Candace A. Beck

Progesterone receptor and the mechanism of action of progesterone antagonists.

Currently available progesterone antagonists have been suggested to fall into two categories based on differences in how they interact with and inactivate the progesterone receptor (PR). The anti-progestin ZK98299 (Type I) impairs PR association with DNA, while Type II compounds (RU486, ZK112993, ZK98734) promote PR binding to DNA. Type II agents, therefore, appear to inhibit receptor activity at a step downstream of DNA binding, presumably failing to induce conformational changes in PR structure requird for enhancement of transcription. This paper discusses both published and unpublished data supporting the concept of two types of progestin antagonists. Using PR-mediated induction of reporter genes in breast cancer cells as an assay for biological response, both types of anti-progestins, after correction for difference in steroid binding affinity, inhibit progestin induction substoichiometrically. However, Type II anti-progestins are more potent, inhibiting at lower ratios of antagonist to agonist than ZK98299. This suggests that in addition to behaving by classical competitive mechanisms these compounds (in particular Type II) may exhibit additional activity as transrepressors of PR in the same cell bound to hormone agonist. Transrepression may occur by the combined mechanisms of heterodimerization and competition for binding to DNA. In support of this, mixed ligand dimers form readily in solution between a PR subunit bound to agonist and another bound to either type of anti-progestin, whereas these mixed ligand dimers bind poorly, if at all, to specific progesterone response elements (PREs) in vitro. Additionally, when added as a single ligand, Type II agents increase PR dimerization in solution and PR affinity for PREs as compared with single ligand dimers formed by progestin agonist. This contrasts with ZK98299, when given as a single ligand, which reduces PR affinity for PREs without disrupting solution dimerization. Thus the higher affinity of PR for PREs may account for the greater biological potency of Type II compounds as compared with ZK98299. As a further distinction between types of antiprogestins, ZK98299 minimally stimulates phosphorylation of PR whereas RU486 increases site-specific phosphorylation of PR in a manner indistinguishable from that of hormone agonist. Additionally, ZK98299 is not susceptible in vivo to functional switching to a partial agonist by cross talk with cAMP signal transduction pathways, as occurs with Type II compounds. Thus, ZK98299 under certain conditions may be a more pure antagonist than Type II compounds.

Phosphorylation and Progesterone Receptor Function

Four phosphorylation sites have been identified in the chicken progesterone receptor. Two of these sites exhibit basal phosphorylation which is enhanced upon treatment with hormone and two of the sites are phosphorylated in response to hormone. Mutation of one of these hormone dependent sites, Ser530 to Ala530, causes a decrease in transcriptional activation at low concentrations of hormone, but the activity is unaffected at high concentrations. However, the hormone binding of the mutant is unaffected suggesting that phosphorylation of Ser530 plays a role in facilitating the response of the receptor to low concentrations of hormone. The chicken progesterone receptor can be activated by modulators of kinases in the absence of hormone. The finding that signals initiated by tyrosine phosphorylation (through treatment with EGF) or through the dopamine receptor suggests that there are multiple means of activating chicken progesterone receptor. In contrast, the human progesterone receptor does not exhibit ligand independent activation; however, its activity in the presence of the agonist R5020 is enhanced by treatment with 8-Br-cAMP, an activator of protein kinase A, and treatment with 8-Br-cAMP causes the antagonist, RU486, to act as an agonist.

