Abstract
Binding of the bromodomain and extra-terminal domain proteins (BETs) to acetylated histone residues is critical for gene transcription. This study sought to determine the anti-fibrotic efficacy and potential mechanisms of BET inhibition in systemic sclerosis (SSc). Blockade of BETs was done using a pan BET inhibitor JQ1, BRD2 inhibitor BIC1, or BRD4 inhibitors AZD5153 or ARV825. BET inhibition, specifically BRD4 blockade, showed anti-fibrotic effects in an animal model of scleroderma and in patient-derived diffuse cutaneous (dc)SSc fibroblasts. Transcriptome analysis of JQ1-treated dcSSc fibroblasts revealed differentially expressed genes related to extracellular matrix, cell cycle, and calcium signaling. The anti-fibrotic effect of BRD4 inhibition was at least in part mediated by downregulation of Ca2+/calmodulin-dependent protein kinase II α (CaMKII-α) and reduction of intracellular calcium concentrations. These results suggest that targeting calcium pathways or BRD4 might be novel therapeutic approaches for progressive tissue fibrosis.
Introduction
Systemic sclerosis (scleroderma, SSc) is an autoimmune disease characterized by vascular dysfunction as well as excessive synthesis and deposition of extracellular matrix (ECM) in affected organs. Activation of the immune system and vasculopathy precede fibrosis, in which fibroblast activation and subsequent myofibroblast transdifferentiation are necessary events. At present, SSc is not curable; current treatments focus on managing disease manifestations in an effort to ease the progression of tissue fibrosis.
Although the exact etiology of the disease is not known, a growing body of literature has been pointing to the critical involvement of epigenetic mechanisms in SSc pathogenesis (1). Epigenetic regulation affects chromatin dynamics and thereby modulates gene transcription. In addition to DNA methylation and non-coding RNAs, histone changes are implicated in SSc fibroblast activation (2–4). Acetylation of histones on lysine residues is one of the most common histone modifications that relaxes the chromatin structure by loosening the histone-DNA interaction, which results in increased chromatin accessibility for transcription. Dysregulation of histone acetylation could result in aberrant gene expression leading to pathogenic consequences. These histone marks are therefore tightly regulated by a set of histone acetyltransferases and histone deacetylases. They are also controlled by proteins containing the bromodomain module, such as the bromodomain extra-terminal domain (BET) family proteins. The four members of the BETs, BRD2, BRD3, BRD4, and the testis-specific BRD-t, share a common domain consisting of two N-terminal bromodomains, BD1 and BD2, that bind to acetylated lysine residues on histones. These histone readers provide scaffolds toattract components of the transcriptional machinery to histone acetylation marks. Pharmacological inhibition of BET proteins results in repression of downstream gene expression, thereby modulating various physiological conditions. Indeed, prototype BET inhibitors such as JQ1 or I-BET attenuated various types of cancer and tissue fibrosis (5–9).
Considering the potential anti-fibrotic properties of BET inhibition, we investigated whether BET inhibitor JQ1 can modulate fibrogenesis in an animal model of SSc, as well as in dermal fibroblasts isolated from patients with diffuse cutaneous SSc (dcSSc). We posit that BET inhibition impedes the expression of pro-fibrotic genes and blocks myofibroblast differentiation in SSc, thereby improves fibrosis. Through whole transcriptomic analysis we revealed a novel mechanism by which BET inhibition exerts its anti-fibrotic effect in SSc. Further, we demonstrated the involvement of BRD4 in mediating the pro-fibrotic effect of BETs and identified BRD4 as a novel therapeutic target for this disease. The novelty of our study is that we performed a comprehensive analysis by employing both functional assays and transcriptomic analysis that allowed us to pinpoint a novel mechanism where BET inhibition affects intracellular calcium and related signaling in SSc fibroblasts. We also showed the anti-fibrotic properties of two novel BRD4 inhibitors, one (AZD5153) currently evaluated in a clinical trial (NCT03205176). The effect of these inhibitors has not been examined in other fibrotic models before.
Results
The anti-fibrotic effects of JQ1 in bleomycin-treated mice
To investigate the anti-fibrotic effect of BET inhibition, we used a pan-BET inhibitor, JQ1, in an animal model of fibrosis. We observed approximately 3.5% weight reduction in the bleomycin/vehicle group and a 11.4% weight reduction in the bleomycin/JQ1 group. All animals were active without apparent distress throughout the course of the experiment. Daily injections of bleomycin in a defined area in the back of mice increased dermal thickness and collagen accumulation (Figure 1A). Daily JQ1 oral gavage prevented skin fibrosis in bleomycin-treated mice, as significantly attenuation of dermal thickness and collagen was observed. In addition, immunofluorescent staining revealed increased αSMA- and F4/80-positive cells in bleomycin-treated mice, with F4/80 staining reaching statistical significance (Figure 1B). In addition, JQ1 treatment reduced αSMA- and F4/80-positive cells, with αSMA staining reaching statistical significance. These results were also reflected at the mRNA level, as pro-fibrotic genes including Acta2 and Col1a1 that were significantly elevated in bleomycin-treated mice, were downregulated in the presence of JQ1 (Figure 1C). Bleomycin also induced significant increase in Il6 and Ccl2, and both were significantly downregulated with JQ1 treatment. In contrast, Ctgf and Cxcl10 were not significantly altered by bleomycin or JQ1.
