nerve growth factors. Two steroids, androgens and glucocorticoids, greatly influence the growth, differentiation, and secretory activity of GCT cells. Because glucocorticoids can partially mimic or potentiate androgen effects, it has been thought that glucocorticoids act via androgen receptors. Since the presence of glucocorticoid receptors is a prerequisite for glucocorticoid action, we have investigated the presence and cellular distribution of glucocorticoid receptors within the rat submaxillary gland. Binding experiments using [3H]dexamethasone revealed the presence of high affinity binding sites in rat submaxillary tissue homogenates. Most of these sites were specifically competed by dexamethasone, corticosterone, and a pure glucocorticoid agonist RU 28362. Neither testosterone nor dihydrotestosterone competed for glucocorticoid binding. The cellular distribution of glucocorticoid receptors within the submaxillary gland was investigated by immunocytochemistry, using two highly specific glucocorticoid receptor antibodies. The receptor was localized in the GCT cells, but not in the acinar cells of rat and mouse submaxillary tissue sections. In GCT cells, the glucocorticoid receptor colocalized with several secretory polypeptides, including epidermal growth factor, nerve growth factor, alpha 2u-globulin, and atrial natriuretic factor. These data suggest that the submaxillary gland is a target for glucocorticoid action and that at least part of the glucocorticoid effects on this tissue are mediated by bona fide glucocorticoid receptors.

Regulation of glucocorticoid receptor expression: I. Use of a specific radioimmunoassay and antiserum to a synthetic peptide of the N-terminal domain.

In order to study glucocorticoid receptor (GR) gene expression at the protein level, we have produced an anti-serum to the GR using a 14 amino acid peptide (14-mer) of amino terminus domain of the human GR, and established a simple and specific RIA to quantitate both the human and rat GR. The antibody was raised in rabbits to the 14-mer coupled to either BSA or keyhole limpet hemocyanin. This antibody immunoblots the Mr = 94,000 bona fide GR in tissue extracts and localizes the GR at the subcellular level by immunocytochemistry. In addition, cytosolic GR, previously labeled by the affinity ligand, [3H]dexamethasone mesylate, was immunoprecipitated by the peptide antibody. The 14-mer was iodinated at its tyrosine residue and used in a standard RIA. The binding of the antibody to the 125I-14-mer was displaced by increasing concentrations of either the 14-mer (standard curve) pure GR or tissue cytosol containing native GR. This RIA reliably detects glucocorticoid receptor level between 20 and 500 fmol/tube in human, rat, and mouse tissues. In two well established cell line systems and their subclones (human CEM and in rat hepatoma tissue culture cells transfected or not with GR cDNA) the GR level, as assessed by this RIA, was compared to GR values using the classical radioreceptor or previously published mRNA assays. The relative amount of GR in wild-type cells and in subclones, as assessed by the novel RIA, was identical to the above-mentioned assays. Using the RIA, we demonstrated the down-regulation of GR level in liver following glucocorticoid administration and its up-regulation following adrenalectomy. This study, which constitutes the first description of an RIA for a steroid receptor using a synthetic peptide, provides a powerful tool for a standardized, sensitive, and simple assay for the GR in human and animal tissues.

The atrial natriuretic peptide system in rat ovaries.

The atrial natriuretic peptide (ANP) gene is expressed in several extracardiac tissues where ANP is thought to be involved in autocrine or paracrine regulation. The current studies were designed to characterize the ANP system in rat ovaries. ANP content in rat ovaries was estimated by RIA to be 240 +/- 70 pg/mg protein. HPLC revealed the presence of the 28-amino acid circulating peptide as well as the 126-amino acid prohormone, suggesting that the ovaries are a site of ANP synthesis. Indeed, ANP messenger RNA was detected in this tissue by RNase mapping. ANP present in ovarian extracts displaced [125I]ANP from bovine adrenal receptors (R1 class) in a dose-dependent manner and in parallel to the synthetic peptide, indicating that it possesses biological activity. Immunocytochemical studies localized ANP to interstitial cells surrounding the follicles; weaker but specific staining was also observed in the ovum. High affinity ANP receptors (dissociation constant, 0.30 +/- 0.06 nM; maximum binding capacity, 160 +/- 40 fmol/mg protein) were identified in ovarian membranes. Unlabeled ANP but not c-atrial natriuretic factor (a specific agonist of ANP clearance receptors) competed with binding of [125I]ANP to ovarian membranes in a dose-dependent manner, suggesting that ovarian ANP receptors are predominantly of the R1 class. This was confirmed by cross-linking studies with [125I]ANP, which detected a single protein band with a molecular size of about 120 kilodaltons, corresponding to that of the guanylate cyclase-coupled R1 class of receptor. Consistent with the presence of biologically active receptors, ANP markedly enhanced cGMP accumulation (by 15-fold) in ovarian cells. The presence of both local ANP synthesis and high affinity transducing receptors in the ovaries indicates that the peptide plays a local role in ovarian growth or steroidogenesis.

