Glucocorticoid inhibition of lymphokine secretion by alloreactive T lymphocyte clones

The effect of glucocorticoids on lymphokine production by T lymphocytes was examined by using long-term alloreactive T cell clones that secreted one or more of the lymphokines interleukin 2 (IL 2), interferon-gamma, macrophage-activating factor (MAF), and colony-stimulating factor when stimulated by an antigen or a mitogen. Production of all of these four lymphokines was inhibited when glucocorticoids were added at physiologic concentrations (10(-8) to 10(-6) M) to clones stimulated with concanavalin A (Con A). Clones were heterogeneous with respect to their sensitivity to glucocorticoid inhibition of MAF production; cytolytic clones were generally more resistant than noncytolytic clones. The glucocorticoid dexamethasone (Dex) and an IL 2-containing supernatant exerted opposing effects on clonal MAF production. Kinetics experiments showed that Dex inhibited MAF production by reducing the rate of secretion without causing a compensatory increase in the duration of secretion, whereas the IL 2 source increased the rate and the total amount of MAF secretion. Dex abrogated the effect of IL 2. Inhibition by Dex was apparent from the earliest time of detectable MAF production (about 4 hr after stimulation) and increased with longer exposure until production ceased (12 to 24 hr). Pre-exposure and removal of Dex before Con A stimulation also inhibited MAF release. Effects of Dex on lymphokine secretion by clones could be dissociated from effects on their growth in response to stimulator cells and IL 2. Factor production by the 16 clones tested was inhibited to some degree. Proliferation, however, by two of these clones (both cytolytic) was unaffected by Dex, whereas proliferation of two noncytolytic clones was strongly inhibited even in the presence of a saturating dose of IL 2.     

The suppression of cellular proliferation in SV40-transformed 3T3 cells by glucocorticoids

Glucocorticosteroids, when added two hours after cell plating to SV40-transformed, 3T3 mouse fibroblasts in low serum (0.3% v/v), biotin-supplemented medium, suppress cellular proliferation by 24 hours. While some cell death probably occurs, the growth inhibition is not primarily due to cytotoxicity and cytolysis. This conclusion is supported by the following: 1) both dead and viable cell numbers are suppressed, 2) little cell debris is evident in the medium, and 3) very high concentrations of glucocorticoids do not cause an increase in the dead cell count. Furthermore, this growth suppression, which is specific for glucocorticoids since several non-glucocorticoid steroids have no inhibitory effect, is not permanent nor irreversible. Removal of the glucocorticoid and replacement with 10% serum restore rapid proliferation. Although higher concentrations (1% and 10%) of serum afford some protection against glucocorticoid inhibition, this protection is not simply a consequence of faster growth rates. SV3T3 cells can be grown in serum-free medium supplemented with biotin, transferrin, insulin, and epidermal growth factor (EGF). Under these conditions growth rates are comparable to high serum media, yet glucocorticoids are still powerful inhibitors. However, the omission of insulin from serum-free, glucocorticoid cultures does result in observable cell death and lysis. Flow microfluorometry and autoradiographic studies have determined that glucocorticoid-inhibited cells are partially blocked in G1. The proportions of S phase and G2 + M cells are greatly reduced with an accompanying accumulation of G1 cells. These results suggest that glucocorticoids regulate a biochemical step(s) in G1 which is critical for DNA initiation.     

Mechanism of the glucocorticoid regulation of growth of the androgen-sensitive prostate-derived R3327H-G8-A1 tumor cell line

The R3327H-G8-A1 cell line derived from the Dunning rat prostate adenocarcinoma contains both androgen and glucocorticoid receptors. Following steroid deprivation, androgens specifically increase the concentration of their receptors in these cells by approximately 2-fold within 6 h and 3-4-fold in 24 h. In the presence of potent glucocorticoids, androgen receptor augmentation is reduced by 40-50% in the first 6 h and completely inhibited during the subsequent 24 h. This event, which is specific for glucocorticoids, appears to be due to an inhibition of androgen receptor synthesis. Furthermore, glucocorticoids inhibit proliferation of these cells by inhibiting the release of growth factors and arresting them in the G0 or A state of the cell cycle. This inhibition can be overcome by addition of low concentrations of either epidermal growth factor or platelet-derived growth factor; however, the inhibitory effect of the glucocorticoid on androgen receptor augmentation is not released. These results suggest that glucocorticoids arrest cellular proliferation by altering the autoregulation of growth and that this event is not dependent upon inhibition of androgen receptor augmentation.     

