Effects of dexamethasone and 5-bromodeoxyuridine on protein synthesis and secretion during in vitro pancreatic development

Protein synthesis and secretion during in vitro pancreatic development and after treatment with the glucocorticoid dexamethasone and the thymidine analog 5-bromodeoxyuridine (BrdU) was monitored using two-dimensional gel electrophoresis. At 14 days gestation, the synthesis of more than 200 proteins and the secretion of a complex set of proteins was detected. The relative rate of synthesis and secretion of the majority of this set of proteins decreased dramatically during development; after 6 days of culture most were no longer detected. In contrast, the synthesis and secretion of pancreas-specific exocrine proteins amylase, a Sepharose binding protein (protein 2), and chymotrypsinogen first detected after one day in culture, increased throughout the 6-day culture period. Other pancreatic digestive (pro)enzymes normally found in the adult such as the basic form of chymotrypsinogen, lipase, ribonuclease, and trypsinogen were not detected during the culture period. Thus at least two distinct regulatory events are involved in the expression of the exocrine genes during development. Dexamethasone treatment during the 6-day culture period selectively increased the synthesis of amylase and several other minor secretory proteins. BrdU treatment caused major changes in the protein synthetic and secretory patterns of the pancreas as well as in morphogenesis. BrdU treated pancreases showed greatly reduced synthesis of amylase, protein 2, and chymotrypsinogen and prolonged synthesis of many proteins normally detected only at early stages of pancreatic development. BrdU treatment also stimulated the secretion of a set of proteins ostensibly associated with duct cells. Thus, BrdU specifically alters the developmental program of the pancreas.     

Interaction of glucocorticoid hormones and cyclic nucleotides in induction of tyrosine aminotransferase in cultured hepatoma cells.

Reproducible induction of the enzyme tyrosine aminotransferase by dibutyryl cAMP (Bt2cAMP) in a line of HTC hepatoma cells in suspension culture requires that the cells be preinduced with dexamethasone, a synthetic glucocorticoid which itself induces tyrosine aminotransferase. Concentrations of dexamethasone that do not induce tyrosine aminotransferase fail to support Bt2cAMP induction, removal of the steroid from the medium leads to a loss of the Bt2cAMP effect, and an HTC cell line whose aminotransferase is not steroid-inducible does not respond to the cyclic nucleotide. We show that the further induction of tyrosine aminotransferase by Bt2cAMP in dexamethasone-treated cells is due to an increased rate of enzyme synthesis. The cyclic nucleotide has no effect on aminotransferase synthesis in cells grown in the absence of steroid. Several lines of evidence suggest that dexamethasone acts at a step beyond the activation of protein kinase by cAMP: (a) basal levels of cAMP are not altered by growth of HTC cells in dexamethasone; (b) accumulation of cAMP from the medium is not enhanced; (c) the glucocorticoid does not induce cAMP-dependent protein kinase in HTC cells; and (d) there is no augmentation of cAMP binding to the regulatory protein, nor is there any change in cAMP activation of protein kinase caused by growth in dexamethasone. These results help define a system that should be useful in studying the interaction of cyclic nucleotides and steroid hormones.     

Dexamethasone-mediated induction of mouse mammary tumor virus RNA: a system for studying glucocorticoid action.

We have investigated the mechanisms by which dexamethasone (a synthetic glucocorticoid) stimulates the production of mouse mammary tumor virus (MMTV) by cell cultures derived from mammary carcinomas of GR mice. Treatment of these cells with dexamethasone stimulates a rapid accumulation of intracellular virus-specific RNA which is dependent upon RNA synthesis but not upon DNA or protein synthesis. The effect of dexamethasone is probably mediated by a specific and saturable glucocorticoid receptor. We conclude that the accumulation of MMTV RNA is a primary response to dexamethasone and that the rate of synthesis of MMTV RNA is probably accelerated by treatment with dexamethasone.     