Glucocorticoid receptor mediated inhibition of interleukin-1 stimulated neutral metalloprotease synthesis in normal human chondrocytes.

Glucocorticoids play an important role in the therapy of arthritic diseases. We sought, firstly, to identify, characterize and localize glucocorticoid receptors (GR) in normal human chondrocytes and, secondly, to determine whether glucocorticoid suppression of human recombinant interleukin-1 beta (rhIL-1 beta)-stimulated metalloproteases (MPs) synthesis by chondrocytes requires GR occupancy. Radioligand binding studies with cultured chondrocytes revealed the presence of high affinity-low capacity [3H]dexamethasone (DEX) binding sites with the following kinetic parameters: Kd = 12.5 +/- 1.4 nmol/L, Nmax = 57,560 +/- 3,960 sites per cell. Competition studies indicated that the DEX binding site was glucocorticoid specific and the competitive hierarchy established was: DEX greater than RU-26988 greater than RU-486 greater than cortisol greater than progesterone much greater than testosterone greater than estradiol-17 beta. Immunocytochemical studies using a specific anti-human GR antiserum identified immunoreactive material primarily in the cytoplasm with cells cultured in the absence of glucocorticoids. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis-Western immunoblotting analysis of chondrocyte cytosol detected the presence of a macromolecular species comigrating with a standard protein possessing a molecular weight of 94 kilodalton. rhIL-1 beta provoked the synthesis and secretion of the MPs stromelysin and collagenase from human chondrocytes in a saturable, coordinate, and dose-dependent fashion. DEX and cortisol inhibited the cytokine-stimulated MP synthesis in similar dose-dependent fashions: DEX, IC50 for stromelysin and collagenase suppression was 1.12 X 10(-8) mol/L and 1.26 X 10(-9) mol/L, respectively and the IC50 for cortisol was 6.3 X 10(-7) mol/L and 4.9 X 10(-8) mol/L, respectively. rhIL-1 beta failed to stimulate metalloprotease synthesis and release from chondrocytes pretreated with 10 nmol/L DEX, even after 20 days of incubation. The antiglucocorticoid, RU-486 completely reversed the DEX induced suppression of MP synthesis at 10(-7) mol/L. RU-486 alone had no effect on MP synthesis. We believe there is a biochemical rationale for the therapeutic efficacy of glucocorticoid administration in the management of arthritic diseases such as osteoarthritis and rheumatoid arthritis, and cytokines such as IL-1 are likely to be involved in the increase in MP synthesis.

Reduced expression of glucocorticoid receptor levels in human osteoarthritic chondrocytes. Role in the suppression of metalloprotease synthesis.

Glucocorticoid occupancy of a large percentage of glucocorticoid receptor (GR) is necessary for the suppression of matrix metalloprotease synthesis by human articular chondrocytes. In this study, we evaluated the levels of GR binding, cellular GR protein, and messenger RNA expression in both normal and osteoarthritic (OA) human articular chondrocytes and compared the degree of suppression of collagenase synthesis by glucocorticoids in cultures of the two cell types in order to investigate whether or not the GR system played an important role in the pathophysiology of OA. By radioreceptor binding assay, we recorded 56,320 +/- 8,230 sites per cell (mean +/- SE, n = 9) in primary cultures of normal chondrocytes and 27,480 +/- 14,240 sites in OA cells (n = 10, P < 0.0001). Equilibrium dissociation constant (Kd) values did not vary between normal (12.4 +/- 1.4 nmol/L) and OA (13.0 +/- 1.8 nmol/L). Subculturing of primary OA chondrocytes resulted in the up-regulation of the number of GR binding sites per cell to values comparable to those obtained in normal chondrocytes. Analysis of protein-immuno dot-blots of cytosolic extracts from normal (n = 4) and OA chondrocytes (n = 4) revealed that the former cytosols contained a 1.9 +/- 0.2 (P < 0.05) higher relative density of GR protein than the latter. By comparing the optical densities of GR-polymerase chain reaction products generated from normal (n = 6) and OA (n = 9) chondrocyte total RNA (normalized using an internal standard, glyceraldehyde phosphate dehydrogenase), we established a relative ratio, normal/OA, of 1.4. Experiments comparing the biological responsiveness of normal and OA chondrocytes to glucocorticoid suppression of interleukin-1-stimulated metalloprotease synthesis showed that dexamethasone inhibited collagenase synthesis in a dose-dependent manner with an IC50 of 6.3 +/- 1.2 x 10(-10) mol/L (n = 5) in normal cells while an IC50 of 5.0 +/- 0.4 x 10(-9) mol/L (P < 0.05) was recorded using OA (n = 5) chondrocytes. The results suggest that OA chondrocytes express fewer GR than normal cells as a result of a decrease in specific gene expression. The decreased responsiveness of OA cells to circulating glucocorticoids may be among the factors responsible for an increased level of metalloprotease synthesis by chondrocytes in OA cartilage.

