Analyses of steroid receptors are important for understanding molecular details of transcriptional control, as well as providing insight as to how an individual transacting factor contributes to cell identity and function. These studies have led to the identification of a superfamily of regulatory proteins that include receptors for thyroid hormone and the vertebrate morphogen retinoic acid. Although animals employ complex and often distinct ways to control their physiology and development, the discovery of receptor-related molecules in a wide range of species suggests that mechanisms underlying morphogenesis and homeostasis may be more ubiquitous than previously expected.

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The steroid and thyroid hormone receptor superfamily

We have cloned a member of the steroid hormone receptor superfamily of ligand-activated transcription factors. The receptor homologue is activated by a diverse class of rodent hepatocarcinogens that causes proliferation of peroxisomes. Identification of a peroxisome proliferator-activated receptor should help elucidate the mechanism of the hypolipidaemic effect of these hepatocarcinogens and aid evaluation of their potential carcinogenic risk to man.

Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators

Gene regulation by steroid hormones.

Bone density achieved in early adulthood is the major determinant of risk of osteoporotic fracture. Up to 60% of women suffer osteoporotic fractures as a result of low bone density, which is under strong genetic control acting through effects on bone turnover. Here we show that common allelic variants in the gene encoding the vitamin D receptor can be used to predict differences in bone density, accounting for up to 75% of the total genetic effect on bone density in healthy individuals. The genotype associated with lower bone density was overrepresented in postmenopausal women with bone densities more than 2 standard deviations below values in young normal women. The molecular mechanisms by which bone density is regulated by the vitamin D receptor gene are not certain, although allelic differences in the 3' untranslated region may alter messenger RNA levels. These findings could open new avenues to the development and targeting of prophylactic interventions. It follows that other pathophysiological processes considered to be subject to complex multifactorial genetic regulation may also be modulated by a single gene with pleiotropic transcriptional actions.

Prediction of Bone-Density from Vitamin-D Receptor Alleles

We have provided a historical perspective on a body of steroid receptor research dealing with the structure and physiological significance of the untransformed 9S receptor that has often confused both novice and expert investigators. The frequent controversies and equivocations of earlier studies were due to the fact that the native, hormone-free state of these receptors is a large multiprotein complex that resisted description for many years because of its unstable and dynamic nature. The untransformed 9S state of the steroid and dioxin receptors has provided a unique system for studying the function of the ubiquitous, abundant, and conserved heat shock protein, hsp90. The hormonal control of receptor association with hsp90 provided a method of manipulating the receptor heterocomplex in a manner that was physiologically meaningful. For several steroid receptors, binding to hsp90 was required for the receptor to be in a native hormone-binding state, and for all of the receptors, hormone binding promoted dissociation of the receptor from hsp90 and conversion of the receptor to the DNA-binding state. Although the complexes between tyrosine kinases and hsp90 were discovered earlier, the hormonal regulation or steroid receptor association with hsp90 permitted much more rapid and facile study of hsp90 function. The observations that hsp90 binds to the receptors through their HBDs and that these domains can be fused to structurally different proteins bringing their function under hormonal control provided a powerful linkage between the hormonal regulation of receptor binding to hsp90 and the initial step in steroid hormone action. Because the 9S receptor hsp90 heterocomplexes could be physically stabilized by molybdate, their protein composition could be readily studied, and it became clear that these complexes are multiprotein structures containing a number of unique proteins, such as FKBP51, FKBP52, CyP-40, and p23, that were discovered because of their presence in these structures. Further analysis showed that hsp90 itself exists in a variety of native multiprotein heterocomplexes independent of steroid receptors and other 'substrate' proteins. Cell-free systems can now be used to study the formation of receptor heterocomplexes. As we outlined in the scheme of Fig. 1, the multicomponent receptor-hsp90 heterocomplex assembly system is being reconstituted, and the importance of individual proteins, such as hsp70, p60, and p23, in the assembly process is becoming recognized. It should be noted that our understanding of the mechanism and purpose of steroid receptor heterocomplex assembly is still at an early stage. We can now speculate on the roles of receptor-associated proteins in receptor action, both as individuals and as a group, but their actual functions are still vague or unknown. We can make realistic models about the chaperoning and trafficking of steroid receptors, but we don't yet know how these processes occur, we don't know where chaperoning occurs in the cell (e.g. Is it limited to the cytoplasm? Is it a diffuse process or does chaperoning occur in association with structural elements?), and, with the exception of the requirement for hormone binding, we don't know the extent to which the hsp90-based chaperone system impacts on steroid hormone action. It is not yet clear how far the discovery of this hsp90 heterocomplex assembly system will be extended to the development of a general understanding of protein processing in the cell. Because this assembly system is apparently present in all eukaryotic cells, it probably performs an essential function for many proteins. The bacterial homolog of hsp90 is not an essential protein, but hsp90 is essential in eukaryotes, and recent studies indicate that the development of the cell nucleus from prokaryotic progenitors was accompanied by the duplication of genes for hsp90 and hsp70 (698). (ABSTRACT TRUNCATED)

