Estradiol regulation of secretory component in the uterus of the rat: evidence for involvement of RNA synthesis

The present studies were undertaken to characterize the response of uterine secretory component (SC) to estradiol. Administration of estradiol for 3 days to ovariectomized rats before incubation of uterine tissues resulted in a marked accumulation of SC in the incubation media. When uteri from ovariectomized rats treated with progesterone or testosterone were incubated, very little SC accumulated in the media, indicating that the estradiol-stimulated increase is hormone-specific. When uteri from rats that received estradiol for 6 days were compared with uteri from 3-day treated rats, SC release during a 24-hr incubation period was the same. This finding indicates that in the presence of prolonged estradiol exposure, SC production continues. The estradiol-induced accumulation of SC in culture is not due to the release of pre-formed uterine SC. When tissue SC levels were measured after 3 days of estradiol treatment, very little tissue SC was found relative to that released into culture media during 24 hr of incubation. The addition of actinomycin D to the incubation media markedly inhibited SC release by uteri from estradiol-treated rats. The release of SC was also inhibited by alpha-amanitin, a known inhibitor of Type II polymerase. These studies demonstrate that estradiol stimulation of SC is markedly reduced by inhibitors of RNA synthesis, and suggest that estradiol regulation of SC is mediated through uterine mRNA synthesis.     

Cooperative effect of thyroid and glucocorticoid hormones on the induction of hepatic phosphoenolpyruvate carboxykinase in vivo and in cultured hepatocytes

The present study investigates the effect and interaction of glucocorticoid and thyroid hormones on the induction of phosphoenolpyruvate carboxykinase (PEPck) mRNA and enzyme protein under in vivo conditions and in serum-free cultured hepatocytes from hypothyroid rats. In hypothyroid/adrenalectomized rats T3 significantly enhanced the cAMP induced PEPck mRNA activity within 3-6 h. This effect was further enhanced by the presence of glucocorticoids. The half-life of PEPck mRNA, as determined after administration of cordycepin, was not affected by hypothyroidism or hyperthyroidism (t 1/2 approximately equal to 45 min), but considerably prolonged by the absence of glucocorticoid hormones (t 1/2 less than 80 min). In hepatocytes in culture Bt2cAMP (0.2 mM) provoked an increase in translatable PEPck mRNA within 2 h incubation time. Preincubation with either T3 (0.1 microM) or dexamethasone (0.1 microM) for 4 h significantly enhanced the cAMP response on PEPck mRNA. Addition of both, T3 plus dexamethasone further enhanced this Bt2cAMP-mediated effect. By measurement of PEPck synthesis corresponding findings were observed. It is concluded that glucocorticoid and thyroid hormones predominantly enhance the cAMP-provoked induction of hepatic PEPck mRNA and, consequently, of PEPck synthesis. Their effect is rapid, significant and additive, indicating an independent action. While glucocorticoids, in addition, accelerate PEPck mRNA degradation, the PEPck mRNA decay rate is similar in the presence and absence of thyroid hormones.     

Estradiol and progesterone regulation of immunoglobulin A and G and secretory component in cervicovaginal secretions of the rat

The present study examined the influence of hormones on the levels of immunoglobulins A (IgA) and G (IgG) and secretory component (SC) in cervicovaginal secretions of ovariectomized rats. Administration of estradiol to ovariectomized rats resulted in a significant decline in cervicovaginal content of IgA, IgG and SC. This response was dose dependent and was not prevented by administration of dexamethasone, a potent synthetic glucocorticoid, with estradiol. Treatment of ovariectomized rats with progesterone also lowered the levels of IgA and SC in cervicovaginal secretions. In contrast, dexamethasone had no apparent vaginal effect. The action of estradiol on cervicovaginal IgA, IgG and SC appears to be independent of uterine influence. This conclusion is based upon our observation that estrogen treatment of rats with ligations at their uterocervical junction still have decreased cervicovaginal IgA and SC levels. In parallel with this inhibitory effect, estradiol administration stimulated the accumulation of IgA and SC in uterine secretions. These findings indicate that the sex hormones play a role in regulating IgA, IgG and SC content in cervicovaginal secretions. In addition, it suggests that hormonal balance in females may influence the immune response of the reproductive tract to infectious disease.     