Synthesis and immunocytochemical localization of alpha 2u globulin in the duct cells of the rat submaxillary gland.

Alpha 2u globulin, a protein of unknown function so far believed to be synthesized exclusively in the male liver under multihormonal control, is now shown to be localized by immunocytochemistry in the granular convoluted tubules of the adult male submaxillary gland. In addition, using Northern blot analysis, we have shown specific alpha 2u globulin mRNA sequences in the RNA extracted from the submaxillary gland. Thus, it is evident that the protein is being synthesized therein. Alpha 2u globulin was also detected in the submaxillary gland duct cells of adult female and immature animals of both sexes, all of which are known not to synthesize alpha 2u globulin in their livers. The present data have established that alpha 2u globulin is synthesized in the rat submaxillary gland and indicate that the control of alpha 2u globulin gene expression in the rat liver and in the submaxillary gland is different.

Glucocorticoids inhibit bone resorption by isolated rat osteoclasts by enhancing apoptosis.

We have studied the effects of glucocorticoids on the activity and viability of neonatal rat osteoclasts in vitro. In the bone slice assay, glucocorticoids caused a dose-dependent decrease in the amount of bone resorbed, which was accompanied by a parallel decrease in osteoclast number. Loss of osteoclasts was due to their death, which occurred by the process of apoptosis. Evidence for the latter was obtained by a range of techniques, including time-lapse video microscopy, acridine orange staining, DNA fragment detection and transmission electron microscopy. Immunocytochemistry revealed the presence of glucocorticoid receptors in osteoclasts, and glucocorticoid-induced cell death could be prevented by the glucocorticoid receptor antagonist, RU486. These observations suggest that glucocorticoids promote receptor-mediated apoptosis of rat osteoclasts in vitro. This finding may help to explain recent data indicating that, in sharp contrast with their effects on the human skeleton, glucocorticoids inhibit bone resorption and increase bone mass in rats in vivo.

Corticostatic peptides.

In the last four years corticostatic (anti-ACTH) peptides have been isolated from human, rabbit, guinea pig and rat tissues. These peptides do not act via the cAMP cell signalling system but rather via the inhibition of the binding of ACTH to its receptor most probably through direct competition with the 14-18 sequence of ACTH for receptor binding. ACTH has specific high affinity receptors on adrenal cells but rabbit corticostatin I (CSI) has high capacity, low affinity receptors which are competed for by unlabelled excess CSI but not by excess ACTH. This indicates the presence of specific CSI adrenal cell receptors. The rabbit pituitary, hypothalamus, thalamus, adrenals, lungs and placenta contain sizeable amounts of immunoassayable CSI. Immunochemical localization of CSI indicates that it is present in the large macrophages and in neutrophils in rabbit lung, in macrophages and "supporting" endothelial cells in the spleen and in the adrenals in the cells of the zona reticularis. We have also isolated and identified new peptides which contain 12 cysteines from immune cells of humans, rats and a teleost, the carp. The functions of these peptides are now being determined. This large family of peptides may have many other, yet unidentified functions but at present we can only describe a small number of these.

Expression of atrial natriuretic factor gene in heart ventricular tissue.

A novel peptide hormone, atrial natriuretic factor (ANF), was recently isolated and characterized in mammalian atria. This hormone has potent natriuretic diuretic and vasorelaxant activities. Since ANF bioactivity was initially found in atria but not in ventricles, it was assumed that the ANF gene is specifically expressed in atria. We now report that ANF mRNA is present in ventricular tissue as well as in atria. This is clearly demonstrated by in situ hybridization and by Northern blot analysis. Rat ventricular ANF mRNA concentration is a hundred-fold lower than in atria. As in atria, the 126 amino acids precursor form of ANF is predominant in ventricles and it is present at a thousand-fold lower concentration. The ten-fold discrepancy in the ratio of ANF mRNA to immunoreactivity between atria and ventricles could reflect a higher rate of peptide release in the latter. Thus, ventricular ANF production may be physiologically significant in view of the much larger ventricular mass.