The influence of glucocorticoid on albumin synthesis and its messenger RNA in rat in vivo and in hepatocyte suspension culture

Corticosteroids are known to stimulate the synthesis of a number of liver-specific proteins. The reports regarding the effect of glucocorticoid on albumin synthesis in vivo and in vitro are controversial. In an attempt to determine the mechanism by which glucocorticoid exerts its influence on hepatic albumin synthesis and to find an explanation for the conflicting data, we have studied the effect of dexamethasone disodium phosphate on albumin synthesis and albumin messenger RNA as determined by the molecular hybridization technique in hepatocytes in rat in vivo and in suspension culture. In hepatocyte suspension culture, addition of 0.48 μM dexamethasone in medium at zero time led to a significant increase (20%) in incorporation of labeled precursor into albumin as compared to control experiments; this was accompanied by a maintainance of the initial level of full-length albumin mRNA for a 9 h period. In hepatocytes cultured without dexamethasone in the medium there was a progressive loss of albumin mRNA content. Despite this finding, dexamethasone was not able to increase the albumin mRNA content in hepatocyte to a level higher than the initial value. Moreover, administration of this hormone either intraperitoneally or intravenously into rats did not lead to enhanced cell-free albumin synthesis or to an increased level of albumin mRNA. These findings suggest that glucocorticoid does not play an essential role in the regulation of albumin synthesis in vivo. In vitro, however, glucocorticoid leads to a preservation of the initial level of albumin mRNA and thus plays a role in the control of spontaneous dedifferentiation of liver cells in culture.     

Dexamethasone modulates the metabolism of type IV collagen and fibronectin in human basement-membrane-forming fibrosarcoma (HT-1080) cells.

The effect of dexamethasone on the synthesis and degradation of type IV collagen was studied in human fibrosarcoma cells, HT-1080. A dexamethasone concentration as low as 0.1 microM markedly increased collagen synthesis in HT-1080 cells labelled with [14C]proline. The increase in type IV collagen synthesis was not specific, since total protein synthesis was also increased. Further studies indicated that part of the increase was due to an increase in the specific radioactivity of the intracellular proline pool, after dexamethasone treatment. In fact, with dexamethasone concentrations of 0.1-10 microM the relative collagen synthesis was decreased, indicating that synthesis of other protein was increased more than that of type IV collagen. This was also confirmed by measuring the relative amount of type IV collagen RNA by using recombinant plasmid cDNA specific for the human procollagen pro alpha l (IV) RNA. The results indicated that relative collagen synthesis and the relative amount of type IV collagen messenger RNA was decreased similarly, indicating that dexamethasone affected type IV collagen synthesis at the pre-translational level. The dexamethasone-induced effect on total protein and collagen synthesis was maximal after 12-24 h. Dexamethasone induced a marked accumulation of collagen into the cell layer, leading to diminished deposition of soluble collagen into the medium. Since bacterial-collagenase treatment of the cell layer drastically decreased the collagen content of the dexamethasone-treated cells, this indicates that dexamethasone caused an accumulation of collagen into the extracellular matrix of the cell layer. In contrast, the amount of fibronectin was markedly increased in the medium. Dexamethasone decreased the type IV collagen-degrading activity in HT-1080 cells. The HT-1080 cells contained glucocorticoid receptors, as demonstrated by two different methods: by a whole-cell binding assay and by using a cytosol-gel-filtration method. The number of specific binding sites was similar to that in human skin fibroblasts. In conclusion, glucocorticoids affect the metabolism of type IV collagen and fibronectin in HT-1080 cells, and, since these cells contain specific glucocorticoid receptors, the effects are apparently receptor-mediated.     

Regulation of secretory component by glucocorticoids in primary cultures of rat hepatocytes

The present study examined the effects of steroid hormones on the production of secretory component (SC) by rat hepatocytes in cell culture. When hepatocytes were incubated in the presence of cortisol (10(-6) M), the levels of SC in media increased significantly after 2 days of incubation. This response was dose-dependent and specific for glucocorticoids because progesterone, dihydrotestosterone, and estradiol had no effect. When estradiol was added to the incubation media along with dexamethasone, a known potent synthetic glucocorticoid, it diminished the glucocorticoid response. The addition of cycloheximide to incubation media significantly decreased the effect of dexamethasone on SC accumulation. These findings suggest that glucocorticoid regulation of hepatocyte SC most likely involves stimulation of its synthesis. In addition, our results suggest that endogenous glucocorticoids may play a role in enhancing the clearance of IgA from blood into bile in the intact animal.     

Dexamethasone is a potent stimulator of growth hormone-releasing factor-induced cyclic AMP accumulation in the adenohypophysis

The stimulatory effect of maximal concentrations of synthetic human pancreatic growth hormone (GH)-releasing factor (GRF)(1-40)NH on cyclic AMP accumulation in rat anterior pituitary cells in culture is 4.5-fold increased following a 48-h preincubation with the potent glucocorticoid dexamethasone while the sensitivity of GRF action is increased by approximately 4-fold. Dexamethasone pretreatment, on the other hand, has no effect on basal cyclic AMP levels but approximately doubles both basal and GRF-induced GH release. The present data suggest that the potent stimulatory effect of glucocorticoids on GH secretion is exerted on the adenylate cyclase system at a step preceding cyclic AMP formation.     

