Regulation of gene expression by steroid hormones.

Regulation of Gene Expression by Steroid Hormones ANDREW C. B. CATO,~ HELMUT PONTA AND PETER HERRLICH Kernforschungszentrum Karlsruhe lnstitut f u r Genetik and Toxikologie D-7500 KarEsruhe 1 , Germany I. Identification of cis-Acting DNA Sequences That Mediate Steroid Hormone Action by Gene-Transfer Experiments. . . . . . . . 11. Receptor-DNA Interaction. .................................. 111. The Structure of Steroid Hormone Receptors .................. IV. Induction of Gene Expression by Steroid Hormone Receptors . . V. Positive Regulation of Gene Expression by Steroid Hormones through the Modulating Activity of Different Transcription Factors ..................................................... VI. Negative Action of Steroid Hormones ......................... VII. Repression of Steroid Hormone Action by Transcription Factors ..................................................... VIII. Positive and Negative Action of Steroid Antagonists . . . . . . . . . . . . . . . . . . . ...................... IX. Concluding Remarks.. ....................................... References.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3 6 10 15

17 20 26

27 30 31

Steroid hormones are a group of naturally occuring cyclopentanophenanthrene compounds that control a number of developmental processes in higher euKaryotes and mediate physiological responses to a number of stimuli. Three classes of steroids have been identified, based on their biological function. These are adrenal steroids (cortisol and aldosterone), sex steroids (estrogen, progesterone, and testosterone), and calcitriol (the vitamin D3 metabolite, la,25-dihydroxycholecalciferol). The adrenal steroids influence body homeostasis, mediate stress response, control carbohydrate metabolism, promote retention of water and sodium in the body, and facilitate the excretion of potassium. They also have widespread effects on the immune and nervous systems, reduce inflammation, and influence the differentiation To whom correspondence should be addressed. 1

Progress in Nucleic Acid Research and Molecular Biology.Val. 43

Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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ANDREW C. B. C A T 0 ET AL.

of cultured cells. The sex hormones are responsible for embryonic development and the acquisition of secondary sex characteristics. Calcitriol is required for calcium metabolism. The first hint as to how these steroids work was the discovery, some 30 years ago, of intracellular receptors in target cells that specifically bind steroids and coordinate their biological responses through modulation of the expression of specific genes (for reviews, see 1-3). However, these receptors are present at such low concentrations that enormous problems were encountered in their isolation. Some of these problems were overcome by the use of synthetic steroids that could be labeled to high specific activity, and that possess higher affinity for the receptors than most natural ligands. Labeling the receptors in uiuo or in uitro allowed their identification and purification by classical biochemical techniques. By the beginning of the 1980s, most of the steroid receptors, including the estrogen, progesterone, and glucocorticoid receptors (ER, PR, and GR),' had been purified to homogeneity. The purification of these receptors and identification of DNA elements that mediate their induction of gene expression by molecular-biology techniques set the pace for studies into the mechanisms of action of steroid hormones. Steroid hormones, upon binding their corresponding receptors in target cells, conformationally alter the receptors (receptor activation or transformation), conferring on them the ability to bind to DNA or chromatin. Protein-DNA-binding studies show that steriod receptors activated in uitro bind to discrete sequence motifs in DNA. The receptor-binding sites, when linked to otherwise hormone-insensitive promoters, convey hormone responsiveness to these promoters. Such studies show not only that steroid hormone receptors bind DNA, but that the receptor-DNA complexes are functionally active in the induction of gene expression. These inducing cis-acting elements were termed hormone-response elements (HREs). Some DNA-regulatory elements that confer negative regulation by steroid hormones have recently been identified. These elements are also targets for receptor binding in uitro. They have therefore been termed negative HREs (nHREs) by analogy to the above-described HREs for the positive regulation of gene expression by steroid hormones. Ahbl-eviutions: .4R, androgen receptor; DBD;DNA-binding domain; ER, estrogen receptor; ERE; estrogen-response element; GR, glucocorticoid receptor; GRE, glucocorticoid-response element; HBD, hormone-binding domain; HRE, hormone-response element; LTR, long terminal repeat; MhlTV, mouse mammary tumor virus; MR, mineralocorticoid receptor; MRE, mineralocorticoid-response element; PR, progesterone receptor; PRE, progesterone-response element; tk, thymidine kinase.

GENE EXPRESSION BY STEROID HORMONES

3

There is yet another type of regulatory element, the composite HRE (cHRE), which can either negatively or positively regulate the expression of genes. As the name implies, this element is a composite of a classical steroid receptor-binding site and a binding site for a non-receptor protein. The functional activity of the cHRE is therefore controlled by the simultaneous binding to this regulatory element of the non-receptor and receptor proteins as well as through proteinprotein interaction of the two regulatory factors. Such interactions of steroid hormone receptors with other cellular proteins, culminating in negative or positive regulation of gene expression, introduce a large degree of versatility into the action of steroid hormones. Such flexibility in the action of steroid hormones is also observed when synthetic steroids replace natural hormones in endocrine therapy. Therefore, it is necessary to fully understand how these positive and negative actions of steroids are manifested, for effective therapeutic use of steroids. In this essay, we describe some of the early work that led to the identification of HREs, and discuss some more recent experiments that have necessitated the action of these regulatory elements to be broadened to accommodate positive and negative regulators of gene expression. We further discuss observations showing that synthetic steroids known to antagonize the action of the natural steroids also positively and negatively regulate gene expression in a promoterspecific manner.

1. Identification of &Acting DNA Sequences That Mediate

Steroid Hormone Action by Gene-Transfer Experiments An important discovery in the way steroid hormones regulate gene expression was the identification of DNA sequences in inducible genes that mediate steroid hormone action. These studies were made possible by gene-transfer experiments. Regulatory elements of hormone-inducible genes were linked to promoters regulated by polymerase I1 driving the expression of reporter genes. These chimeric constructs were then stably or transiently transfected into recipient cells that contained functional steroid hormone receptors. Treatment of the transfected cells with steroids led to alteration in the expression of the reporter genes (4-9). Mutational analyses of the regulatory region of the inducible genes helped delineate relatively short nucleotide sequences that are necessary and sufficient for the steroid hormone response (5-7,9).

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ANDREW C. B. CAT0 ET AL.

As most established cell lines contain GRs, gene-transfer experiments of the type described above were most successful with glucocorticoids. One of the first experiments of this kind identified a 143-bp (-202 to -59) glucocorticoid-response element (GRE) upstream from the start of transcription on the long terminal repeat (LTR) region of the DNA of mouse mammary tumor virus (MMTV) (10).The MMTV DNA thus became a paradigm for the study of glucocorticoid-regulated expression of genes transcribed by polymerase 11.

A. The Hormone-Response Element The 143-bp sequence on the MMTV LTR that mediates glucocorticoid response was characterized as an HRE (10). This HRE, when separated from the MMTV LTR promoter and cloned in front of an otherwise hormone-insensitive gene such as the herpes simplex thymidine-kinase (tk) gene, confers hormone inducibility upon this gene (10).Although the HRE of the MMTV LTR was initially identified as an element conferring glucocorticoid inducibility upon the MMTV DNA, later studies showed that this element is not specific for glucocorticoids. Chimeric constructs encompassing the HRE of the MMTV responded to other steroids when introduced into cells possessing receptors for these steroids. For example, in the human mammary tumor cell line T47D, which contains receptors for gonadal steroids, transfected chimeric constructs containing the HRE of the MMTV respond to progestins and androgens (11, 12, and Fig. 1).In feline kidney cell lines that contain GRs and mineralocorticoid receptors (MRs), the HRE of the MMTV LTR responds to both glucocorticoids and mineralocorticoids (14). As the mineralocorticoid aldosterone can also bind the GR (15), it was imperative to show that indeed this hormone induces MMTV expression through its corresponding receptor. This was achieved using the mineralocorticoid antagonist RU-28318 (16),which specifically blocks aldosterone action mediated by the MR. The antagonist obliterates aldosterone action in the feline cells without any significant effect on the action of the GR. This indicates that the effect of aldosterone indeed occurs through the MR ( 1 4 ) . The ability of aldosterone to induce MMTV expression through the MR was again demonstrated in an independent experiment involving the transfer of cloned MR cDNA expression vector with MMTV HRE constructs into simian CVI cells that contain no functional steroid hormone receptors other than the MR (17,18). Thus, the HRE of the MMTV LTR responds to a number of steroids. However, there are

