Rather than being listed chronologically, the papers are grouped by subject area.
Goolsby, K.M. and Shapiro, D.J. (2003) RNAi-mediated depletion of the 15 KH domain protein, vigilin, induces death of dividing and non-dividing human cells but does not initially inhibit protein synthesis. Nucleic Acids Res. In Press.
Abstract: Vigilin/Scp160p/DDP1 is a ubiquitous and highly conserved protein containing 15 related, but non-identical, K-homology (KH) nucleic acid binding domains. While its precise function remain unknown, proposed roles for vigilin include chromosome partitioning at mitosis, facilitating translation and tRNA transport, and control of mRNA metabolism, including estrogen-mediated stabilization of vitellogenin mRNA. To probe sites of vigilin action in vertebrate cells, we performed nucleic acid binding and RNA interference studies. Consistent with a potential role in chromosome partitioning, human vigilin exhibits a higher affinity for Drosophila dodecasatellite single-stranded DNA than for vitellogenin mRNA 3’-UTR. Direct observation and flow cytometry in non-mitotic, serum-starved, HeLa cells showed that RNAi-mediated vigilin knockdown is rapidly lethal, indicating an essential function for vigilin distinct from its proposed role in chromosome partitioning. Pulse labeling experiments revealed that rates of protein synthesis and degradation are unaffected by the several fold reduction in vigilin levels early in siRNA knockdown indicating that vigilin is not a global regulator of translation. These data show that vigilin is an essential protein in human cells, support the view that vigilin’s most essential functions are neither chromosome partitioning nor control of translation, and are consistent with vigilin playing a critical role in cytoplasmic mRNA metabolism.
Dodson, R.E. and Shapiro D.J. (2002) Regulation of Pathways of mRNA Destabilization and Stabilization. In K. Moldave Ed. Progress in Nucleic Acids Res. and Molec. Biol. 72: 129-164
(Although a chapter, this refereed work contains substantial unpublished data as well as our model for interaction of vigilin with nucleic acids.)
Abstract: This chapter provides a short overview of pathways of eukaryotic mNA degradation and their importance. The primary focus is on our studies of the pathway for regulated mRNA degradation mediated by mRNA binding proteins and nucleases that cleave within the body of the mRNA. The estrogen-mediated stabilization of vitellogenin mRNA represents a potential model for this type of pathway. In vitro genetic analysis of a large pool of poential targets and analysis of selected mRNA segments indicates that while vigilin has some capacity to bind to a variety of mRNA sequences, it exhibits strong preferences for binding to specific sequences. Vigilin does not contain a single ucleation site that initiates RNA binding. Segments of vigilin containing sub-sets of its 15 KH domains retained the ability to bind to diverse RNAs, albeit with a reduced affinity. Our data supports a model for vigilin-RNA interactions in which initial binding of a sub-set of vigilin’s 15 KH domains to a relatively high affinity site in an RNA target is followed by a processive binding, or “zippering”, process in which close proximity then allows additional regions of the highly flexible nucleic acid template to make weak contacts with additional vigilin domains. BY summing up the individually weak interactions of all of its many KH domains with RNA, vigilin achieves high affinity binding to nucleic acids.
Cunningham, K., Dodson, R.E., Nagel., M.A., Shapiro, D.J. and Schoenberg. D.R. (2000) Vigilin Binding Selectively Inhibits Cleavage of the Vitellogenin mRNA 3'-Untranslated Region by the mRNA Endonuclease Polysomal Ribonuclease 1. Proc. Natl. Acad. Sci. USA 97, 12498-12502.
Abstract:The regulation of mRNA stability is an important control mechanism which helps govern cellular mRNA levels. Most eukaryotic mRNAs contain a 5'-cap and a 3'-poly(A) tail which impede RNase digestion from the ends of the mRNA. For some mRNAs, cleavage within the 3'-untranslated region provides a way to remove the entire poly(A) tail and make the mRNA susceptible to rapid degradation. To investigate mRNA stabilization, we use cultured Xenopus hepatocytes. Addition of estrogen to the hepatocyte culture medium results in a 30 fold increase in the stability of the mRNA encoding the egg yolk precursor protein, vitellogenin, and destabilizes serum albumin mRNA. We identified vigilin as an estrogen-inducible protein binding to a segment of the vitellogenin mRNA 3'-untranslated region, (3'-UTR) implicated in the control of mRNA stability. Our collaborators identified PMR-1, a sequence-selective mRNase implicated in the degradation of albumin mRNA. The availability of recombinant vigilin and purified PMR-1 enabled us to test the hypothesis that mRNAs can be protected against endonuclease cleavage by the differential affinity of an RNA binding protein for a particular mRNA. We showed that vigilin binds with much higher affinity to the vitellogenin mRNA 3'-UTR segment than to the segment of albumin mRNA containing the PMR-1 cleavage sites. In biochemical experiments vigilin binds the vitellogenin mRNA 3'-UTR segment with high affinity and protects it against cleavage by PMR-1. Because vigilin binds the albumin mRNA segment with low affinity under the same conditions it is unable to protect albumin mRNA from cleavage by PMR-1. These studies provide direct evidence for differential susceptibility to endonuclease-mediated mRNA decay, resulting from the differential affinity of an RNA binding protein for mRNA stability determinants.
