Ligand-induced glucocorticoid receptor (GR) activation has recently been linked to the inhibition of cell proliferation via the transcriptional induction of p21(WAF1/Cip1), which functions as a universal inhibitor of cyclin-dependent protein kinases. Herein, we identify a Ser/Thr protein phosphatase (PP5) that promotes cellular proliferation by inhibiting both glucocorticoid and p53-mediated signaling pathways leading to p21(WAF1/Cip1)-mediated growth arrest. The suppression of PP5 expression (1) markedly increases the association of GR with its cognate DNA-binding sequence, (2) induces GR transcriptional activity without the addition of hormone, and (3) increases dexamethasone-mediated induction of GR reporter activity to a level that is approximately 10 times greater than the maximal response obtainable in the presence of PP5. PP5 has no apparent effect on the binding of hormone to the GR, and dexamethasone-mediated growth arrest correlates with an increase in p53 phosphorylation. Comparative studies in p53-wild-type, p53-defective, and p53-deficient cell lines indicate that either (1) p53 participates in GR-mediated induction of p21(WAF1/Cip1), with the hyperphosphorylation of basal p53 induced by glucocorticoids sufficient for the propagation of an antiproliferative response when PP5 expression is inhibited, or (2) PP5 acts where p53-mediated and GR-induced signaling networks converge to regulate the transcriptional induction of p21(WAF1/Cip1). Thus, aberrant PP5 expression may have an additive effect on the development of human cancers by promoting cell proliferation via the inhibition of a GR-induced antiproliferative signaling cascade, and facilitating neoplastic transformation via the inhibition of a growth-arresting p53-mediated response that guards against genomic instability.
The proliferation of many estrogen receptor (ER)-positive breast cancer cells depends on estradiol, and tumors arising from these cells are often responsive initially to treatment with selective ER modulators, which produce an antiestrogen effect. However, tumors that are refractory to the antiestrogenic effects of selective ER modulators often reemerge, and the prognosis for these patients is poor because of the lack of additional effective therapy. Accordingly, deciphering the cellular events associated with estrogen-dependent growth and the subsequent outgrowth of tumors with an estrogenindependent phenotype is of considerable interest. Here we show that the expression of PP5, an evolutionarily conserved Ser/Thr phosphatase that functions as an inhibitor of glucocorticoid-and p53-induced signaling cascades leading to growth suppression, is responsive to 17-estradiol (E 2 ) in ER-positive human breast carcinoma cells (MCF-7). Northern analysis revealed that E 2 -induced PP5 expression is blocked by treatment with tamoxifen, and a consensus ER recognition element was identified in the PP5 promoter. The PP5-ER recognition element associates with human ERs and confers E 2 -induced transcriptional activation to reporter plasmids. The specific inhibition of PP5 expression ablates E 2 -mediated proliferation in MCF-7 cells without having an apparent effect on E 2 -induced expression of c-myc or cyclin D1. Thus, although critical for cell growth, PP5 likely acts either downstream or independently of c-Myc and Cyclin D1. To further characterize the role of PP5 in E 2 -regulated growth control, we constructed stable MCF-7 cell lines in which the expression of PP5 was placed under the control of tetracycline-regulated transactivator and operator plasmids. Studies with these cells revealed that the constitutive overexpression of PP5 affords E 2 -dependent MCF-7 cells with the ability to proliferate in E 2 -depleted media. Together, these studies indicate that E 2 -induced PP5 expression functions to enhance E 2 -initiated signaling cascades leading to cell division and that aberrant PP5 expression may contribute to the development of MCF-7 cells with an estrogen-independent phenotype.Breast cancer is a leading cause of cancer mortality among women with an annual rate as high as 1.04/1,000 women in the United States (1). Approximately 50% of all breast cancer cells express receptors that recognize estrogen, and in these estrogen receptor (ER 1 )-positive cells 17-estradiol (E 2 ) has a potent mitogenic effect (2). The growth-promoting effects of E 2 influence both the initiation and progression of cell growth, stimulating resting noncycling (G 0 phase) cells to enter the cell cycle and accelerating G 1 -S phase progression (3-6). Proliferation is initiated by the activation of ERs, which act as hormone-regulated transcription factors that control the expression of estrogen-responsive genes (7-8). Several ER-responsive genes (e.g. c-myc and cyclin D1) have been linked to the propagation of a proliferative response, and a...
