Background
Osteoarthritis (OA) is a common multifactorial disorder of the joints and is mainly characterized by the progressive degeneration of articular cartilage. Matrix-degrading enzymes including matrix metalloproteinase (MMP) 1, MMP3, MMP13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 4, and ADAMTS5 played important roles in cartilage degradation in OA [
1]. Among these matrix-degrading enzymes, MMP1, MMP3, MMP13, and ADAMTS4 can be upregulated by proinflammatory cytokines, such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α. Signal transduction pathways, such as the nuclear factor-κB (NF-κB), Jun N-terminal kinase (JNK), and mitogen-activated protein kinase (MAPK) pathways, were involved in the process of activating matrix-degrading enzymes by proinflammatory cytokines [
1‐
3].
Recently, a small molecular inhibitor which could block bromodomain and extraterminal domain (BET) proteins from recognizing acetylated histones (I-BET151) was generated [
4], and we found I-BET151 could abrogate the induction of MMP1, MMP3, MMP13, and ADAMTS4 by IL-1β or TNF-α in chondrocytes [
5]. In mammals, the BET family of proteins consists of ubiquitously expressed bromodomain-containing protein (Brd)2, Brd3, Brd4, and testes/oocyte-specific Brdt. All members of BET proteins contain two conserved tandem bromodomains capable of recognizing acetylated histones, which are involved in transcription regulation through the recruitment of transcriptional regulatory complexes to acetylated chromatin [
6,
7]. There were studies which reported that IL-1β and TNF-α could increase the acetylation of histones within the promoter regions of different genes [
8‐
10]. We suspected that enhanced recruitment of BET proteins to acetylated histones at the promoter regions was responsible for the induction of these matrix-degrading enzymes by IL-1β or TNF-α.
In the present study, we examined the role and mechanisms of BET proteins in the transcriptional regulation of matrix-degrading enzyme genes. We demonstrated that recruitment of BET proteins to acetylated chromatin is a crucial step for the induction of these matrix-degrading enzymes by IL-1β or TNF-α, and only Brd3 and Brd4 had a positive effect in this process.
Discussion
In our study, we found that the depletion of Brd4 and Brd3 by siRNA-mediated knockdown resulted in the reduction of IL1β- or TNFα-induced transcription of MMP1, MMP3, MMP13, and ADAMTS4 in chondrocytes, but depletion of Brd2 did not show such effects. It suggested that both Brd3 and Brd4 were responsible for the IL1β- or TNFα-induced transcription of these genes in chondrocytes.
Increasing amounts of evidence suggest that the recruitment of BET proteins to chromatin is closely associated with their roles in transcriptional regulation [
6,
7]. Here, we found enhanced recruitment of Brd3 and Brd4 to the promoter regions of
MMP1,
MMP3,
MMP13, and
ADAMTS4 after the treatment of IL-1β or TNF-α in chondrocytes, and the recruitment could be alleviated by I-BET151. The recruitment of Brd3 and Brd4 is consistent with the expression of these four genes after the treatment of IL-1β or TNF-α in chondrocytes. So, we suspected that the recruitment of Brd3 and Brd4 to the promoter regions was essential for the induction of
MMP1,
MMP3,
MMP13, and
ADAMTS4 by IL-1β or TNF-α in chondrocytes.
There have been studies which reported that Brd4 could directly interact with positive transcription elongation factor b (p-TEFb) [
14,
15]. p-TEFb is composed of the CDK9 and cyclin T1 (CycT1) subunits, and CDK9 could phosphorylate Ser2 of the RNA polymerase II C-terminal domain (RNAP II CTD), leading to the release of paused RNAP II during transcriptional elongation [
16]. In parallel with enhanced recruitment of Brd3 and Brd4 to the promoter regions of
MMP1,
MMP3,
MMP13, and
ADAMTS4, we found enhanced recruitment of CDK9 and Ser2 phosphorylation of RNAP II CTD at the promoter regions of all the four genes after the treatment of IL-1β or TNF-α in chondrocytes. When we inhibited the combination of BET proteins and acetylated histones by using I-BET151 in chondrocytes, we found that the IL-1β- or TNF-α-induced recruitment of Brd3, Brd4, and CDK9 to the promoter regions and Ser2 phosphorylation of RNAP II CTD at the promoter regions of these genes were abrogated synchronously. Our results conformed to the previous reports that Brd4 could directly interact with p-TEFb and p-TEFb phosphorylated Ser2 of the RNAP II CTD. The results suggested that the recruitment of Brd4, Brd3, and pTEFb to the chromatin and subsequent Ser2 phosphorylation of RNAP II CTD are correlated with the IL-1β- or TNF-α-induced transcriptional expression of
MMP1,
MMP3,
MMP13, and
ADAMTS4 genes in human chondrocytes.
