Ernest Bloom

Cellular Pharmacology and Molecular Biology of the Trabecular Meshwork Inducible glucocorticoid Response Gene Product

Studies of the effects of glucocorticoid (GC) and oxidative stress stimuli in differentiated cultures of human trabecular meshwork (HTM) cells have provided the rationale for our studies of a major new gene termed TIGR (trabecular meshwork inducible GC response). The TIGR clone was isolated by differential library screening using selection criteria based on the induction pattern of a new protein/glycoprotein found in HTM cultures after prolonged but not brief exposure to GCs. This GC induction pattern matched the time course and dose response required for intraocular pressure elevation in patients receiving corticosteroids. The very large, progressive induction of TIGR combined with specific structural features of its cDNA suggested that TIGR should be considered a candidate gene for outflow obstruction in glaucoma. Among the properties of TIGR cDNA were a signal sequence for secretion, several structural features for interactions with glycosaminoglycans and other glycoproteins and putative sites for cell surface interactions. In addition, the leucine zippers in the structure were related to TIGR-TIGR oligomerization that was shown to occur with native and recombinant TIGR protein. The verification that TIGR was a major stress response protein in HTM cells following hydrogen peroxide (or phorbol esters) exposure provided a potential link between GC and oxidative mechanisms thought to be involved in glaucoma pathogenesis. Pharmacological evaluation showed that basic fibroblast growth factory and transforming growth factor beta decreased the GC induction of TIGR, and certain nonsteroidal anti-inflammatory drugs protected against both GC- and oxidation-induced stress responses in HTM cells. Our recent studies of TIGRs genomic structure have shown motifs in the promoter region that suggest a basis by which multiple hormonal/environmental stimuli can regulate TIGR production and by which putative genetic alterations could lead to an overexpression of the protein. Further application of cell biology/biochemistry, molecular biology, genetic and histological approaches will be helpful in understanding the role of TIGR in different glaucoma syndromes.

Nuclear binding of glucocorticoid receptors: Relations between cytosol binding, activation and the biological response

Abstract To better understand the early steps in glucocorticoid hormone action from cytoplasmic receptor-steroid binding to nuclear binding of the resulting complexes, we have in the current studies examined in cultured hepatoma (HTC) cells the kinetics of the nuclear binding of glucocorticoid receptors, the activation of the receptor-steroid complex required for this binding and the nature of the nuclear binding sites. The quantitative relationships of these events have also been compared to the glucocorticoid response. When intact cells are incubated with dexamethasone at 0°C, cytosol binding of the steroid occurs readily but nuclear binding proceeds very slowly. After heating the cells at 37°C for 40s and then chilling them, the kinetics of the nuclear binding at 0°C are markedly increased. Thus, in the intact cell at 0°C. like cell-free systems, the heat-sensitive activation and not the nuclear binding itself appears to occur and be rate-limiting for nuclear binding of the complexes. Nuclear binding measured at 37°C was similar at 30–40 min, and 16 h, by which time induction of tyrosine aminotransferase is maximal. Thus, unlike the case with most other classes of hormones, in this system glucocorticoids do not affect the concentration of their receptors. Nuclear binding was linearly related to the content of cytoplasmic reccptor-glucocorticotd complexes and a Scatchard plot (nuclear-bound over cytosol-bound vs nuclear-bound dexamethasone) was parallel to the abscissa. These observations suggest that in the cell the nuclear acceptors are far from saturated with receptors. It has been proposed that there are multiple orders of affinity for binding receptors in the nucleus. Salt sensitivity of bound receptors has been used on one criterion. Indeed, whereas most of the nuclear-bound receptors can be extracted with 0.5 M KCl, about 20% resist this extraction. In the current studies, the proportion of salt-extractable and salt-resistant nuclear-bound receptors was found to be constant with time (to 16 h) and cytosol receptor-steroid complex concentration. Thus, the thermodynamics of receptor association with the salt-labile and salt-resistant nuclear-bound receptors appear similar and the evidence does not support the idea that these receptors have fundamental differences; the incomplete extraction of the receptors with salt may reflect some property of the solubilization process rather than two types of nuclear acceptors. The content of nuclear-bound receptors was found to be linearly related to the induction of tyrosine aminotransferase; this suggests that the number of glucocorticoid receptors, and not some distal event, is the limiting factor in the glucocorticoid hormone response and that “spare receptors” are not present. These data also do not support the idea that in these cells, a physiological “acceptor” that binds receptors with an affinity much higher than the observed nuclear-binding (“high-affinity operators”) mediates glucocorticoid action; these hypothetical sites would have been more saturated at the receptor concentrations achieved. Instead, the data are consistent with the possibility that a nuclear acceptor population with an affinity that does not vary greatly from the observed nuclear binding is responsible for the glucocorticoid response. The data are discussed in terms of a model in which receptors bind similarly to acceptors present in excess, many of which are located at sites where a physiological response is not elicited. By this model, the probability of obtaining a glucocorticoid response is proportional to the chance association of receptors with those acceptors located at important loci.

Skin Barrier Properties in the Newborn

Relative permeabilities of newborn and adult skin to small molecules were determined by measuring emission of carbon dioxide and water vapor through skin. Transepidermal water loss (TEWL) measured on

Topical FK506: suppression of allergic and irritant contact dermatitis in the guinea pig

Topical FK506 has recently been shown to have an anti-inflammatory effect in vivo in humans. In this study its effects in contact dermatitis were studied in the guinea pig model. Topical FK506 suppressed both irritant and allergic patch-test reactions. The most prominent suppressive effect was seen when skin sites were pretreated with FK506. Topical FK506 did not impair the induction of contact allergy as assessed by challenges, although it suppressed local lymph node cell accumulation during contact allergy induction. Topical FK506 may hold promise as a treatment for skin disorders that respond to oral FK506 or cyclosporin A.