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FIG. 1. The HRE of the MMTV LTR is a hormone-inducible enhancer element. MMTV LTR sequences (-202 to -59) were cloned in two different orientations (a and b) in front of the tk promoter driving transcription of the CAT gene. The HRE-tk-CAT and tk-CAT constructs were transfected into human mammary tumor T47D cells and treated with 1niM ofthe progestin R-5020 and 10 nM ofthe androgen DHT. The transfected cells were harvested and CAT activity was determined.

hormones that do not alter expression at the MMTV LTR promoter, namely, estradiol and vitDg (12,13).The response of the MMTV LTR promoter to varius steroids can therefore be used to classify steroid hormones into two functional groups: those that induce expression at the MMTV promoter (glucocorticoids, progestins, mineralocorticoids, and androgens) and those that do not (estradiol and vitDg). It is not only the HRE of the MMTV DNA that responds to multiple steroid hormones. The HRE found in the human metallothionein-IIa gene responds to glucocorticoids and progestins (19,20), whereas the HRE of the chicken vitellogenin-I1 gene mediates estrogen, glucocorticoid, androgen, and progestin responses (22,23).

HREs as Conditional Enhancers The activating function of HREs in gene expression is reminiscent of the action of enhancer elements; hence, HREs could be considered conditional enhancers. They confer hormone inducibility to promoters that are otherwise hormone-insensitive. In addition, they enhance

6

ANDREW C . B. CAT0 ET AL.

transcription independent of their orientation to the promoter on which they act (10,27).For example, progestin and androgen responsiveness is conferred upon the thymidine-kinase promoter of herpes simplex by cloning the HRE of the MMTV LTR in two different orientations in front of this promoter (Fig. 1). Such enhancer-like properties of the HRE are not only evident in chimeric genes, but also occur in nature. A compilation of naturally occurring HREs in a number of steroid hormone-inducible genes in Fig. 2 shows clearly that these sequences are not located at fixed positions with respect to the promoters they control, nor do they occur in any one fixed orientation. I n one example, a GRE is even present in the first intron at positions 86 to 115 3' from the promoter of the human growth-hormone gene (28).

II. Receptor-DNA Interaction Protein-DNA-binding studies in vitro and in uivo show that the action of the HRE is functionally controlled by the binding of trunsacting factors (steroid receptors). These results were obtained in studies using one or more of the experimental approaches described below and summarized in Table I.

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FIG.2. Orientation and location relative to the transcription start site (CAP) of hormone receptor-binding sites at different promoters. The promoters shown are those of the MhslTV LTR, human metallothionein-IIa (hMTIIa), rabbit uteroglobin (rUG), chicken vitellogenin (chVitII), and Xenopus laeois vitellogenin A2 (Xenopus VitAZ). The receptor-binding sites are indicated as boxes. G , P, and E stand for the glucocorticoid, progesterone, and estrogen receptor-binding sites. The orientations of these binding sites to the CAP site are indicated by horizontal arrows.

7


TABLE I STUDYOF INTERACTION OF STEROID HORMONE RECEPTORS WITH VARIOUS HORMONE-INDUCIBLE GENES

DIFFERENTMETHODS USEDIN

THE

Gene

Reference

MMTV

Glucocorticoid

Rabbit uteroglobin

Progesterone Glucocorticoid Progesterone

Human metallothionein IIa Chicken vitellogenin I1 Xenopus vitellogenin A2 Xenopus vitellogenin B2 c3(u Synthetic ERE Moloney murine sarcoma virus Tyrosine aminotransferase

Glucocorticoid Progesterone Progesterone Estrogen Estrogen Estrogen Androgen Estrogen Glucocorticoid Glucocorticoid

DNase-I footprinting DNA-cellulose assay Nitrocellulose-filter binding Electron microscopy Electron microscopy DNase-I footprinting Electron microscopy DNase-I footprinting DNase-I footprinting DNase-I footprinting DNase-I footprinting DNA-cellulose assay DNA-cellulose assay Electron microscopy Gel retardation Gel retardation DNase-I footprinting DNase-I footprinting

33 34 30,31 35 36 37 36 38,39 19 20 22 40 21 41 42 43 4i 45

A. DNA-Cellulose Assay This assay is based on niodification of a technique described several years ago for determining the DNA-binding activity of proteins (29). In the adaptation of this method, the receptor is labeled with a radioactive ligand, incubated with DNA from calf thymus or salmon testis, immobilized on cellulose. The binding of the labeled receptor to the immobilized DNA was competed with different sets of DNA fragments, among which were those perferentially recognized by the receptor. The amount of radioactive receptor bound in the absence of competitor DNA was arbitrarily assigned 100%. Preference of the receptor for any specific free DNA fragment was measured by the ability of that fragment to serve as a better competitor for the receptor than nonspecific free calf-thymus DNA. In this assay, as one measures the amount of labeled receptor released from the immobilized DNA, there is no need for prior purification of the receptor. However, this procedure is indirect and is interfered with by many parameters that influence receptor release. Thus, there is a need to include a number of controls when this assay is used.

8


B. Nitrocellulose-FilterBinding Assay I n this method, DNA fragments are end-labeled with 32P, incubated with partially purified receptor, and passed through nitrocellulose filter disks (30,31).DNA bound to the receptor sticks to the filter disks, whereas free labeled DNAs are excluded. The filters are then washed, and the bound DNA is released by detergent or high-salt treatment followed by gel electophoresis and autoradiography. As a partially purified receptor preparation is required, this method is handicapped by prior purification of the receptor.

C. DNase-l Footprinting Assay This method offers direct identification of the nucleotide sequences bound by proteins (32).Under appropriate conditions, DNase I cleaves an end-labeled DNA fragment statistically once per molecule to yield a continuous “ladder” of bands on a sequencing gel. If a receptor molecule is bound to a defined region on the DNA fragment prior to the DNase-I digestion, the bound region is protected from the action of the enzyme. This region can be identified as a gap in the ladder sequence of DNA bands. By chemical cleavage of the same DNA fragment, precise determination of the nucleotides bound by the receptor is possible. The limitation of this method is that the receptor concentration should be so high that 60-70% of the DNA binding sites are covered by the receptor. Such high concentrations of receptor can only be achieved by extensive purification.

D. DNA Methylation Protection or Interference Assay Information on the specific nucleotides crucial for receptor binding can be obtained by chemical modification of specific bases at the recognition sequence before or after receptor binding. Such modifications are usually carried out with dimethyl sulfate, which methylates guanine residues in DNA at the N-7 position. If such residues are required for receptor binding, methylation prevents or interferes with the binding reaction. Usually such sites are identified on sequencing gels after a strand-cleavage reaction with piperidine. In some cases, certain guanine residues are hypermethylated after receptor binding. These sites are pockets of hydrophobic regions generated after receptor binding. The resulting conformational change in the DNA structure might trap dimethyl sulfate, leading to hypermethylation. The DNA methylation protection or interference assay has been used to determine contact points for a number of steroid hormone


9

receptors on different genes, including the MMTV (46),human metallothionein-IIa (20),and rabbit-uteroglobin genes (37).

E. Gel-Retardation or Band Shift Assay In this assay, the DNA fragment to be studied is radioactively end-labeled and incubated with unpurified receptor preparation. DNA fragments of about 100 bp or, preferably, shorter oligonucleotides are appropriate for this assay. The reaction products are electrophoresed on a low-percentage polyacrylamide gel, which separates receptorbound DNA fragments from nonbound fragments. Control experiments, in which the receptor preparations are incubated with or without hormone, demonstrate the requirement of the steroid ligand for DNA binding. Incubation of the DNA-receptor complex with antireceptor antibody demonstrates the presence of receptor in the protein preparation used. This method has shown that progesterone receptor from the human mammary tumor cell line T47D binds an oligonucleotide consisting of a progesterone-response element (PRE). The receptor isoforms A and B bind as dimers of AA, AB, and BB (47-50).

