Bromodomain and Extra-terminal (BET) Protein Inhibitors Suppress Chondrocyte Differentiation and Restrain Bone Growth

Abstract

Edited by Xiao-Fan WangSmall molecule inhibitors for bromodomain and extra-terminal (BET) proteins have recently emerged as potential therapeutic agents in clinical trials for various cancers. However, to date, it is unknown whether these inhibitors have side effects on bone structures. Here, we report that inhibition of BET bromodomain proteins may suppress chondrocyte differentiation and restrain bone growth. We generated a luciferase reporter system using the chondrogenic cell line ATDC5 in which the luciferase gene was driven by the promoter of Col2a1, an elementary collagen of the chondrocyte. The Col2a1-luciferase ATDC5 system was used for rapidly screening both activators and repressors of human collagen Col2a1 gene expression, and we found that BET bromodomain inhibitors reduce the Col2a1-luciferase. Consistent with the luciferase assay, BET inhibitors decrease the expression of Col2a1. Bromodomain and extra-terminal (BET)2 proteins (containing four mammalian members, BRD2, BRD3, BRD4, and BRDT) function as key epigenetic readers by recognizing histone acetylation through binding to ⑀-N-lysine acetylation motifs of histone, such as Lys-5, Lys-12, and Lys-16 residues of H4 histone (1, 2) and Lys-27 residues of H3 histone (3). The BET proteins interact with the positive transcription elongation factor P-TEF␤ and RNA polymerase II (Pol II) to facilitate gene transcription (4 -6). A list of drugs, such as I-BET151, I-BET762, PFI-1, and (ϩ)-JQ1, were discovered to target BET proteins and block the interaction between BET proteins and acetyl-lysine of histones (2, 7).BET proteins and BET inhibitors have been reported to be involved in a variety of biological processes. Pharmacological inhibition of BET proteins lowers the expression of key transcription factors such as oncogene c-MYC (8), clamps the transductions of PI3K signaling (9), inhibits Gli1 transcription and Hedgehog pathway (10), blocks transcription in neurons (11), represses VEGF-induced angiogenesis and vascular permeability (12), reduces cell viability of osteosarcoma cells and inhibits osteoblastic differentiation (13), and restrains osteoclastogenesis (14). Meanwhile, potent BET inhibitors have been identified as showing antitumor efficacy in a number of preclinical cancer models in recent years, including leukemia, multiple myeloma, lymphoma, melanoma, and gastric cancer (15,16). This led to clinical studies focusing mostly on the treatment of leukemia and lymphoma. As a result of these studies, the first encouraging signs of efficacy have already been reported (17). Furthermore, previous studies showed that BET inhibitors also control neuronal differentiation and cause an autism-like syndrome (18). However, BET inhibitors were not examined extensively in these studies to determine their side effects on skeletal bone structures.The long bone is mainly formed through endochondral bone formation, which starts with the formation of a cartilage template from condensed mesenchymal cells. The chondrocytes of the cartilage template proliferate axial...

Role of Epigenomics in Bone and Cartilage Disease

“…The proteins that “read” the histone PTMs are also important for cell differentiation. Proteins from the bromodomain and extra‐terminal domain (BET) family recognize and bind to acetylated lysine on histones, forming a scaffold for protein complexes involved in gene transcription . The inhibition of BET proteins leads to a suppression of osteoclast differentiation and activity in vitro .…”

Epigenetic‐Based Mechanisms of Osteoblast Suppression in Multiple Myeloma Bone Disease

“…In these experiments, CPI203 prevented BMSC‐mediated protection from the cytotoxic effects of the drug as well as the increased proliferation of MM cells usually found in BMSC cocultures. Interestingly, BETi suppressed chondrocyte differentiation in vitro and reduced bone growth in vivo in a zebra fish model . BRD4 binds to and upregulates expression of OB‐specific enhancers and matrix‐specific genes during lineage commitment during OB differentiation.…”

Bi-directional regulation of cartilage metabolism by inhibiting BET proteins—analysis of the effect of I-BET151 on human chondrocytes and murine joints

“…It may because of different sources of the chondrocytes in two studies. The chondrocytes in our study were achieved from OA patients who underwent knee replacement surgery, and the chondrocytes in the previous study were achieved from newborn mouse [ 15 ]. Our result suggested that repression of SOX9 contributed to the regulation of I-BET151 on the expression of matrix genes of chondrocytes, but other mechanisms might also occur in this procedure.…”

“…We have found that inhibiting BET led to repression of ACAN and COL2A1 in chondrocytes; it may be another reason for the attenuation of protecting effect at 8 weeks after treatment. The previous report stated that inhibiting BET can restrain bone growth, and there have been some reports indicated the importance of subchondral bone remodeling in etiology of OA [ 15 , 28 , 29 ], so the effect on subchondral bone by inhibiting BET might affect OA progress and needed further evaluation.…”

“…BET protein inhibitors show therapeutic efficacy in cancer and inflammation diseases [ 8 – 12 ]. Recent reports revealed that I-BET151, a BET family protein inhibitor, suppressed the expression of inflammatory genes induced by IL-1β and TNF-α in rheumatoid arthritis synovial fibroblasts and inhibiting BET proteins can ameliorate K/BXN serum-induced arthritis [ 13 , 14 ], while another study suggested that inhibiting BET proteins can suppress chondrocyte differentiation [ 15 ]. There have been reports that inhibiting the inflammatory response induced by IL-1β and TNF-α had protecting effect on OA progress in surgical mouse model of OA [ 16 , 17 ], and anti-inflammatory drugs, such as COX-2 inhibitor and diacerein, can be the common treatment for both rheumatoid arthritis and OA [ 18 , 19 ].…”