Glucocorticoids inhibit serum depletion-induced apoptosis in T lymphocytes expressing Bcl-2.

Depletion of growth factors and glucocorticoids are known to induce apoptosis and inhibit growth in T lymphocytes. We have examined the effect of Bcl-2 expression on the cellular response to growth factor depletion in the presence or absence of glucocorticoids. Cell growth was determined by cell counting and viability was quantitated by dye exclusion. Apoptosis was evaluated by flow cytometry, analysis of DNA integrity, and enzymatic determination of caspase-3-like activity. Serum depletion and glucocorticoid administration inhibited cell growth and stimulated apoptosis in Bcl-2 negative cells. Cotreatment with both stimuli had additive effects on apoptosis but not on inhibition of cell growth. Bcl-2 expression abrogated the repressive effect of glucocorticoids on apoptosis but not on cell growth. In contrast, neither apoptosis nor growth inhibition induced by serum depletion of cells was blocked by Bcl-2 expression. However, glucocorticoid treatment of Bcl-2-overexpressing cells protected them from apoptosis induced by serum depletion. Glucocorticoid protection of Bcl-2-overexpressing cells from serum depletion-induced apoptosis was mimicked by other inducers of apoptosis, which act to inhibit protein synthesis. These data suggest that Bcl-2 expression can switch the effect of glucocorticoids from proapoptotic to antiapoptotic when lymphocytes expressing Bcl-2 are exposed to other apoptotic stimuli.     

Glucocorticoids stimulate the release of a viral tumor marker (MMTV gp52) while inhibiting tumor cell growth

The influence of glucocorticoid treatments on the release of mouse mammary tumor virus (MMTV) envelope antigen (gp52) has been studied in C3H mammary tumor cell cultures and compared to treatment-mediated effects on tumor cell growth. Simultaneous assessment of extracellular viral antigen levels and tumor cell growth has indicated that both are coordinately affected by glucocorticoid treatment. While gp52 release is stimulated by treatment, this effect is accompanied by an inhibition of tumor cell growth. These stimulatory and inhibitory effects are mediated by dexamethasone (DEX) in a dose-dependent fashion, and both effects are more pronounced with the synthetic glucocorticoids DEX or triamcinolone acetonide (TA). Quantitation of media gp52 levels by RIA revealed the following hierarchy of glucocorticoid enhancement: TA greater than DEX greater than prednisolone greater than hydrocortisone greater than triamcinolone. A similar order of activity was observed in terms of inhibition of cell growth. The ability of TA to enhance gp52 release was 2.4-2.7 times greater than DEX, a previously proven stimulator of MMTV expression. Cell density of B9 mammary tumor cells was reduced 73% following 72 h of 10(-8) MTA treatment while C3H Mm5mt/cl mammary tumor cells were reduced by 53%. Hormone-mediated changes in in vitro gp52 release suggest that hormones might also influence plasma levels of MMTV gp52 as a systemic marker for the presence and status of murine mammary tumors. Coordinate stimulatory and inhibitory effects suggest that glucocorticoids may play a complex role in murine mammary tumorigenesis and subsequent mammary disease.     