Inhibition of microcystin-induced release of cyclooxygenase products from rat hepatocytes by anti-inflammatory steroids

We showed previously that exposure to microcystin causes eicosanoid release. That study was extended further to test the effect of glucocorticoids on microcystin-induced release of [14C]arachidonic acid and its metabolites from rat hepatocytes previously treated with [14C]arachidonic acid. Release of total radioactivity was 4-fold greater from hepatocytes after 2-hr incubation with 1 microM microcystin than after incubation with control medium. Fluocinolone pretreatment decreased the microcystin-induced synthesis and release of prostacyclin by 24 +/- 2.6% (P less than 0.05) and thromboxane B2 by 39 +/- 3% (P less than 0.025). Treatment of hepatocyte cultures with either microcystin (1 microM) or steroids had no effect on cell viability or total cell protein. Total radioactivity released into the incubation medium was not affected by glucocorticoid alone. Under these conditions, the quantities of both prostaglandin F2 alpha and prostaglandin E2 released were not significantly different when control and microcystin-treated cultures were compared. The half-maximal inhibition (IC50) values obtained from the dose-response data for the inhibition of arachidonic acid release by steroids were comparable with normal cortisol levels in humans. Dose-response curves gave the following rank order of inhibitory potency: fluocinolone greater than dexamethasone greater than hydrocortisone. These results suggest that glucocorticoid therapy might be beneficial in microcystin toxicosis.     

Stimulation and inhibition of cellular functions by glucocorticoids. Correlations with rapid influences on chromatin structure.

The effect of media conditions on the glucocorticoid response has been examined in three types of cultured cells. In rat pituitary tumor cells (GC cells) growth hormone production was stimulated by glucocorticoids provided fresh culture media was present (enriched media conditions). In contrast, dexamethasone either failed to induce or deinduce growth hormone synthesis if added to cultures which had not received fresh media for 3 days (depleted media condition). With human skin fibroblasts, cortisol stimulated [3H]thymidine incorporation in the enriched condition but inhibited this response in the depleted condition. In mouse lymphoma (S49) cells the enriched media conditions significantly delayed the killing response to glucocorticoids (20% killing after 24 h versus 90% killing after 24 h for the depleted condition). Thus, the magnitude and in some cases, the direction of the glucocorticoid response are sensitive to the conditions to which the cells are exposed. In all three cell types the steroid also rapidly (detectable by 15 min, maximal by 2 h) altered chromatin structure as detected by a change in the number of initiation sites for Escherichia coli RNA polymerase assayed under cell-free conditions. This early nuclear response could be in a positive or negative direction and was also affected by the culture conditions; enriched media favored a positive or less negative effect on the initiation sites by the steroid, while depleted media favored a steroid-induced inhibition of this chromatin function. In S49 and GC cells the kinetics and magnitude of the change in chromatin closely followed receptor . glucocorticoid complex binding to nuclei while removal of dexamethasone from the culture media resulted in a rapid (t 1/2 = approximately 20 min) disappearance of the effect which paralleled loss of bound hormone from the nucleus. The glucocorticoid effect on chromatin was not observed in two lines of glucocorticoid-resistant mutant S49 cells. One line (R-) lacks detectable glucocorticoid receptors; the other line (Nti) has receptors that bind the hormone normally, but the receptor . glucocorticoid complexes bind more avidly to the nucleus. These results suggest that the receptor is involved in both the stimulatory and the inhibitory effects on chromatin. The findings in the Nti cells and of a slight lag between nuclear binding of receptors and initiation site alteration implies that some receptor property, in addition to nuclear binding per se, is responsible for the influence on chromatin. These results are discussed in terms of a model in which steroid hormones initiate their actions by influencing a reaction that modifies chromatin structure. The direction and magnitude of the reaction, and its effect on the expression of specific genes, are dictated by the metabolic state and differentiation of the cell.     

