Lee E. Faber

Nuclear localization of two steroid receptor-associated proteins, hsp90 and p59.

It has been proposed that the unliganded nontransformed form of steroid hormone receptor is a heterooligomer comprising, in addition to the hormone-binding subunit, two associated proteins: a heat shock protein of MW 90,000 (hsp90) and another protein of MW 59,000 (p59). Using monoclonal antibodies, we demonstrate immunocytochemically the presence of both hsp90 and p59 in cell nuclei of progesterone target cells of the rabbit uterus. While steroid receptors (e.g., progesterone receptors) appear to be exclusively nuclear, we find p59 predominantly in the cell nuclei and hsp90 in both the nucleus and the cytoplasm. In addition, Western blotting of high-salt extracts of nuclear proteins detects the presence of hsp90 and p59 in the nuclei of rabbit uterus. These observations are consistent with the presence of the untransformed heterooligomeric form of steroid hormone receptors in the nuclei of target cells.

Rabbit FKBP59-heat shock protein binding immunophillin (HBI) is a calmodulin binding protein.

FKBP59-HBI, a heat shock protein hsp90-binding immunophilin that was originally detected in heterooligomer forms of steroid receptors, is retained on Calmodulin (CAM)-Sepharose 4B in the presence of 2 mM Ca2+ and is eluted by EGTA, demonstrating a specific p59-CAM interaction. The p59 amino acid sequence reveals the presence of two putative CAM binding sites in a helix regions of the protein, as well as PEST sequences which are generally present in CAM-binding proteins. In vitro proteolysis by calpain II (a Ca(2+)-activated neutral protease), another feature of CAM-binding proteins, generates shorter peptides revealed by the mAb EC1, but not by the pAb 173 which recognizes the C-terminal of the protein. The potential function of CAM binding by the hsp90-binding immunophilin is discussed.

Estrogen and rapamycin effects on cell cycle progression in T47D breast cancer cells.

The contribution of estrogen (and progesterone) in driving cell cycle progression of hormone dependent breast cancer cells is well documented, however, the roles of the various relevant signal transduction pathways remain unclear. The immunosuppressant rapamycin is a potent inhibitor of cell cycle progression and has been used to define signal transduction pathways. In this study we have determined rapamycins effects on cell cycle progression in estrogen dependent breast cancer cells using a novel method of inducing S-phase. In this method estradiol-17-β alone induced S-phase without mitogen support. In our studies the T47D cells were quite sensitive to estradiol-17-β, with half-maximal induction in the picomolar range, indicating that the estrogen can induce S-phase in the absence of mitogens such as insulin. The estrogen response does not seem to be particularly specific because estriol estrone and estradiol-17-β-BSA were about as effective as estradiol-17-β. R5020, a progestin also induced S-Phase, while rapamycin blocked steroid driven transition of cells from G1 to S-phase.

Epidermal growth factor suppresses induction by progestin of the adhesion protein desmoplakin in T47D breast cancer cells

IntroductionAlthough the effects of progesterone on cell cycle progression are well known, its role in spreading and adhesion of breast cancer cells has not attracted much attention until recently. Indeed, by controlling cell adhesion proteins, progesterone may play a direct role in breast cancer invasion and metastasis. Progesterone has also been shown to modulate epidermal growth factor (EGF) effects in neoplasia, although EGF effects on progesterone pathways and targets are less well understood. In the present study we identify an effect of EGF on a progesterone target, namely desmoplakin.MethodsInitially flow cytometry was used to establish the growing conditions and demonstrate that the T47D breast cancer cell line was responding to progesterone and EGF in a classical manner. Differential display RT-PCR was employed to identify differentially expressed genes affected by progesterone and EGF. Western and Northern blotting were used to verify interactions between EGF and progesterone in three breast cancer cell lines: T47D, MCF-7, and ZR-75.ResultsWe found the cell adhesion protein desmoplakin to be upregulated by progesterone – a process that was suppressed by EGF. This appears to be a general but not universal effect in breast cancer cell lines.ConclusionOur findings suggest that progesterone and EGF may play opposing roles in metastasis. They also suggest that desmoplakin may be a useful biomarker for mechanistic studies designed to analyze the crosstalk between EGF and progesterone dependent events. Our work may help to bridge the fields of metastasis and differentiation, and the mechanisms of steroid action.

Tamoxifen decreases progesterone and nuclear androgen receptors in the human prostate

Androgen (AR) and progesterone (PR) receptors were measured in resected prostate tissues of patients with benign prostatic hypertrophy. One group of patients received an anti-estrogen, tamoxifen (Tm 20 mg b.i.d.) for 10 days prior to prostate resection; a second group served as controls and were untreated. Plasma levels of Tm were 200-500 pmol/ml at the time of surgery. Statistically significant decreases (P less than 0.05) were found in cytosol PR (154 fmol/mg DNA +/- 33 SE in 14 Tm-patients vs 266 +/- 40 SE in 13 untreated patients) and in nuclear AR (103 fmol/mg DNA +/- 70 SE in 18 Tm-patients vs 257 +/- 62 SE in 17 controls). Cytosol AR was not significantly different in Tm-treated patients (257 fmol/mg DNA +/- 79 SE in 15 Tm-patients vs 346 +/- 130 SE in 17 controls, P greater than 0.6). Although receptor recycling is one of several possible explanations, these decreases in progesterone and nuclear androgen receptors in Tm-treated patients suggest that estrogen has a role in the biological regulation of steroid receptors in the human prostate.