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Steroid receptor interactions with heat shock protein and immunophilin chaperones.

Complementary DNA clones encoding the human vitamin D receptor have been isolated from human intestine and T47D cell cDNA libraries. The nucleotide sequence of the 4605-base pair (bp) cDNA includes a noncoding leader sequence of 115 bp, a 1281-bp open reading frame, and 3209 bp of 3' noncoding sequence. Two polyadenylylation signals, AATAAA, are present 25 and 70 bp upstream of the poly(A) tail, respectively. RNA blot hybridization indicates a single mRNA species of approximately equal to 4600 bp. Transfection of the cloned sequences into COS-1 cells results in the production of a single receptor species indistinguishable from the native receptor. Sequence comparisons demonstrate that the vitamin D receptor belongs to the steroid-receptor gene family and is closest in size and sequence to another member of this family, the thyroid hormone receptor.

https://www.pnas.org/content/pnas/85/10/3294.full.pdf

Cloning and expression of full-length cDNA encoding human vitamin D receptor.

We have used a series of human estrogen receptor (ER) mutants to evaluate the cell- and promoter-specific transcriptional activities of the TAF1 and TAF2 transactivation regions within the human ER. We show that the manifestation of TAF1 or TAF2 function depends strongly upon promoter context; on certain promoters, both the TAF1 and TAF2 activators are required for wild-type transcriptional activity, whereas on other promoters, the TAF1 and TAF2 activators function independently. Using these constructs, we show that the antagonist activity of the triphenylethylene-derived antiestrogens, e.g. tamoxifen, arises from their intrinsic inability to activate ER TAF2 function. However, on certain promoters, these antiestrogens efficiently activate gene transcription through ER. Consistent with this observation, the TAF2 function of the ER is not required on all promoters. In these TAF2-independent promoter contexts, TAF2 function may be provided by a separate transcription factor bound to the promoter. These data suggest that 1) TAF1 may be the major transcriptional activator of the ER; and 2) TAF2 functions as a transcriptional facilitator. On promoters where TAF2 function is provided independently of the ER, the TAF1 function of the ER can function independently of TAF2 activity, allowing triphenylethylene-derived antiestrogens to demonstrate partial agonist activity. These observations provide a possible molecular explanation for the tissue-specific partial agonist properties of tamoxifen and related triphenylethylene antiestrogens observed in vivo.

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Human estrogen receptor transactivational capacity is determined by both cellular and promoter context and mediated by two functionally distinct intramolecular regions.

Hypocalcemic vitamin D-resistant rickets is a human genetic disease resulting from target organ resistance to the action of 1,25-dihydroxyvitamin D3. Two families with affected children homozygous for this autosomal recessive disorder were studied for abnormalities in the intracellular vitamin D receptor (VDR) and its gene. Although the receptor displays normal binding of 1,25-dihydroxyvitamin D3 hormone, VDR from affected family members has a decreased affinity for DNA. Genomic DNA isolated from these families was subjected to oligonucleotide-primed DNA amplification, and each of the nine exons encoding the receptor protein was sequenced for a genetic mutation. In each family, a different single nucleotide mutation was found in the DNA binding domain of the protein; one family near the tip of the first zinc finger (Gly----Asp) and one at the tip of the second zinc finger (Arg----Gly). The mutant residues were created in vitro by oligonucleotide directed point mutagenesis of wild-type VDR complementary DNA and this cDNA was transfected into COS-1 cells. The produced protein is biochemically indistinguishable from the receptor isolated from patients.

http://science.sciencemag.org/content/sci/242/4886/1702.full.pdf

Point mutations in the human vitamin D receptor gene associated with hypocalcemic rickets.