Krieg, A.J., Krieg, S.A. Ahn, B.S. and Shapiro, D.J. (2003) Interplay Between Estrogen Response Element Sequence and Ligands Controls In Vivo Binding of Estrogen receptor to Regulated Genes. J. Biol. Chem., Provisionally accepted, Subject to minor revision.
Abstract: To examine the role of the estrogen response element (ERE) sequence in binding of liganded estrogen receptor (ER) to promoters, we analyzed in vivo interaction of liganded ER with the imperfect ERE in the pS2 gene and the composite Estrogen Responsive Unit (ERU) in the Proteinase Inhibitor 9 (PI-9) gene. In transient transfections of ER positive HepG2ER7 cells, PI-9 was strongly induced by the estrogen, moxestrol (MOX), and by 4-hydroxytamoxifen (OHT). PI-9 was not induced by raloxifene (RAL) or ICI 182,780. Quantitative RT-PCR showed that cellular PI-9 mRNA was strongly induced by moxestrol and moderately induced by OHT. PS2 mRNA was strongly induced by moxestrol and weakly induced by OHT. Chromatin immunoprecipitation (ChIP) experiments demonstrated rapid and continuous association of MOX-hERα with the PI-9 ERU and with the pS2 ERE. Although MOX- hERα bound strongly to the PI-9 ERU and less well to the pS2 ERE in ChIP, gel shift assays showed that estrogen-hERa binds with higher affinity to the deproteinized pS2 ERE, than to the PI-9 ERU. Across a broad range of OHT concentrations, OHT-hERα associated strongly with the pS2 ERE and weakly with the PI-9 ERU. ICI-hERα bound poorly to the PI-9 ERU and effectively to the pS2 ERE. Raloxifene-hERα bound to the PI-9 ERU and the pS2 ERE about as well as MOX-hERα. These studies demonstrate that ER ligand and ERE sequence work together to regulate in vivo binding of ER to estrogen responsive promoters.
de Haan, G., Chusacultanachai, S., Mao, C., Katzenellenbogen, B.S. and Shapiro, D.J. (2000) Estrogen Receptor-KRAB Chimeras are Potent Ligand Dependent Repressors of Estrogen Regulated Gene Expression. J. Biol. Chem. 275: 13493-13501
Abstract: As an approach to targeted repression of any gene of interest we combined large scale genetic selection using P22 challenge phage with small molecule regulated ER chimeras. We prepared human estrogen receptor alpha (hER)-KRAB repressor domain chimeras which are potent ligand-dependent repressors of the transcription of estrogen response element (ERE) containing promoters and analyzed their mechanisms of action. Repression by the KRAB domain was dominant over transactivation mediated by the ERs activation domains. One or more EREs was required for repression. An ER ligand was required for repression. 17beta-estradiol, the Selective Estrogen Receptor Modulator 4-hydroxytamoxifen (the acitve metabolite of tamoxifen) and the "pure" antagonist ICI 182,780 were all highly effective in promoting repression.Studies with several promoters and cell lines demonstrated that the presence of EREs, rather than the capacity for estrogen induction determines the potential for repression of a gene by the KRAB-ER-KRAB (KERK) chimera. A single consensus ERE was sufficient for repression. Although wild-type ER effectively induces expression from the imperfect ERE in the native pS2 promoter,the KERK chimera was unable to suppress transcription from a promoter containing the pS2 ERE. These data suggest that repression by KRAB chimeras and transactivation by wild-type ER are quite different. Our data suggests that tight binding to the ERE, resulting in continued presence of the ER-KRAB chimera on the promoter is critical for repression, but not for transactivation. We recently reported the use of the P22 challenge phage system to select mutations which enhance binding of a steroid receptor DNA binding domain to the ERE (Chusacultanachai et al. 1999). Introducing one sety of these mutations into wild-type ER enhanced transactivation from the pS2 ERE. Insertion of these mtuations into KERK created the novel repressor, KERK-3M, which was a potent repressor of both ER-induced and basal transcription from a promoter containing the pS2 ERE. Using a genetically selected set of up-binding mutations strongly potentiates the ability of KRAB-ER chimeras to act as ligand-dependent repressors of transcription from a naturally occurring imperfect ERE. These modified ER-KRAB chimeras should prove useful as new tools for the functional analysis and repression of ER-regulated genes.