F. Electron-MicroscopeTechniques Electron-microscope techniques used to determine the binding of proteins to DNA are also instrumental in determining the sites bound by the receptor on DNA. Fragments of the DNA of interest after in vitro binding with the receptor are fractionated by gel filtration to remove the unbound receptor from the receptor-DNA complexes. The complexes are fixed by glutaraldehyde and spread on positively charged carbon grids. The grids are then washed by floatation on distilled water and stained with uranyl formate. They are then shadowed, and the position of the receptor on the DNA fragments is determined (35,36),As it is not easy to determine accurately the lengths of DNA molecules and the position bound by the receptor, such a method cannot locate the receptor-binding sites precisely. The degree of uncertainty of this procedure is about 250 bp. Furthermore, it is not easy to determine whether the proteins bound are receptor molecules when an impure preparation of receptor is used. Thus, a highly purified receptor protein is required. Results from these different receptor-DNA studies have helped in identifying the DNA recognition sequence for almost all steroid hormone receptors. In the HRE of the MMTV, the GR binds to sequences that contain the motif (5')TGTTCT(3'),present four times on the 143bp enhancer element (33).These same sequences are also bound by the PR (51,52).

10


The binding of different receptors to the same sequence can also be demonstrated with the HRE of genes such as those for human metallothionein IIa or rabbit uteroglobin, which are bound by both the GR and PR (19,20,37-39). The binding site of the androgen receptor (AR) on the C3(1) gene (42)is identical to the sequence bound by the GR and PR. The MR may also bind the same sequence recognized by GR, PR, and AR (18). Studies on a large number of binding sites for the GR and PR allowed the delineation of a 15-bp palindromic consensus sequence for the GR and PR (Fig. 3A). This sequence is also expected to be recognized by the AR and MR, as discussed above. In contrast, the ER binds a distinct sequence, but with a high degree of homology to the sequence bound by the other receptors. A comparison of the sequence of the GRE/PRE/ARE with that of the ERE consensus shows that the ERE only differs from the other response elements by two nucleotide changes on each arm of the palindromic sequence (Fig. 3B). The reason that GR, PR, and AR bind to identical sites was enigmatic for a while, but became understandable upon isolation, cloning, and determination of the primary amino-acid sequences of the steroid receptors.

111. The Structure of Steroid Hormone Receptors

Molecular cloning and determination of the cDNA seauences of steroid hormone receptors have revealed their relationship to one another. They belong to a large family of ligand-binding transcriptional factors made up principally of three major domains (2,3,53):a conserved DNA-binding domain (DBD) joined to a C-terminal hormone-binding domain (HBD) by a hinge region and a hypervariable N-terminal domain. The DBD and the HBD are joined by a short hinge region. A.

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B.

GGTCA

NNN TGACC

consensus binding site for ER (9-21)

FIG. 3. Binding sites for various steroid hormone receptors. N, Nonconserved nucleotides.

11


A. The DNA-Binding Domain (DBD) The DBD of the steroid hormone receptors has the highest degree of sequence homology in this large family of ligand-binding transcription factors. It contains two zinc atoms tetrahedrally coordinated by conserved cysteine residues required for proper folding and sequencespecific DNA binding (54).From the degree of homology of amino acids in the DBD of the cloned receptors, two groups of receptors can be distinguished. One group consists of the GR, MR, PR, and AR (2,3); the second group consists of the ER and vitDg, retinoic, and thyroid receptors as well a large collection of related proteins whose ligands have not been identified ( 2 , 3 ) .The high degree of sequence homology in the DBD explains why different receptors, despite their various physiological effects, nevertheless bind sequences with a common motif. Recently, the structures in solution of the DBD of the ER and GR have been studied using a number of physicochemical techniques (55,56).The three-dimensional structure of the DBD of the GR determined by nuclear magnetic resonance spectroscopy and distance geometry shows that it consists of a globular body from which the two coordinated zinc residues extend as fingers (56).These findings have led to the establishment of a model of a dimeric complex between the DBD of the GR and the GRE. This model is consistent with other biochemical analyses that show that the GR binds cooperatively to the two GRE half-sites (57). It further indicates that specific amino-acid residues of the DBD are involved in protein-DNA and proteinprotein interactions. The essential features of this model are summarized below using the DBD of the rat GR as a reference (Fig. 4).

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FIG. 4. The amino-acid sequence of the DNA-binding domain of the rat glucocorticoid receptor, showing two zinc atoms tetrahedrally coordinated by conserved cysteine residues in the form of zinc fingers.

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ANDREW C . B. C A T 0 ET AL.

i. Residues 458, 459, and 462 near the first zinc coordination site discriminate between the GRE and ERE (58-60). ii. .4n a-helix encompassing residues Ser459to G ~ is the u “recog~ ~ nition helix” located in the major groove of the DNA, with residues Gly458, Ser459,and Val4@ in close proximity to the middle AT base-pairs of the GRE (56). iii. to Asp481 ( D box) may recognize spacing between the palindromic half-sites through protein-protein interaction. Substitutions of this D box with the corresponding segment of thyroid receptor yields a protein that mediates expression from a thyroid-response element. [The thyroid-response element is similar to an ERE, but without the three nucleotides between the half-sites (58).]

Other features of the model indicate possible electrostatic interactions between charged amino acids such as Arg, Lys, and His residues with the DNA phosphates. Furthermore, positions 488, 489, and 491 within the second zinc coordination site in the model extend away from the DNA and are therefore potentially available to interact with other proteins. Such predictions agree with mutagenesis analyses in which point mutations at these three positions generate mutant receptors that bind normally to GRE but fail to enhance transcription i n u k o

(61).

6. The Hormone-Binding Domain (HBD) A number of functions have been assigned to the HBD of the steroid hormone receptor superfamily. Apart from binding various steroids, this region contains a dimerization signal, a sequence for interaction with the heat-shock protein hsp90, a nuclear localization signal, and pait of the transcriptional activation function. A region has been identified within the HBD of the mouse ER that is necessary for both receptor dimerization and high-affinity DNA binding (62).Previous studies on the binding of the human ER to DNA suggested the existence of two dimerization signals in the ER (63):a constitutive dimerization signal in the DBD and a “strong” estrogeninducible dimerization function in the HBD (63).The hormone therefore plays an important role in the formation of stable ER dimers that bind tightly to the ERE. Analysis of the sequences in the HBD required for hormone binding of the mouse ER revealed a heptad repeat of hydrophobic residues conserved in all members of the nuclear receptor superfamily (62).

~


13

Single amino-acid substitution of residues in the HBD of the mouse

E R in the N-terminal, but not the C-terminal, half of the heptad repeat

prevented receptor dimerization. While point mutations in the center of the conserved region abolished steroid binding (62))there were a number of receptor mutants that failed to bind estradiol, but dimerized and bound DNA with high affinity (62). These mutants prove that, contrary to other experimental reports, steroid binding is not an absolute requirement for specific DNA binding in vitro. Further proof of this comes from results of experiments on in vitro manipulations of both the PR and GR that led to tight DNA binding in the absence of steroids (38,50,64).These findings raise questions as to the functional significance of the steroid ligand in vivo. I n uuivo, steroid receptors are noncovalently associated with such proteins as the heat-shock protein hsp90 in the absence of hormones. These proteins, together with the steroid receptors, form a complex with a sedimentation rate of 8-9 S that does not bind DNA (65-67). This complex is disrupted when the hormone binds to its receptor. Thus, the steroid triggers in viwo the disintegration of the complex resulting in the generation of a DNA-binding receptor from a nonDNA-binding component. This function of the steroid can be replaced in vitro by other physical treatments that can equally result in converting the receptor into a DNA-binding protein. The region of the steroid receptors involved in the formation of the inactive complex with hsp90 is the HBD. This was demonstrated by transfection experiments in Cos-7 cells using mutated cDNAs of the human GR and ER (68, 69). Detailed analyses of sequences in the HBD of the human ER show the involvement of amino acids between 251 and 271 in the interaction with hsp90 (69). Interestingly, this region includes an unusually large number of positively charged amino-acid residues that possibly interact with negatively charged regions in the hsp90 described as the “A region” (70). The HBD of the steroid receptors is also involved in transactivation of the receptors (71-73) and contains nuclear localization signals. Two distinct nuclear localization signals (NL1 and NL2) have been identified in the C-terminal region of the rat GR (74).NL1 maps to a 28-amino-acid segment close to the DBD, whereas the main signal directing the receptor into the nucleus (NL2) resides within a 256amino-acid region in the HBD (74).Like the GR, the PR also has two nuclear localization signals (75).Its main signal is located in the DBD. It contains sequences rich in basic amino acids (lysines and arginines), and it is homologous to the nuclear localization signal of the SV40 T-antigen as well as to NL1 of the GR (75).

