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Estrogenically Regulated LRP16 Interacts with Estrogen Receptor a and Enhances the Receptor’S Transcriptional Activity

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Estrogenically Regulated LRP16 Interacts with Estrogen Receptor a and Enhances the Receptor’S Transcriptional Activity Endocrine-Related Cancer (2007) 14 741–753 Estrogenically regulated LRP16 interacts with estrogen receptor a and enhances the receptor’s transcriptional activity W-D Han1, Y-L Zhao1, Y-G Meng 2, L Zang 3, Z-Q Wu1,QLi1, Y-L Si1, K Huang 2, J-M Ba3, H Morinaga4, M Nomura4 and Y-M Mu3 Departments of 1Molecular Biology, Institute of Basic Medicine, 2Obstetrics and Gynecology and 3Endocrinology, Chinese PLA General Hospital, Beijing 100853, China 4Department of Medicine and Bioregulatory Science, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan (Correspondence should be addressed to Y-M Mu; Email: [email protected]) Abstract Previous studies have shown that leukemia related protein 16 (LRP16) is estrogenically regulated and that it can stimulate the proliferation of MCF-7 breast cancer cells, but there are no data on the mechanism of this pathway. Here, we demonstrate that the LRP16 expression is estrogen dependent in several epithelium-derived tumor cells. In addition, the suppression of the endogenous LRP16 in estrogen receptor a (ERa)-positive MCF-7 cells not only inhibits cells growth, but also significantly attenuates the cell line’s estrogen-responsive proliferation ability. However, ectopic expression of LRP16 in ERa-negative MDA-MB-231 cells has no effect on proliferation. These data suggest the involvement of LRP16 in estrogen signaling. We also provide novel evidence by both ectopic expression and small interfering RNA knockdown approaches that LRP16 enhances ERa-mediated transcription activity. In stably LRP16-inhibitory MCF-7 cells, the estrogen-induced upregulation of several well-known ERa target genes including cyclin D1 and c-myc is obviously impaired. Results from glutathione S-transferase pull-down and coimmuno- precipitation assays revealed that LRP16 physically interacts with ERa in a manner that is estrogen independent but is enhanced by estrogen. Furthermore, a mammalian two-hybrid assay indicated that the binding region of LRP16 localizes to the A/B activation function 1 domain of ERa. Taken together, these results present new data supporting a role for estrogenically regulated LRP16 as an ERa coactivator, providing a positive feedback regulatory loop for ERa signal transduction. Endocrine-Related Cancer (2007) 14 741–753 Introduction (ERa and b; Mangelsdorf et al. 1998, McDonnell & Estrogen plays a crucial role in the control of Norris 2002). The activation of an ER results in an development, sexual behavior, and reproductive altered expression of its direct transcriptional targets, functions. Its effects have been linked to the thereby affecting downstream secondary biological progression of the majority of human breast cancers activities. It is believed that 75% of carcinoma and acts as a potent mitogen for many breast in situ breast lesions and similar percentages of cancer cell lines (Masood 1992, Prall et al. invasive breast cancers express elevated levels of 1998a). Mechanistic studies on estrogen-induced ERa protein; whereas in normal breast epithelial mitogenesis have revealed multiple cell cycle cells, only 10% of cells express ERa and only at low regulatory pathways activated by estrogen (Foster levels (Allred et al. 2001). Patients expressing & Wimalasena 1996, Planas-Silva & Weinberg 1997, elevated levels of ERa are therefore suitable Prall et al. 1997). The biological actions of estrogen candidates for hormonal therapy, which aims to are manifest through the transcriptional activation block estrogen stimulation of breast cancer cells of the ligand-dependent estrogen receptor a and b (Deroo & Korach 2006). Endocrine-Related Cancer (2007) 14 741–753 Downloaded from Bioscientifica.comDOI:10.1677/ERC-06-0082 at 09/27/2021 01:57:07AM 1351–0088/07/014–741 q 2007 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.orgvia free access W-D Han et al.: Modulation of ERa signaling by LRP16 ERa contains six domains named A through F. The of G1 to S (Han et al. 2003). In primary breast cancer structure of ERa is similar to that of other nuclear samples, our data indicated that LRP16 mRNA was receptors (NRs), having three separate functional overexpressed in nearly 40% of all samples. The domains; a variable activation function 1 domain clinicopathologic characteristics, including ERa/PR (AF-1) in the N-terminal A/B domain, a central DNA- status, tumor diameter, and the involvement of axillary binding domain (LBD, ligand-binding domain) located lymphoid nodes were tightly linked with LRP16 in the C domain and a C-terminal LBD, domain E that mRNA overexpression (Liao et al. 2006). Taken contains AF-2 (Nilsson et al. 2001). The AF-1 domain together, these data implied that LRP16 may play an is regulated by growth factors and its activity depends important role in carcinogenesis and/or progression of on the cellular and promoter context, while the