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

Ningning Niu,Rui Shao,Guang Yan,Weiguo Zou

doi:10.1074/jbc.m116.749697

Abstract

Small 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 Furthermore, we constructed a zebrafish line in which the enhanced green fluorescent protein (EGFP) expression was driven by col2a1 promoter. The transgenic (col2a1-EGFP) zebrafish line demonstrated that BET inhibitors I-BET151 and (+)-JQ1 may affect EGFP expression in zebrafish. Furthermore, we found that I-BET151 and (+)-JQ1 may affect chondrocyte differentiation in vitro and inhibit zebrafish growth in vivo Mechanistic analysis revealed that BET inhibitors influenced the depletion of RNA polymerase II from the Col2a1 promoter. Collectively, these results suggest that BET bromodomain inhibition may have side effects on skeletal bone structures.

Highlights

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]
To explore novel activators or repressors of chondrogenic differentiation, we generated an ATDC5 cell line that includes the 3-kb human Col2a1 promoter ligated to the open reading frame of firefly luciferase (Fig. 1A)
We checked the responses of Col2a1-luc to the heterocyclic molecule kartogenin (KGN) and transcriptional factor SOX9, which have been widely recognized to promote the expression of Col2a1 and chondrocyte differentiation [24, 25]

Summary

BET Inhibitors Induce Cartilage Destruction

Cytes will switch from the synthesis of the cartilage-characteristic collagens to the synthesis of type I collagen (Col1a1), an osteoblast-characteristic collagen that organizes a mineralizing bone matrix [20, 21]. To further examine the underlying mechanisms for chondrocyte proliferation and growth inhibition, we established a chondrogenic ATDC5 cell line with the Col2a1 promoter (Col2a1-luc ATDC5 system) and screened a list of epigenetic compounds. Six different BET inhibitors were shown to reduce Col2a1-luc, consistent with the decrease of Col2a1 mRNA and protein. We found that BET inhibitors blocked the differentiation of chondrocyte culture. Consistent with this, BET inhibitors I-BET151 and (ϩ)-JQ1 could induce retardation of the growth of zebrafish. Taken together, these data suggest that the Col2a1-luc ATDC5 system may be used to screen chondrogenic factors. These data suggest that the Col2a1-luc ATDC5 system may be used to screen chondrogenic factors These data suggest that anticarcinogenic BET inhibitors may potentially injure chondrocytes and the bones of patients

Results

Histone acetyltransferase

Discussion

Experimental Procedures