14


A second nuclear localization signal of the PR with limited activity resides in HBD (75). Thus, although the GR and the PR contain two nuclear localization signals, the PR differs from the GR in that it resides in the nucleus in the absence of hormone. This difference most likely comes from the different contributions of the two nuclear localization signals of these receptors. While the main signal of the PR functions constitutively (in the absence of hormone), that of the GR is hormone-dependent.

C. The N-Terminal Domain The least well-conserved domain of the steroid hormone receptors is the N-terminal region, which ranges in length from 603 amino acids in the human MR to 24 amino acids in the human vitDg receptor (3). The N-terminal region of the human GR contains amino-acid sequences rich in negative charges, which are apparently important for the transcriptional activation function of this receptor (72, 76). The negatively charged character of the residues in this region of the GR has given them the name “acidic activating regions.” They appear to function cooperatively with other trans-activating regions elsewhere in the receptor molecule (71, 77). In the human GR, for example, the N-terminal acidic activating region functions cooperatively with a 30amino-acid peptide identified in the C terminus of the receptor. This oligopeptide stretch also contains a high percentage of acidic amino acids (71,72). Thus, negatively charged amino-acid residues constitute an important feature of the trans-activating domain of the GR. Like the GR, the PR and the AR also possess trans-activating functions in their N-terminal regions (49, 78-80), which are also rich in negatively charged amino acids (79-82). The M R and the ER are exceptions to this rule. The MR has no truns-activating function at its N-terminal end (18)and it induces transcription of indicator genes like the MMTV at only one-tenth of the level induced by the GR (18). However, this receptor can be made to function as efficiently as the GR by the exchange of its N-terminal sequences with the acidic activating region of the GR (A. C. B. Cato, unpublished). In the case of the ER, chimeric constructs lacking the HBD but containing the N-terminal region stimulate transcription with only 1-5% of the efficiency of the wild-type receptor in HeLa cells (73). These results suggest that the N terminus of the ER contains a weak autonomous activator that acts cooperatively with the trans-activating region in the HBD (73). Sequence analyses of the N-terminal region did not identify highly acidic regions, nor were such regions identified in the HBD (83).It is conceivable therefore that the N terminus and the HBD of the human ER contain transcription activation functions dis-


15

tinct from those of acidic domains. In fact, further studies show that the N-terminal region of the human ER contains an independent transcription activation function that is cell-type-specific. It activates transcription efficiently in chicken embryo fibroblasts, but poorly in HeLa cells (84).The N-terminal region of the chicken PR also controls specificity of the receptor for target genes in a cell-specific manner (85).How these N-terminal sequences confer the cell-type specificity of action to these receptors has yet to be identified. With the cloning and analyses of the structure of the steroid hormone receptors as well as identification of the regulatory elements that mediate steroid hormone action, the way is now clear for a number of questions to be answered. Some of these are related to how interaction of the receptors with DNA is transduced in a positive or negative way to changes in transcriptional activity.

IV. Induction of Gene Expression by Steroid Hormone Receptors

The general model of activation of gene expression by steroid hormones is the conversion of the steroid hormone receptor from a nonDNA-binding complex into a DNA-binding protein (2, 3 ) . This activated receptor then binds to hormone-regulated genes (which, on average, contain at least one receptor-binding site) to induce transcription. There are genes that contain more than one copy of such steroid HREs in fairly close proximity. On the whole, such genes are more highly inducible than are those with a single response element. In the HRE of the MMTV DNA that contains four GR/PR-binding sites, gene-transfer experiments show that mutation of any one of the four receptor-binding sites drastically reduces the overall steroid hormone response at this promoter (86-89). These results suggest a concerted action of the receptor-binding sites. In uitro, GR- and PR-binding studies provide no evidence for cooperativity of binding of steroid receptors to the HRE of the MMTV DNA (52, 90). Thus, there is a discrepancy in the in vitro receptor-DNAbinding studies and the in vivo gene-transfer experiments. This discrepancy could be due to the absence in the in vitro studies of other proteins that bind to the regions between the receptor-binding sites that are required in vivo to modulate the steroid hormone response. Alternatively, other proteins may be involved in interaction with the receptor in vivo without necessarily requiring the need to bind to DNA. Recent studies show the former to be true. Ubiquitous transcrip-

16


tion factors such as CTF/NFI and OTF-I, through their interaction with sequences in the HRE, modulate the steroid response at the MMTV LTR promoter (86,87,91-93; E. Hartig, S. Mink and A. C. B. Cato, unpublished). As well as the ubiquitous transcription factors mentioned above, additional nuclear proteins binding to the HRE of the MMTV LTR outside the receptor-binding sites have been postulated to differentially modulate the steroid hormone response at this promoter (86). This prediction was based on results from studies of the induction of MMTV expression by glucocorticoid, progestin, and androgen in genetransfer experiments using MMTV LTR constructs with mutations outside the receptor-binding sites (86). Mutations around -200 upstream from the start of transcription at the MMTV LTR promoter had virtually no influence on progestin response, but glucocorticoid and androgen responses were significantly reduced (86). Mutations at - 144 of the same promoter decreased the androgen response without a significant effect on the progestin action (86). Gel-retardation and DNase-1 footprinting experiments show that these sites, in their nonmutated form, bind nuclear factors from a number of cell lines (E. Hartig, S. Mink and A. C. B. Cato, unpublished). However, how these factors differentially influence the steroid hormone response at the MMTV LTR promoter must await purification and characterization of their interaction with their cognate sites in in vivo footprinting experiments. Studies on the effect of ubiquitous transcription factors on different steroid responses at simple promoters show no differences in the modulating activity of these factors on the steroid response (94). All the transcription factors studied functioned synergistically with either the GR or the PR (94). When an ERE or a GRE was placed immediately upstream from the TATA box of the tk promoter, these single response elements conferred glucocorticoid or estradiol inducibility upon this promoter (95). However, when a GRE was inserted 351 b p upstream from the start of transcription of the tyrosine aminotransferase gene, hormone inducibility was attained only by a combination of GHEs or by a single GRE in association with the binding site of other transcription factors (95).These studies provide an explanation for the need for more than one receptor-binding site in many inducible genes, particularly those in which the steroid-response elements are at considerable distance from the inducible promoters (37,45, 96). Synergistic action of multiple steroid HREs need not be restricted to one class of steroid receptors. In fact, receptors of one class can function synergistically with the receptors of another class. For exam-