Adrenergic and cholinergic receptors in isolated non-pigmented ciliary epithelial cells

Methods were developed to isolate non-pigmented ciliary epithelium from bovine eyes (BCBE) free from other cell types in the ciliary process. Once background binding due to pigment was minimized, it was possible to assay specific beta adrenergic receptors in these preparations. The BCBE beta adrenergic receptors showed appropriate stereospecificity and an order of binding affinities compatible with a beta 2 adrenergic selectivity. Interestingly, specific high affinity muscarinic cholinergic receptors were detected in both BCBE and primate non-pigmented ciliary epithelium. These muscarinic receptors showed an appropriate affinity and selectivity for agonists and antagonists. Further evaluations of the receptors and responses of non-pigmented ciliary epithelial cells may prove useful in understanding the effects of adrenergic and cholinergic agents on aqueous humor inflow.

Increased proliferation of skin cells by sublethal doses of sodium lauryl sulfate.

Most quantitative in vitro approaches to determine irritancy have examined the potential of compounds to decrease biological functions or inhibit growth of cells. Irritants, however, are known to generally have the opposite effect in vivo, i.e. to stimulate cell division. This property has not been directly studied in vitro. We examined the ability of sublethal concentrations of sodium lauryl sulfate (SLS) to stimulate cultured keratinocyte and fibroblast proliferation in vitro. The growth of keratinocytes, without added growth factors, continuously exposed to SLS for 4 days was stimulated approximately 89% compared to control. Keratinocytes exposed to SLS for 1 or 18 h were stimulated 36 and 12%, respectively, over the next 4 days of growth. Subconfluent fibroblasts were also stimulated approximately 38%. Confluent fibroblasts were stimulated 40%. All stimulations were maximal between 10(-8) and 10(-5) M added SLS. Media conditioned by keratinocytes exposed to 10(-8) M SLS were able to increase the growth of naive keratinocytes by 117%. In all experiments doses of SLS > 10(-5) M inhibited cell growth. We conclude that sublethal doses of SLS can stimulate the growth of cultured keratinocytes and fibroblasts. The stimulation of growth seen may be related to the stimulation observed in in vivo irritation.

A local lymph-node assay validation study of a structure-activity relationship model for contact allergens

A structure-activity relationship model for prediction of contact allergenic potential of chemicals had previously been developed. The model had been shown to be able to classify known allergens and nonallergens using data on physicochemical and reactivity parameters of functional groups by discriminant two-value multiple regression analysis. To investigate the model, six selected chemicals which had not been previously investigated for allergenicity were studied with both the model and a murine local lymph-node assay. The same compounds were predicted to be allergens (3-bromo-2-coumaranone, 1-nitrocyclohexene and alpha-acryloyloxy-beta, beta-dimethyl-beta-butyrolactone) and nonallergens (1-carbethoxy-4-piperidone, 6,7-dimethoxy-2-tetralone and 9-acetylanthracene) by both the model and the local lymph-node assay.

Skin Penetration and Metabolism: Comparative Evaluation of Skin Equivalent, Cell Culture, and Human Skin

AbstractUsing an organotypic model of human skin, living skin equivalent (LSE) and its homogenate, monolayer cell culture, and human skin, we have studied the simultaneous transport and metabolic fate of two compounds. The LSE was maintained in an assay culture medium. When the model compounds were applied to LSE at dosages of 9.0 ± 1.2 µg/cm2, the transport of salicylic acid through human skin was 0.12 ±0.1µg/cm2/hr. Salicylic acid flux was 5.6-fold greater in LSE than in human skin. Shorter lag time of absorption was observed in LSE (∼ 1.5 hr) than in human skin (7–9 hr). Percutaneous transport across LSE was accompanied by metabolism of the compounds and there were quantitative and qualitative similarities between the metabolites produced by the LSE and human skin. When compounds were added to homogenates (LSE or skin) and to human cell cultures, the activities of the LSE and human fractions were similar. Data from the present study demonstrate that although LSE is more permeable than human skin, the a...

Adrenergic Drug Effects on Cyclic AMP in Cultured Human Trabecular Meshwork Cells

Cyclic AMP production in the presence or absence of various adrenergic agonists and antagonists was determined in cultured human trabecular cells using a gammaflow automated radioimmunoassay that allowed multiple simultaneous experiments and reproducible results. Adrenergic agonists and antagonists showed activation and inhibition constants consistent with the presence of β2-receptors: Ka of isoproterenol < epinephrine < norepinephrine < phenylephrine; Ki of timolol < betaxolol < celiprolol < atenolol. Selective ICI antagonists showed β2-specificity.

Effect of topical cis-urocanic acid on local lymph node activation during contact sensitization in mouse, rat and guinea-pig

Summary Cis‐urocanic acid (cUCA) has been suggested as a mediator of impairment of contact hypersensitivity induction by ultraviolet B (UVB) irradiation. We ascertained whether topical cUCA influences local lymph node activation during induction of contact hypersensitivity. Topical cUCA or vehicle was applied during the local lymph node assay to oxazolone. Local lymph node weight and cell number were assessed in all animals. Additionally, cell proliferation rate was studied in Hartley guinea‐pigs and CHA/Ca mice, whereas activation of antigen‐presenting cells was quantified in NMRI mice and Wistar rats. Topical cUCA suppressed all parameters of local lymph node activation due to oxazolone application in guinea‐pigs. No effect, with the exception of a suppression of antigen‐presenting cell activity, was seen in mice. No effect was seen in rats. The study shows that topical cUCA may suppress local lymph node activation during contact sensitization and suggests that differences between the effect of cUCA in different animal species may exist.