Kanan-Thulasiraman, P., and Shapiro, D.J. (2002) Modulators of Inflammation use Nuclear Factor-κB and Activator Portein-1 Sites to Induce the Caspase-1 and Granzyme B Inhibitor, Proteinase Inhibitor 9. J. Biol. Chem., 277: 41230-41239.
Abstract: Proteinase inhibitor 9 (PI-9) inhibits caspase-1 (IL-1β converting enzyme) and granzyme B, thereby regulating production of the pro-inflammatory cytokine IL-1β and susceptibility to granzyme B-induced apoptosis. We find that the PI-9 promoter region contains functional imperfect nuclear factor-κB (NF-κB) sites at –135 and –88 and a consensus activator protein 1 (AP-1) site at –308. Purified NF-κB p50 subunit bound to a putative PI-9 NF-κB site. Nuclear extracts from IL-1β-treated HepG2 cells exhibited strong, IL-1β inducible, binding to the NF-κB sites and to the AP-1 site. Using transient transfections to assay PI-9 promoter mutations, we demonstrate that mutational ablation of the AP-1 site, or of either NF-κB site, reduces IL-1β induced expression of PI-9 by ~60%. Mutational ablation of the two NF-κB sites, and of the AP-1 site, nearly abolishes both basal and IL-1β-induced expression of PI-9. Estrogens, which induce PI-9, do not interfere with IL-1β induction, but induction by estrogens and IL-1β was not synergistic. IL-1β induction of the transiently transfected PI-9 promoter-luciferase reporter closely parallels IL-1β induction of cellular PI-9 mRNA, indicating that IL-1β induction of PI-9 mRNA is transcriptional. Cellular PI-9 mRNA and protein are potently induced by IL-1β, LPS, TNF-α and TPA. Our surprising finding that the pro-inflammatory cytokine IL-1β strongly induces PI-9, suggests a novel mechanism for regulating inflammation and apoptosis through a negative feedback loop controlling expression of the anti-inflammatory and anti-apoptotic protein, PI-9.
Krieg, S.A., Krieg, A.J., and Shapiro D.J. (2001) A unique Downstream Estrogen Responsive Unit Mediates Estrogen induction of proteinase Inihibitor-9, A Cellular inhibitor of IL-1beta Converting Enzyme (Capsase 1). Molec. Endocrinol., 15: 1971-1982.
Abstract: Recently, proteinase inhibitor 9 (PI-9) was identified as the first endogenous inhibitor of caspase 1 (Interleukin-1β Converting Enzyme, ICE Protease). The regulation of PI-9 expression therefore has broad importance in control of inflammatory processes. We reported that PI-9 mRNA and protein are rapidly and directly induced by estrogen in human liver cells. Using transient transfections to assay PI-9 promoter truncations and mutations, we demonstrate that this strong estrogen induction is mediated by a unique downstream estrogen responsive unit (ERU) ~200 nucleotides downstream of the transcription start site. This ERU consists of an imperfect ERE palindrome immediately adjacent to a direct repeat containing two consensus ERE half sites separated by 13 nucleotides (DR13). In transient transfections, all four of the ERE half sites in the imperfect ERE and in the DR13 were important for estrogen inducibility. Transfected COUP-TF I and II down-regulated estrogen-mediated expression from the ERU. Electrophoretic mobility shift assays using purified recombinant hERα demonstrate high affinity binding of two ER complexes to the ERU. DNase I footprinting showed that purified ER protected all four of the half sites in the imperfect ERE and in the DR13. By showing that a direct repeat of the ERE half site can function in a composite element to confer estrogen inducibility on a native gene, these studies extend the repertoire of DNA sequences able to function as estrogen responsive elements.
Kanamori, H., Krieg, S., Mao, C., Di Pippo V.A., Wang, S., Zajchowski, D.A. and Shapiro, D.J. (2000) Proteinase Inhibitor 9, an Inhibitor of Granzyme B-mediated Apoptosis, Is a Primary Estrogen-inducible gene in Human Liver Cells. J. Biol. Chem. 275, 5867-5873.