17


ple, a piece of DNA from the chicken vitellogenin-I1 gene containing an ERE and a PRE/GRE, when cloned in front of the tk promoter, mediates the synergistic action of estrogen and glucocorticoid or estrogen and progestin at this promoter (22,23).The synergistic action of these steroids correlates with the ability of their respective receptors to bind to the vitellogenin DNA. Point mutations in the PRE that destroy the interaction of PR to its binding site but leave the ERE intact destroy the synergistic action of estrogen and progestin (22).Similarly, a point mutation in the ERE destroying ER binding no longer mediates action of estrogen and progestin (22). the -- -synergistic .. . . In an attempt to understand the molecular basis of the synergistic action of steroid hormones, the in vivo function of paired palindromic and non-palindromic EREs spaced differently from an indicator promoter was studied (97). It was observed that perfectly paired palindromic EREs acted additively and independently of their spacing when positioned close to a TATA box. However, they acted synergistically when moved 175 bp further upstream from the TATA box (97). Imperfect palindromic EREs, on the other hand, acted synergistically even when positioned close to the TATA box (97). In none of these cases could the functional synergism of the EREs be correlated with cooperative DNA binding by the ER to dimeric perfect or imperfect EREs (97,98). Cooperative binding was only observed on an isolated perfect ERE, in agreement with the finding that the ER binds an ERE as a homodimer (63).So far, there is only one report of cooperative binding of the ER to a tandem imperfect ERE of the Xenopus vitellogenin-B1 gene (99). Thus, it still remains to be clarified how the synergistic action of steroid hormones is brought about. It is now becoming increasingly clear that steroid hormone receptors are not the only components involved in mediating the increased expression of genes by steroid hormones. Other as yet ill-defined factors have also been implicated in the positive effects of steroids on gene expression. The identification of these factors will greatly clarify some of the contradictory findings in this field.

V. Positive Re ulation of Gene Expression by Steroic? Hormones throu h the Modulating Activity of Dif erent Transcription Factors

P

Although estrogen response is mediated by either perfect or imperfect EREs through synergistic action of the ER, there are genes regulated by estrogen that do not carry any of the above-mentioned charac-

18

ANDREW C . B. C A T 0 ET AL.

teristic regulatory elements. For instance, in the chicken ovalbumin gene, two separate sequences of GGTCA have been identified that mediate at least part of the hormonal regulation of this gene (100).One of these two elements binds the COUP3 transcription factor that is classified as a member of the steroid-receptor superfamily, as its primary amino-acid sequence is homologous with other members of the steroid hormone-receptor superfamily (101,102).The other regulatory element is preceded by the sequence TG to generate TGGGTCA, and it is a functional mediator of estrogen action. In contrast to the classical ERE already described, the sequence TGGGTCA does not bind the ER. A careful scrutiny of this sequence indicates a strong sequence similarity to a canonical AP1 recognition site (TGAGTCA) (103, 104), thus indicating that it could bind the transcription factor AP1. Indeed, in gel-retardation analyses, together with antibody studies, this site binds the heterodimeric ,4Pl complex, which is composed of the oncoproteins Fos and Jun (100).In the gel retardation assay, the complex bound was not altered by the presence of the ER. However, in uiuo, the ER coactivates the ovalbumin promoter with c-Fos and c-Jun. This coactivation does not require the DBD of the ER. ER mutants that lack the DBD functionally coactivate with c-fos and c-jun coding plasmids with an efficiency similar to that of the wild-type ER (100). It is not clear how this coactivation is achieved without the ER’s binding to DNA. Either the ER alters in uiuo binding of AP1 (genomic footprinting has not been performed) or ER, Fos, and Jun cooperate through protein-protein interaction. It has also not been ruled out that the ER needs to activate another gene, whose product then mediates Fos-Jun function. A further possibility is that the ER titrates a putative Fos-Jun inhibitor. Recently, a novel mechanism of modulation of the functional activity of another type of steroid hormone response b y polypeptide growth factors such as EGF, TGFa, or IGF-I was identified (105).In human mammary T47D cells transfected with an MMTV CAT construct, EGF and TGFa increase CAT activity induced by the progestins, R-5020, norethisterone, or medroxyprogesterone acetate (105).The growth factors influence the level of MMTV CAT mRNA in progestin-treated cells, as shown by S1-nuclease mapping experiments using mRNA from T47D cells stably transfected with pSV2neo and MMTV CAT constructs (Fig. 5).Transcription at the SV40 promoter was not affected by progestins or the growth factors. IGF (Fig. 5) and EGF (not shown) on their own had no effect on MMTV expression in the absence of COUP = chicken ovalbumin upstream promoter

19


+

M

1

2

3

4

&

5

FIG.5. Epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) potentiate the progesterone response. S1-nuclease mapping of the SV40 and MMTV LTR transcripts in T47D cells stably transfected with MMTV CAT and SV40 neo constructs. The cells are routinely cultured in RPMI medium supplemented with 10% fetal calf serum (FCS) and 0.6 pgiml insulin at 37°C. For the S1-nuclease mapping experiment, the cells were cultured in the absence of FCS and insulin, and treated with the hormone and growth factors for 16 hr. The autoradiogram shows S1-nuclease mapping products with RNAs from (1)cells treated without hormone, (2) cells treated with IGF-I, (3) cells treated with the progestin R-5020, (4)cells treated with R-5020 and IGF-I, and (5) cells treated with R-5020 and ECF. The concentrations used were as follows: R-5020, M ; EGF, 20 ng/ml; ICF-I, 50 ngiml. The SV40 and MMTV transcripts have been indicated. M, Marker.

hormone. However, they potentiated the progestin-induced expression at this promoter (Fig. 5). The receptors for these growth factors possess tyrosine kinase activity and it appears that this enzyme is necessary for the effect of the growth factors. This idea is supported by the finding that Tyrphostin

20

ANDREW C. B. C A T 0 ET AL.

RG-50863, an inhibitor of the EGF tyrosine kinase activity, inhibits the EFG/TGFa response without any effect on progestin action (105).It is conceivable that activation of the tyrosine kinase activity of the EGF receptor phosphorylates the PR. Studies on the phosphorylation of the PR have so far shown that although the state of phosphorylation of the PR is inceased upon hormone binding, this level is not further influenced by the presence of the growth factors EGF or TGFa (105).The affinity of the steroid receptor for DNA upon binding the hormone was also not altered by these growth factors (105).At the moment, it is not clear how EGF, TGFa, or IGF-I influence the action of the PR. The options are that different sites of the PR are phosphorylated in the presence of the steroid hormone and the growth factors, or that phosphorylation of other proteins modulates the activity of the PR. In another type of modulation of steroid hormone-receptor action, agents that activate CAMP-dependent protein-kinase A functionally activate the chicken PR in the absence of steroid binding (106).This raises yet again the question of the functional significance of the steroid if other agents are equally proficient in activating the receptor. Undoubtedly, steroid hormone research is still open to surprises in the future. Taken together, activation of gene expression by steroid hormones proceeds through diverse pathways in which dependent and independent DNA-binding activities of the steroid receptors are required. The receptors bind to single or multiple response elements for activation. In some instances, receptor-binding sites function in synergy or in concert with the binding sites of other transcription factors. In these cases, protein-protein interaction of the receptors and the nonreceptor proteins may play a role in the activation. There are other mechanisms by which the steroid receptor does not need to bind to DNA but induces gene expression through interaction with other DNA-bound factors. Finally, there are some mechanisms that require modification of the steroid receptor or of proteins that interact with the receptor. All these diverse pathways come together in the positive regulation of gene expression by steroid hormones.

VI. Negative Action of Steroid Hormones A. Feedback Effect Many physiological systems use negative regulatory pathways that repress expression by establishing a close regulatory loop and returning the systems to their starting points. This feedback mechanism is


21

also exhibited by steroid hormones. This occurs (1)through repression of the timely production of steroids by other components of the endocrine system as well as the central nervous system (107),and (3) through negative regulation of expression of the steroid hormone receptor by their own ligands and receptors. Glucocorticoid and progesterone both repress the RNA and protein levels of their receptors (108-113). How this inhibition is achieved is not clear. In the case of the GR, it is thought to be mediated by binding of the activated GR to 3’-untranslated regions, of the DNA coding for the receptor (110).A sequence in the promoter of the PR that mediates down-regulation of expression of this receptor by progestins has recently been identified (114). This sequence maps to positions + 698/+ 723, overlapping the initiation of translation of the rabbit PR. It closely resembles an ERE, and indeed it is bound by the ER (114). This suggests that PR may down-regulate its own expression through protein-protein interaction with either the ER or other proteins binding to the ER recognition sequence (114). Further evidence that the ER may be involved in the autoregulation of the PR comes from observations that the negative effect on the PR is cell-typedependent. It is observed in epithelial and stromal cells in the endometrium that contain ER (115),but not in the myometrium or cells that lack ER (116-118). Apart from the feedback effect of steroid hormones on their own response, other types of negative action of steroids on the expression of a number of genes have been reported. Glucocorticoids, for instance, are known to inhibit the expression of the human plasminogen activator gene (119) and the induction of neural-specific genes (157) by mechanisms yet to be investigated. However, other negative actions of glucocorticoids on gene expression have been investigated, and these can be classified mechanistically as “overlapping binding sites” and “protein-protein interference.”

