Examination of ERα Signaling Pathways in Bone of Mutant Mouse Models Reveals the Importance of ERE-Dependent Signaling

Abstract

The mechanisms of estrogen receptor (ER)-α activity can be categorized into those involving direct (classical) or indirect (nonclassical) DNA binding. Although various mouse models have demonstrated the importance of ERα in bone, the specific gene expression patterns affected by these modes of ERα action are unknown. In this report, the gene expression patterns of ERα-deficient (ERKO) mice and nonclassical ER knock-in (NERKI) mice, which can function only by nonclassical means, were analyzed. Three-month-old mice were ovariectomized and implanted with estrogen pellets for 1 month to normalize estrogen levels. Microarray analysis of flushed cortical bone revealed 28% (210 of 763) of the genes differentially expressed in ERKO mice were altered in NERKI mice, suggesting estrogen response element-dependent regulation of these genes in bone. Pathway analysis revealed alterations in genes involved in focal adhesion and extracellular matrix interactions. However, the majority of genes regulated in ERKO mice (72%) were unique (i.e. not altered in NERKI mice), suggesting these are regulated by nonclassical mechanisms. To further explore the pathways affected in ERKO mice, we performed focused quantitative PCR arrays for genes involved in various aspects of bone physiology. Genes involved in bone formation, senescence, apoptosis, and autophagy were significantly regulated. Overall, the majority of the genes regulated by ERα in bone are via nonclassical pathways. However, because NERKI mice display an osteoporotic phenotype, it can be deduced that the minority of the estrogen response element-dependent genes/pathways play critical roles in the regulation of bone physiology. These data demonstrate the importance of classical ERα signaling in regulating bone metabolism.

27-Hydroxycholesterol, an endogenous selective estrogen receptor modulator

“…(2) Mice harboring a mutant ERα that fails to bind DNA but should retain its non-genomic actions (non-classical ER knock-in, NERKI). Although both approaches suggest the potential importance of non-classical ER signaling, it should be noted that they do not always agree [18, 19], and this continues to be an emerging field. In vitro studies in support of non-classical ER signaling indicate that ERs can interact with and modulate the activities of MAPK, adaptor protein Shc, caveolins, c-Src protein kinase complex and the regulatory subunit of phosphoinositide-3 kinase (p85) [20, 21].…”

Iron overload increases osteoclastogenesis and aggravates the effects of ovariectomy on bone mass

“…Estrogen is the predominant female steroid hormone and a pivotal regulator in bone formation and bone resorption processes (Chokalingam et al 2012). …”

The molecular mechanisms underlying the pharmacological actions of estrogens, SERMs and oxysterols: Implications for the treatment and prevention of osteoporosis

“…The same polymeric form of estradiol was able to prevent ovariectomy-associated loss of cortical bone but not trabecular bone in mice, indicating a potential role for non-genomic signaling of E2 in this compartment [55]. However, studies in mice transgenic for a mutant ERα that fails to bind DNA but should retain its non-genomic actions (nonclassical ER knock-in, NERKI), had an osteoporotic phenotype [56]. Further, the gene expression profiles between traditional ERα knockout mice and the NERKI mice were very similar, most likely due to the failure to bind EREs and modulate transcription [56].…”

“…However, studies in mice transgenic for a mutant ERα that fails to bind DNA but should retain its non-genomic actions (nonclassical ER knock-in, NERKI), had an osteoporotic phenotype [56]. Further, the gene expression profiles between traditional ERα knockout mice and the NERKI mice were very similar, most likely due to the failure to bind EREs and modulate transcription [56]. Therefore, it is clear that further studies are required to clarify the precise roles of non-genomic actions of ER in bone.…”