The human-derived promyelocytic leukemia cell line, HL-60, is known to differentiate into mature myeloid cells in the presence of 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) We investigated differentiation by monitoring 1,25(OH)2D3-exposed HL-60 cells for phagocytic activity, ability to reduce nitroblue tetrazolium, binding of the chemotaxin N-formyl-methionyl-leucyl-[3H]phenylalanine, development of nonspecific acid esterase activity, and morphological maturation of Wright-Giemsa-stained cells 1,25(OH)2D3 concentrations as low as 10(-10) M caused significant development of phagocytosis, nitroblue tetrazolium reduction, and the emergence of differentiated myeloid cells that had morphological characteristics of both metamyelocytes and monocytes These cells were conclusively identified as monocytes/macrophages based upon their adherence to the plastic flasks and their content of the macrophage-characteristic nonspecific acid esterase enzyme The estimated ED50 for 1,25(OH)2D3-induced differentiation based upon nitroblue tetrazolium reduction and N-formyl-methionyl-leucyl-[3H]phenylalanine binding was 57 X 10(-9) M HL-60 cells exhibited a complex growth response with various levels of 1,25(OH)2D3: less than or equal to 10(-10) M had no detectable effect, 10(-9) M stimulated growth, and greater than or equal to 10(-8) M sharply inhibited proliferation We also detected and quantitated the specific receptor for 1,25(OH)2D3 in HL-60 and HL-60 Blast, a sub-clone resistant to the growth and differentiation effects of 1,25(OH)2D3 The receptor in both lines was characterized as a DNA-binding protein that migrated at 33S on high-salt sucrose gradients Unequivocal identification was provided by selective dissociation of the 1,25(OH)2D3-receptor complex with the mercurial reagent, p-chloromercuribenzenesulfonic acid, and by a shift in its sedimentation position upon complexing with anti-receptor monoclonal antibody On the basis of labeling of whole cells with 1,25(OH)2[3H]D3 in culture, we found that HL-60 contains approximately 4,000 1,25(OH)2D3 receptor molecules per cell, while the nonresponsive HL-60 Blast is endowed with approximately 8% of that number The concentration of 1,25(OH)2D3 (5 X 10(-9) M) in complete culture medium, which facilitates the saturation of receptors in HL-60 cells, is virtually identical to the ED50 for the sterol's induction of differentiation This correspondence, plus the resistance of the relatively receptor-poor HL-60 Blast, indicates that 1,25(OH)2D3-induced differentiation of HL-60 cells to monocytes/macrophages is occurring via receptor-mediated events

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1,25-Dihydroxyvitamin D3-induced differentiation in a human promyelocytic leukemia cell line (HL-60): receptor-mediated maturation to macrophage-like cells

Osteoblast-specific expression of the bone protein osteocalcin is controlled at the transcriptional level by the steroid hormone 1 alpha,25-dihydroxyvitamin D3. As this protein may represent a marker for bone activity in human disease, we examined the regulation of its expression at the molecular level by evaluating human osteocalcin gene promoter function. We describe regions within the promoter that contribute to basal expression of the gene in osteoblast-like cells in culture. Further, we define a 21-base-pair DNA element with the sequence 5'-GTGACTCACCGGGTGAACGGG-3', which acts in cis to mediate 1 alpha,25-dihydroxyvitamin D3 inducibility of the osteocalcin gene. This response element bears sequence similarity with other short DNA segments, particularly those for estrogen and thyroid hormone, which act together with their respective trans-acting receptors to modulate gene transcription.

J W Pike

Papers

Cloning and expression of full-length cDNA encoding human vitamin D receptor.

Human estrogen receptor transactivational capacity is determined by both cellular and promoter context and mediated by two functionally distinct intramolecular regions.

Point mutations in the human vitamin D receptor gene associated with hypocalcemic rickets.

1,25-Dihydroxyvitamin D3-induced differentiation in a human promyelocytic leukemia cell line (HL-60): receptor-mediated maturation to macrophage-like cells

Sequence elements in the human osteocalcin gene confer basal activation and inducible response to hormonal vitamin D3.