Regulation and Function of FOXC1 in Osteoblasts.

Estrogens, which bind to estrogen receptor alpha (ERα), are important for proper bone mineral density. When women go through menopause, estrogen levels decrease, and there is a decrease in bone quality, along with an increased risk for fractures. We previously identified an enhancer near FOXC1 as the most significantly enriched binding site for estrogen receptor alpha (ERα) in osteoblasts. FOXC1 is a transcription factor belonging to a large group of proteins known as forkhead box genes and is an important regulator of bone formation. Here, we demonstrate that 17β-estradiol (E2) increases the mRNA and protein levels of FOXC1 in primary mouse and human osteoblasts. GATA4 is a pioneer factor for ERα and it is also recruited to enhancers near Foxc1. Knockdown of Gata4 in mouse osteoblasts in vitro decreases Foxc1 expression as does knockout of Gata4 in vivo. Functionally, GATA4 and FOXC1 interact and regulate osteoblast proteins such as RUNX2, as demonstrated by ChIP-reChIP and luciferase assays. The most enriched motif in GATA4 binding sites from ChIP-seq is for FOXC1, supporting the notion that GATA4 and FOXC1 cooperate in regulating osteoblast differentiation. Together, these data demonstrate the interactions of the transcription factors ERα, GATA4, and FOXC1 to regulate each other's expression and other osteoblast differentiation genes.