Interactions of glucocorticoids with the AtT-20 cell: effect on protein accumulation

In order to investigate the mechanism through which glucocorticoids downregulate the number of their own receptors in the AtT-20 cell, the effect of glucocorticoids on cell protein metabolism was studied. Glucocorticoids were found to inhibit cellular protein accumulation when included in long-term cultures. The concentrations of agonists that cause a mid-maximal effect are similar to those needed to half-saturate the glucocorticoid receptor, suggesting that the growth-inhibiting effect is receptor-mediated. Two-dimensional electrophoresis of cytosolic extracts of treated and control cells suggested that the effect reflected a general suppression of overall protein accumulation rather than a selective effect on certain classes. Comparison of the protein to DNA ratio of control and dexamethasone-treated cells showed that the latter have higher ratios suggesting that cell composition may be altered by agonists. However, time-course studies of this effect indicated that this is basically an expression of a glucocorticoid effect on cell growth rather than a selective effect on protein metabolism. It is concluded that glucocorticoids inhibit overall AtT-20 cell growth and that this, in turn, manifests itself as a decrease in the rate of protein accumulation. It is suggested that this change in protein metabolism may be a minor component in the mechanism through which glucocorticoids decrease AtT-20 cell ACTH secretion and glucocorticoid receptor number.     

Glucocorticoid receptor regulation: the effects of adrenalectomy, exogenous glucocorticoid, and stress on hepatic receptor number in male and female mice

Although glucocorticoids are known to regulate their own receptor number, the physiologic significance of this process is not known. In order to assess this process in intact animals the effects of adrenalectomy, stress, and exogenous glucocorticoid on the number of hepatic glucocorticoid receptors in Swiss-Webster mice were evaluated. In males 24 hr after adrenalectomy there was a clear 2- to 2.5-fold increase (upregulation) in glucocorticoid receptor number. Conversely, 24 hr after the ip administration of exogenous corticosterone there was a clear downregulation of receptor number. In each case (upregulation and downregulation) female mice were much less responsive than males. Three stressors were used to evaluate the effect of the endogenous secretion of glucocorticoids on downregulation. Male mice were exposed to ether, vibration, and confinement either once or daily for periods up to 3 days. Animals were sacrificed 24 hr after the last stress and hepatic receptor number was compared to an unstressed control. Cytosolic receptor number was not influenced by any of these stimuli. It is concluded that although glucocorticoids clearly regulate glucocorticoid receptor number, as demonstrated by adrenalectomy and the administration of steroids to adrenalectomized animals, the physiologic significance of this process is uncertain as receptor number does not appear to be changed by stimuli of adrenal glucocorticoids in the intact animal.     

Glucocorticoids enhance the gamma-interferon augmentation of human monocyte immunoglobulin G Fc receptor expression

At physiologic and therapeutic concentrations, glucocorticoids decrease the number of Fc receptors for IgG (Fc gamma R) on human monocyte-like cell lines. In comparison, gamma-interferon (IFN-gamma) increases Fc gamma R expression on both human monocytes and monocyte-like cell lines. In this study, we examined the combined effects of glucocorticoids and IFN-gamma on human monocyte expression of the high affinity (72 kDa) Fc gamma R. Mononuclear cells prepared from heparinized venous blood of normal donors were treated for up to 90 hr with or without recombinant IFN-gamma and/or steroids. Monocyte Fc gamma R were measured by Scatchard analysis of the binding of human monomeric 125I-IgG1; indirect immunofluorescence plus flow cytometry, utilizing a monoclonal antibody (MoAb 32) which is specific for the high affinity Fc gamma R; and direct immunofluorescence using fluorescein isothiocyanate-labeled human monomeric IgG1 and flow cytometry quantitated using U-937 cells as a standard. Cultured monocytes incubated in the presence of both glucocorticoids and IFN-gamma for 18 hr had significantly higher (p less than 0.01) Fc gamma R levels than monocytes treated with IFN-gamma alone. The effect of combined treatment reached a plateau by 42 hr of incubation without increasing expression of other surface markers tested. Treatment with glucocorticoids alone did not consistently decrease monocyte Fc gamma R levels after either 18 or 42 hr of culture. Only glucocorticoids augmented the IFN-gamma increase in Fc gamma R; other steroids tested had no effect on IFN-gamma action. Furthermore, the effect was observed after treatment with only one type of interferon, IFN-gamma. These results describe a glucocorticoid immunoregulatory effect that may explain why combined IFN-gamma plus glucocorticoid treatment enhances mononuclear phagocyte Fc-mediated functions.     

