Estrogen receptor-related receptor alpha (ERR alpha) regulates osteopontin expression through a non-canonical ERR alpha response element in a cell context-dependent manner

We previously demonstrated that the orphan nuclear receptor, estrogen receptor-related receptor alpha (ERR alpha) 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 ERR alpha of the gene for osteopontin (OPN), an extracellular matrix protein required in bone remodeling, and a potential direct target mediating ERR alpha effects in bone. We therefore readdressed OPN gene regulation by ERR alpha in both osteoblastic (rat osteosarcoma ROS 17/2.8 cells) and non-osteoblastic (HeLa) cell lines using a mouse proximal 2 kb OPN promoter fragment. A minimal OPN promoter fragment spanning from -56 to +9 bp is activated in HeLa cells but repressed it in ROS 17/2.8 cells. Adenine scanning mutagenesis revealed the presence of a non-canonical ERR alpha, response element in this minimal promoter. Surprisingly, prototypical inactivating mutations in the activation function 2 (AF2) domain ora naturally occurring allelic variant of ERR alpha (ERR alpha H408) were all better activators than wild-type ERR alpha in HeLa cells, activities that were generally paralleled by repression in ROS 17/2.8 cells. Finally, we found that the N-terminus of ERR alpha harbors a repressor domain that acts in a cell context-dependent manner. We conclude that OPN is an ERR alpha target gene whose promoter is regulated by ERR alpha in a cell context-dependent manner and that a predicted silencing mutation in AF2 or a more flexible helix 12 increases ERR alpha transcriptional activity, effects with implications for ERR alpha as a therapeutic target in bone.