Glucocorticoids inhibit estradiol-mediated uterine growth: possible role of the uterine estradiol receptor

Stress-related activation of the hypothalamic-pituitary-adrenal axis (HPA) is associated with suppression of the reproductive axis. This effect has been explained by findings indicating that corticotropin-releasing hormone suppresses hypothalamic gonadotropin-releasing hormone (GnRH) secretion via an opioid peptide-mediated mechanism, and that glucocorticoids suppress both GnRH and gonadotropin secretion and inhibit testosterone and estradiol production by the testis and ovary, respectively. To evaluate whether glucocorticoids suppress the effects of estradiol on its target tissues, we examined the ability of dexamethasone to inhibit estradiol-stimulated uterine and thymic growth in ovariectomized rats. Estradiol alone, given daily for 5 days, caused dose-dependent uterine and thymic growth. Dexamethasone alone, given daily for 5 days, caused a dose-dependent decrease in body weight gain and in thymic growth. When estradiol and dexamethasone were administered simultaneously, however, body weight gain and thymic growth were also inhibited (p less than 0.05). Dexamethasone decreased estradiol-induced uterine cytosolic and nuclear estrogen receptor concentrations (E2 R0, p less than 0.05; E2nR0, respectively), but had no effect on estradiol-induced progesterone receptor concentrations (P4R0, p greater than 0.05). Levels of uterine glucocorticoid receptors were not affected by estrogen and/or dexamethasone treatment. These findings suggest that stress levels of glucocorticoids, administered over a 5-day interval, block the estradiol-stimulated growth of female sex hormone target tissues. This effect may be partially mediated by a glucocorticoid-induced decrease of the estradiol receptor concentration. Thus, another mechanism by which the HPA may influence reproductive function during stress is by a direct effect of glucocorticoids on the target tissues of sex steroids.     

Glucocorticoids and the isolated rat hepatocyte

Following incubation at 37 degrees C with tritiated glucocorticoids isolated hepatocytes prepared from non-adrenalectomized rats show rapid uptake of label. Uptake is non-saturable, and non-linear over the first 60 sec of exposure to steroids. HPLC separation of aqueous extracts of cells and incubation medium shows that polar metabolites of the natural steroid, corticosterone, appear within 10 sec, whereas the synthetic glucocorticoid, dexamethasone, is not altered. Our results suggest that diffusion is the most important process by which glucocorticoids enter liver cells, and that the predominant fate of corticosterone is rapid metabolism.     

Glucocorticoid inhibition of estrus in ovariectomized pigs: Relationship to progesterone action

Synthetic glucocorticoids and progesterone were evaluated for their inhibitory action on estrus in ovariectomized pigs treated with estrogen. Triamcinolone acetonide (˜70 μg/kg BW), and dexamethasone (˜140 μg/kg BW) inhibited the estrous response to estradiol benzoate when these glucocorticoids were given during a period of 5 days before to 4 days after estradiol benzoate. The minimum effective dosage of progesterone that would inhibit estrus when given concurrently with estradiol benzoate was 600 (μg/kg BW. When triamcinolone acetonide (30 μg/kg BW) or dexamethasone (125 or 150 μg/kg BW) was given as a single injection in combination with progesterone (100 μg/kg BW), estrous response to estradiol benzoate was again inhibited. These steroids at these dosages had no significant effect when either was administered alone. Based on these results, the inhibitory action of these glucocorticoids on estrus in pigs is additive to the action of progesterone, and we suggest that triamcinolone acetonide and dexamethasone inhibit estrus through mechanisms related to those of progesterone.     

Differential control of eosinophil survival by glucocorticoids

Glucocorticoids are effective drugs for eosinophil-related disorders, such as asthma and allergy. Previous studies have demonstrated that glucocorticoids increase eosinophil apoptosis and block the survival effect of submaximal concentrations of interleukin-5 (IL-5). We investigated the effect of glucocorticoids on eosinophil survival in the presence of a higher concentration of IL-5 (1 ng/ml), comparable to IL-5 levels in bronchoalveolar lavage and sputum specimens from patients with asthma. In contrast to incubation in the presence of submaximal concentrations of IL-5, the addition of dexamethasone (DEX) to media containing 1 ng/ml IL-5 led to a significant increase in eosinophil cell viability from 58 ± 6.9% to 87 ± 2.4% (p < 0.005) after 72 hours in culture. We found that RU486 blocked the DEX effect on cell viability confirming that glucocorticoid receptor functions are required. We investigated the possibility that the glucocorticoid enhancement of eosinophil survival may be due to an effect on IL-5 receptor expression. Our results show that the IL-5 associated decrease in IL-5 receptor -subunit expression was blocked significantly after 24 hrs in culture with media containing IL-5 plus DEX compared to IL-5 alone. It is tempting to speculate that the observed glucocorticoid enhancement of eosinophil survival in the presence of elevated concentrations of IL-5 could be a mechanism that contributes to glucocorticoid resistance in asthma.     

