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Kathryn B. Horwitz - One of the best experts on this subject based on the ideXlab platform.

  • 0888-8809/06/$15.00/0 Molecular Endocrinology 20(11):2656–2670 Printed in U.S.A. Copyright © 2006 by The Endocrine Society doi: 10.1210/me.2006-0105 Progesterone Receptors (PR)-B and-A Regulate Transcription by Different Mechanisms: AF-3 Exerts Regul
    2013
    Co-Authors: Lin Tung, Jennifer K Richer, Carol A Sartorius, Glenn S Takimoto, Tianjie Shen, Hany Abdel-hafiz, Djuana M. E. Harvell, Lisa K. Nitao, Kathryn B. Horwitz
    Abstract:

    The two, nearly identical, isoforms of human Progesterone Receptors (PR), PR-B and-A, share activation functions (AF) 1 and 2, yet they possess markedly different transcriptional profiles, with PR-B being much stronger transactivators. Their differences map to a unique AF3 in the B-upstream segment (BUS), at the far N terminus of PR-B, which is missing in PR-A. Combined mutation of two LXXLL motifs plus tryptophan 140 in BUS, to yield PR-BdL140, completely destroys PR-B activity, because strong AF3 synergism with downstream AF1 and AF2 is eliminated. This synergism involves cooperative interactions among receptor multimers bound at tandem hormone response elements and is transferable to AFs of other nuclear Receptors. Other PR-B functions—N-/C-terminal First Published Online June 8, 200

  • Progesterone Receptors their isoforms and Progesterone regulated transcription
    Molecular and Cellular Endocrinology, 2012
    Co-Authors: Britta M Jacobsen, Kathryn B. Horwitz
    Abstract:

    Abstract This review discusses mechanisms by which Progesterone Receptors (PR) regulate transcription. We examine available data in different species and tissues regarding: (1) regulation of PR levels; and (2) expression profiling of progestin-regulated genes by total PRs, or their PRA and PRB isoforms. (3) We address current views about the composition of Progesterone response elements, and postulate that PR monomers acting through “half-site” elements are common, entailing cooperativity with neighboring DNA-bound transcription factors. (4) We summarize transcription data for multiple progestin-regulated promoters as directed by total PR, or PRA vs. PRB. We conclude that current models and methods used to study PR function are problematical, and recommend that future work employ cells and Receptors appropriate to the species, focusing on analyses of the effects of endogenous Receptors targeting endogenous genes in native chromatin.

  • Progesterone independent effects of human Progesterone Receptors prs in estrogen receptor positive breast cancer pr isoform specific gene regulation and tumor biology
    Molecular Endocrinology, 2005
    Co-Authors: Britta M Jacobsen, Jennifer K Richer, Stephanie A Schittone, Kathryn B. Horwitz
    Abstract:

    Progesterone Receptors (PRs) are prognostic markers in breast cancers irrespective of the patient's progestational status. However, there are two PR isoforms, PR-A and PR-B, that are equimolar in the normal breast but dysregulated in advanced disease. Postmenopausal, tamoxifen-treated patients with estrogen receptor (ER)-positive, PR-A-rich tumors have much faster disease recurrence than patients with PR-B-rich tumors. To study the mechanisms we engineered ER+ breast cancer cells that express each PR isoform under control of an inducible promoter. We identified 79 genes regulated by Progesterone (P), mainly by PR-B, and 51 genes regulated without Progesterone, mainly by PR-A. Only nine genes were regulated with and without ligand, leading to definition of three classes: I) genes regulated only by liganded PR; II) genes regulated only by unliganded PR; III) genes regulated by both. Unliganded PR-A and PR-B differentially regulate genes that coordinate extracellular signaling pathways and influence tumor cell biology. Indeed, in the absence of P, compared with ER+/PR-B+ or PR- cells, ER+, PR-A+ cells exhibit an aggressive phenotype, are more adhesive to an extracellular matrix, and are more migratory. Additionally, unliganded PR-A and PR-B both inhibit cell growth and provoke resistance to Taxol-induced apoptosis. We propose that PR-A:PR-B ratios, even in the absence of P, influence the biology and treatment response of ER+ tumors, that PR-A isoforms are functionally dominant in P-deficient states, and that PR-A rich tumors are especially aggressive.

