Gary L. Firestone

Characterization of sgk, a novel member of the serine/threonine protein kinase gene family which is transcriptionally induced by glucocorticoids and serum.

A novel member of the serine/threonine protein kinase gene family, designated sgk, for serum and glucocorticoid-regulated kinase, was identified in a differential screen for glucocorticoid-inducible transcripts expressed in the Con8.hd6 rat mammary tumor cell line. sgk encodes a protein of 49 kDa which has significant sequence homology (45 to 55% identity) throughout its catalytic domain with rac protein kinase, the protein kinase C family, ribosomal protein S6 kinase, and cyclic AMP-dependent protein kinase. sgk mRNA is expressed in most adult rat tissues, with the highest levels in the thymus, ovary, and lung, as well as in several rodent and human cell lines. sgk mRNA was stimulated by glucocorticoids and by serum within 30 min, and both inductions were independent of de novo protein synthesis. The transcriptional regulation by glucocorticoids is a primary response, since the promoter of sgk contains a glucocorticoid response element consensus sequence 1.0 kb upstream of the start of transcription which is able to stimulate chloramphenicol acetyltransferase reporter gene activity in a dexamethasone-dependent manner. Antibodies that specifically recognize sgk-encoded protein on an immunoblot were generated. This protein was shown to increase in abundance with glucocorticoid treatment in a manner which paralleled the mRNA accumulation. This is the first report of a presumed serine/threonine protein kinase that is highly regulated at the transcriptional level by glucocorticoid hormones and suggests a novel interplay between glucocorticoid receptor signalling and a protein kinase of the second messenger family.

Serum and glucocorticoid-inducible kinase (SGK) is a target of the PI 3-kinase-stimulated signaling pathway

Serum and glucocorticoid‐inducible kinase (SGK) is a novel member of the serine/threonine protein kinase family that is transcriptionally regulated. In this study, we have investigated the regulatory mechanisms that control SGK activity. We have established a peptide kinase assay for SGK and present evidence demonstrating that SGK is a component of the phosphoinositide 3 (PI 3)‐kinase signaling pathway. Treatment of human embryo kidney 293 cells with insulin, IGF‐1 or pervanadate induced a 3‐ to 12‐fold activation of ectopically expressed SGK. Activation was completely abolished by pretreatment of cells with the PI 3‐kinase inhibitor, LY294002. Treatment of activated SGK with protein phosphatase 2A in vitro led to kinase inactivation. Consistent with the similarity of SGK to other second‐messenger regulated kinases, mutation of putative phosphorylation sites at Thr256 and Ser422 inhibited SGK activation. Cotransfection of PDK1 with SGK caused a 6‐fold activation of SGK activity, whereas kinase‐dead PDK1 caused no activation. GST‐pulldown assays revealed a direct interaction between PDK1 and the catalytic domain of SGK. Treatment of rat mammary tumor cells with serum caused hyperphosphorylation of endogenous SGK, and promoted translocation to the nucleus. Both hyperphosphorylation and nuclear translocation could be inhibited by wortmannin, but not by rapamycin.

Estrogen Receptor β Inhibits Human Breast Cancer Cell Proliferation and Tumor Formation by Causing a G2 Cell Cycle Arrest

Studies indicate that estrogen receptor (ER) α mediates breast cancer-promoting effects of estrogens. The role of ERβ in breast cancer is unknown. Elucidating the role of ERβ in the pathogenesis of breast cancer is important because many human breast tumors express both ERα and ERβ. We show that adenovirus-mediated expression of ERβ changes the phenotype of ERα-positive MCF-7 cells. Estradiol increases cell proliferation and causes tumor formation of MCF-7 cells expressing only ERα. In contrast, introducing ERβ into MCF-7 cells causes an inhibition of proliferation in vitro and prevents tumor formation in a mouse xenograft model in response to estradiol. ERβ inhibits proliferation by repressing c-myc, cyclin D1, and cyclin A gene transcription, and increasing the expression of p21Cip1 and p27Kip1, which leads to a G2 cell cycle arrest. These results demonstrate that ERα and ERβ produce opposite effects in MCF-7 cells on cell proliferation and tumor formation. Natural or synthetic ERβ-selective estrogens may lack breast cancer promoting properties exhibited by estrogens in hormone replacement regimens and may be useful for chemoprevention of breast cancer.