Glucocorticoids have opposite effects on ornithine decarboxylase and cell growth in pancreatic acinar AR42J cells.

This paper reviews the relationships between the effects of glucocorticoids on rat pancreatic acinar AR42J cell polyamine levels and cellular growth and differentiation. Glucocorticoids inhibit the growth of AR42J cells. Glucocorticoids either stimulate or inhibit the formation of polyamines in a variety of cell types. Cells require polyamines for normal growth. Therefore, we tested the hypothesis that polyamines mediate the effects of glucocorticoids on AR42J cells. First, to confirm that AR42J cells required polyamines for growth we examined the effects of inhibiting ornithine decarboxylase (ODC). ODC is the most important and generally rate-limiting enzyme in the synthesis of the polyamines. As expected, the ODC inhibitor difluoromethylornithine (DFMO) inhibited AR42J cell DNA synthesis, and the addition of exogenous putrescine reversed this effect. The levels of growth inhibition by glucocorticoids and DFMO treatment were similar. Second, we examined the effects of glucocorticoids on ODC. Surprisingly, glucocorticoids increased levels of AR42J cell ODC mRNA, ODC activity, and putrescine. Glucocorticoids increased these parameters over a similar time-course as they decreased DNA synthesis. Analog specificity studies indicated that a glucocorticoid receptor mediated both the growth inhibitory and ODC stimulatory effects. Dose-response studies indicated, however, that growth inhibition was more sensitive to dexamethasone (DEX) than were ODC levels. Therefore, polyamines do not account for the effects of glucocorticoids on AR42J cell growth. In these cells, glucocorticoids have opposite and independent effects on ODC and growth.     

Interleukin 1 production by human lung tissue. II. Inhibition by anti-inflammatory steroids

Glucocorticoids block the localized accumulation of leukocytes as sites of inflammation by preventing their adherence to vascular endothelium. This implies that glucocorticoids are acting either on the leukocytes, endothelium, or cells which produce adherence-promoting factors (such as interleukin 1 (IL-1)). Previous studies have shown that dexamethasone (DEX) treatment of either polymorphonuclear leukocytes (PMN) or human umbilical vein vascular endothelial cells (VEC) or both in vitro does not prevent adherence induced by thrombin or formylmethionyl-leucyl-phenylalanine (f-met peptide). We now show that pretreatment of PMN and/or VEC for 24 hr with 0.1 microM DEX had no effect on adherence of PMN to VEC activated with IL-1 (2 U/ml), lipopolysaccharide (1 microgram/ml), or 12-O-tetradecanoylphorbol-13-acetate (30 ng/ml) suggesting that glucocorticoids may inhibit adherence in vivo by blocking formation of IL-1 and other adherence-inducing stimuli. We have recently established that cultured human lung fragments produce IL-1 in vitro. To investigate whether glucocorticoids could inhibit the production of adherence-inducing factors, we examined the effect of glucocorticoids on IL-1 production from human lung tissue. Treatment of human lung fragments in vitro for 18 hr with glucocorticoids such as DEX and hydrocortisone resulted in dose dependent inhibition of IL-1 production; these and other glucocorticoids, at concentrations ranging between 0.1 and 1 microM, produced greater than 50% inhibition of IL-1 release. Nonglucocorticoid steroids including testosterone and beta-estradiol (1 microM) had no effect. Inhibition of IL-1 production occurred after a lag period 5 of 16 hr, and the relative glucocorticoid potencies agreed with their known anti-inflammatory potencies in vivo (beta-methasone approximately triamcinolone acetonide greater than DEX greater than fludrocortisone greater than prednisolone greater than hydrocortisone). Inhibition of IL-1 production in vivo may, in part, explain the remarkable ability of glucocorticoids to prevent the adherence of leukocytes to endothelium and their accumulation at an inflammatory site.     