HPAC,a new human glucocorticoid-sensitive pancreatic ductal adenocarcinoma cell line

Summary A new human pancreatic cancer (HPAC) cell line was established from a nude mouse xenograft (CAP) of a primary human pancreatic ductal adenocarcinoma. In culture, HPAC cells form monolayers of morphologically heterogenous, polar epithelial cells, which synthesize carcinoembryonic antigen, CA 19-9, CA-125, cytokeratins, antigens for DU-PAN-2, HMFG1, and AUA1, but do not express chromogranin A or vimentin indicative of their pancreatic ductal epithelial cell character. In the presence of serum, HPAC cell DNA synthesis was stimulated by insulin, insulin growth factor-I, epidermal growth factor, and TGF-α but inhibited by physiologic concentrations of hydrocortisone and dexamethasone. Dose-dependent inhibition of DNA synthesis was limited to steroids with glucocorticoid activity. The inhibitory effect of dexamethasone was abolished by the glucocorticoid antagonist RU 38486. Binding of [³H]dexamethasone to cytosolic proteins was specific and saturable at 4° C. Scatchard analysis of binding data demonstrated a single class of high-affinity binding sites (K_d=3.8±0.9 nM; B_max=523±128 fmol/mg protein). Western blot analysis revealed a major protein band that migrated at a M_r of 96 kDa. Northern blot analysis identified an mRNA of approximately 7 kilobases which hybridized with a specific glucocorticoid receptor complementary DNA probe (OB7). These findings support a role for glucocorticoids in the regulation of human malignant pancreatic cell function.     

Nonactivated and activated glucocorticoid receptor complexes from human salivary gland adenocarcinoma cell line

[3H]Triamcinolone acetonide glucocorticoid receptor complexes from human salivary gland adenocarcinoma cells (HSG cells) were shown to be activated with an accompanying decrease in molecular weight in intact cells, as analyzed by gel filtration, DEAE chromatography, the mini-column method and glycerol gradient centrifugation. Glucocorticoid receptor complexes consist of steroid-binding protein (or glucocorticoid receptor) and non-steroid-binding factors such as the heat-shock protein of molecular weight 90,000. To determine whether the steroid-binding protein decreases in molecular weight upon activation, affinity labeling of glucocorticoid receptor in intact cells by incubation with [3H]dexamethasone 21-mesylate, which forms a covalent complex with glucocorticoid receptor, was performed. Analysis by gel filtration and a mini-column method indicated that [3H]dexamethasone 21-mesylate-labeled receptor complexes can be activated under culture conditions at 37 degrees C. SDS-polyacrylamide gel electrophoresis of [3H]dexamethasone 21-mesylate-labeled steroid-binding protein resolved only one specific 92 kDa form. Furthermore, only one specific band at 92 kDa was detected in the nuclear fraction which was extracted from the cells incubated at 37 degrees C. These results suggest that there is no change in the molecular weight of steroid-binding protein of HSG cell glucocorticoid receptor complexes upon activation and that the molecular weight of nuclear-binding receptor does not change, although the molecular weight of activated glucocorticoid receptor complexes does decrease. Triamcinolone acetonide induced an inhibitory effect on DNA synthesis in HSG cells. Dexamethasone 21-mesylate exerted no such effect and blocked the action of triamcinolone acetonide on DNA synthesis. These results suggests that dexamethasone 21-mesylate acts as antagonist of glucocorticoid in HSG cells. The fact that dexamethasone 21-mesylate-labeled receptor complexes could be activated and could bind to DNA or nuclei as well as triamcinolone acetonide-labeled complexes suggests that dexamethasone 21-mesylate-labeled complexes can not induce specific gene expression after their binding to DNA.     