  • functional properties of the n terminal region of Progesterone Receptors and their mechanistic relationship to structure
    The Journal of Steroid Biochemistry and Molecular Biology, 2003
    Co-Authors: Glenn S Takimoto, Jennifer K Richer, Britta M Jacobsen, Carol A Sartorius, Lin Tung, Hany A Abdelhafiz, Michael G Abel, David L Bain, Kathryn B. Horwitz
    Abstract:

    Progesterone Receptors (PR) are present in two isoforms, PR-A and PR-B. The B-upstream segment (BUS) of PR-B is a 164 amino acid N-terminal extension that is missing in PR-A and is responsible for the functional differences reported between the two isoforms. BUS contains an activation function (AF3) which is defined by a core domain between residues 54-154 whose activity is dependent upon a single Trp residue and two LXXLL motifs. We have also identified sites both within and outside of BUS that repress the strong synergism between AF3 and AF1 in the N-terminal region and AF2 in the hormone binding domain. One of these repressor sites is a consensus binding motif for the small ubiquitin-like modifier protein, SUMO-1 (387IKEE). The DNA binding domain (DBD) structure is also important for function. When BUS is linked to the glucocorticoid receptor DBD, AF3 activity is substantially attenuated, suggesting that binding to a DNA response element results in allosteric communication between the DBD and N-terminal functional regions. Lastly, biochemical and biophysical analyses of highly purified PR-B and PR-A N-terminal regions reveal that they are unstructured unless the DBD is present. Thus, the DBD stabilizes N-terminal structure. We propose a model in which the DBD through DNA binding, and BUS through protein-protein interactions, stabilize active receptor conformers within an ensemble distribution of active and inactive conformational states. This would explain why PR-B are stronger transactivators than PR-A.

  • mitogen activated protein kinase regulates nuclear association of human Progesterone Receptors
    Molecular Endocrinology, 2003
    Co-Authors: Ming Qiu, Kathryn B. Horwitz, Abby L Olsen, Emily J Faivre, Carol A. Lange
    Abstract:

    Breast cancers often have increased MAPK activity; this pathway may drive breast cancer cell growth by targeting steroid hormone Receptors. MAPK phosphorylates human Progesterone Receptors (PRs) on Ser294, thus regulating several aspects of PR activity. To study the role of PR Ser294 phosphorylation on subcellular distribution, we stably expressed wild-type (wt) or S294A (Ser294 to Ala) PR-B in several cell types. PRs phosphorylated on Ser294 were nuclear. Activation of MAPK induced Ser294 phosphorylation and rapid nuclear translocation of wt, but not S294A, PR-B; both Receptors concentrated in the nucleus after progestin treatment. The MAPK kinase inhibitor, U0126, blocked epidermal growth factor but not progestin-induced Ser294 phosphorylation and translocation of wt PR, indicating a novel mechanism for nuclear localization. After progestin treatment, wt PR-B underwent ligand-dependent down-regulation, while S294A PR-B persisted in nuclei. Prolonged treatment with U0126 or the nuclear export inhibitor, leptomycin B, promoted nuclear accumulation of wt PR-B and blocked ligand-dependent PR down-regulation, suggesting that PR degradation occurs in the cytoplasm and requires MAPK-dependent nuclear export. Stabilization of PRs by leptomycin B also blocked PR transcriptional activity, indicating a link between nucleocytoplasmic shuttling, receptor stability, and function. These results support a regulatory role for MAPK in nuclear steroid hormone receptor subcellular localization and coupling to multiple PR functions.

Carol A. Lange - One of the best experts on this subject based on the ideXlab platform.