AF-2 hormone-dependent breast cancer. domain is dependent on ligand binding (Lees et al. The goal of this study was to gain an understanding 1989, Shao & Brown 2004). The two activating of the functional role of LRP16 in hormone-sensitive domains act synergistically to generate maximum breast cancer cells. To do this, we investigated the transcriptional activity of ERa (Danielian et al. 1992, expression of LRP16, its interaction with ERa and its Me´tivier et al. 2001). ERa-mediated transcription is a effect on ERa-mediated signaling. First, we showed highly complex process involving multiple of coregu- that both mRNA and protein expression levels of latory factors (Hall et al. 2001, Hall & McDonnell LRP16 are positively linked with estrogen action in 2005). The cofactors can be broadly divided into several breast cancer cell lines. Next, the proliferation subgroups of either coactivator which augments the rate of ERa-positive MCF-7 cells, but not ERa- function of an activated receptor or corepressors which negative MDA-MB-231 cells, was shown to be related sustain the inactivated state of a receptor. Transcrip- to LRP16 expression levels. Furthermore, we showed tional activation of ERa involves interactions with that LRP16 enhanced the transcriptional activity of coactivator molecules and the basal transcription ERa. This provided evidence that LRP16 is not only a machinery in a sequential and cyclic fashion at the target gene of ERa but also a novel ERa coactivator, promoter region of responsive genes (Glass & thus creating a positive regulatory feedback loop. Rosenfeld 2000, Shang et al. 2000, McKenna & Therefore, LRP16 may play an important role in the O’Malley 2002, Me´tivier et al. 2003). The balance of proliferation of ERa-positive breast cancer cells by ERa cofactors in target cells, coupled with different amplifying the function of ERa. concentrations of ERa and its ligand, allows fine- tuning of target gene transcription in response to estrogen. Over the past decade, more than 20 Materials and methods coactivator molecules have been identified using Cell culture biochemical approaches as well as yeast two-hybrid All cell lines were purchased from American Type screens (Klinge 2000, Shao & Brown 2004). Most of Culture Collection (ATCC, Rockville, MD, USA) and the coactivators bind to the ERa AF-2 domain in a cultured according to the instructions. Pure estrogen ligand-dependent fashion. Only two related coactiva- antagonist ICI 182 780 was provided by Dr Qinong Ye tors specific to ERa AF-1 have been identified to date, (College of Military Medicine Science of China). E p68 and p72 RNA helicases (Endoh et al. 1999, 2 was purchased from Sigma. Phenol red-free DMEM Watanabe et al. 2001). The LRP16 gene was originally and estrogen-deprived fetal calf serum (FCS) were isolated from human lymphocyte cells, and was prepared as described previously (Zhao et al. 2005). identified as an estrogen-responsive gene by our group (Han et al. 2003). We have previously Cell proliferation assay documented that 17b-estradiol (E2) upregulated the leukemia related protein 16 (LRP16) mRNA level in an Cells were seeded at 1!104 per well in 24-well plates. ERa-dependent manner in MCF-7 human breast After cell attachment, the medium was replaced with cancer cells (Han et al. 2003). One-half of the estrogen 1 ml fresh DMEM supplemented with 1% FCS. For each response element (ERE)/GC-rich Sp1-binding site and cell line, cells from three dishes were counted daily to several GC-rich Sp1-binding sites within the promoter generate proliferation curves. For the E2-stimulated region of LRP16 gene were identified as the main proliferation experiment, a total of 5000 cells were contributors to the E2-responsive regulatory seeded and cultured in each well of a 24-well plate in mechanism (Zhao et al. 2005, Si et al. 2006). phenol red-free DMEM supplemented with charcoal- Overexpression of LRP16 significantly stimulated stripped FCS (5%). After 12 h, cells were treated with E2 MCF-7 cell proliferation by promoting the transition in the same fresh medium for 24 h. Cell proliferation rate Downloaded from Bioscientifica.com at 09/27/2021 01:57:07AM via free access 742 www.endocrinology-journals.org Endocrine-Related Cancer (2007) 14 741–753 was quantified using CellTiter 96 Aqueous One Solution pACT-ERa-DEF vectors. All the constructions Cell Proliferation Assay (Promega). Each experiment were confirmed by restriction enzyme mapping and was performed in triplicate and repeated on three DNA sequencing. occasions. Viral production and infection of target cells Plasmids 293GP2 cells were plated into 10 cm dishes and The mU6pro vector containing the mouse U6 snRNA cultured until reaching 80–90% confluency. pLPC- promoter was kindly provided by Dr Turner from based plasmids (7.5 mg/well) and VSVG expression University of Michigan (Yu et al. 2002). Mammalian vector (2.5 mg/well) were transiently cotransfected into expression plasmid for ERa (pS5G-hERa)was 293GP2 cells using Superfect transfection reagent provided by Prof. Hajime Nawata at Kyushu (Qiagen) according to manufacturer’s protocol.
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