Corticosteroid binding by fetal rat and rabbit lung in organ culture

To further characterize glucocorticoid action in fetal lung cells, we investigated corticosteroid metabolism and binding in explants of fetal rat and rabbit lung. Cortisone (E) was concerted to cortisol (F) and bound by receptor with a time course only somewhat slower than for F. Production of F (0.243 pmol/min/mg DNA) was the same in male and female rabbits and was not affected by prior exposure to glucocorticoid in utero or in culture. The t 1/2 for dissociation of nuclear-bound [3H]F was 84 min on changing the culture medium and 21 min on addition of excess non-labeled dexamethasone. Dissociation of [3H]dexamethasone was approx 5-fold slower by both procedures. The KD for nuclear binding of dexamethasone, F, E, and corticosterone in rabbit lung were 0.7, 7.3, 6.8 and 70.6 nM, respectively. In rat lung, the KD for dexamethasone was 6.8 nM. The concentrations of dexamethasone and F required for half-maximal stimulation of phosphatidylcholine synthesis were similar to the KD values. Dexamethasone binding capacity (sites/mg DNA) increased with age in both rat (+103% increase from day 16 to 22) and rabbit (+47% between day 23 and 30). Receptor concentration was the same in both sexes, and there were no developmental changes in non-specific binding, nuclear:cytoplasmic distribution, or KD. In 27-day rabbit fetuses, the rate of choline incorporation was higher in lungs with greater binding capacity. We conclude that (1) E is rapidly converted to F in rabbit lung to become an active glucocorticoid, whereas corticosterone probably has little physiologic activity, (2) there is a species difference in the affinity of dexamethasone binding which is reflected in responsiveness (3) there is no difference between sexes in E conversion, receptor capacity, or phosphatidylcholine synthesis, and (4) the concentration of binding sites per lung cell increases during fetal development. We suggest that developmental increases in both F production and receptor may be important factors in the expression of endogenous glucocorticoid effects.     

Effect of glucocorticoids on C3 gene expression by the A549 human pulmonary epithelial cell line.

The third component of C, C3, is the key opsonin of the C cascade and is produced locally within the lung by pulmonary epithelial cells, macrophages, and fibroblasts. Because glucocorticoids regulate the maturation and expression of several physiologically important genes in pulmonary epithelial cells, we examined the effects of glucocorticoids on C3 mRNA expression and C3 synthesis by the human pulmonary epithelial cell line, A549. Treatment with dexamethasone enhanced C3 production in a time- and dose-dependent fashion such that concentrations of dexamethasone greater than or equal to 0.001 microM significantly increased C3 production on day 3 of culture. Natural glucocorticoids, corticosterone, cortisol, and 11-deoxycortisol also increased C3 concentrations in A549 supernatants. Both cycloheximide and the glucocorticoid receptor antagonist, RU486, individually inhibited the effect of dexamethasone on C3 production. Northern analysis demonstrated that the steady state 5.2-kb C3 message increased in A549 cells within 10 h of treatment with dexamethasone. RU486 inhibited the effect of dexamethasone on C3 mRNA expression. The integrity of the C3 thiolester bond, as measured by [3H]iodoacetic acid titration and hemolytic assay, was not disrupted by dexamethasone. We conclude that glucocorticoids such as dexamethasone enhance the expression of C3 mRNA and increase the production of functionally active C3 by A549 cells by a mechanism that is mediated by the intracellular glucocorticoid receptor.     

Cortisol 21-mesylate exerts glucocorticoid action on the induction of milk protein synthesis in cultured mammary gland

Cortisol 21-mesylate, an alkylating derivatives of cortisol, was previously shown to exert an anti-glucocorticoid action in rat hepatoma cell culture (Simons, Thompson and Johnson 1980). In this study the effect of cortisol 21-mesylate on milk protein synthesis induced in cultured mouse mammary gland by glucocorticoid, insulin, and prolactin was investigated. Addition of cortisol 21-mesylate at concentrations ranging from 10(-8) M to 10(-6) M produced no inhibition of casein synthesis that was induced by glucocorticoid, insulin and prolactin in mammary explants from midpregnant mice. On the other hand, cortisol 21-mesylate in combination with insulin and prolactin stimulated casein synthesis in cultured tissue. The potency of cortisol mesylate was about 1/10 to 1/30th of that of cortisol. Cortisol 21-mesylate, like cortisol, also augmented the accumulation of alpha-lactalbumin in midpregnant rat mammary tissue cultured in the presence of insulin and prolactin. A cell-free competition study of glucocorticoid receptors using cytoplasmic extracts from mouse mammary tissue showed that cortisol 21-mesylate competitively inhibited the binding of dexamethasone on glucocorticoid receptors. The apparent affinity of cortisol 21-mesylate for glucocorticoid receptors is about 1/10th of that of cortisol. These results indicate that cortisol 21-mesylate acts as a glucocorticoid but not as an antiglucocorticoid in the mammary gland.     