  • Progesterone Receptors induce foxo1 dependent senescence in ovarian cancer cells
    Cell Cycle, 2013
    Co-Authors: Caroline H Diep, Nathan J Charles, Blake C Gilks, Steve E Kalloger, Peter A Argenta, Carol A. Lange
    Abstract:

    Loss of nuclear Progesterone Receptors (PR) and low circulating Progesterone levels are associated with increased ovarian cancer (OC) risk. However, PR are abundantly expressed in a significant percentage of serous and endometrioid ovarian tumors; patients with PR+ tumors typically experience longer progression-free survival relative to those with PR-null tumors. The molecular mechanisms of these protective effects are poorly understood. To study PR action in OC in the absence of added estrogen (i.e., needed to induce robust PR expression), we created ES-2 OC cells stably expressing vector control or GFP-tagged PR-B (GFP-PR). Progestin (R5020) stimulation of ES-2 cells stably expressing GFP-PR induced cellular senescence characterized by altered cellular morphology, prolonged survival, senescence-associated β-galactosidase activity, G1 cell cycle arrest and upregulation of the cell cycle inhibitor, p21, as well as the Forkhead-box transcription factor, FOXO1; these results repeated in unmodified ER+/PR+ P...

  • phosphorylated and sumoylation deficient Progesterone Receptors drive proliferative gene signatures during breast cancer progression
    Breast Cancer Research, 2012
    Co-Authors: Todd P Knutson, Andrea R Daniel, Kevin A T Silverstein, Kyle R Covington, Suzanne A W Fuqua, Carol A. Lange
    Abstract:

    Introduction Progesterone Receptors (PR) are emerging as important breast cancer drivers. Phosphorylation events common to breast cancer cells impact PR transcriptional activity, in part by direct phosphorylation. PR-B but not PR-A isoforms are phosphorylated on Ser294 by mitogen activated protein kinase (MAPK) and cyclin dependent kinase 2 (CDK2). Phospho-Ser294 PRs are resistant to ligand-dependent Lys388 SUMOylation (that is, a repressive modification). Antagonism of PR small ubiquitin-like modifier (SUMO)ylation by mitogenic protein kinases suggests a mechanism for derepression (that is, transcriptional activation) of target genes. As a broad range of PR protein expression is observed clinically, a PR gene signature would provide a valuable marker of PR contribution to early breast cancer progression.

  • phosphorylated and sumoylation deficient Progesterone Receptors drive proliferative gene signatures during breast cancer progression
    Breast Cancer Research, 2012
    Co-Authors: Todd P Knutson, Andrea R Daniel, Kevin A T Silverstein, Kyle R Covington, Suzanne A W Fuqua, Danhua Fan, Carol A. Lange
    Abstract:

    Progesterone Receptors (PR) are emerging as important breast cancer drivers. Phosphorylation events common to breast cancer cells impact PR transcriptional activity, in part by direct phosphorylation. PR-B but not PR-A isoforms are phosphorylated on Ser294 by mitogen activated protein kinase (MAPK) and cyclin dependent kinase 2 (CDK2). Phospho-Ser294 PRs are resistant to ligand-dependent Lys388 SUMOylation (that is, a repressive modification). Antagonism of PR small ubiquitin-like modifier (SUMO)ylation by mitogenic protein kinases suggests a mechanism for derepression (that is, transcriptional activation) of target genes. As a broad range of PR protein expression is observed clinically, a PR gene signature would provide a valuable marker of PR contribution to early breast cancer progression. Global gene expression patterns were measured in T47D and MCF-7 breast cancer cells expressing either wild-type (SUMOylation-capable) or K388R (SUMOylation-deficient) PRs and subjected to pathway analysis. Gene sets were validated by RT-qPCR. Recruitment of coregulators and histone methylation levels were determined by chromatin immunoprecipitation. Changes in cell proliferation and survival were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and western blotting. Finally, human breast tumor cohort datasets were probed to identify PR-associated gene signatures; metagene analysis was employed to define survival rates in patients whose tumors express a PR gene signature. 'SUMO-sensitive' PR target genes primarily include genes required for proliferative and pro-survival signaling. DeSUMOylated K388R Receptors are preferentially recruited to enhancer regions of derepressed genes (that is, MSX2, RGS2, MAP1A, and PDK4) with the steroid receptor coactivator, CREB-(cAMP-response element-binding protein)-binding protein (CBP), and mixed lineage leukemia 2 (MLL2), a histone methyltransferase mediator of nucleosome remodeling. PR SUMOylation blocks these events, suggesting that SUMO modification of PR prevents interactions with mediators of early chromatin remodeling at 'closed' enhancer regions. SUMO-deficient (phospho-Ser294) PR gene signatures are significantly associated with human epidermal growth factor 2 (ERBB2)-positive luminal breast tumors and predictive of early metastasis and shortened survival. Treatment with antiprogestin or MEK inhibitor abrogated expression of SUMO-sensitive PR target-genes and inhibited proliferation in BT-474 (estrogen receptor (ER)+/PR+/ERBB2+) breast cancer cells. We conclude that reversible PR SUMOylation/deSUMOylation profoundly alters target gene selection in breast cancer cells. Phosphorylation-induced PR deSUMOylation favors a permissive chromatin environment via recruitment of CBP and MLL2. Patients whose ER+/PR+ tumors are driven by hyperactive (that is, derepressed) phospho-PRs may benefit from endocrine (antiestrogen) therapies that contain an antiprogestin.