The effect of dexamethasone on the cytotoxic and enzymatic response of cultured endothelial cells to radiation

Experiments were conducted to determine (1) whether glucocorticoids directly protected endothelial cells (EC) from radiation and (2) if angiotensin converting enzyme (ACE) activity, known to be increased by glucocorticoid, played a role in the EC response to radiation. Confluent monolayers of EC cultured from bovine aorta EC were treated with dexamethasone (10(-6) M); after irradiation (5.0 Gy, 60Co gamma), ACE and lactate dehydrogenase (LDH) activities, DNA and protein contents, and nuclei number were measured. Twenty-four hours after 5 Gy, there was increased cell loss (-40%, P less than 0.001), greater LDH release (greater than 100%, P less than 0.001), more LDH activity per cell (+40%, P less than 0.001), and unchanged ACE activity compared to sham-irradiated control EC. However, 48 hr after 5 Gy, ACE activity per cell was decreased (-24%, P less than 0.005). A 48-hr exposure to dexamethasone alone was accompanied by a slight cell loss (-10%, P less than 0.001) and increased cellular ACE activity (+40-140%, P less than 0.001), but a 24-hr dexamethasone exposure was not cytotoxic and did not change ACE activity. Dexamethasone exposure for 48 hr before and after irradiation did not attenuate cell loss or LDH release. However, combined dexamethasone treatment and radiation increased cellular ACE activity at a time when neither agent alone had an effect (24-hr dexamethasone exposure before 5 Gy and assayed 24 hr after 5 Gy). This interaction between radiation and dexamethasone treatment suggests that the glucocorticoid modifies the cell's response to injury. Although this interaction does not ameliorate radiation cytotoxicity, maintenance of ACE levels in injured vessels by hormones may have physiological significance in the hemodynamics of irradiated tissues.     

Effects of progesterone, promegestone and RU 486 on glucocorticoid receptor levels in primary cultures of mouse mammary epithelial cells

Mammary epithelial cells isolated from midpregnant mice and cultured on collagen gels contain glucocorticoid receptors whose levels are modulated by a variety of steroids. In the absence of any added steroid to the cell culture medium, the levels of glucocorticoid receptors in the cells decline during culture, which is counteracted by the addition of a variety of glucocorticoid agonists. The effectiveness of the glucocorticoid in preventing the loss of glucocorticoid receptors is in turn counteracted by the addition of the synthetic progestin promegestone and the synthetic antiglucocorticoid RU 486. Of the two, RU 486 is the most potent in antagonizing the effect of cortisol on the GR levels. Promegestone antagonizes the effect of cortisol, too, although higher concentrations are necessary. Progesterone was without a clear effect either as a glucocorticoid agonist or an antagonist. Progesterone, however, was extensively metabolized by mammary epithelial cells in culture. Based on these observations we conclude that in mammary epithelial cells glucocorticoids positively regulate the metabolism of their own receptors and that antiglucocorticoids, such as RU 486 and progestins, can antagonize that effect.     

Glucocorticoids induce a drastic inhibition of proliferation and stimulate differentiation of adult rat fat cell precursors

The effects of physiological glucocorticoids such as cortisol and corticosterone, as well as dexamethasone, on proliferation and differentiation of rat fat cell precursors kept in primary culture were analyzed. In serum-containing medium (10%), glucocorticoids markedly decreased cell proliferation, either on subconfluent or on confluent cultures. This effect was independent of the presence of insulin. In contrast, acute amplification of adipose conversion was observed mainly when glucocorticoids and insulin were added simultaneously. Morphological quantification of lipid-containing cells confirmed acceleration of the maturation process, and an early and specific reorganization of the cytoskeleton was detected at the ultrastructural level. In the presence of insulin, glucocorticoids also enhanced the main marker enzymes, lipoprotein lipase, and glycerol phosphate dehydrogenase. Glucocorticoid effects on precursor proliferation and differentiation were clearly dose-dependent, dexamethasone being 10 times more potent than cortisol and corticosterone. Similar results were obtained in serum-free medium, as well as in preadipocyte cultures derived from different fat deposits. This study demonstrates that in addition to an acute inhibition of precursor growth, glucocorticoids exert a clear stimulation of adipose conversion, which depends mainly on the presence of insulin and the glucocorticoid concentration.     