Anti-fibrotic properties of BET inhibition in dermal fibroblasts
To further evaluate the effect of BET proteins in SSc fibrosis, we treated dcSSc fibroblasts with JQ1 at various doses (Supplemental Figure 1). Given that the highest dose (22 µM) had minimal effect on CTGF while upregulated TGFB1, suggesting a potential off-targeteffect, we found that 1 μM appears to be the optimal dose to use. We evaluated the expression of pro-fibrotic genes COL1A1, ACTA2, CTGF, and TGFB1, as well as collagen-degrading enzyme MMP1 and its inhibitor TIMP3. Inhibition of BET by JQ1 led to a dose-dependent decrease in fibrotic markers including COL1A1, ACTA2, and CTGF (Supplemental Figure 1 and Figure 2A). In addition, JQ1 also increased MMP1, which is critical for collagen turnover, while it had minimal effect on TIMP3. Interestingly, JQ1 significantly increased TGFB1. This might be an off-target effect of the inhibitor at high doses.
To further characterize the anti-fibrotic effect of BET inhibition in dcSSc fibroblasts, we performed several functional assays. Treatment with JQ1 dose-dependently reduced migration and proliferation of dcSSc fibroblasts (Figure 2B and 2C). The mechanism of reduction of cell proliferation by JQ1 might be in part due to its effect on inducing apoptosis, as JQ1 dose dependently increased apoptotic cells as indicated by green fluorescence released by activated caspase-3/7 using IncuCyte® (Supplemental Figure 2A). However, this effect appears to be delayed, as apoptotic cells appeared after 40 hours while differences in cell proliferation became evident after 30 hours of JQ1 treatment (Figure 3C right panel vs. Supplemental Figure 2A). This suggests that other mechanisms are involved in the effect of JQ1 on cell growth. To further analyze the effect of JQ1 on cell growth, we used Vybrant DyeCycle Violet stain to analyze cell cycle distribution in dcSSc fibroblasts. JQ1 induced a reduction in cells in the S-phase and accumulation of cells in the G1/G0 phase (Supplemental Figure 2B). Treatment of dcSSc fibroblasts with JQ1 also resulted in decrease in gel contraction (Figure 2D), with 0.5 µM being the most effective concentration.
Since TGFβ is critical in promoting fibrosis in SSc, we treated normal dermal fibroblasts with TGFβ to induce a myofibroblast phenotype. This was confirmed with an increase in cell migration, proliferation, as well as gel contraction in these TGFβ-treated normal fibroblasts compared to non-treated controls (Figure 2E-G). Co-treatment with JQ1 significantly reduced TGFβ-induced migration, proliferation, and gel contraction in normal dermal fibroblasts.
Transcriptome analysis
To further explore the mechanisms involved in BET inhibition in SSc fibrosis, mRNA-seq was performed on dcSSc fibroblasts in the absence or presence of 1μM JQ1. This analysis identified 2113 upregulated and 3499 downregulated genes affected by JQ1 (Figure 3A and Supplemental Figure 3). The excess number of downregulated genes with BET inhibition indicates that BET proteins primarily act as transcription activators in SSc fibroblasts, echoing the findings in other tissues indicating that histone readers are indeed transcription activators. The top 20 upregulated and downregulated genes are shown in Figure 3B. In addition, RNA-seq results confirmed expression changes observed using qPCR in fibrosis-related genes (Figure 2A), where ACTA2 and COL1A1 were significantly downregulated while TGFB1 and BRD2 were significantly upregulated at 1μM of JQ1 treatment (Supplemental Figure 4). Further pathway enrichment analysis of the differentially expressed genes regulated by JQ1 revealed that these genes are enriched in KEGG pathways including calcium signaling pathway, cytokine-cytokine receptor pathway, MAPK and Rap1 signaling pathways, as well as metabolic pathways, among others (Figure 3C and Supplemental Table 1). In addition, gene ontology analysis showed that BET proteinsare involved in a wide spectrum of cellular components, biological processes, and molecular functions known to play critical roles in fibroblast activation (Figure 3D). These include ECM, plasma membrane, and signaling receptor activity. To better visualize the network of functional categories represented by JQ1-associated changes, we performed additional functional enrichment analysis using ClueGO/CluePedia by incorporating the KEGG, REACTOME, and WikiPathways. As shown in Figure 3E, networks related to cell cycle regulation, ECM organization, signaling by Rho GTPases, diseases of glycosylation, ferroptosis, and homeostasis, among others, are significantly enriched. We also examined the subcellular localization of the enriched pathways using the cerebral layout tool implemented in Cytoscape. The subcellular localization of JQ1-affected pathways was skewed toward extracellular and plasma membrane (Supplemental Figure 5). This is not surprising, as