Glucocorticoid induction of plasminogen activator and plasminogen activator-inhibitor messenger RNA in rat hepatoma cells

HTC rat hepatoma cells synthesize and secrete both tissue-type plasminogen activator (tPA) and type 1 plasminogen activator-inhibitor (PAI-1). Incubation with the synthetic glucocorticoid dexamethasone causes a rapid decrease in tPA activity which is secondary to a 5-fold increase in PAI-1 antigen and activity. Paradoxically, dexamethasone increases tPA antigen by 50%. We have analyzed HTC cell RNA by Northern and slot blot analysis, using as probes radiolabeled human PAI-1 and rat tPA cDNAs. HTC cells have a single species of PAI-1 mRNA of approximately 3.2 kilobases, which is increased 4-fold upon incubation with dexamethasone. Maximal induction occurs after 8-10 h of incubation. Half-maximal induction occurs at 5 nM dexamethasone. Dexamethasone also transiently increases the 2.8 kilobase tPA mRNA. The protein synthesis inhibitor cycloheximide does not affect accumulation of PAI-1 mRNA and does not block its induction by dexamethasone. In contrast, cycloheximide alone causes an increase in tPA mRNA, and in combination with dexamethasone, no further increase is observed. Induction of both mRNAs is prevented by actinomycin D. We conclude that the dexamethasone-induced increase in HTC cell PAI-1 activity and antigen is the result of a direct effect on accumulation of PAI-1 mRNA.     

Characterization of the inhibitory effect of glucocorticoids on the DNA replication of adult rat hepatocytes growing at various cell densities

Dexamethasone inhibited the basal and EGF-stimulated DNA synthesis of adult rat hepatocytes in primary culture. The inhibition was glucocorticoid-specific: It was shown by dexamethasone and hydrocortisone, but not by progesterone, testosterone, or estradiol; and was counteracted by the glucocorticoid antagonist RU-38486 in a concentration-dependent manner. Dexamethasone acted by decreasing the rate of entry into S-phase (kG1/S), while cell cycle parameters were unaffected. The steroid was able to decrease the kG1/S severalfold even when added more than 20 hr after EGF, half-maximal effect occurring 11 hr after the addition of dexamethasone. Densely populated areas were much more sensitive to the inhibition by dexamethasone than sparsely populated areas within the same culture dish: A moderate (10 nM) concentration of dexamethasone nearly abolished the DNA synthesis in densely populated areas of hepatocyte cultures with only marginal effect on sparsely populated cells.     

Glucocorticoid dexamethasone reversibly complements EJ-RAS oncogene to transform mouse embryo BALB-3T3 cells

EJ-A is a Balb-3T3 transfectant cell line that bears a small number of EJ-ras oncogene copies/cell, has low EJ-ras expression, and resembles the parental cell line in displaying a non-transformed phenotype. The glucocorticoid hormone dexamethasone reversibly induces transformation traits in EJ-A cells, namely: 1) morphological transformation; 2) increased growth rate and saturation density; 3) reduced G1 length; and 4) independence of the FGF requirement to initiate DNA synthesis. Western blot analysis revealed that dexamethasone does not increase the p21ras protein intracellular level. beta-IFN, added to the culture medium, does not suppress the dexamethasone-induced growth stimulation and morphological transformation. Therefore, glucocorticoid hormones can complement low EJ-ras expression to transform Balb-3T3 cells, by a mechanism that is likely to be independent of p21ras increase and beta-IFN decrease.     

Glucocorticoids affect the synthesis of pulmonary fibroblast-pneumonocyte factor at a pretranslational level

Glucocorticoids accelerate fetal lung maturation by acting on the fetal lung fibroblast to induce the synthesis of fibroblast-pneumonocyte factor which in turn stimulates pulmonary surfactant synthesis by the alveolar type II cell. We have studied the site of glucocorticoid regulation of fibroblast-pneumonocyte factor synthesis in primary cultures of fetal rat lung fibroblasts. Conditioned media from fetal rat lung fibroblasts exposed to cortisol stimulate [Me-3H]choline incorporation into saturated phosphatidylcholine by primary cultures of fetal rat lung alveolar type II cells. This effect is blocked by the presence of actinomycin D during the first, but not the second, 24 h of incubation of the fibroblasts with cortisol. Cycloheximide blocks this effect if present during either the first or second 24 h of incubation. We fractionated mRNA from fetal rat lung fibroblasts incubated in the presence or absence of dexamethasone and observed that cell-free translation products from a fraction of approximately 500 bases possess biological activity in the bioassay. Such activity is only present in cell-free translation products of mRNA isolated from fibroblasts treated with dexamethasone. These results suggest that glucocorticoids act at a pretranslational level to induce production of fibroblast-pneumonocyte factor and that the primary translation products are biologically active.