Faculty Bibliography

The glucocorticoid antagonist 17 alpha-methyltestosterone inhibits binding of the agonist [3H]triamcinolone acetonide ot the glocucorticoid receptor in cytosol prepared from rat pituitary tumor GH1 cells. Competitive binding studies indicate that the dissociation constant for 17 alpha-methyltestosterone is about 1 microM. After incubation of intact GH1 cells with 10 nM [3H]triamcinolone acetonide at 37 C and subsequent cell fractionation at 4 C, three glucocorticoid receptor forms are observed: cytosolic 10 S receptor, cytosolic 4 S receptor, and nuclear receptor. Concurrent incubation with 17 alpha-methyltestosterone reduces the amount of [3H]triamcinolone acetonide bound to each of these receptor forms. Ligand-exchange assays performed at 0 C in intact cells using [3H]triamcinolone acetonide show that the exchangeable antagonist is associated predominantly with cytosolic 10 S receptor. Immunochemical analysis using monoclonal antibody BuGR2 indicates that 17 alpha-methyltestosterone does not cause substantial accumulation of glucocorticoid receptors in GH1 cell nuclei and, when present together with agonist, reduces nuclear accumulation of receptor seen with agonist alone. Results from dense amino acid labeling studies show that unlike [3H]triamcinolone acetonide, 17 alpha-methyltestosterone does not reduce the total amount of cellular glucocorticoid receptor and does not reduce receptor half-life. These results are consistent with a model for glucocorticoid receptor transformation in which binding of agonist promotes the dissociation of an oligomeric 10 S cytosolic receptor protein to its DNA-binding 4 S subunit. The antagonist 17 alpha-methyltestosterone competes with agonist for binding to the 10 S cytosolic receptor but does not appear to promote dissociation of the oligomer, thus inhibiting agonist-mediated nuclear actions of the glucocorticoid receptor

Chicken c-erbA (Ck-c-erbA) cDNA (1250 base pairs), a cellular homologue of the avian erythroblastosis virus v-erbA gene, encodes a 408 amino acid protein which binds L-T3 and its analogs with affinities similar to that of endogenous thyroid hormone nuclear receptors. By analogy with steroid receptors, Ck-c-erbA(Met1-Val408) contains an A and B domain (amino acids 1-50); a putative DNA binding C domain (amino acids 51-118); a hydrophilic D domain (amino acids 119-189); and a putative ligand binding E domain (amino acids 187-408). To further characterize the ligand binding region of Ck-c-erbA, two deletion mutations were constructed: Ck-c-erbA(Met120-Val408) which encodes a 289 amino acid protein lacking regions A, B, and C; and Ck-c-erbA-(Met199-Val408) which encodes a 210 amino acid protein lacking regions A, B, C, D, and the first 12 amino acids of the E region. The in vitro translation products ([35S]methionine) of cDNA transcripts of a human placental c-erbA, Ck-c-erbA (Met1-Val408), and Ck-c-erbA(Met120-Val408) efficiently bind L-[125I]T3, whereas Ck-c-erbA(Met199-Val408) does not bind L-[125I]T3. In frame substitution of the last 14 C-terminal amino acids of Ck-c-erbA(Met1-Val408) for the last 7 C-terminal amino acids of v-erbA reduces but does not eliminate L[125I]T3 binding. These results indicate that a broad region of the E domain is important for ligand binding.(ABSTRACT TRUNCATED AT 250 WORDS)

A report of two patients in which a soft tissue mass, initially regarded as a malignant tumor, was shown to be the result of calcium pyrophosphate deposition disease. The first case, a woman aged 71 years, presented with a mass involving the right fifth finger. In the second case, also a woman aged 71 years, the lesion involved the tissues adjacent to the right hip. Each lesion consisted of a mass of highly cellular tissue containing deposits of calcium pyrophosphate dihydrate crystals. The clinical, radiological, and pathological features of the two cases are compared with those of seven similar cases reported in the literature.

Regulation of gene expression by the thyroid hormones is thought to be mediated by a nuclear-associated receptor found in a wide variety of cells and tissues. Cellular homologues of the avian erythroblastosis virus oncogene, v-erbA, encode proteins which bind thyroid hormone with similar affinities as thyroid hormone receptors. However, it has not been shown that any of the c-erbA proteins can function as receptor and modulate thyroid hormone responsive genes. In this study, using transient expression of chimeric reporter constructs, we document that the chick fibroblast c-erbA-alpha and the human placental c-erbA-beta modulate cis-acting regulatory sequences of two thyroid hormone responsive genes; rat GH and PRL. From these results we conclude: 1) in a receptor deficient cell line (235-1) both c-erbA subtypes act as hormone-dependent modulators of PRL gene expression and hence function as thyroid hormone receptors, 2) in two different receptor containing cell lines (GH4C1 and GH1), both c-erbA proteins act in a hormone-independent fashion to regulate PRL and GH expression. This suggests that events other than ligand binding can result in formation of a c-erbA protein that modulates transcription of thyroid hormone responsive genes, 3) no qualitative functional differences were detected between alpha- and beta-c-erbA subtypes, and 4) depending on the cell-type, L-T3 acts through its endogenous receptor to stimulate (GH4C1) or suppress (GH1) expression of a chimeric PRL construct. In these cells, c-erbA expression results in the same positive or negative response as the endogenous receptor except that the response occurs in the absence of hormone. These results suggest that the endogenous receptor and the c-erbAs act by augmenting the effect of transcription factors which can positively or negatively control gene expression

The elements involved in mediating cell-specific and thyroid hormone stimulation of rat growth hormone gene expression have been defined by transfection studies and by nuclease footprinting. 5'-Flanking DNA extending to -104 can mediate cell-specific expression, and this is enhanced 3- to 4-fold with DNA extending to -145. Cell-specific factors, found only in rat growth hormone producing cells, bind within the -137/-107 and -95/-65 regions, and competition studies suggest that the same factor binds to both sites. The sequence A (A or T) TAAAT is found at the center of both footprints at -80 and -122, suggesting that it is a core component of the recognition sequence of the cell-specific factor. Disruption of the spatial and/or distance relationships between the two regions eliminates the enhanced level of cell-specific expression, suggesting a cooperative interaction of the proteins which bind to these elements. Sequences located between -208 and -178 can confer thyroid hormone-regulated expression when linked in either orientation in close proximity to one or both cell-specific elements. The thyroid hormone and cell-specific elements function as an enhancer-like unit and are both required to confer regulated expression to heterologous promoters. We propose that thyroid hormone acts via its receptor to enhance the function of the cell-specific element by forming a more 'active' transcription complex which stimulates the level of gene expression