functions related to ECM and homeostasis, as well as signaling pathways, were among the most enriched (as demonstrated by the size of the nodes). Indeed, when the differentially expressed genes were overlaid on the KEGG: 04512 ECM-receptor interaction pathway, many ECM genes and their binding partners were affected by JQ1, with majority of these genes downregulated (Supplemental Figure 6). In the intracellular compartment and the nucleus, cell cycle regulation is the main enriched pathway (Supplemental Figure 5). These results echo our functional data shown in Figure 2 and Supplemental Figure 2, as JQ1 showed strong anti-proliferative and anti-fibrotic effects in these cells. Finally, as expected, the predicted upstream regulator analysis showed JQ1 as the most significant upstream chemical, as 1521 genes from our transcriptomic analysis overlapped with reported JQ1-target genes (Supplemental Figure 7). This not onlyvalidates the JQ1 inhibition condition used and sequencing analysis employed in this study, but also shows that genes affected by BET inhibition are common among different biological systems and cell types.
BET inhibition affects the calcium signaling pathways in dcSSc fibroblasts
As shown in Figure 3C, the most significant enrichment of the differentially expressed genes in the KEGG pathways was the calcium signaling pathway. To better visualize the location of the differentially expressed genes, we overlaid them in the pathway. JQ1 appears to affect genes involved in calcium (Ca2+) channels on the plasma membrane, Ca2+ receptors that are located in the endoplasmic/sarcoplasmic reticulum, Ca2+-related signaling pathways, as well as Ca2+-dependent downstream effectors (Figure 4A). All of the differentially expressed genes enriched in this pathway were listed in Figure 4B. 15 out of the 59 genes were upregulated by JQ1, again pointing BET proteins to be transcriptional activators. As calcium signaling affects fibroblast function and myofibroblast transformation (10), we examined whether JQ1 affects intracellular Ca2+ concentration in dcSSc fibroblasts. As shown in Figure 4C, JQ1 significantly reduced intracellular Ca2+ levels in dcSSc fibroblasts.
JQ1 mediates its anti-fibrotic effect in part by affecting calcium signaling in dcSSc fibroblasts
To further examine the involvement of intracellular Ca2+ in the anti-fibrotic effect of JQ1, we cultured dcSSc fibroblasts in the presence or absence of Ca2+, and treated the cells with or without JQ1. Comparing the non-treated groups in the presence or absence of Ca2+ in culture media, the expression of ACTA2 and COL1A1was significantly lower in cells cultured in media without Ca2+ (Figure 5A). JQ1 significantly downregulated ACTA2 and COL1A1 in Ca2+-containing media, while its effect on COL1A1 was diminished in media without Ca2+. Using BAPTA-AM, an intracellular Ca2+-chelating reagent, we also showed that depleting Ca2+ in cells decreased ACTA2 and COL1A1 expression, and the inhibitory effect of JQ1 on these genes was blocked (Figure 5B). To further demonstrate the involvement of Ca2+ in the anti-fibrotic effect of JQ1, we focused on CAMK2A, which encodes Ca2+/calmodulin-dependent protein kinase II α (CaMKII-α), in subsequent studies. This gene is central for Ca2+-mediated effects in cells and was significantly downregulated by JQ1 (Figure 4A-B). We further confirmed the effect of JQ1 on CAMK2A expression by qPCR (Figure 5C). Overexpression of CAMK2A in dcSSc fibroblasts blocked the inhibitory effect of JQ1 on COL1A1 expression (Figure 5C) but not on ACTA2. Since CaMKII is involved in cell cycle (11), we examined whether CAMK2A is involved in JQ1-mediated anti-proliferative effect in dcSSc fibroblasts. Indeed, we showed that CAMK2A appears to be involved, at least in part, in the anti-proliferative effect of JQ1, as overexpression of CAMK2A resulted in approximately 25% reduction in cell growth by JQ1 compared to 50% in the control group (Figure 5D). Notably, CAMK2A overexpression in dcSSc fibroblasts not only resulted in elevated levels of ACTA2 and COL1A1 but also increased cell proliferation, suggesting that this gene plays a critical and non-redundant role in promoting SSc fibrosis. These results support the involvement of Ca2+ and Ca2+-related pathways in the anti-fibrotic effect of JQ1, specifically on collagen expression and cell proliferation.BET expression in SSc. We examined the expression of BET proteins in dermal fibroblasts. Out of the four BETs, BRD-t is expressed predominantly in the testis and therefore excluded from our study. At the mRNA level, only BRD4 was significantly upregulated in dcSSc fibroblasts compared to normal fibroblasts (Supplemental Figure 8A). At the protein level, however, the expression of the BETs was variable in dcSSc fibroblasts but no difference between normal and dcSSc was seen (Supplemental Figure 8B).