Insulin inhibits the glucocorticoid-mediated increase in hepatocyte EGF binding

Hydrocortisone and dexamethasone produced a time-dependent increase [125I]epidermal growth factor [( 125I]EGF) binding in primary cultures of isolated rat hepatocytes. Maximally effective doses of glucocorticoids resulted in a 70-100% increase in binding. The effect was similar when hepatocytes were maintained on collagen-coated plates or directly on culture dishes. The glucocorticoid-mediated increase in [125I]EGF binding could be detected after 4 h exposure to glucocorticoid and was substantial by 8 h. The major effect of glucocorticoid appeared to be to increase the number of EGF receptors. While insulin (100 nM) had no effect on basal [125I]EGF binding, it significantly inhibited the increase produced by the glucocorticoid. Since the inhibitory effect of insulin was only observed when insulin was added with the inducing glucocorticoid, insulin appears to inhibit an early hydrocortisone-mediated event.     

Glucocorticoid inhibition of vascular smooth muscle cell proliferation: influence of homologous extracellular matrix and serum mitogens

We examined the influence of glucocorticoid hormones on the proliferation of cultured adult bovine aortic smooth muscle cells (BASM) using both primary mass cultures and a cloned strain. Cloned BASM cells maintained on plastic culture dishes were inhibited by approximately 40% by dexamethasone treatment but showed no inhibition when grown of homologous extracellular matrix (ECM) coated dishes. Dexamethasone inhibited growth of primary cultures by 73% on plastic and by 45% on ECM. The inhibitory effect was specific for the glucocorticoids, dexamethasone, corticosterone, and cortisol and was not observed with progesterone, aldosterone, estradiol or 17-alpha OH progesterone. In cloned cells, the abolition of glucocorticoid inhibition by ECM was independent of seeding density and serum concentration. The inhibition on plastic was dependent on serum concentrations greater than 1% and resulted in both a slow rate of proliferation and a lower saturation density. A specific subset of peptides detected on two-dimensional gels was induced by glucocorticoids under growth inhibitory conditions but was not induced when the cells were grown on ECM. Primary cultures grown on ECM and exposed to Dulbecco's modified Eagle's Medium (DME) containing high density lipoprotein and transferrin grew at 40% of the rate observed for cultures exposed to DME with 10% serum. Both conditions showed growth inhibition of 70% in the presence of dexamethasone. The addition of epidermal and platelet-derived growth factors in DME containing high density lipoprotein and transferrin to cells grown on ECM resulted in growth rates comparable to that observed with cultures exposed to 10% serum and were inhibited 45% by dexamethasone. These results suggest that glucocorticoids inhibit smooth muscle proliferation by decreasing the sensitivity of the cells to mitogenic stimulation by high density lipoprotein when the cells are maintained on a homologous substrate.     

Dexamethasone modulates ErbB tyrosine kinase expression and signaling through multiple and redundant mechanisms in cultured rat hepatocytes

Glucocorticoids paradoxically exert both stimulatory and inhibitory effects on the proliferation of cultured rat hepatocytes. We studied the effects of dexamethasone, a synthetic glucocorticoid, on the proliferation of cultured rat hepatocytes. The timing of growth factor addition modified the action of high-dose dexamethasone (10(-6) M) on DNA synthesis. When we added transforming growth factor-alpha at the time of plating, 10(-6) M dexamethasone weakly stimulated DNA synthesis by 26% relative to cells cultured in dexamethasone-free media. When we delayed growth factor addition until 24-48 h after plating, 10(-6) M dexamethasone inhibited DNA synthesis by 50%. Using immunological methods, we analyzed the expression and signaling patterns of the ErbB kinases in dexamethasone-treated cells. High-dose dexamethasone stabilized the expression of epidermal growth factor receptor (EGFr) and ErbB3, and it suppressed the de novo expression of ErbB2 that occurs during the third and fourth day of culture in 10(-8) M dexamethasone. High-dose dexamethasone by 72 h suppressed basal and EGF-associated phosphorylation of ERK and Akt. The reduction in ERK1/2 phosphorylation correlated with suppression of a culture-dependent increase in Son-of sevenless 1 (Sos1) and ERK1/2 expression. High-dose dexamethasone in hepatocytes stabilized or upregulated several inhibitory effectors of EGFr/ErbB2 and ERK, including receptor-associated late transducer (RALT) and MKP-1, respectively. Thus 10(-6) M dexamethasone exerts a time-dependent and redundant inhibitory effect on EGFr-mediated proliferative signaling in hepatocytes, targeting not only the ErbB proteins but also their various positive and negative effectors.     