  • Progesterone Receptors induce cell cycle progression via activation of mitogen activated protein kinases
    Molecular Endocrinology, 2005
    Co-Authors: Andrew J Skildum, Emily J Faivre, Carol A. Lange
    Abstract:

    Progestins induce proliferation of breast cancer cells and are implicated in the development of breast cancer. The effects of progestins are mediated by Progesterone Receptors (PRs), although it is unclear whether proliferative effects are delivered through activities as ligand-activated transcription factors or via activation of cytoplasmic kinases. We report that progestin induces S phase entry of T47D cells stably expressing either wild-type (wt) PR-B or a transcriptionally impaired PR-B harboring a point mutation at Ser294, a ligand-dependent and MAPK consensus phosphorylation site (S294A). Both wt and S294A PR are capable of activating p42/p44 MAPKs and promoting proliferation. However, cells expressing wt, but not S294A PR, exhibited enhanced proliferation in response to combined epidermal growth factor and progestin. S phase progression correlated with up-regulation of cyclin D1. The PR antagonist RU486 also induced MAPK activation, increased cyclin D1 expression, and stimulated S phase entry, which was blocked by inhibition of either p42/p44 or p38 MAPKs, whereas proliferation induced by R5020 was sensitive only to p42/p44 MAPK inhibition. MCF-7 cells stably expressing a mutant PR unable to bind c-Src and activate MAPK failed to support progestin-induced proliferation. These data suggest that PR mediate cell cycle progression primarily through activation of cytoplasmic kinases and independently of direct regulation of transcription, whereas the coordinate regulation of both aspects of PR action are required for enhanced proliferation in response to progestins in the presence of growth factors. Targeting the ability of steroid Receptors to activate MAPKs may be beneficial for breast cancer patients.

  • mitogen activated protein kinase regulates nuclear association of human Progesterone Receptors
    Molecular Endocrinology, 2003
    Co-Authors: Ming Qiu, Kathryn B. Horwitz, Abby L Olsen, Emily J Faivre, Carol A. Lange
    Abstract:

    Breast cancers often have increased MAPK activity; this pathway may drive breast cancer cell growth by targeting steroid hormone Receptors. MAPK phosphorylates human Progesterone Receptors (PRs) on Ser294, thus regulating several aspects of PR activity. To study the role of PR Ser294 phosphorylation on subcellular distribution, we stably expressed wild-type (wt) or S294A (Ser294 to Ala) PR-B in several cell types. PRs phosphorylated on Ser294 were nuclear. Activation of MAPK induced Ser294 phosphorylation and rapid nuclear translocation of wt, but not S294A, PR-B; both Receptors concentrated in the nucleus after progestin treatment. The MAPK kinase inhibitor, U0126, blocked epidermal growth factor but not progestin-induced Ser294 phosphorylation and translocation of wt PR, indicating a novel mechanism for nuclear localization. After progestin treatment, wt PR-B underwent ligand-dependent down-regulation, while S294A PR-B persisted in nuclei. Prolonged treatment with U0126 or the nuclear export inhibitor, leptomycin B, promoted nuclear accumulation of wt PR-B and blocked ligand-dependent PR down-regulation, suggesting that PR degradation occurs in the cytoplasm and requires MAPK-dependent nuclear export. Stabilization of PRs by leptomycin B also blocked PR transcriptional activity, indicating a link between nucleocytoplasmic shuttling, receptor stability, and function. These results support a regulatory role for MAPK in nuclear steroid hormone receptor subcellular localization and coupling to multiple PR functions.

