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HEXIM1 Regulates 17B-Estradiol/Estrogen Receptor- A–Mediated Expression of Cyclin D1 in Mammary Cells Via Modulation of P-Tefb

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HEXIM1 Regulates 17B-Estradiol/Estrogen Receptor- A–Mediated Expression of Cyclin D1 in Mammary Cells Via Modulation of P-Tefb Research Article HEXIM1 Regulates 17B-Estradiol/Estrogen Receptor- A–Mediated Expression of Cyclin D1 in Mammary Cells via Modulation of P-TEFb Ndiya Ogba,1 Laura J. Chaplin,1 Yong Qiu Doughman,3 Koh Fujinaga,2 and Monica M. Montano1 1Department of Pharmacology, 2Division of Infectious Diseases and Department of Molecular Biology and Microbiology, Case Western Reserve University; and 3Division of Pediatric Cardiology, Department of Pediatrics, Rainbow Babies and Children’s Hospital, Cleveland, Ohio Abstract tion either by directly binding to estrogen response elements of Estrogen receptor A (ERA) plays a key role in mammary gland target genes (3–5) or indirectly via protein-protein interactions development and is implicated in breast cancer through the with other transcription factors like SP1 or activator protein 1 (AP-1; ref. 6). In both cases, coregulatory proteins are also recruited transcriptional regulation of genes linked to proliferation and apoptosis. We previously reported that hexamethylene bisace- to the promoter, and together ERs and these factors elicit changes tamide inducible protein 1 (HEXIM1) inhibits the activity of in mRNA and protein levels of ER target genes and, ultimately, ligand-bound ERA and bridges a functional interaction a physiologic response (4–6). Because estrogen signaling controls between ERA and positive transcription elongation factor b the balance of growth and apoptosis in normal breast epithelial (P-TEFb). To examine the consequences of a functional cells, a disruption of this balance contributes to abnormal cell HEXIM1-ERA-P-TEFb interaction in vivo, we generated growth and can lead to tumorigenesis (4, 7). Therefore, it is MMTV/HEXIM1 mice that exhibit mammary epithelial-specific important to identify and elucidate the mechanism of action of ERs and doxycycline-inducible expression of HEXIM1. Increased and their coregulators that give better insight into ER-mediated HEXIM1 expression in the mammary gland decreased estro- transcriptional regulation (8). gen-driven ductal morphogenesis and inhibited the expression In eukaryotic transcription, the elongation stage is highly of cyclin D1 and serine 2 phosphorylated RNA polymerase II regulated and important for the generation of full-length mRNA (S2P RNAP II). In addition, increased HEXIM1 expression in transcripts (9–11). One of the positive regulators, positive MCF-7 cells led to a decrease in estrogen-induced cyclin D1 transcription elongation factor b (P-TEFb), has an essential role expression, whereas down-regulation of HEXIM1 expression in RNA polymerase II (RNAP II) transcription elongation (9, 10). led to an enhancement of estrogen-induced cyclin D1 In many human cell types, the predominant form of P-TEFb expression. Studies on the mechanism of HEXIM1 regulation consists of cyclin-dependent kinase 9 (CDK9) and its regulatory on estrogen action indicated a decrease in estrogen-stimulated partner, cyclin T1 (11). It phosphorylates and thereby inhibits the recruitment of ERA, P-TEFb, and S2P RNAP II to promoter and activity of negative elongation factors, NELF and DSIF (DRB coding regions of ERA-responsive genes pS2 and CCND1 with sensitivity inducing factor; ref. 9). It also phosphorylates the increased HEXIM1 expression in MCF-7 cells. Notably, in- COOH-terminal repeat domain (CTD) of the largest subunit of creased HEXIM1 expression decreased only estrogen-induced RNAP II (9, 10). The RNAP II CTD consists of multiple repeats of P-TEFb activity. Whereas there have been previous reports on the heptapeptide sequence, YSPTSPS, phosphorylated at serine 5 HEXIM1 inhibition of P-TEFb activity, our studies add a new by general transcription factor TFIIH during initiation and at dimension by showing that E2/ER is an important regulator of serine 2 by P-TEFb during elongation (9, 10). These phosphor- the HEXIM1/P-TEFb functional unit in breast cells. Together, ylation events are crucial for effective transition from an abortive these studies provide novel insight into the role of HEXIM1 to a productive phase of elongation (11, 12). P-TEFb is essential and ERA in mammary epithelial cell function. [Cancer Res for productive HIV-1 transcriptional elongation and several 2008;68(17):7015–24] studies have shown that various transcription factors bind to and recruit P-TEFb to specific promoters stimulating elongation Introduction (12, 13). Mammary gland morphogenesis and development requires input In previous studies, we identified an ERa-interacting protein, from several genetic and epigenetic pathways regulated by estrogen down-regulated gene-1 [also known as hexamethylene hormones