Aberrant cryptic responsiveness of the pCAT 3- and pGL3-promoter reporter vectors

Transfection analyses are an informative method to assess the activity of specific promoter or enhancer elements in mammalian cells. Commercially available reporter vectors can be extremely useful investigative tools for such studies. This study reports that the pCAT 3- and pGL3-promoter vectors display cryptic responsiveness to androgens when they contain a DNA insert, while the empty vector, a commonly used negative control, is nonresponsive. Our studies initially aimed to characterize novel androgen-responsive DNA sequences in human genomic DNA through transactivational analyses. An isolated DNA fragment, designated ARC-3, contained three putative androgen response element "half-sites" and was androgen-responsive when cloned into the pCAT3-promoter vector. While we originally believed this to be a novel enhancer element, subsequent analyses of this clone revealed that this vector displays cryptic activity in the presence of an androgen. This was confirmed by cloning several unrelated DNA fragments that did not contain any known classic response elements into the pCAT3-promoter vector, all of which were found to be responsive. The empty vector (negative control) was again nonresponsive. The ARC-3 DNA fragment was also weakly responsive to stimulation when cloned into the pGL3-promotor vector, which is identical to the pCAT3-promoter vector, with the exception of an intron located 5' of the chloramphenicol acetyltransferase gene, and the reporter genes. This work demonstrates that both the pCAT3- and pGL3-promoter vectors are inappropriate to assess androgen-responsive enhancers and emphasizes the importance of the careful selection of reporter vectors and controls when conducting transactivational analysis.     

Pharmacological characterization of type II glucocorticoid binding sites in AtT20 pituitary cell culture

Recent evidence indicates that at least two functional glucocorticoid receptors (Type I and Type II) are present in many tissues. It has also become increasingly recognized that, as in other systems, stimulus-response relationships for steroid hormones are often nonlinear. Thus, precise pharmacological parameters are required to establish a functional relationship(s) between binding site and response characteristics. We therefore pharmacologically characterized a glucocorticoid binding site present in AtT20 mouse pituitary cells, a cell line extensively used in studying Type II glucocorticoid receptor function. By several different criteria, glucocorticoids were shown to bind to a single class of binding sites, which, in comparison to available literature, correspond to classical Type II glucocorticoid receptors. No evidence for Type I adrenal steroid binding sites was observed, under the experimental conditions used. Unambiguous Kb values for both glucocorticoid agonists and antagonists were therefore calculated. These parameters should prove of use in elucidating the relationships between glucocorticoid receptor activation and different responses in both AtT20 cells and other glucocorticoid responsive tissues.     

A quantitative comparison of dual control of a hormone response element by progestins and glucocorticoids in the same cell line

Progesterone receptor-containing T47D human breast cancer cells are responsive to progestins but fail to respond to other steroid hormones, in particular dexamethasone, because they have no measurable levels of receptors for estrogens, androgens, or glucocorticoids. To quantitatively study dual responsiveness of the mouse mammary tumor virus (MMTV) promoter to progestins and glucocorticoids, we have stably transfected T47D cells with a glucocorticoid receptor (GR) expression vector. A cloned derivative (A1-2) was isolated that expresses a normal, full length GR, as assessed by steroid binding and Western immunoblot with a monoclonal anti-GR antibody. Moreover, GR is expressed at levels (80,000-100,000 molecules per cell) comparable to the high levels of endogenous progesterone receptor (200,000 molecules per cell). In A1-2 cells transiently transfected with an MMTV-chloramphenicol acetyl transferase reporter gene, induction by glucocorticoid was substantially greater (5-fold) than induction mediated by progestins. These results suggest that glucocorticoids may be the primary regulator of MMTV.     

