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.

Fig. 1.

Comparison of gene expression patterns between ERKO and NERKI flushed bone samples using microarray analysis. A, The Venn diagram represents the intersection the genes regulated in either ERKO or NERKI bone samples, categorized as ERKO unique, NERKI unique, or common, the latter of which represents genes commonly regulated in the ERKO and NERKI data sets. B, The fold changes of the commonly regulated data set in both ERKO and NERKI samples (both relative to WT) were plotted on the x- and y-axes, respectively. Each dot on the graph represents a gene from the commonly regulated list.

Fig. 2.

QPCR analysis of individually significant genes (using Student's t test) within statistically significant pathways (using Hotelling's T squared distribution) for genes in the osteoblast differentiation (A), osteocyte differentiation (B), and apoptosis pathways (C) are graphed. The bars represent the fold change between ERKO and WT ± se and statistically significant differences of P ≤ 0.05 (Student's t test) are indicated with an asterisk.