Stimulus-dependent regulation of serum and glucocorticoid inducible protein kinase (SGK) transcription, subcellular localization and enzymatic activity.

We originally discovered the serum and glucocorticoid inducible protein kinase, SGK, as a novel protein kinase that is under acute transcriptional control by serum and glucocorticoids. An expanding set of cell surface receptor, nuclear receptor, and cellular stress pathways has been shown to target SGK, which has implicated this regulated signaling molecule in a variety of biological functions. Compared to most other protein kinases, a distinguishing feature of SGK is the stringent stimulus-dependent regulation of its transcription, subcellular localization and enzymatic activity. In addition, SGK expression is regulated during discrete developmental stages, and during normal and abnormal physiological function. An analysis of the SGK promoter reveals many potential transcription factor sites that potentially account for the stimulus-dependent changes in SGK transcript expression observed in a variety of cell systems, although, the direct stimulus regulation of SGK promoter activity has been established only for glucocorticoids, p53 tumor suppressor protein, hyperosmotic stress and follicle stimulating hormone. In the systems tested to date, hormones, growth factors and environmental cues induce expression of a catalytically active SGK. It is now well established that the enzymatic activity of SGK is controlled by the PI 3-kinase cascade which produces a hyperphosphorylated active SGK. A critical third level of regulation is the stimulus-dependent control of SGK subcellular localization. The nuclear-cytoplasmic shuttling of SGK is regulated by a nuclear localization signal (NLS) that binds to the importin-alpha nuclear import receptor. Modeling of the 3-D structure of the central region of SGK that includes the kinase domain predicts that the NLS is located at an external surface of the molecule. Thus, multiple signal transduction pathways converge on SGK to control its availability, function and access to its substrates and non-substrate targets.

Hyperosmotic Stress Stimulates Promoter Activity and Regulates Cellular Utilization of the Serum- and Glucocorticoid-inducible Protein Kinase (Sgk) by a p38 MAPK-dependent Pathway

We have established that the serum- and glucocorticoid-inducible protein kinase (Sgk) is a new component of the hyperosmotic stress response. Treatment of NMuMg mammary epithelial cells with the organic osmolyte, sorbitol, caused the stable accumulation of Sgk transcripts and protein after an approximately 4-h lag. Transient transfection of a series of sgk-CAT reporter plasmids containing either 5′ deletions or continuous 6-base pair substitutions identified a hyperosmotic stress-regulated element that is GC-rich and is necessary for the sorbitol stimulation ofsgk gene promoter activity. Gel shift analysis identified four major DNA-protein complexes in the hyperosmotic stress-regulated element that, by competition with excess consensus wild type and mutant oligonucleotides and by antibody supershifts, contains the Sp1 transcription factor. Several lines of evidence suggest that the p38 MAPK signaling pathway mediates the hyperosmotic stress stimulation of sgk gene expression. Treatment with pharmacological inhibitors of p38 MAPK or with a dominant negative form of MKK3, an upstream regulator of p38 MAPK, significantly reduced or ablated the sorbitol induction ofsgk promoter activity or protein production. Using anin vitro peptide transphosphorylation assay, sorbitol treatment activates either endogenous or exogenous Sgk that is localized to the cytoplasmic compartment. Thus, we propose that the stimulated expression of enzymatically active Sgk after sorbitol treatment is a newly defined component of the p38 MAPK-mediated response to hyperosmotic stress.

