Estrogen receptor-related receptor a (ERRa) regulates osteopontin expression through a non-canonical ERRa response element in a cell context-dependent manner Ralph A Zirngibl, Janet S M Chan and Jane E Aubin Department of Molecular Genetics, Faculty of Medicine, University of Toronto, 1 Kings College Circle, Medical Sciences Building Room 6230, Toronto, Ontario M5S 1A8, Canada (Correspondence should be addressed to J E Aubin; Email: jane.aubin@utoronto.ca) Abstract We previously demonstrated that the orphan nuclear receptor, estrogen receptor-related receptor a (ERRa) is highly expressed in osteoblasts and osteoclasts, regulates osteogenesis and expression of osteoblast-associated markers in the rat calvaria cell differentiation system, and is dysregulated in the rat ovariectomy model of postmenopausal osteoporosis. There are conflicting published data on the transcriptional regulation by ERRa of the gene for osteopontin (OPN), an extracellular matrix protein required in bone remodeling, and a potential direct target mediating ERRa effects in bone. We therefore readdressed OPN gene regulation by ERRa in both osteoblastic (rat osteosarcoma ROS17/2.8 cells) and non-osteoblastic (HeLa) cell lines using a mouse proximal 2 kb OPN promoter fragment. A minimal OPN promoter fragment spanning from K56 to C9 bp is activated in HeLa cells but repressed it in ROS17/2.8 cells. Adenine scanning mutagenesis revealed the presence of a non-canonical ERRa response element in this minimal promoter. Surprisingly, prototypical inactivating mutations in the activation function 2 (AF2) domain or a naturally occurring allelic variant of ERRa (ERRaH408) were all better activators than wild-type ERRa in HeLa cells, activities that were generally paralleled by repression in ROS17/2.8 cells. Finally, we found that the N-terminus of ERRa harbors a repressor domain that acts in a cell context-dependent manner. We conclude that OPN is an ERRa target gene whose promoter is regulated by ERRa in a cell context-dependent manner and that a predicted silencing mutation in AF2 or a more flexible helix 12 increases ERRa transcriptional activity, effects with implications for ERRa as a therapeutic target in bone. Journal of Molecular Endocrinology (2008) 40, 61–73 Introduction Nuclear receptors (NRs) are structurally and functionally modular with the N-terminal A/B domain regulating transcriptional activity in a ligand-indepen- dent fashion (activation function 1 (AF1)) and a conserved centrally located DNA-binding domain (DBD) composed of two zinc fingers. The DBD allows the NR to control transcription directly by binding to cognate response elements found in a subset of promoters or indirectly by tethering to other transcrip- tion factors (Kushner et al. 2000, Safe et al. 2004). The C-terminal ligand/hormone-binding domain (HBD) is composed of 12 a-helices that form a centrally located ligand-binding pocket, with helix 12 harboring the ligand-dependent AF2 (Bourguet et al. 2000). NRs are controlled by a large and increasing number of co-repressors and co-activators, the balance of which determines transcriptional activity (Privalsky 2004, Spiegelman & Heinrich 2004). Generally, co-repressors bind to a hydrophobic surface in the HBD and are released due to a conformational shift involving helices 3, 5, and 12 upon ligand binding, which repositions the AF2 domain, closing the ligand-binding pocket to form a new hydrophobic binding surface allowing co-activa- tors to bind (Nettles & Greene 2005). The binding of the co-activator to the NR is greatly stabilized by a charge clamp involving a conserved lysine (K) in helix 3 and either a glutamic acid (E) or aspartic acid (D) found in the AF2 domain (Li et al. 2003). Recent evidence has pointed to an important role for the A/B domain in binding the co-activators and interactions with the C-terminus of the NR (Schaufele et al. 2005). The estrogen receptor-related receptor (ERR) subfamily of NRs comprises three distinct genes (ERRa (nuclear receptor 3b;NR3B1), ERRb (NR3B2), and ERRg (NR3b3)) most closely related to the estrogen receptor (ER) subfamily (ERa (NR3A1) and ERb (NR3A2)) with 68% identity in their DBD (Nuclear Receptors Nomenclature Committee 1999, Giguere 2002). ERRs bind to a response element (ERRE) with the consensus sequence 5 0 -TCAAGGTCA-3 0 , while the ER response element (ERE) comprises two inverted repeats of 5 0 -AGGTCA-3 0 separated by three nucleotides 61 Journal of Molecular Endocrinology (2008) 40, 61–73 DOI: 10.1677/JME-07-0114 0952–5041/08/040–061 q 2008 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 02/18/2023 09:43:21AM via free access