Endocrinology, Vol 122, 1532-1540, Copyright © 1988 by Endocrine Society

ARTICLES

Progesterone receptor regulation in T47D human breast cancer cells: analysis by density labeling of progesterone receptor synthesis and degradation and their modulation by progestin

AM Nardulli and BS Katzenellenbogen
Department of Physiology and Biophysics, University of Illinois, Urbana 61801.

We have examined the effect of progestin on the regulation of cellular progesterone receptor (PR) levels and have used dense amino acid- density shift experiments to determine the mechanism by which progestin markedly decreases PR. We have utilized T47D human breast cancer cells which contain high levels of PR and are progestin responsive. When these cells are exposed to the progestin R5020, there is a time- and concentration-dependent decrease in PR levels. Experiments with different concentrations of R5020 reveal that the rate and extent of PR decrease reflect the time course of receptor occupancy and the fractional saturation of receptor. With a high concentration of ligand (20 nM) that labels all receptors rapidly, reductions in PR levels (processing) occur immediately and proceed rapidly to levels that are 15-20% of the initial; at lower concentrations (5 nM), where it takes several hours to achieve full saturation of receptors, there is a delay before the maximal rate of processing develops and then continues to achieve final receptor levels that are 15-20% of the initial; with a low concentration of ligand (0.5 nM), binding is even slower and never reaches full receptor saturation, with the consequence that processing is not only delayed but also less complete. Immunochemical detection of PR with a monoclonal antibody (B39) reveals a good correspondence between the loss of immunoreactive and hormone binding PR, and analysis of the A (Mr 85,000) and B (Mr 115,000) receptor forms on Western blots demonstrates that both A and B receptor forms are reduced after exposure to R5020. Density labeling of PR by biosynthetic incorporation of 2H, 13C, 15N (dense) amino acids reveals that PR turns over with a half-life of 21 h in control cells. In cells exposed to 20 nM R5020, PR levels decline and receptor half-life is reduced to 6 h. In addition, there is also a time-dependent decrease in the rate constant of PR synthesis, k8, which decreases to less than 10% of its initial value after 24 h of R5020 exposure. Thus, the R5020-evoked reduction in PR levels in this progestin-sensitive cell line is due both to a marked increase in the rate of receptor degradation as well as a dramatic decrease in the rate of receptor synthesis.

This article has been cited by other articles:

				A. Samalecos and B. Gellersen Systematic Expression Analysis and Antibody Screening Do Not Support the Existence of Naturally Occurring Progesterone Receptor (PR)-C, PR-M, or Other Truncated PR Isoforms Endocrinology, November 1, 2008; 149(11): 5872 - 5887. [Abstract] [Full Text] [PDF]

				W.-Y. Chen, J.-H. Weng, C.-C. Huang, and B.-c. Chung Histone Deacetylase Inhibitors Reduce Steroidogenesis through SCF-Mediated Ubiquitination and Degradation of Steroidogenic Factor 1 (NR5A1) Mol. Cell. Biol., October 15, 2007; 27(20): 7284 - 7290. [Abstract] [Full Text] [PDF]

				E. T. Alarid Lives and Times of Nuclear Receptors Mol. Endocrinol., September 1, 2006; 20(9): 1972 - 1981. [Abstract] [Full Text] [PDF]

				J. C. Condon, D. B. Hardy, K. Kovaric, and C. R. Mendelson Up-Regulation of the Progesterone Receptor (PR)-C Isoform in Laboring Myometrium by Activation of Nuclear Factor-{kappa}B May Contribute to the Onset of Labor through Inhibition of PR Function Mol. Endocrinol., April 1, 2006; 20(4): 764 - 775. [Abstract] [Full Text] [PDF]

				L. K. Pierson-Mullany and C. A. Lange Phosphorylation of Progesterone Receptor Serine 400 Mediates Ligand-Independent Transcriptional Activity in Response to Activation of Cyclin-Dependent Protein Kinase 2 Mol. Cell. Biol., December 15, 2004; 24(24): 10542 - 10557. [Abstract] [Full Text] [PDF]

				C. A. Lange Making Sense of Cross-Talk between Steroid Hormone Receptors and Intracellular Signaling Pathways: Who Will Have the Last Word? Mol. Endocrinol., February 1, 2004; 18(2): 269 - 278. [Abstract] [Full Text] [PDF]

				I. U. Agoulnik, W. C. Krause, W. E. Bingman III, H. T. Rahman, M. Amrikachi, G. E. Ayala, and N. L. Weigel Repressors of Androgen and Progesterone Receptor Action J. Biol. Chem., August 15, 2003; 278(33): 31136 - 31148. [Abstract] [Full Text] [PDF]

				S. L. Grimm, T. N. Seagroves, E. B. Kabotyanski, R. C. Hovey, B. K. Vonderhaar, J. P. Lydon, K. Miyoshi, L. Hennighausen, C. J. Ormandy, A. V. Lee, et al. Disruption of Steroid and Prolactin Receptor Patterning in the Mammary Gland Correlates with a Block in Lobuloalveolar Development Mol. Endocrinol., December 1, 2002; 16(12): 2675 - 2691. [Abstract] [Full Text] [PDF]

				N. Takamoto, B. Zhao, S. Y. Tsai, and F. J. DeMayo Identification of Indian Hedgehog as a Progesterone-Responsive Gene in the Murine Uterus Mol. Endocrinol., October 1, 2002; 16(10): 2338 - 2348. [Abstract] [Full Text] [PDF]

				B. He, N. T. Bowen, J. T. Minges, and E. M. Wilson Androgen-induced NH2- and COOH-terminal Interaction Inhibits p160 Coactivator Recruitment by Activation Function 2 J. Biol. Chem., November 2, 2001; 276(45): 42293 - 42301. [Abstract] [Full Text] [PDF]

				T. Shen, K. B. Horwitz, and C. A. Lange Transcriptional Hyperactivity of Human Progesterone Receptors Is Coupled to Their Ligand-Dependent Down-Regulation by Mitogen-Activated Protein Kinase-Dependent Phosphorylation of Serine 294 Mol. Cell. Biol., September 15, 2001; 21(18): 6122 - 6131. [Abstract] [Full Text] [PDF]

				J. L. Turgeon and D. W. Waring Progesterone Regulation of the Progesterone Receptor in Rat Gonadotropes Endocrinology, September 1, 2000; 141(9): 3422 - 3429. [Abstract] [Full Text] [PDF]

				C. A. Lange, T. Shen, and K. B. Horwitz Phosphorylation of human progesterone receptors at serine-294 by mitogen-activated protein kinase signals their degradation by the 26S proteasome PNAS, February 1, 2000; 97(3): 1032 - 1037. [Abstract] [Full Text] [PDF]

				J. L. Turgeon, S. M. Van Patten, G. Shyamala, and D. W. Waring Steroid Regulation of Progesterone Receptor Expression in Cultured Rat Gonadotropes Endocrinology, May 1, 1999; 140(5): 2318 - 2325. [Abstract] [Full Text]

				J. D. Graham and C. L. Clarke Physiological Action of Progesterone in Target Tissues Endocr. Rev., August 1, 1997; 18(4): 502 - 519. [Abstract] [Full Text] [PDF]