Cell cycle and hormonal control of nuclear-cytoplasmic localization of the serum- and glucocorticoid-inducible protein kinase, Sgk, in mammary tumor cells. A novel convergence point of anti-proliferative and proliferative cell signaling pathways.

The serum- and glucocorticoid-inducible kinase (sgk) is a novel serine/threonine protein kinase that is transcriptionally regulated in rat mammary tumor cells by serum under proliferative conditions or by glucocorticoids that induce a G1 cell cycle arrest. Our results establish that the subcellular distribution of Sgk is under stringent cell cycle and hormonal control. Sgk is localized to the perinuclear or cytoplasmic compartment as a 50-kDa hypophosphorylated protein in cells arrested in G1 by treatment with the synthetic glucocorticoid dexamethasone. In serum-stimulated cells, Sgk was transiently hyperphosphorylated and resided in the nucleus. Laser scanning cytometry, which monitors Sgk localization and DNA content in individual mammary tumor cells of an asynchronously growing population, revealed that Sgk actively shuttles between the nucleus (in S andG 2/M) and the cytoplasm (in G1) in synchrony with the cell cycle. In cells synchronously released from the G1/S boundary, Sgk localized to the nucleus during progression through S phase. The forced retention of exogenous Sgk in either the cytoplasmic compartment, using a wild type sgkgene, or the nucleus, using a nuclear localization signal-containingsgk gene (NLS-Sgk), suppressed the growth and DNA synthesis of serum-stimulated cells. Thus, our study implicates the nuclear-cytoplasmic shuttling of sgk as a requirement for cell cycle progression and represents a novel convergence point of anti-proliferative and proliferative signaling in mammary tumor cells.

Role of the CCAAT/Enhancer Binding Protein-α Transcription Factor in the Glucocorticoid Stimulation of p21 waf1/cip1 Gene Promoter Activity in Growth-arrested Rat Hepatoma Cells

The preceding paper (Cha, H. H., Cram, E. J., Wang, E. C., Huang, A. J., Kasler, H. G., and Firestone, G. L. (1998) J. Biol. Chem. 273, 0000–0000(478563) defined a glucocorticoid responsive region within the promoter of the p21 CDK inhibitor gene that contains a putative DNA-binding site for the transcription factor CCAAT/enhancer binding protein-α (C/EBPα). Wild type rat BDS1 hepatoma cells as well as as4 hepatoma cells, which express antisense sequences to C/EBPα and ablate its protein production, were utilized to investigate the role of this transcription factor in the glucocorticoid regulation of p21 gene expression. The stimulation of p21 protein levels and promoter activity, as well as inhibition of CDK2-mediated retinoblastoma protein phosphorylation, by the synthetic glucocorticoid, dexamethasone, required the expression of C/EBPα. Overexpression of C/EBPα in as4 cells rescued the dexamethasone responsiveness of the p21 promoter. Site-directed mutagenesis of the p21 promoter revealed that dexamethasone stimulation of p21 promoter activity required the C/EBP consensus DNA-binding site. Furthermore, in glucocorticoid receptor-defective EDR1 hepatoma cells, dexamethasone failed to stimulate C/EBPα and p21 protein expression and promoter activities. Our results have established a functional link between the glucocorticoid receptor signaling pathway that mediates a G1 cell cycle arrest of rat hepatoma cells and the transcriptional control of p21 by a cascade that requires the steroid induction of C/EBPα gene expression.

Anticancer activities of artemisinin and its bioactive derivatives.

Artemisinin, a sesquiterpene lactone derived from the sweet wormwood plant Artemisia annua, and its bioactive derivatives exhibit potent anticancer effects in a variety of human cancer cell model systems. The pleiotropic response in cancer cells includes growth inhibition by cell cycle arrest, apoptosis, inhibition of angiogenesis, disruption of cell migration, and modulation of nuclear receptor responsiveness. These effects of artemisinin and its derivatives result from perturbations of many cellular signalling pathways. This review provides a comprehensive discussion of these cellular responses, and considers the ramifications for the potential development of artemisinin-based compounds in anticancer therapeutic and preventative strategies.