Glucocorticoids are double-edged sword in the treatment of COVID-19 and cancers

Glucocorticoids are important steroid hormones. As an outstanding scientific discovery, the scientist who discovered glucocorticoids was awarded the Nobel Prize in Physiology and Medicine in 1950. Cortisone (hydrocortisone) is a natural glucocorticoid, which is secreted with circadian rhythm by the cortical cells of adrenal glands. Physiologically, about 10-20 mg of hydrocortisone are secreted each day for maintaining homeostasis. Since the biological half-life of natural glucocorticoid is short, scientists developed various synthetic glucocorticoids including prednisone, prednisolone, methylprednisolone, triamcinolone, dexamethasone, betamethasone, and so on. These synthetic glucocorticoids are generated by modifying some structures based on the cortisone backbone, leading to extension of their biological half-life with stronger activities. In the face of severe infection, allergy, shock, trauma, pain, and other stresses, the demand for glucocorticoids increases dramatically. It is critical to supplement extra glucocorticoids to protect the biological functions of vital organs. However, the amount and duration of glucocorticoid administration need to be carefully adjusted, because a series of side effects may occur after long-term or high-dose usage of glucocorticoids. This review article will discuss the application of glucocorticoids in the treatment of patients with severe or critical COVID-19 and solid tumors of advanced stage. The controversy of using glucocorticoid in medical community will also be discussed. This review article will help doctors and basic researchers better understand the practical application of glucocorticoids.     

Parenchymal cells purified from Xenopus liver and maintained in primary culture synthesize vitellogenin in response to estradiol-17 beta and serum albumin in response to dexamethasone.

Xenopus liver was disaggregated by perfusion with collagenase. The released cells were separated according to their equilibrium densities on gradients of Metrizamide. Highly purified populations of parenchymal cells, sinusoidal cells, and melanocytes were obtained. All the parenchymal cells in an animal, before, during, and after hormone stimulation, are contained within a single density peak and subpopulations are not observed. Normal female parenchymal cells are less dense than normal male cells and estrogen stimulation causes a decrease in the peak density of the parenchymal cell population. Electron microscopic examination revealed the appearance and disappearance of lipid vacuoles in parenchymal cells following estrogen treatment. We hypothesize that the changing lipid content of these cells accounts for the shift in cell density. Immunofluorescent staining revealed that all liver parenchymal cells contain either vitellogenin or serum albumin, depending upon the prior treatment with hormones in vivo. Primary cultures of purified parenchymal and sinusoidal cells were established and were found to secrete different sets of proteins. The addition of estradiol-17β to cultures of parenchymal cells from either male or female frogs induces the synthesis and secretion of vitellogenin and two other polypeptides. Furthermore, the synthesis and secretion of polypeptides normally produced by parenchymal cells are curtailed as a result of estrogen treatment. Addition of glucocorticoids to a similar population of cells produces an increase in the synthesis and secretion of polypeptides, one of which is serum albumin. These results demonstrate that every parenchymal cell is responsive to two different classes of steroid hormones, estrogens and glucocorticoids, and in response to these hormones the same cells can synthesize and secrete alternate sets of polypeptides.     

Different regions of the estrogen receptor are required for synergistic action with the glucocorticoid and progesterone receptors.

Estrogen and progesterone or estrogen and glucocorticoid receptors functionally cooperate in gene activation if their cognate binding sites are close to one another. These interactions have been described as synergism of action of the steroid receptors. The mechanism by which synergism is achieved is not clear, although protein-protein interaction of the receptors is one of the favorite models. In transfection experiments with receptor expression vectors and a reporter gene containing estrogen and progesterone-glucocorticoid receptor binding sites, we have examined the effects that different portions of the various receptors have on synergism. N-terminal domains of the chicken progesterone and human glucocorticoid receptors, when deleted, abolished the synergistic action of these receptors with the estrogen receptor. Deletion of the carboxy-terminal amino acids 341 to 595 of the estrogen receptor produced a mutant receptor that could not trans-activate on its own. This mutant receptor did not affect the action of the glucocorticoid receptor but functioned synergistically with the progesterone receptor. We therefore conclude that the synergistic action of the receptors for estrogen and progesterone is mechanistically different from the synergistic action of the receptors for estrogen and glucocorticoid.