6. Overlapping Binding Sites Negative regulation of expression of genes by glucocorticoids has been identified in cases in which the GRE overlaps the binding sites for factors required for positive regulation. Examples of such a mode of negative regulation by glucocorticoids can be found in the CAMPinducible chorionic gonadotropin a-subunit gene (120), the rat afetoprotein gene (122,123),the human osteocalcin gene (124),and the genes for bovine prolactin (125)and rat pro-opiomelanocortin (126). Expression at the CAMP-inducible chorionic gonadotropin a-subunit promoter is controlled by a 168-bp fragment that contains a CAMP-

22


response element cooperating with a tissue-specific element (120). Elevation of CAMP levels enhances expression at this promoter, while glucocorticoid represses both the basal and enhanced levels of expression. This is achieved b y the activated GR binding to the CAMPresponse element and inhibiting its action as well as that of the tissuespecific element (120). The action of the GR is independent of its N-terminal region but dependent on the DBD and HBD (120).Removal of the HBD of the human GR in contransfection experiments resulted in a receptor variant with minimal repressor activity (121).The HBD of the GR could therefore bring about repression by blocking or neutralizing transactivation of the tissue-specific factor through protein-protein interaction. Alternatively, it could interfere with the activity of the tissuespecific factor through a mechanism of steric hindrance. A single amino-acid change of the human GR at position 442 from Lys to Gly generates a receptor that binds hormone and DNA in vitro with nearly normal activity (77), but activates reporter genes to only 1%of the wild-type level (121).This mutation still has 68% of full function in the repression of the gene encoding the a-subunit of the chorionic gonadotropin (121).This shows that although DNA binding is required for both positive and negative action of the GR, the negative activity of the receptor is mechanistically different from its positive counterpart. Glucocorticoids negatively regulate gene expression not only through the binding of the GR to a GRE overlapping tissue-specific element, but also to other regulatory elements. The overlapping sequence could be other transcription factors such as AP1, as in the case of the rat a-fetoprotein gene (122, 123), or even a TATA box that determines initiation of transcription, as in the human osteocalcin gene (124).As the binding site for the GR in these cases is a classical GRE, transfection of chimeric genes containing this sequence in cells that lack the interfering factor should lead to positive gene expression. For example, in the absence of c-Fos and c-Jun (the components of APl), the GR positively regulates the expression of a chimeric construct containing the a-fetoprotein-regulatory element (123).Similarly, c-fos and c-jun expression vectors positively regulate the expression of this construct in the absence of the GR (123). This is not always the case, as, in some instances, the GR is not able to induce gene expression in the absence of c-Fos and c-Jun, although c-Fos and c-Jun induce expression in the absence ofGR. Such a case is encountered in the regulation of expression of a chimeric construct containing a 25-bp sequence from -254 to -230 bp 5' upstream from


23

the transcriptional start site of the proliferin gene (132).This element, described as “composite” GRE (cGRE), consists of a binding site for AP1 overlapping a binding site for the GR, and it is indeed bound by AP1 and the GR (132).However, in contrast to the classical GREs that positively regulate gene expression, this GRE is different in that it is incapable of positively regulating the expression of a heterologous tk promoter in F9 cells that lack c-Fos and C-Jun (132).Positive regulation by GR is only achieved in the presence of c-jun expression vectors. In the presence of c-Sun, c-Fos, and GR, there is antagonism of promoter activity (132),possibly due to the strong DNA-binding activity of this heterodimeric complex, which disrupts the positive effect of the GR and c-Jun (132).Thus, depending on the availability of c-Jun or C-FOS,cGRE can mediate a positive or negative response in the presence of GR and glucocorticoid. This occurs without the need to change regulatory element, promoter, receptor, or hormone. So far, the selectors for the positive and negative responses of cGRE are c-Jun and c-Fos. However, it is conceivable that other selectors may also be available. Thus, cGREs, because of their regulatory versatility, may prove a prevalent mode of regulation of gene expression by glucocorticoids. In any case, cGREs remain just a model, as there is no evidence that such gene regulation, demonstrated with chimeric constructs, indeed occurs at the proliferin promoter. Glucocorticoids also repress gene expression through sequences termed “negative GREs” (nGREs) in the bovine prolactin and rat pro-opiomelanocortin genes (125, 126). The nGREs were identified through gene-transfer experiments and an in vitro receptor-binding (DNase-I footprinting) assay as sequences that, on their own, confer repression by glucocorticoid (125, 126). nGREs enhance promoter activity in the absence of glucocorticoid and the GR, presumably through the action of proteins binding to the same sequences to which the GR binds. In the presence of activated GR, expression is repressed, possibly by competition of the GR for the same regulatory sites. A strong consensus sequence for receptor binding within the nGRE has not yet been defined. However, it is apparent from transfection experiments that nGREs differ functionally from the classical GREs (125,

126).

It is not clear whether, in the presence of glucocorticoids, the activated GR and the factor that binds to the nGREs occupy this site simultaneously or whether repression is achieved by the replacement of this factor by the GR. Whatever the mechanism, the principle of this negative regulation by glucocorticoids is based on competition for overlapping binding sites.

24

ANDREW C. B. CAT0 ET .4L.

C. Protein-Protein Interaction Negative action of glucocorticoid hormones is also achieved without the need for the GR to bind to DNA. An example of this type of regulation is the rat prolactin gene (127). This gene is negatively regulated by glucocorticoids, but positively regulated by estrogen through elements located at - 1582 to - 1568 upstream from the start of transcription of this gene. Deletion of the positive acting cis-regulatory elements for the ER generates a mutated construct that is inhibited by both glucocorticoids and estrogen, although there are no obvious binding sites for these steroid receptors at the promoter of the prolactin gene (127). In an attempt to investigate how the receptor of these steroids inhibits expression of the prolactin gene, it was discovered that cis-acting sequences that bind a tissue-specific transcription factor, Pitl,4 was the target for the repression (127). The ER inhibits expression through this element in a non-DNA-binding fashion. Mutations in the ER showed that the hinge region of the receptor is required for the inhibition. Exchange of this hinge region with the corresponding hinge region of the GR generated a chimeric ER/GR that also inhibited expression ofthe prolactin gene (127).It is not clear how the inhibition is brought about, but it is thought to involve the hinge region of the steroid receptors and the Pitl transcription factor. However, no direct interaction of these factors has been demonstrated, and, if such an interaction occurs, it is not clear whether it results in diminished or altered binding of the Pitl factor to its cognate binding site. There are many reports that the GR and AP1 inhibit each other’s effect on gene expression through a mechanism that does not require the DNA-binding activity of the GR (128-132,134). The GR does not bind an APl-binding site, and similarly, c-Fos and c-Jun interact with neither the classical GREs nor nGREs. The fact that these two transcription factors interfere with each other’s activity presupposes an interaction at the protein-protein level. Three different models can be proposed as a consequence of such an interaction: (i) titration of a common positive factor required for AP1-induced activity by the GR and vice versa; (ii) repression of the binding activity of AP1 by the GR or repression of the binding activity of the GR by AP1; and (iii)repression of trans-activating activity ofAPl by the GR or of the GR by APl without affecting the DNA-binding activities of these factors. 4Pit = pituitary transcriptional activator.