Effect of buffers and electrolytes on the stability of uterine progesterone receptors

Abstract We have investigated the effects of buffers and electrolytes on the stability of the 7S progesterone-receptor complex of guinea pig and rabbit uterine cytosol. Receptor half-life was greater in 5 m m phosphate than in the cationic or zwitterionic buffers tested. The best medium contained 5 m m phosphate, 10% glycerol, and 10 m m monothioglycerol, and was degassed under vacuum. Exposure to 100 m m sodium phosphate, like exposure to 300 m m KCl, resulted in the disappearance of the 7S progesterone binding component and a concurrent increase in binding to a 4 to 5S component. However, Scatchard analyses suggest that 300 m m KCl led to a reduction in the apparent association constant, whereas the “Ka” observed in 5m m phosphate was maintained in 100 m m phosphate.

The 56 kDa protein of human genital skin fibroblasts is identical to that radiolabelled by [3H]dihydrotestosterone 17β-bromoacetate

Analysis of soluble proteins from human genital skin fibroblasts by two-dimensional polyacrylamide gel electrophoresis reveals an abundant protein doublet of mol. wt 56,000 with isoelectric points (pI) of 6.7 and 6.5. This protein is absent in non-genital skin fibroblasts as well as in genital skin fibroblasts of most patients with complete forms of androgen insensitivity. The protein specifically binds androgen. A protein of similar estimated molecular weight (58,000) from human genital skin fibroblasts has recently been found to be covalently radiolabelled by the affinity ligand dihydrotestosterone 17 beta-bromoacetate (DHT-BA). In the present study these proteins have been found to be indistinguishable on one- and two-dimensional gel electrophoresis. Antibodies raised against the 56 kDa pI 6.7/6.5 protein also recognized the protein covalently radiolabelled by DHT-BA. A third protein of estimated mol. wt 59,000 has been found to be associated with several steroid hormone receptor complexes but has no known ligand binding activity. This protein was found to be clearly separable from the 56/58 kDa protein on two-dimensional gel electrophoresis as it has a more acidic pI of approximately 5.4. Furthermore, antibodies against the 59 kDa protein do not recognize the 56 kDa species, and vice versa.

Effects of Progestins on the Progesterone Receptor in Guinea Pig Uterus

We examined the effects of progesterone and some synthetic progestins and other steroids on the physical properties of the progesterone receptor of guinea pig uterine cytosol and on the binding of the receptor by nuclei. Progestational potency seemed to correlate with the ability to keep the receptor in the 7S form and to prevent dissociation into smaller subunits. The rate of activation prior to nuclear binding was slower with steroids of increasing progestational activity. Therefore activation in vitro may be unrelated to biological activity. Concentration of the cytosol led to a decrease in the equilibrium association constant. The extent of the decrease was less with progesterone than with its metabolite, 5 alpha-pregnanedione. When cytosol and nuclei were incubated in the absence of ligand measureable progesterone receptor was bound by the nuclei. The uncomplexed nuclear receptor bound [3H]-progesterone of [3H]-R5020 rapidly at 0 degrees, but progesterone-receptor complexes exchanged [3H]-progestin slowly at 0 degrees. Progesterone increased the amount of nuclear receptor at concentrations of 10(-9) and 10(-8)M, but decreased binding at higher concentrations. 5 alpha-Pregnanedione had the same effect as progesterone, but other metabolites of progesterone that had little affinity for the 7S progesterone receptor in cytosol had no effect on nuclear binding at any concentration. Glucocorticoids, testosterone and estradiol-17 beta increased the nuclear binding of the progesterone receptor when present at concentration of 10(-8)M and greater.

Mammalian Progesterone Receptors: Biosynthesis, Structure and Nuclear Binding

Because estrogen and progesterone receptors may be involved in the endocrine sensitivity of certain breast and endometrial carcinomas (1,2), considerable interest has been generated in the study of steroid receptors in hormonally sensitive tumors. In this chapter, we will review some recent progress on three aspects of receptor biology as applied to mammalian progesterone receptors: regulation of receptor biosynthesis, structure of the receptor and nuclear binding of the steroid-receptor complex.

Use of FK506 and Its Analogs in Studying Progesterone Signaling

Acting through their receptors, steroids play important roles in the regulation of growth, differentiation, reproduction, and metabolism. In the absence of hormone, receptors are complexed with several nonsteroid binding proteins. Included among these are hsp90, p59, and hsp70 (Sanchez et al, 1985; Tai et al, 1986; Kost et al, 1989). Following steroid binding, the receptors are found dissociated from the heteromeric complexes and phosphorylated, a step referred to as transformation (for a review, see Pratt, 1987). Subsequently, they bind specific DNA segments, to regulate gene expression at the transcriptional level. Much recent work on steroid receptors has focused on receptor-DNA interaction, a late step in the steroid signal transduction pathways. On the other hand, mechanisms that regulate the early events, such as phosphorylation and dissociation, remain obscure (Orti et al, 1992).