Abstract:A major mechanism by which cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells destroy target cells that are neoplastic or infectedwith intracellular pathogens is induction of apoptosis by granzyme B containing granules. On entry into target cells, granzyme B induces apoptosis by proteolytically cleaving and activating caspases. Proteinase inhibitor 9 (PI-9) is a potent inhibitor of granzyme B and of granzyme B mediated apoptosis. We used modified differential display to identify PI-9 mRNA as strongly induced by estrogen in ER positive human liver cell lines. Since PI-9 was also induced by estrogen in a human liver biopsy specimen, PI-9 is a genuine estrogen-regulated human gene. Several types of data demonstrate that the 30-40 fold estrogen induction of PI-9 mRNA is a rapid direct effect of estrogen on PI-9 gene transcription. PI-9 protein is also strongly induced by estrogen in these liver cells. In chronic aggressive heaptitis, estrogen reportedly exerts a protective effect in protecting liver cells from the severe liver damage which follows immulogically mediated attack on hepatitis virus-infected liver cells. The estrogen induction of PI-9 is therefore of significant biological interest. These studies extend the area of estrogen action to a new class of proteins importan t in immune function and in apoptosis. This work describes a potential point of intersection between the actions of sex steroid hormones at the gene level and a key fiunction of the immune system, induction of apoptosis.
Obrero, M., Yu, D.V. and Shapiro, D.J. (2002) Estrogen receptor-dependent and Estrogen Receptor-independent Pathways for Tamoxifen and 4-Hydroxytamoxifen-induced programmed cell Death. J. Biol. Chem., 277: 45695-45703.
Abstract: Estrogens promote development of breast cancers through interactions with estrogen receptors (ERs). The therapeutic efficacy of the selective estrogen receptor modulator tamoxifen (TAM) in cancer therapy is thought to arise primarily from its ability to compete with estrogens for binding to the ER. We show that TAM and its active metabolite, 4-hydroxytamoxifen (OHT), induce programmed cell death through distinct ER-dependent and ER-independent pathways. The ER-independent pathway requires 10-20 μM TAM or OHT and occurs in ER-negative cells. Nanomolar concentrations of OHT activate an ER-dependent pathway. 17β-estradiol, raloxifene, and ICI 182,780 are all effective competitors, which block OHT-ER and Tam-ER-dependent cell death. Since the p38-specific inhibitor SB203580 blocks OHT-ER induced cell death, stress kinase pathways are likely involved. ER-independent cell death triggers classic caspase-dependent apoptosis. However, while OHT-ER triggers some hallmarks of apoptosis, including Bax translocation and cytochrome c release, the absence of PARP cleavage or DNA laddering indicate caspase-independent programmed cell death. The divergence from classical apoptosis appears to be at the level of caspase 9 activation. This study describes a unique ER-dependent, programmed cell death-promoting activity of TAM and OHT that is distinct from their ability to compete with estrogens for binding to the ER.
Zhang, C.C. and Shapiro, D.J. (2000) Activation of the p38 Mitogen-activated Protein Kinase Pathway by Estrogen or by 4-Hydroxytamoxifen Is Coupled to Estrogen Receptor-induced Apoptosis. J. Biol. Chem. 275, 479-486.
Abstract: 17beta-Estradiol (E2) or the antiestrogen, 4-hydroxytamoxifen (OHT), induce apoptosis in stably transfected estrogen receptor (ER)-positive HeLa-ER5 cells. p38 mitogen-activated protein kinase is implicated in cellular processes involving apoptosis. The p38 kinase inhibitor, SB203580, partially protects HeLa-ER5 cells against apoptosis induced by E2 or by OHT. E2 induces the p38 pathway 12-36-fold in ER-positive cell lines, while OHT induces p38 activity 2-5-fold. In an ER-positive cell line selected for resistance to E2-induced apoptosis, E2 no longer induced p38, and the ER no longer bound to the estrogen response element, while OHT induced both p38 and apoptosis. In cells selected for resistance to OHT-induced apoptosis, OHT no longer induced p38, while E2 induced p38 and apoptosis, and transactivated an estrogen response element-containing reporter gene. In MCF-7 cells, whose growth is stimulated by estrogen, E2 did not induce p38 or apoptosis, while OHT induced both p38 and apoptosis, and SB203580 protected against OHT-induced apoptosis. This work shows that E2 and OHT activate the p38 pathway, suggests that they use different pathways for p38 activation, and links activation of the p38 pathway to apoptosis induced by E2 and by OHT.
Last Updated: 08/12/03