The two faces of a steroid antagonist: When an antagonist isn't

Activation of protein kinase A potentiates the transcriptional response mediated by the glucocorticoid receptor in responsive fibroblasts and in mammary carcinoma cells. This potentiation is ligand-dependent and occurs in responsive fibroblasts and in mammary carcinoma cells. This potentiation is ligand-dependent and occurs without detectable change in the phosphorylation of receptor. The transcriptional response to glucocorticoid or progestin agonists can be blocked by potent antagonists like RU 486. However, upon activation of protein kinase A, the antagonist action of RU 486 on both receptors is blunted. Indeed, RU 486 can itself activate transcription of a hormone-responsive promoter. The conditional agonist activity is observed with type II antagonists, those which recapitulate many of the early steps of ligand-dependent receptor activation, but not type I antagonists, which do not. These studies have now been extended to antimineralocorticoids. In COS-1 cells transfected with a mineralocorticoid receptor expression vector, treatment with 8-BromocAMP potentiates the response to the agonist aldosterone and elicits additional agonist activity in mineralocorticoid antagonists. A model is proposed wherein type II antagonist-receptor complexes occupy receptor binding sites on the genome. The antagonist, however, fails to promote a receptor conformation that can interact productively with a coactivator mediating the communication between receptor and the basal transcription apparatus. Activation of protein kinase A results in the recruitment or activation of a coactivator that permits recovery of receptor-mediated activation function.(ABSTRACT TRUNCATED AT 250 WORDS)

Two Types of Anti-progestins Have Distinct Effects on Site-specific Phosphorylation of Human Progesterone Receptor

Human progesterone receptor (PR) is phosphorylated on multiple serine residues; three sites (Ser102, Ser294, and Ser345) are inducible by hormone agonist, while at least six others are basally phosphorylated and exhibit a general increase in response to hormone. In this study we have used high performance liquid chromatography phosphopeptide mapping and manual peptide sequencing to investigate how two different progestin antagonists, RU486 and ZK98299, affect site-specific phosphorylation of PR isolated from T47D breast cancer cells. As compared to the progestin agonist R5020, RU486 stimulated a similar increase in overall incorporation of [32P]phosphate per PR molecule (2.5-2.6-fold for PR-A and 2.1-fold for PR-B), and at the site-specific level, RU486 stimulated both the basal and inducible sites to the same extent as R5020. In contrast, ZK98299 produced only a minimal increase in overall phosphorylation (1.2-fold for PR-A and 1.1-fold for PR-B) which was due to a reduced stimulation of the basal sites and failure to induce any of the three hormone-dependent sites. No inappropriate phosphorylation sites were detected in response to either RU486 or ZK98299. In cotreatment studies, ZK98299 blocked the increase in overall phosphorylation of PR induced by R5020, demonstrating that the failure of this antagonist to stimulate specific phosphorylation sites is not due to an inefficient interaction with PR in the intact cell. These results indicate that the biological effects of RU486 are not mediated by an alteration in the phosphorylation state of PR, whereas failure to promote phosphorylation of certain sites may contribute to the antagonist action of ZK98299. Additionally these results support the concept of two mechanistic classes of anti-progestins that affect PR differently in vivo.

Stoichiometry and Site-specific Phosphorylation of Human Progesterone Receptor in Native Target Cells and in the Baculovirus Expression System

Human progesterone receptor (PR) in T47D breast cancer cells is phosphorylated on nine different serine residues; three are hormone-inducible (Ser102, Ser294, and Ser345), while others are basal but hormone-stimulated. In the present study, we have compared the phosphorylation state of native and recombinant PR expressed in a baculovirus insect cell system. Stoichiometric measurements showed that unliganded native PR in T47D cells was approximately 50% phosphorylated (≈4 phosphates/PR) and became essentially 100% phosphorylated (≈9 phosphates/PR) when bound to hormone. Unliganded PR expressed in Sf9 insect cells was phosphorylated with a similar stoichiometry (≈3 phosphates/PR), but the phosphate content did not change with hormone addition. Site-specific phosphorylation analyzed by tryptic phosphopeptide mapping and manual peptide sequencing revealed that expressed PR bound to hormone in the Sf9 insect cells was phosphorylated on all the same sites as hormone-treated PR in T47D cells. Only minor differences were detected in the relative proportion of three sites (two basal sites and Ser345) and phosphorylation did not occur on alternate sites. Interestingly, unliganded baculovirus-expressed PR was constitutively phosphorylated on hormone inducible sites and was phosphorylated on basal sites to the same extent as hormone treated PR. Thus, in the absence of hormone, the phosphorylation state of baculovirus-expressed PR resembled that of the hyperphosphorylated native PR. In contrast to native PR, the expressed receptor in cytosols of Sf9 cells did not form a large oligomeric complex suggesting that hyperphosphorylation may be due to dissociation of the complex in the absence of hormone. This study demonstrating phosphorylation on correct sites with a stoichiometry similar to that of native PR indicates that overexpressed PR in the baculovirus system is suitable for in vitro structure/function studies.