Yogeshwar Dayal - One of the best experts on this subject based on the ideXlab platform.

Selim M Nasser - One of the best experts on this subject based on the ideXlab platform.

Jennifer K Richer - One of the best experts on this subject based on the ideXlab platform.

  • 0888-8809/06/$15.00/0 Molecular Endocrinology 20(11):2656–2670 Printed in U.S.A. Copyright © 2006 by The Endocrine Society doi: 10.1210/me.2006-0105 Progesterone Receptors (PR)-B and-A Regulate Transcription by Different Mechanisms: AF-3 Exerts Regul
    2013
    Co-Authors: Lin Tung, Jennifer K Richer, Carol A Sartorius, Glenn S Takimoto, Tianjie Shen, Hany Abdel-hafiz, Djuana M. E. Harvell, Lisa K. Nitao, Kathryn B. Horwitz
    Abstract:

    The two, nearly identical, isoforms of human Progesterone Receptors (PR), PR-B and-A, share activation functions (AF) 1 and 2, yet they possess markedly different transcriptional profiles, with PR-B being much stronger transactivators. Their differences map to a unique AF3 in the B-upstream segment (BUS), at the far N terminus of PR-B, which is missing in PR-A. Combined mutation of two LXXLL motifs plus tryptophan 140 in BUS, to yield PR-BdL140, completely destroys PR-B activity, because strong AF3 synergism with downstream AF1 and AF2 is eliminated. This synergism involves cooperative interactions among receptor multimers bound at tandem hormone response elements and is transferable to AFs of other nuclear Receptors. Other PR-B functions—N-/C-terminal First Published Online June 8, 200

  • Progesterone independent effects of human Progesterone Receptors prs in estrogen receptor positive breast cancer pr isoform specific gene regulation and tumor biology
    Molecular Endocrinology, 2005
    Co-Authors: Britta M Jacobsen, Jennifer K Richer, Stephanie A Schittone, Kathryn B. Horwitz
    Abstract:

    Progesterone Receptors (PRs) are prognostic markers in breast cancers irrespective of the patient's progestational status. However, there are two PR isoforms, PR-A and PR-B, that are equimolar in the normal breast but dysregulated in advanced disease. Postmenopausal, tamoxifen-treated patients with estrogen receptor (ER)-positive, PR-A-rich tumors have much faster disease recurrence than patients with PR-B-rich tumors. To study the mechanisms we engineered ER+ breast cancer cells that express each PR isoform under control of an inducible promoter. We identified 79 genes regulated by Progesterone (P), mainly by PR-B, and 51 genes regulated without Progesterone, mainly by PR-A. Only nine genes were regulated with and without ligand, leading to definition of three classes: I) genes regulated only by liganded PR; II) genes regulated only by unliganded PR; III) genes regulated by both. Unliganded PR-A and PR-B differentially regulate genes that coordinate extracellular signaling pathways and influence tumor cell biology. Indeed, in the absence of P, compared with ER+/PR-B+ or PR- cells, ER+, PR-A+ cells exhibit an aggressive phenotype, are more adhesive to an extracellular matrix, and are more migratory. Additionally, unliganded PR-A and PR-B both inhibit cell growth and provoke resistance to Taxol-induced apoptosis. We propose that PR-A:PR-B ratios, even in the absence of P, influence the biology and treatment response of ER+ tumors, that PR-A isoforms are functionally dominant in P-deficient states, and that PR-A rich tumors are especially aggressive.

