Role of transcription factor E4bp4 in glucocorticoid-mediated gene regulation in osteoblasts

Glucocorticoids (GCs) play important roles in the regulation of bone metabolism and the control of inflammatory disease. However, chronic GC treatment inhibits bone formation, induces osteoblast apoptosis, and is a leading cause of osteoporosis. Elucidation of the molecular pathways mediating GC action in osteoblasts is necessary for the development of novel patient therapies to treat inflammatory disease with fewer side effects. Previous studies have shown that GCs induce transcription factor E4bp4 in GC-induced lymphoid cell apoptosis. E4bp4 is a transcriptional repressor and primary response gene in osteoblasts. Studies have also shown that E4bp4 attenuates promoter activity of Cox-2, a GC-repressed gene, in osteoblasts. Thus, we aimed to study E4bp4 function in osteoblasts, shed light on the molecular details of its significance in bone, and perhaps reveal a novel regulatory pathway of GC effects on osteoblasts. Shown here is evidence that GCs induce E4bp4 expression. In turn, E4bp4 plays a role in mediating GC-regulated basal and inflammatory gene expression in osteoblasts and regulating osteoblast differentiation, function, and apoptosis. GCs induced sustained E4bp4 expression by Northern blot and real-time RT-PCR analysis in vitro, ex vivo, and in vivo. E4bp4 is a GC-induced primary response gene, and its induction was dose-dependent. GC-induced specific nuclear protein binding to the E4bp4 response element (EBPRE) contained E4bp4 by EMSAs and supershift assays. GCs attenuated expression of a promoter containing EBPRE sites in osteoblasts. E4bp4 overexpression inhibited osteogenic differentiation in primary osteoblasts and stable MC3T3-E1 cells in alkaline phosphatase assays and decreased expression of extracellular matrix proteins by real-time RT-PCR analysis. E4bp4-overexpressing primary osteoblasts failed to mineralize by Von Kossa staining. E4bp4 increased osteoblast apoptosis and cell death. E4bp4 knockdown by siRNA transfection attenuated GC-induced osteoblast apoptosis and cell death. Furthermore, microarray analysis demonstrated E4bp4 regulation of basal extracellular matrix gene expression in osteoblasts. E4bp4 downregulated inflammatory-induced osteoblastic Cox-2, IL-6, and iNOS expression, and E4bp4 directly bound to EBPRE-like sites in these promoters. We conclude that E4bp4 plays a critical role in osteoblast function and mediates GC effects on osteoblasts.