Modulators of cellular protein phosphorylation alter the trans-activation function of human progesterone receptor and the biological activity of progesterone antagonists.

SummaryAddition of progesterone to breast cancer cellsin vivo increases phosphorylation of human progesterone receptor (PR), suggesting that phosphorylation has a regulatory role in producing the activated form of receptor. Kinetic analysis indicates that hormone-dependent phosphorylation is sequential and that early stages of phosphorylation(s) are closely associated with enhancement of PR-DNA binding while later stages are associated with atrans-activation function. Various agents that stimulate cellular protein phosphorylation (8-Br cAMP, okadaic acid, TPA) functionally synergize with progesterone to enhance progesterone-dependent PRtrans-activation in intact cells. These results suggest that protein phosphorylation does have a role in modulating thetrans-activation function of PRin vivo. They also demonstrate cross-talk between second messenger signal transduction pathways and nuclear steroid receptors. Whether the phosphorylated target that provides the link between these two signal transduction pathways is PR itself or another protein involved in PR-mediated gene transcription is not known. Positive cooperative interactions were also observed between cAMP signaling pathways and the progesterone antagonist RU486, that resulted in RU486 exerting substantial agonist activities. This ability of cross-talk between second messenger and steroid receptor signal transduction pathways to override the antagonistic effects of RU486 suggests a novel mechanism to explain the problem of resistance to clinically important steroid antagonists.

Mechanisms Controlling Steroid Receptor Binding to Specific DNA Sequences

Mammalian progesterone receptors activated by hormone binding in nuclei of intact cells exhibit substantially higher binding activity for specific DNA sequences than receptors bound with hormone and activated in cell-free cytosol. Differences in DNA-binding activity occur despite the fact that both activated receptor forms sediment at 4S on sucrose gradients and are apparently dissociated from the heat shock protein 90. This suggests that hormone-induced release of heat shock protein 90 from receptors is necessary, but not sufficient for maximal activation of DNA binding. This report is a review of studies from our laboratories that have examined the role of receptor interaction with other nuclear protein factor(s), and receptor dimerization in solution, as additional regulatory steps involved in the process of receptor activation and binding to specific gene sequences.

Progesterone receptors in breast cancer.

Steroid hormone receptors are members of a gene family of liganddependent transcriptional activators that also includes receptors for vitamin D, thyroid hormone, retinoic acid, and a number of transcriptional activators with as yet unknown ligands. Cloning of receptor genes and mutagenesis studies have revealed that steroid receptors are modular proteins organized into the functional domains shown in Figure 1. A highly conserved DNA binding domain (region C) located in the central portion of the molecule contains two zinc finger structures. The carboxyl terminal region (E) harbors domains for steroid binding and receptor dimerization. The amino terminus (A/B) is highly variable, both with respect to amino acid sequence and length, and is important for maximal transcriptional activity [1, 2, 3, 4]. The progesterone receptor (PR) in breast cancer cells and in several normal reproductive tissues is produced as two steroid-binding proteins of different lengths, termed PR-A and PR-B [5, 6]. The two proteins arise from a single gene either by initiation of translation at two different start sites within the same RNA transcript or from separate mRNAs produced by the use of alternate promoters [7, 8, 9]. PR-A is a truncated version of PR-B, missing 165 amino acids present in the N terminus of PR-B (Figure 1). The two PR forms, therefore, are identical in sequence in their hormone and DNA binding domains. In human breast cancer cells, PR-A and PR-B have apparent molecular weights of ≈94,000 and ≈120,000 respectively [6]. This property of the progesterone receptor is unique among the sex steroid and glucocorticoid receptors. Receptors for estrogen (ER), androgens (AR), and glucocorticoids (GR) are all synthesized as single-sized proteins. The functional role of PR-A and PR-B are not known. They have been reported to exhibit some differences in target gene specificities, suggesting that expression of two PR forms from the same gene may provide a mechanism to expand functional diversity [10].