Brd4 Inactivation Increases Adenoviral Delivery of BMP2 for Paracrine Stimulation of Osteogenic Differentiation as a Gene Therapeutic Concept to Enhance Bone Healing

Abstract

Bromodomain (BRD) proteins are histone code interpreters that recognize acetylated lysines and link the dynamic state of chromatin with the transcriptional machinery. Here, we demonstrate that ablation of the Brd4 gene in primary mouse bone marrow-derived mesenchymal stem cells via a conditional Brd4 fl/fl allele suppresses osteogenic lineage commitment. Remarkably, loss of Brd4 function also enhances expression of genes in engineered adenoviral vectors, including Cre recombinase and green fluorescent protein (GFP). Similarly, vector-based expression of BMP2 mRNAs and proteins levels are enhanced upon Brd4 depletion in MC3T3-E1 osteoblasts transduced with an adenoviral vector that expresses BMP2 (Ad-BMP2). Importantly, Brd4 depletion in MC3T3-E1 and human adipose-derived mesenchymal stem cells (AMSCs) transduced with Ad-BMP2 enhances osteogenic differentiation of naïve MC3T3-E1 cells via paracrine mechanisms based on transwell and conditioned medium studies. Our studies indicate that Brd4 depletion enhances adenoviral transgene expression in mammalian cells which can be leveraged as a therapeutic strategy to improve viral vector-based gene therapies.

The epigenetic regulator BRD4 is required for myofibroblast differentiation of knee fibroblasts

“…Because we previously showed that BRD4 is required for osteogenic and chondrogenic differentiation and skeletal formation, [37][38][39] we sought to determine whether the effects of BET inhibition (i.e., JQ1) on myofibroblast differentiation can be mirrored by BRD4 depletion (Figure 5A). Robust siRNAmediated BRD4 depletion is confirmed by RT-qPCR (Figure 5B) and western blot analysis (Figure 6, Supporting Information: Figure 2B) 4 days after transfection in all three primary cell lines.…”

“…While BET-targeting agents such as JQ1 alter activities of several BET proteins, our present target validation focused on BRD4 as our recent studies revealed a unique role for this protein in mesenchymal lineage differentiation and skeletal formation. [37][38][39] Although BRD2 mRNA levels are higher in preosteoblasts, BRD4 depletion was more robust at inhibiting osteogenic commitment when compared to BRD2 depletion, 38 suggesting that BRD4 is a critical epigenetic regulator of lineage allocation of mesenchymal progenitors. The contribution of BRD4 on the differentiation of mesenchymal progenitors is further supporter by studies showing the allocation of BRD4 to lineage-specific genes after the administration of differentiation stimuli.…”

“…[34][35][36] We previously showed that BRD4 supports chondrocyte and osteoblast differentiation and its activity is required for proper skeletal formation. [37][38][39] These findings provided the opportunity to examine whether BRD4 activity is required for the myofibroblastic differentiation of knee fibroblasts, which similar to osteoblasts and chondrocytes are of mesenchymal origin, and support formation of the collagen-rich tissue that is observed in the knees of patients affected by arthrofibrosis.…”

“…Within this class of roughly 40 BRD proteins, a subset of 4 proteins known as the bromodomain and extraterminal (BET) proteins (i.e., BRD2, BRD3, BRD4, and BRDT) bind acetylated lysines, interact with the transcriptional machine, and are readily targeted by small compounds knows as BET inhibitors 34–36 . We previously showed that BRD4 supports chondrocyte and osteoblast differentiation and its activity is required for proper skeletal formation 37–39 . These findings provided the opportunity to examine whether BRD4 activity is required for the myofibroblastic differentiation of knee fibroblasts, which similar to osteoblasts and chondrocytes are of mesenchymal origin, and support formation of the collagen‐rich tissue that is observed in the knees of patients affected by arthrofibrosis.…”