and growth factors including estrogens (1, 2). Estrogens inducible protein 1 (HEXIM1)], and found that it is an inhibitor of mediate their actions through estrogen receptors (ER), ERa and ERa transcription and breast cell growth (14). Additionally, we ERh, nuclear steroid receptors that classically regulate transcrip- showed that HEXIM1 expression was lower in human breast tumors when compared with adjacent normal tissue, suggesting a role for HEXIM1 in breast tumorigenesis (14). Concurrent studies identified HEXIM1 as a P-TEFb–interacting factor that also inhibits Note: Supplementary data for this article are available at Cancer Research Online P-TEFb activity (15, 16). Studies have also shown that HEXIM2, (http://cancerres.aacrjournals.org/). Requests for reprints: Monica M. Montano, Department of Pharmacology, a paralogue of HEXIM1, has the same inhibitory effect on P-TEFb Case Western Reserve University School of Medicine, H.G. Wood Building W307, (17, 18). We showed that HEXIM1 modulates a functional 2109 Adelbert Road, Cleveland, OH 44106. Phone: 216-368-3378; Fax: 216-368-3395; interaction between ERa and cyclin T1 in breast epithelial cells E-mail: [email protected]. I2008 American Association for Cancer Research. and inhibits the recruitment of ligand-bound ERa (E2/ERa) to the doi:10.1158/0008-5472.CAN-08-0814 pS2 gene promoter (19). We also found that cyclin T1 seemed to be www.aacrjournals.org 7015 Cancer Res 2008; 68: (17). September 1, 2008 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2008 American Association for Cancer Research. Cancer Research necessary for E2 induction of cyclin D1 protein expression (19). reverse transcription-PCR (RT-PCR) analyses as described in Supplementary data. Cyclin D1 (CCND1) is a D-type cyclin that regulates G1-S cell cycle Western blot analyses. Western blot analyses using extracts from progression during cell proliferation (20). CCND1 is also E2/ERa responsive and is thought to play major roles in mammary gland mammary glands and MCF-7 cells are described in Supplementary data. RNA interference. A Pol II promoter–driven miRNA expression vector development and breast cancer (1, 21). However, the contribution system (Invitrogen) was used. To make pcDNA-HEXIM1 miR, miRNA oligos a of E2/ER to HEXIM1 action in breast cells is not well defined. (see Supplementary Table S3 for list and sequences) were annealed and In addition, the precise mechanism by which P-TEFb regulates cloned into the pcDNA 6.2 GW/EmGFP vector (Invitrogen) according to the CCND1 transcription and how this can be linked to mammary manufacturer’s instructions. MCF-7 cells were transfected with pcDNA gland development and tumorigenesis need to be further defined. 6.2-GW/EmGFP-miR expression vectors containing either the HEXIM1 To address these questions, we developed a transgenic mouse miRNA insert or a control LacZ miRNA insert. Following blasticidin model in our laboratory that is doxycycline inducible and selection, cells expressing the highest level of green fluorescent protein selectively expresses HEXIM1 in the mammary gland under the (GFP) were flow-sorted and expanded. During experiments, cells were control of the mouse mammary tumor virus long terminal repeat treated with ethanol vehicle or 100 nmol/L E2 for 3 h and harvested as (MMTV-LTR) promoter previously described (22). Using this model, described above for Western blot analyses. Chromatin immunoprecipitation assays. MCF-7 cells were plated on we show that increased HEXIM1 expression decreased E -induced 2 150-mm plates and transfected as described with pCMV-TAG2B or pCMV- CCND1 and serine 2 phosphorylated (S2P) RNAP II expression in TAG2B-HEXIM1. Forty-eight hours later, cells were treated with ethanol the mouse mammary gland. In addition, increasing HEXIM1 vehicle or 100 nmol/L E2 for 45 and 90 min. Chromatin immunoprecip- expression inhibits E2-induced recruitment of ERa, P-TEFb, and itation assays were carried out as previously described (19). See S2P RNAP II to ER-responsive genes, pS2 and CCND1, in MCF-7 Supplementary data for further details of immunoprecipitation and cells. Finally, we observed that E2 stimulates P-TEFb activity and analyses. that HEXIM1 inhibits only E2-induced, and not basal, P-TEFb CTD kinase assays. Kinase assays were done according to previously activity. These results elucidate the functional consequences of described protocols with some modifications (23, 24). See Supplementary data for complete description of assay. modulating HEXIM1 expression on E2/ERa–driven transcription in the mammary gland and its implications for estrogen-dependent Data analyses. Statistical significance was determined using Student’s t test comparison for unpaired data and was indicated as follows: *, P < 0.05; breast cancer. **, P < 0.005. Materials and Methods Results Materials and antibodies. 17h-Estradiol (E2) and CDK9 inhibitor 5,6-dichloro-1-h-D-ribofuranosylbenzimidazole
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