Opposing effects of dexamethasone on the clonal growth of granulocyte and macrophage progenitor cells and on the phagocytic capability of mononuclear phagocytes at different stages of differentiation

Dexamethasone, a synthetic glucocorticosteroid, was shown to modulate the colony-stimulating factor-dependent clonal growth of myeloid progenitor cells in semisolid agar cultures, enhancing the formation of granulocyte colonies (50–100%) and suppressing the formation of macrophage colonies (75–97%). Modulation of the pattern of myeloid colony formation by dexamethasone (12–125 nM) was brought about when the steroid was administered to 6-day cultures at the time of culture initiation and up to 72 hr later. Dexamethasone inhibited myeloid cell proliferation when administered to 5-day liquid cultures at culture initiation and up to 96 hr later. Dexamethasone (12–250 nM) also enhanced the phagocytic activity of bone marrow-derived mononuclear phagocytes toward heat-killed (HK) yeast cells (up to 100%) and IgG-coated sheep red blood cells (up to 60%). Enhancement of the phagocytic capability depended critically on the stage in culture at which dexamethasone was administered. Exposure to dexamethasone for 28 hr up to 96 hr of 96-hr cultures of bone marrow cells did not lead to a modulation of phagocytic activity of the developing mononuclear phagocytes. The presence of dexamethasone during the critical period of 96 hr to 120 hr after culture initiation led to an enhanced phagocytic capability, which was statistically significant already 12 hr after the administration of the glucocorticoid. Dexamethasone induced an enhanced phagocytic activity when administered at any time after culture initiation provided that it was in culture during this critical period. When added at 120 hr of culture, dexamethasone no longer enhanced the phagocytic capability of mononuclear phagocytes and when added later than 156 hr of culture suppressed it. Dexamethasone also suppressed (up to 68%) the phagocytic capability of resident and elicited peritoneal macrophages. The results suggest that glucocorticoids shift the balance of granulocyte vs. macrophage formation at early stages of precursor cell differentiation. Reduction in mononuclear phagocyte growth and enhancement of its phagocytic capability might reflect accelerated differentiation/maturation steps. The inhibitory effect of dexamethasone on macrophage formation and on the phagocytic capability of mature mononuclear phagocytes and peritoneal macrophages might be a relevant aspect of the in vivo immune suppression encountered after glucocorticoid administration.     

Lymphocytolytic activity of glucocorticoids in vitro: 51Cr release under equilibrium conditions.

Cytolytic activity of glucocorticoids in vitro is assessed by measuring radiochromium release from steroid-treated thymic lymphocytes under the equilibrium conditions provided by a continuous-labeling technique. Isotope release is a glucocorticoid-specific effect produced at physiological concentrations and is virtually abolished by inhibitors of RNA and protein synthesis. The relative lytic potencies of the steroids tested are comparable to those reported for glucocorticoids as measured by other methods. This procedure not only possesses the advantages typical of isotopic techniques in general, but, in addition, circumvents the problem of "spontaneous" label release associated with the pulse-labeling method. It is a useful alternative to the morphologic examination of cells or the estimation of cell viability for determination of glucocorticoid cytolytic activity.     