BET proteins directly interact with several different modifications of acetylated histones, including H4 acetylated at Lys5 (H4K5Ac), H4 acetylated at Lys8 (H4K8Ac), and H4 acetylated at Lys12 (H4K12Ac) [
17‐
19]. Here, we also found increased levels of acetylated H4K5/8/12 and enhanced recruitment of Brd3 and Brd4 to the promoter regions of
MMP1,
MMP3,
MMP13, and
ADAMTS4 after the treatment of IL-1β or TNF-α in chondrocytes. Our results suggested that increased levels of acetylated H4K5/8/12 at promoter regions might be responsible for the enhanced recruitment of Brd3 and Brd4 to the promoter regions of these genes after the treatment of IL-1β or TNF-α in chondrocytes.
Acetylation of histones was reported to be involved in the induction of matrix-degrading enzyme genes by proinflammatory cytokines. There were several reports which suggested NF-κB, JNK, and MAPK pathways were involved in the acetylation of histones [
10,
20,
21], and NF-κB, JNK, and MAPK pathways were well known to be involved in the process of activating matrix-degrading enzymes by proinflammatory cytokines [
1‐
3]. TNF could increase the acetylation of histone H3 at the promoter region of another matrix-degrading enzyme, MMP9, and then promote the transcription of
MMP9 [
10]. But the inhibitor of HDAC, which could retain the acetylation of histones, was well known to repress the induction of MMPs by proinflammatory cytokines [
22‐
24]. The effect of the inhibitor of HDAC might be mediated by acetylation of α-tubulin, acetylation of NF-κB p65, and so on [
23,
25]. So, the effect of the acetylation of histones on the induction of matrix-degrading genes by proinflammatory cytokines is still debatable and complicated, and much of the mechanism remains unknown. Our results suggested that increased levels of acetylated H4K5/8/12 at promoter regions of
MMP1,
MMP3,
MMP13, and
ADAMTS4 genes were associated with enhanced transcriptional expression of these genes in chondrocytes after the treatment of IL-1β or TNF-α.
We found the BET proteins, which acted as “readers” of the histone acetylation, were involved in this process. Brd3 and Brd4 were both shown to be essential for the inducible transcription of MMP1, MMP3, MMP13, and ADAMTS4 in chondrocytes after the treatment of IL-1β or TNF-α. Enhanced recruitment of Brd3 and Brd4 to the promoter regions could lead to the enhanced recruitment of p-TEFb to the promoter regions and the subsequent Ser2 phosphorylation of RNAP II CTD, and then lead to the enhanced transcription of these genes after the treatment of IL-1β or TNF-α.
Some limitations should be noted in our study. First, because of the limited test times and variety of ChIP assay result, significance was not detected in several comparisons although the trend and fold change were apparent. And we did not perform an electrophoresis assay to get a qualitative ChIP data, which could show visual differences among different treatment groups. Second, we only checked the transcriptional level of the target genes, and a protein level presentation could make the effect of treatments more convincing in this study. And we only used one effective siRNA for each Brd protein to perform the knockdown treatment, although the knockdown effects have been confirmed by Western assay. Third, we suggested the association between increased levels of acetylated H4K5/8/12 and the enhanced recruitment of Brd3 and Brd4 to the promoter regions depending on the previous reports that BET proteins could directly interact with several different modifications of acetylated histones, including acetylated histones H4 [
17‐
19]. But we did not specifically inhibit the acetylation of histones H4 to evaluate the necessity of acetylated histones H4 in this process. Besides, we cannot deny the enhanced acetylation of other histones at the promoter region or any other mechanisms increasing the affinity of BET proteins to the promoter region as the reason for the enhanced recruitment of Brd3 and Brd4 to the promoter regions of these genes in chondrocytes. The exact binding sites of Brd3 and Brd4 were still unknown in our study, and further structural analysis was needed to understand the exact mechanisms.