  • functional properties of the n terminal region of Progesterone Receptors and their mechanistic relationship to structure
    The Journal of Steroid Biochemistry and Molecular Biology, 2003
    Co-Authors: Glenn S Takimoto, Jennifer K Richer, Britta M Jacobsen, Carol A Sartorius, Lin Tung, Hany A Abdelhafiz, Michael G Abel, David L Bain, Kathryn B. Horwitz
    Abstract:

    Progesterone Receptors (PR) are present in two isoforms, PR-A and PR-B. The B-upstream segment (BUS) of PR-B is a 164 amino acid N-terminal extension that is missing in PR-A and is responsible for the functional differences reported between the two isoforms. BUS contains an activation function (AF3) which is defined by a core domain between residues 54-154 whose activity is dependent upon a single Trp residue and two LXXLL motifs. We have also identified sites both within and outside of BUS that repress the strong synergism between AF3 and AF1 in the N-terminal region and AF2 in the hormone binding domain. One of these repressor sites is a consensus binding motif for the small ubiquitin-like modifier protein, SUMO-1 (387IKEE). The DNA binding domain (DBD) structure is also important for function. When BUS is linked to the glucocorticoid receptor DBD, AF3 activity is substantially attenuated, suggesting that binding to a DNA response element results in allosteric communication between the DBD and N-terminal functional regions. Lastly, biochemical and biophysical analyses of highly purified PR-B and PR-A N-terminal regions reveal that they are unstructured unless the DBD is present. Thus, the DBD stabilizes N-terminal structure. We propose a model in which the DBD through DNA binding, and BUS through protein-protein interactions, stabilize active receptor conformers within an ensemble distribution of active and inactive conformational states. This would explain why PR-B are stronger transactivators than PR-A.

  • association of the ku autoantigen dna dependent protein kinase holoenzyme and poly adp ribose polymerase with the dna binding domain of Progesterone Receptors
    Journal of Molecular Endocrinology, 2000
    Co-Authors: Carol A Sartorius, Jennifer K Richer, Glenn S Takimoto, Lin Tung, Kathryn B. Horwitz
    Abstract:

    Ligand-activated Progesterone Receptors (PR) bind to DNA at specific Progesterone response elements by means of a DNA binding domain (DBDPR) containing two highly conserved zinc fingers. DNA-bound PRs regulate transcription via interaction with other nuclear proteins and transcription factors. We have now identified four HeLa cell nuclear proteins that copurify with a glutathionineS-transferase‐human DBD PR fusion protein. Microsequence and immunoblot analyses identified one of these proteins as the 113 kDa poly(ADPribose) polymerase. The three other proteins were identified as subunits of the DNA-dependent protein kinase (DNA-PK) holoenzyme: its DNA binding regulatory heterodimers consisting of Ku70 and Ku86, and the 460 kDa catalytic subunit, DNA-PKCS. DNA-PK that was ‘pulleddown’ by DBDPR on the affinity resin was able to

  • convergence of Progesterone with growth factor and cytokine signaling in breast cancer Progesterone Receptors regulate signal transducers and activators of transcription expression and activity
    Journal of Biological Chemistry, 1998
    Co-Authors: Jennifer K Richer, Nicole G Manning, Roger L Powell, Carol A. Lange, Gareth Owen, Kathryn B. Horwitz
    Abstract:

    Abstract STATS (signal transducers and activators of transcription) are latent transcription factors activated in the cytoplasm by diverse cell surface signaling molecules. Like Progesterone Receptors (PR), Stat5a and 5b are required for normal mammary gland growth and differentiation. These two proteins are up-regulated during pregnancy, a period dominated by high levels of Progesterone. We now show that progestin treatment of breast cancer cells regulates Stat5a and 5b, Stat3, and Stat1 protein levels in a PR-dependent manner. In addition, progestin treatment induces translocation of Stat5 into the nucleus, possibly mediated by the association of PR and Stat5. Last, Progesterone pretreatment enhances the phosphorylation of Stat5 on tyrosine 694 induced by epidermal growth factor. Functional data show that progestin pretreatment of breast cancer cells enhances the ability of prolactin to stimulate the transcriptional activity of Stat5 on a β-casein promoter. Progesterone and epidermal growth factor synergize to control transcription from p21WAF1 and c-fospromoters. These data demonstrate the convergence of Progesterone and growth factor/cytokine signaling pathways at multiple levels, and suggest a mechanism for coordination of PR and Stat5-mediated proliferative and differentiative events in the mammary gland.