Mouse embryo fibroblast proliferation and prostaglandin production in medium supplemented with fetal bovine serum or serum substitutes (Ultroser SF and G): role of glucocorticoids

The growth of DBA/2 mouse embryo fibroblasts, as well as their prostaglandin (PG) production, was compared under 3 different culture conditions: RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 2% Ultroser SF (steroid-free) or with 2% Ultroser G (containing steroids). The effect of the absence or presence of glucocorticoids on both parameters was more precisely investigated. In FBS-supplemented cultures, dexamethasone had a stimulatory effect on cells characterized by a slow growth rate, whereas it markedly inhibited proliferation in rapidly growing fibroblasts. The experiments carried out with serum substitutes (Ultroser SF and G) strongly corroborated the role of the absence or presence of glucocorticoids on fibroblast proliferation. Manipulations of glucocorticoid concentrations in Ultroser SF by adding 5 x 10(-8) M dexamethasone or in Ultroser G by adding 10(-6) M RU 486 reversed the effect of the absence of glucocorticoid in the first case, or in the latter case the effect of the presence of glucocorticoid on both cell growth and PG production. Progesterone had no effect by itself. Our results emphasize the importance of performing complete kinetic studies to investigate the effect of a given factor on cell proliferation in vitro, since glucocorticoids may have opposite effects on fibroblast proliferation according to their cell growth pattern in vitro.     

Dexamethasone stimulates thyrotropin-releasing hormone production in a C cell line

The hypothalamic peptide hormone TRH is also found in other tissues, including the thyroid. While TRH may be regulated by T3 in the hypothalamus, other regulators of TRH have not been identified and the regulation of TRH in nonhypothalamic tissues is unknown. We recently demonstrated the biosynthesis of TRH in the CA77 neoplastic thyroidal C cell line. We studied the regulation of TRH by dexamethasone in this cell line because glucocorticoids have been postulated to inhibit TSH secretion by decreasing TRH in the hypothalamus. Furthermore, TRH in the thyroid inhibits thyroid hormone release. Thus by regulating thyroidal TRH, glucocorticoids could also directly affect thyroid hormone secretion. Treatment of CA77 cells for 4 days with dexamethasone produced dose-dependent increases in both TRH mRNA and cellular and secreted TRH. Increases in TRH mRNA and peptide levels could be seen with 10(-9) M dexamethasone. A 4.8-fold increase in TRH mRNA and a 4-fold increase in secreted peptide were seen with 10(-7) M dexamethasone. Dexamethasone treatment did not increase beta-actin mRNA levels or cell growth. These results suggest that glucocorticoids may be physiological regulators of TRH in normal C cells. In addition to their inhibitory effects on TSH, glucocorticoids may decrease thyroid hormone levels by increasing thyroidal TRH. Since the glucocorticoid effects on C cell TRH are the converse of what is expected for hypothalamic TRH, glucocorticoid effects in these two tissues may be mediated by different regulators.     

Glucocorticoids enhance the mitogenic action of insulin in serum-free cultures of chick embryo cells.

Addition of insulin to nonproliferating serum-free cultures of secondary chicken embryo (CE) cells caused a 30% to 50% increase in cell number. Addition of any one of several glucocorticoids (dexamethasone, cortisol, or corticosterone) to the cultures two days before insulin addition increased the mitogenic effect of insulin by about twofold at each insulin concentration tested. This glucocorticoid stimulation of cell proliferation was “permissive” because in the absence of insulin glucocorticoids caused little increase in cell number (usually less than 15%). Glucocorticoids were maximally active at low concentrations (e.g., 10^?10 M dexamethasone). Steroids without glucocorticoid activity were inactive over a wide range of concentrations. Glucocorticoids increased the mitogenic response to insulin largely by increasing the percentage of cells that insulin stimulated to synthesize DNA. The maximum mitogenic effect of insulin upon CE cells rapidly decreased after the cells were serially subcultured. After only nine population doublings (4 passages) in culture, the response to insulin was diminished by about 70%. The mitogenic effect of insulin plus dexamethasone declined similarly during serial subculture, and was always about twofold greater than the effect of insulin alone. The cells maintained their mitogenic responsiveness to serum as these responses decreased. In contrast to the growth promoting influence of glucocorticoids in the presence of insulin, glucocorticoids inhibited the mitogenic response of CE cells to serum. This result may resolve our above findings with reports that glucocorticoids inhibit the proliferation of CE cells.