Glucocorticoids Stimulate p21 Gene Expression by Targeting Multiple Transcriptional Elements within a Steroid Responsive Region of the p21 waf1/cip1 Promoter in Rat Hepatoma Cells

Glucocorticoids can induce a G1arrest in the cell cycle progression of BDS1 rat hepatoma cells. In these cells, dexamethasone, a synthetic glucocorticoid, stimulated a rapid and selective increase in expression of the p21 cyclin-dependent kinase (CDK) inhibitor mRNA and protein and virtually abolished CDK2 phosphorylation of the retinoblastoma protein. Expression of the p27 CDK inhibitor, and other G1-acting cell cycle proteins, remained unaffected. Dexamethasone stimulated p21 promoter activity in a p53-independent manner that required functional glucocorticoid receptors. Transforming growth factor-β, which also induced a G1 cell cycle arrest of the hepatoma cells, failed to elicit this response. Analysis of 5′ deletions of the p21 promoter uncovered a glucocorticoid responsive region between nucleotides −1481 and −1184, which does not contain a canonical glucocorticoid response element but which can confer dexamethasone responsiveness to a heterologous promoter. Fine mapping of this region uncovered three distinct 50–60-base pair transcriptional elements that likely function as targets of glucocorticoid receptor signaling. Finally, ectopic expression of p21 had no effect on hepatoma cell growth in the absence of glucocorticoids but facilitated the ability of dexamethasone to inhibit cell proliferation. Thus, our results have established a direct transcriptional link between glucocorticoid receptor signaling and the regulated promoter activity of a CDK inhibitor gene that is involved in the cell cycle arrest of hepatoma cells.

Artemisinin Blocks Prostate Cancer Growth and Cell Cycle Progression by Disrupting Sp1 Interactions with the Cyclin-dependent Kinase-4 (CDK4) Promoter and Inhibiting CDK4 Gene Expression

Artemisinin, a naturally occurring component of Artemisia annua, or sweet wormwood, is a potent anti-malaria compound that has recently been shown to have anti-proliferative effects on a number of human cancer cell types, although little is know about the molecular mechanisms of this response. We have observed that artemisinin treatment triggers a stringent G1 cell cycle arrest of LNCaP (lymph node carcinoma of the prostate) human prostate cancer cells that is accompanied by a rapid down-regulation of CDK2 and CDK4 protein and transcript levels. Transient transfection with promoter-linked luciferase reporter plasmids revealed that artemisinin strongly inhibits CDK2 and CDK4 promoter activity. Deletion analysis of the CDK4 promoter revealed a 231-bp artemisinin-responsive region between -1737 and -1506. Site-specific mutations revealed that the Sp1 site at -1531 was necessary for artemisinin responsiveness in the context of the CDK4 promoter. DNA binding assays as well as chromatin immunoprecipitation assays demonstrated that this Sp1-binding site in the CDK4 promoter forms a specific artemisinin-responsive DNA-protein complex that contains the Sp1 transcription factor. Artemisinin reduced phosphorylation of Sp1, and when dephosphorylation of Sp1 was inhibited by treatment of cells with the phosphatase inhibitor okadaic acid, the ability of artemisinin to down-regulate Sp1 interactions with the CDK4 promoter was ablated, rendering the CDK4 promoter unresponsive to artemisinin. Finally, overexpression of Sp1 mostly reversed the artemisinin down-regulation of CDK4 promoter activity and partially reversed the cell cycle arrest. Taken together, our results demonstrate that a key event in the artemisinin anti-proliferative effects in prostate cancer cells is the transcriptional down-regulation of CDK4 expression by disruption of Sp1 interactions with the CDK4 promoter.