Glucocorticoid Regulation of Spermidine Acetylation in the Rat Brain

The effect of glucocorticoids on polyamine metabolism has been elucidated further by measuring putrescine, spermidine, and spermine levels as well as ornithine decarboxylase, S-adenosylmethionine decarboxylase, and N1-acetylspermidine transferase activities in the hippocampus, cerebellar cortex, vermis, and deep nuclei of adrenalectomized rats. At 6 h after corticosterone or dexamethasone administration, the specific activities of ornithine decarboxylase and N1-acetylspermidine transferase showed the greatest increases in all brain tissues examined, and at 12 h, S-adenosylmethionine decarboxylase activity was not increased significantly. The hippocampus and cerebellar regions displayed different responses to corticosterone and dexamethasone, corresponding to the distribution of glucocorticoid and mineralocorticoid receptors. Corticosterone and dexamethasone increased ornithine decarboxylase and N1-acetylspermidine transferase activities in a dose-dependent manner, with dexamethasone being more active than corticosterone in all tissues. However, estradiol, progesterone, testosterone, and aldosterone were only active at doses greater than 5 mg/kg. The great increases in ornithine decarboxylase and N1-acetylspermidine transferase activities were accompanied by a marked increase in putrescine level and a small decrease in spermidine level. Our data confirm that the hippocampus and cerebellum are glucocorticoid target tissues and suggest that the increase in the content of putrescine, following acute treatment with glucocorticoids, is dependent on ornithine decarboxylase as well as N1-acetylspermidine transferase induction.     

Activity and secretion of sialyltransferase in primary monolayer cultures of rat hepatocytes cultured with and without dexamethasone

Monolayers of hepatocytes attached on collagen-coated dishes were cultured for 20-24 h and were found suitable to study the activity and secretion of CMP-N-acetylneuraminate:asialo-alpha 1-acid glycoprotein sialyltransferase. A progressive increase of sialyltransferase activity in the culture medium was observed during incubation of the hepatocytes. After 24 h 34-48% of the total sialyltransferase activity of the hepatocyte incubation system was present in the medium. The enzyme activity present in the medium was soluble in nature and could not be stimulated by Triton X-100. The secretion of the enzyme was stimulated about twofold by dexamethasone. The activity of sialyltransferase in the hepatocytes was also increased by dexamethasone. The Km of either hepatocyte or medium sialyltransferase for CMP-sialic acid was only slightly changed by dexamethasone, whereas the Vmax was increased about twofold. The secretion of sialyltransferase could be inhibited partially by the anti-microtubular agent colchicine. The dexamethasone-induced increase of the sialyltransferase activity in cells and media could be eliminated by inclusion of alpha-amanitin in the culture media at 0 h. The inhibiting effect of alpha-amanitin was only partially expressed when the drug was added 4 h after the addition of dexamethasone to the media. The results suggest that isolated rat hepatocytes actively secrete sialyltransferase and that the increase in the sialyltransferase activity in cells and media owing to the synthetic glucocorticosteroid dexamethasone results from increased synthesis of the enzyme molecule. It is supposed that in the intact rat the increased levels of the enzyme activity in serum observed in inflammation may originate from an induction of the synthesis of sialyltransferase in the hepatocytes of rat liver by the increased levels of circulating corticosteroids.     