Serine/threonine phosphatase 5 (PP5) can act as a suppresser of p53-dependent growth suppression and has been reported to associate with several proteins, including the glucocorticoid receptor/heat-shock protein-90 complex. Still, the physiological/pathological roles of PP5 are unclear. To characterize the relationship of PP5, glucocorticoid receptor activation and p53, here we describe the development of chimeric antisense oligonucleotides that potently inhibit human p53 expression. This allowed us to regulate the expression of either p53 (e.g. with ISIS 110332) or PP5 (e.g. with ISIS 15534) in genetically identical cells. Studies with ISIS 110332 revealed that the suppression of p53 expression is associated with a decrease in the basal expression of the cyclin-dependent kinase inhibitor protein, p21 WAF1/Cip1 , and a concomitant increase in the rate of cell proliferation. Suppression of p53 also blocks dexamethasone-induced p21 WAF1/Cip1 expression and G 1 -growth arrest. Furthermore, treatment with ISIS 110332, but not the mismatched controls, ablates the suppression of growth produced by prior treatment with dexamethasone. Additional studies revealed that dexamethasone-dependent p21 WAF1/Cip1 expression occurs without an apparent change in p53 protein levels or the phosphorylation status of p53 at Ser-6, -37, or -392. However, dexamethasone treatment is associated with an increase in p53 phosphorylation at Ser-15. Suppression of PP5 expression with ISIS 15534 also results in the hyperphosphorylation of p53 at Ser-15. Together, these findings indicate that the basal expression of p53 plays a functional role in a glucocorticoid receptor-mediated response regulating the expression of p21 Waf1/Cip1 via a mechanism that is suppressed by PP5 and associated with the phosphorylation of p53 at Ser-15.Serine/threonine phosphatase 5 (PP5) is an okadaic acid/ calyculin A-sensitive phosphatase that is expressed ubiquitously in mammals and has been reported to associate with the atrial natriuretic peptide receptor (1), the heat shock protein 90 (Hsp-90) 1 -glucocorticoid receptor (GR) heterocomplex (2, 3), the CDC16/CDC27 subunits of the anaphase-promoting complex (4), cryptochrome 2 (5), apoptosis signal-regulating kinase1 (6), Hsp90-dependent heme-regulated eIF2␣ kinase (7), and the G␣ 12 /G␣ 13 subunits of heterotrimeric G proteins (8). In estrogen-responsive breast carcinoma cells, the expression of PP5 is induced by treatment with estrogen, and the constitutive expression of PP5 converts MCF-7 cells into an estrogen-independent phenotype (9). Still, determining the physiological/pathological roles of PP5 has proven difficult for many reasons. First, in a crude cell homogenate PP5 exists predominately in an inactive state (for review see Ref. 10). Second, the physiological activators of PP5 are unknown. Third, when activated by the addition of polyunsaturated lipids or protease mediated cleavage of the N-terminal autoinhibitory domain (11, 12), the activity of PP5 cannot be distinguished from that of PP2A and PP1, whi...
Background: In most cells glucocorticoid receptors (GR) reside predominately in the cytoplasm. Upon hormone binding, the GR translocates into the nucleus, where the hormone-activated GRcomplex regulates the transcription of GR-responsive genes. Serine/threonine protein phosphatase type 5 (PP5) associates with the GR-heat-shock protein-90 complex, and the suppression of PP5 expression with ISIS 15534 stimulates the activity of GR-responsive reporter plasmids, without affecting the binding of hormone to the GR.
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