25

Regarding model (i), experiments on the human collagenase gene show that protein synthesis is not required for AP1 activity, nor is protein synthesis required for the inhibition of AP1-induced expression of this gene by the synthetic glucocorticoid dexamethasone (128). This makes it unlikely that a newly formed protein required for AP1 action is titrated by the GR. Although an auxiliary preformed protein may be required for the action of AP1 or the GR, which is titrated by the GR or AP1, respectively, this possibility is very remote and is therefore considered unlikely. Thus, models (ii) and (iii) appear the more likely candidates to explain this mutual inhibitory activity of AP1 and the GR. A number of researchers are currently involved in studying these two models, but so far no clear conclusions have been reached. Cross-linking experiments of c-Fos and c-Jun synthesized in vitro to GR, followed by immunoprecipitation with GR antibodies, showed physical interaction of c-Fos (130) or c-Jun (130,132) with the GR. Similarly, GR synthesized in vitro was also shown to interact with bacterially expressed c-Jun (130,132).Thus, the GR appears to interact physically with c-Fos and/or c-Jun. To investigate whether this interaction destroys DNA-binding activity of the GR or c-Jun, in vitro experiments were performed in which the DNA-binding activity of GR or of AP1 was analyzed in the presence of the interfering factor. These experiments showed that incubation of bacterially expressed c-Jun with GR, or vice versa, destroyed each other’s DNA-binding activity (129,130). Different results were obtained in in vivo footprinting experiments to determine the binding of AP1 to the human collagenase gene (133). In this case, the binding of AP1 to its cognate binding site was not displaced by treating the cells with glucocorticoid. Although a possibility exists that the bound AP1 could have been replaced by an inactive APl, this possibility appears very remote in view of in vitro experiments showing that binding of the c-Fos/c-Jun heterodimeric complex to DNA is not altered by the presence of the GR (133).Thus, it is still not clear how the mutual repression of activity of AP1 or of the GR occurs. Deletion mutations in c-Fos and c-Jun revealed the regions ofthese proteins involved in inhibiting the action of the GR. In c-Jun, deletion of the leucine-zipper region abolished the ability of this protein to repress the GR response from a GRE tk CAT construct (129).Deletion of the c-Jun DBD or the entire N terminus did not alter the ability of this protein to repress the GR response (129). In the case of C-FOS, truncation of the leucine-zipper and basic regions had no influence on

26


its ability to repress the GR response, but sequences at the N-terminal region encompassing amino acids 40-111 were important (131).This region of c-Fos is poorly conserved among the Fos-related proteins Fos-B and Fra-1; consistent with this finding, these two proteins do not participate in the repression of GR-induced gene expression (131). With the GR, mutational analyses show that sequences in and around the DBD are necessary for the inhibition of AP1-induced expression (129-131). The facts that DNA binding of the GR is not required for inhibiting AP1-induced expression, but that the DBD of this receptor is nevertheless important for this function, further emphasize the finding that this region of the receptor is involved in other functions. In gene activation, the GR and the MR both recognize the same response elements and activate expression from promoters that contain classical GREs/MREs. However, in terms of ability to repress AP1induced gene expression, the MR differs from the GR in that it has no negative action on AP1-induced gene expression (A. C. B. Cato, unpublished). Chimeric GR/MR expression vectors localized the inability ofthe MR to trans-repress to the DBD ofthis receptor (A. C. B. Cato, unpublished). Thus, amino-acid sequence differences in the DBD of the MR that distinguish this receptor from the GR may be responsible for the lack of the MR to repress AP1-induced gene expression. There are four such differences, and their systematic mutagenesis allows identification of the appropriate amino acids required for the transrepression.

VII. Repression of Steroid Hormone Action by Transcription Factors

It is not only the action of glucocorticoid that can be repressed by c-Fos and c-Jun. The induction of gene expression by the steroid 1,25dihydroxyvitamin Dd (vitD) is also repressed by these oncoproteins in a mechanism called cross-coupling (135)or phenotype suppression (136).This mechanism is best exemplified by the osteocalcin gene, a gene that codes for a bone-specific protein and marker of mature osteoblasts. The osteocalcin gene is positively regulated by vitD through binding of the vitD receptor to the vitD-response element (137-140). The vitD-response element in both the rat and human osteocalcin genes overlaps a binding site for APl (135,136).Cotransfection of c-fos and c-jun expression vectors in gene-transfer experiments leads to repression of vitD-induced transcription of a chimeric osteocalcin-CAT


27

construct (135).Thus, increased expression of c-fos and c-jun in proliferating osteoblasts is thought to repress vitD-induced transcription of the osteocalcin gene through direct competition with the vitD receptor for binding to the same response element. Such suppression of steroid hormone action is also thought to occur in the alkaline phosphatase gene of the rat that contains a vitD-response element overlapping c-Fos/c-Jun-binding sites (136). Apart from these negative regulations by overlapping binding sites for AP1 and the vitD receptor, AP1 also inhibits estrogen action without the need for this factor to bind to DNA (158).This is mechanistically similar to inhibition of the glucocorticoid response discussed above. Interesting deletion analyses of c-Jun have shown that, as in the case of the repression of glucocorticoid response, the leucine-zipper region is required for trans-repression; in addition, glycine-rich sequences between amino acids 147 and 220 of the c-Jun protein are also required (158). Thus, as in the case of induction of gene expression by steroid hormones, negative action of steroids occurs through different pathways. Some of these require that the steroid receptor bind to DNA, but others do not require the DNA-binding activity of the receptor. In a great number of the examples presented on the repression of gene expression by steroid hormones, the mechanism of repression is based on a principle of mutual interference. For instance, the activated steroid receptor not only represses the expression of genes whose activity is under the control of c-Fos and c-Jun, but the response of certain steroid hormones is also repressed by these transcription factors. These interdependent regulatory circuits present a large number of possibilities by which the expression of various genes could be regulated by steroid hormones.

VIII. Positive and Negative Action of Steroid Antagonists

Many attempts have been made to deliberately inhibit the action of steroid hormones for clinical, diagnostic, and therapeutic purposes. Antagonists of steroid hormone action may exert their effect by interfering with the synthesis or metabolism of a specific steroid or proteins essential to the expression of the physiological response of steroids. Alternatively, steroid antagonists may function by direct competition for the hormone-binding site on the receptor. The latter type of antagonist should have structural features that enable them to bind to a common site on receptors, but at the same time different enough so as

28

ANDREW C. B. C A T 0 ET AL,

not to activate the receptors. Attempts at synthesizing such types of compounds have been geared mainly to modifications on the C and D rings of the cyclopentanophenanthrene nuclei of steroids. Modifications of the C ring have produced useful antagonists, whereas D-ring substitutions have produced steroids with less antagonistic action in vivo (141). Apart from these modifications of the ring structures, the discovery of the epoxide pathway for the introduction of substituents at the l l p position of 19-nor steroids (142) has made it possible to introduce any organic group into this position. Such substitutions produce llpsubstituted 4,g-estradienes and 1,3,5(10)-estratiienes with agonistic and antagonistic activities. Actually, it is the size of a substituent that determines the agonistic or antagonistic activity of a synthetic steroid. Studies on the properties of different l l p substitutions have shown that 11~-(4-dimethylaminophenyl) derivatives are potent antagonists of glucocorticoid and progesterone action. The most popular member in this series is RU-38486. Over the past few years, inuch attention has been paid to a class of nonsteroidal compounds that are derivatives of triphenylethylene and are referred to as pure or nearly pure antiestrogens (143). The most popular member of these is tamoxifen or its active metabolite, 4hydroxytamoxifen. As these compounds do not have the steroid backbone structure, they are not discussed in this essay.