Effect of dexamethasone on the isozyme pattern of adult rat liver parenchymal cells in primary cultures

Summary The influence of dexamethasone on the isozyme patterns of ATP-hexose phosphotransferases, aldolase and pyruvate kinase of adult rat hepatocytes maintained in primary cultures has been studied.A progressive loss of the typical adult liver isozymes glucokinase, pyruvate kinase L and aldolase B, with a simultaneous increase of both pyruvate kinase A and hexokinase activities, was observed in hepatocytes cultured in the absence of added glucocorticoid.When the culture medium was supplemented with 10^–7 M dexamethasone, the adult liver patterns of pyruvate kinase and aldolase were preserved for at least seven days of culture, the initial level of glucokinase was maintained for three days, and the rise of hexokinase activity was delayed and partially blocked.These results are discussed in relation to the known beneficial effect of glucocorticoids on the survival of cultured hepatocytes.     

Glucocorticoids inhibit cytokine-mediated eosinophil survival

Glucocorticoids characteristically induce eosinopenia in vivo and are effective for treating allergic and other eosinophilic disorders. We studied the effect of glucocorticoids on cytokine-induced survival of human eosinophils in vitro. Eosinophils were purified from normal or mildly atopic volunteers by Percoll density gradient and incubated for 4 days in the presence of cytokine plus steroid. Cell viabilities were determined by staining cells with fluorescein diacetate and propidium iodide. In the absence of glucocorticoids, human rIL-5 enhanced eosinophil survival in a dose-dependent manner, from 22 fM for a minimal effect to 2200 fM for maximal effect. When eosinophils were cultured with a submaximal concentration of rIL-5 (220 fM), dexamethasone, methylprednisolone, and hydrocortisone inhibited eosinophil survival in a dose-dependent manner. Inhibition was time-dependent and required at least 2 days' exposure of eosinophils to dexamethasone. Dexamethasone, methylprednisolone, and hydrocortisone at 1000 nM inhibited survival by 88 +/- 2, 66 +/- 9 and 37 +/- 7%. In contrast, estradiol and testosterone (1000 nM) had no effect on eosinophil survival. When eosinophils were incubated with varying concentrations of human rIL-5 and 1000 nM dexamethasone, survival inhibition was reduced at higher concentrations of human rIL-5, and completely abolished by human rIL-5 23,000 fM. Human recombinant granulocyte-macrophage CSF, human rIL-3, and human rIFN-gamma also enhanced eosinophil survival in a dose-dependent manner and dexamethasone (1000 nM) strongly inhibited cell survival when submaximal concentrations of these cytokines were used. The effects of dexamethasone were reversed by higher concentrations of granulocyte-macrophage CSF (10 U/ml) and IL-3 (3 ng/ml). However, even 1000 U/ml IFN-gamma did not overcome dexamethasone inhibition, indicating a difference between the mechanism of eosinophil survival induced by IFN-gamma and other cytokines. These results suggest that glucocorticoids exert a direct, inhibitory effect on eosinophil survival, which may be important in the treatment of allergic and other eosinophilic disorders. Antagonism of this effect by higher amounts of cytokine may be a mechanism for glucocorticoid resistance.     

Glucocorticoid receptors in mouse mammary tumors: Specific binding to nuclear components.

The specific interaction of glucocorticoids with nuclei of mouse mammary tumor was studied in vitro by incubation of the tissue with [3H]dexamethasone at 25 degrees. It was demonstrated that the mammary tumors contain a limited number of specific nuclear binding sites which were saturated with low hormone concentrations (10-8 M)9 The concentrations of specific binding sites in the nuclei were related to the concentration of cytoplasmic binding sites of unincubated tissues and varied between individual tumors. The binding component in the nuclei appeared to be a protein and was easily solubilized with 0.4 M KCl containing buffers. The ability of various corticoids to block the nuclear localization of the steroid correlated well with their glucocorticoid potency. Estradiol and progesterone at concentrations of 10-6 M were also effective in competing for the glucocorticoid receptor binding sites. However, while the glucocorticoids such as hydrocortisone and corticosterone translocated to nuclear sites also specific for dexamethasone, estradiol and progesterone competed for the cytoplasmic binding sites and did not translocate to the nucleus. The possible significance of the interaction of various steroids with the glucocorticoid receptors in mammary tumors is discussed.     

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.