A. Antagonistic Action of RU-486 One of the most extensively studied steroid antagonists is 1lp-(4dimethylaminophenyl)- 17p-hydroxy- 17a-(l-propinyl)-4,9-estradien$one (RU-38486, also known simply as RU-486, or Mifepristone). This antihormone has a strong antiprogestin action as well as a remarkable antiglucocorticoid action (144).Howe\.er, the mechanism of the antagonistic action of this antihormone is not very well understood. As an antigl:~cocorticoid,RU-486 impairs the nuclear accumulation of GR (145-148). Its negative action on the GR has been shown in the chicken oviduct to be due to trapping of the receptor in a nonactivated cytoplasmatic form (146). Compatible with this is the lack of binding by the GR-RU-486 complex to the GREs on the tyrosineaminotransferase gene in in vivo DNase-I-binding experiments (149). The situation is completely different for the antagonistic action of the RU-486-PR complex. In this case, RU-486 binding does not alter the cellular localization of this receptor, as it is already localized in the nucleus. Results of in vitro receptor-binding studies and i n vivs competition experiments indicate that the PR-RU-486 complex binds


29

DNA (38, 48, 49, 51). RU-486 binding to the PR also leads to phosphorylation of the PR, a feature so far attributed to the agonistic action of steroids (48).Thus, in terms of its ability to induce DNA-binding activity and to increase the phosphorylation of the PR, RU-486 would be expected to function as an agonist. It is therefore unclear how RU-486 mediates its antagonistic action on the progestin response. In gel-retardation experiments, a PR-RU-486-DNA complex migrates slightly faster than a PR-progestin-DNA complex (47).Such a change in the migration properties of the PR-RU-486 complex bound to DNA could be due to an altered conformation of the PR induced by the antagonist, and this could account for its negative properties on progestin-induced gene expression.

B.

Agonistic Action of RU-486 Recent results show that RU-486 has specific agonistic effects on at least some promoters (49). On promoters containing a single PRE, RU-486 increases transcription, using the truns-activation domain situated at the N terminus of the human PR (49).In contrast, no transcriptional activation of the human PR was observed in the presence of RU-486 from a complex promoter such as the MMTV LTR promoter (49). A number of in vivo studies involving treatment of animals or post-menopausal patients with RU-486 show morphological changes in cells of the endometrium indicative of an agonistic action of RU-486 (150,151).Whether these results are due to the increased expression of genes with simple HREs remains to be established. However, possibility exists that some agonistic action of RU-486 may not be mediated directly at the transcriptional level. For example, this antihormone stabilizes mRNA of the progestin-induced human fatty-acidsynthetase gene (152).The transcripts of other genes could be similarly affected by this antihormone.

C. identification of an Antiprogestin with True

Antagonistic Activity In a further study of the agonistic effect of RU-486 on PR-regulated gene expression, in vitro cell-free transcription and gel-retardation studies were used to study the properties of a number of llp-arylsubstituted steroids upon interaction with the human PR. In gelretardation experiments, two types of antiprogestins were identified ( 4 7 ) .Type-I antiprogestins, represented by ZK-98299 (Onapristone) (Fig. 6), belongs to a group that does not confer DNA-binding activity upon the PR; type-I1 antiprogestins (e.g., RU-486) induce receptor

30


(RU-38486)

&

Onapristone (ZK-98299)

FIG.6. The structures of RU-38486 (h4ifepristone) and ZK-98299 (Onapristone), showing the change at C-13 from a P-methyl in RU-38486 to the a-methyl epimer in

ZK-98299.

binding to DNA (Fig. 6). Another major difference in the action of type-I and -11 antiprogestins is that, while type I has no agonistic activity in a cell-free transcription assay, type-I1 antiprogestins induce expression from a simple template consisting of a PRE linked directly to a TATA sequence (47,153). In studies on phosphorylation of the PR, the type-I antiprogestins did not phosphorylate the PR, whereas the type-I1 antiprogestins did (S. Krusekopf and A. C. B. Cato, unpublished). Thus, from the differences exhibited by these two types of antiprogestins, type I should be antiprogestins with true antagonistic activity. Incidentally, the type-I antiprogestin ZK-98299 has a rather unusual configuration in that its C-13 methyl group is not in the pposition, as in other natural steroids, but in the a-position. It is therefore likely that this sterochemical configuration may represent a novel feature that confers pure antagonistic properties upon antiprogestins.

IX. Concluding Remarks In this essay, different elements that mediate the steroid hormone response have been described. Different sequences that mediate a positive action of steroid hormones have been presented. Sequences that mediate a negative action of steroid hormones have also been classified into various mechanistic groups. Most of these cis-acting elements were shown to bind the various steroid receptors b y in citro binding studies using “naked” DNA. However, in uiuo, the receptorbinding sites do not occur on naked DNA, but are nucleosomally packaged. There is, therefore, the major question of how steroid hormone receptors recognize their cognate binding sites on chromatin to regulate transcription.


31

Experiments with reconstituted chromatin on a short DNA from the

MMTV LTR show that the GR and the PR bind naked DNA and

reconstituted nucleosomes with equivalent affinity. However, NFI, a transcription factor essential for MMTV promoter activity, binds tightly to naked DNA, but not at all to the in uitro nucleosomally organized promoter (154). This has therefore led to the notion that hormone induction at the MMTV promoter proceeds through displacement of a nucleosome at the HRE by the steroid receptors, allowing transcription factors such as NFI to bind to the promoter (155).This notion is reinforced by exonuclease-I11 footprinting experiments in uiuo, showing that NFI binding is observed only after glucocorticoid treatment of cells transfected with MMTV LTR chimeric constructs (156). However, these findings are not in agreement with in vivo studies with MMTV LTR constructs containing a mutated NFI-binding site that showed that the PR does not require NFI for its response, whereas GR does (86,87). Thus, either the two receptors use different mechanisms in the induction of expression at the MMTV LTR promoter, or there are some flaws in interpretation of the nucleosome displacement experiments. It is also not clear whether this nucleosome displacement model applies only to the HRE of the MMTV LTR or to other hormonally regulated genes. Whatever the case, it will be interesting to find out not only how changes in chromatin influence positive gene expression by steroids, but what contributions they make toward determining the different ways in which steroid hormones negatively regulate the expression of genes. These studies will not be complete without the isolation and characterization of the limiting factors that mediate the steroid hormone response without necessarily binding to DNA. The identification and cloning of these auxiliary factors will be the next major step in elucidating the action of steroid hormones on gene expression.

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Differential regulation of anterior pituitary prodynorphin and gonadotropin-subunit gene expression by steroid hormones.

Regulation of expression of the chicken ovalbumin gene: interactions between steroid hormones and second messenger systems.

Transcription regulation by steroid hormones: a computer simulation study.

Multidrug resistance gene expression is controlled by steroid hormones in the secretory epithelium of the uterus.

Control of globin gene expression by steroid hormones in differentiating Friend leukemia cells.

Promotion of liver tumors by steroid hormones.

Regulation by steroid hormones of phosphorylation of specific protein common to several target organs.

Effects of steroid hormones and related compounds on gene transcription.

Regulation of growth of LNCaP human prostate tumor cells by growth factors and steroid hormones.

Regulation of proliferation of rat cartilage and bone by sex steroid hormones.

Regulation of lactogenic hormone binding in rat liver by steroid hormones.

Dynamic patterns of medial preoptic mu-opiate receptor regulation by gonadal steroid hormones.

Regulation of object recognition and object placement by ovarian sex steroid hormones.

Excitatory amino acid regulation of gonadotropin secretion: modulation by steroid hormones.

Regulation of estrogen receptor messenger ribonucleic acid in rat hypothalamus by sex steroid hormones.

Regulation of gene expression by tumor promoters.

Regulation of gene expression by insulin.

Regulation of angiotensinogen gene expression by estrogen.

Co-expression of CXCR4 and CXCR7 in human endometrial stromal cells is modulated by steroid hormones.

Placental steroid hormones.

[Protein binding of steroid hormones].

Regulation of growth hormone gene expression: synergistic effects of thyroid and glucocorticoid hormones.

Replacement of serum in cell culture by hormones: a study of hormonal regulation of cell growth and specific gene expression.

Inhibition of human lymphocyte stimulation by steroid hormones: cytokinetic mechanisms.