BRD4 is pro-fibrotic
Since JQ1 is a pan-BET inhibitor, we next performed a set of experiments to determine which BET is responsible for the anti-fibrotic effect of JQ1. JQ1 significantly induced BRD2 and decreased BRD4, while it had minimal effect on BRD3 (Supplemental Figure 1 and Figure 2A). We then knocked down BRD2, BRD3, or BRD4 individually and examined the expression of genes involved in fibrosis. As shown in Figure 6A, knockdown of BRD2 resulted in significant elevation of COL1A1, while knockdown of BRD4 led to significant reduction of COL1A1, ACTA2, TGFB1, CTGF, and MMP1. BRD3 knockdown decreased TIMP3 while it had no effect on the other genes examined. In addition, BRD4 knockdown led to significant reduction in gel contraction (Figure 6B), suggesting the BRD4 is pro-fibrotic, and that JQ1, by significantly reducing BRD4 expression in dcSSc fibroblasts, inhibits fibrosis in SSc.
To further confirm the involvement of BRD2 and BRD4 in myofibroblast function, we treated dcSSc fibroblasts with specific BRD2 inhibitor (B1C1) or BRD4 inhibitors (AZD5153 or ARV825). As shown in Figure 7A, inhibition of BRD2 by BIC1 did not affect dcSSc fibroblast proliferation, while BRD4 inhibition by either AZD5153 orARV825 significantly inhibited proliferation at both 1 and 10 µM. In addition, dcSSc fibroblasts treated with BRD4 inhibitors ARV825 or AZD5153 significantly reduced ACTA2 and COL1A1 while BRD2 inhibitor BIC1 did not have any effect (Figure 7B). Similar results were observed at the protein level (Figure 7C). To further determine the anti-fibrotic effect of BRD4 inhibition in vivo, we induced skin fibrosis by bleomycin in mice and dosed the animals with either ARV825 or AZD5153. We found that both BRD4 inhibitors prevented bleomycin-induced skin fibrosis in mice, shown by dermal thickness, αSMA-positive cells, and hydroxyproline content (Figure 7D). These results further confirm the involvement of BRD4 in myofibroblast transformation in dcSSc fibroblasts and in the bleomycin mouse model.
BRD4 mediates its fibrotic properties through Ca2+ signaling in dcSSc fibroblasts
To further investigate whether BRD4 inhibitors affect intracellular Ca2+ in dcSSc fibroblasts similar to what was observed with JQ1, we measured intracellular Ca2+ levels in the presence or absence of the BRD4 inhibitors. We found that both inhibitors decreased intracellular Ca2+ levels after 48 hours of treatment, with AZD5153 reaching statistical significance (Figure 7E). Interestingly the BRD2 inhibitor BIC1 significantly increased intracellular Ca2+, suggesting that this BET isoform could act in an opposite way compared to BRD4 in dcSSc fibroblasts. We further showed that both BRD4 inhibitors downregulated CAMK2A in dcSSc fibroblasts (Figure 7F). In addition, the anti-fibrotic effects of both AZD5153 and ARV825 in dcSSc fibroblasts were dependent on CAMK2A. Overexpression of CAMK2A in these cells appeared to block the inhibitory effect of ARV825 and AZD5153 on fibrotic gene expression, with COL1A1 reachingstatistical significance for AZD5153 (Figure 7F). BRD4 inhibitors also blocked cell proliferation by 50% in control cells but only by approximately 25% in the CAMK2A overexpressing cells, suggesting that CAMK2A is involved in BRD4-mediated effects on cell growth (Figure 7G).
Discussion
The increased understanding of the effect of epigenetic aberrations on gene transcription has led the field to a better appreciation of the role of transcriptional dysregulation in initiating and perhaps maintaining SSc fibrosis. In this study we performed an in-depth examination of BET inhibition in SSc fibrosis. Our in vivo studies showed that administration of JQ1 prevented skin fibrosis induced by bleomycin in mice. We further demonstrated that BET inhibition by JQ1 suppressed expression of many pro-fibotic genes in dcSSc fibroblasts and reversed the established progression of myofibroblast differentiation in vitro. Transcriptomic analysis of JQ1-treated cells not only showed that differentially expressed genes are enriched in ECM- and cell cycle-related pathways but also revealed the involvement of calcium signaling pathway as a novel anti-fibrotic mechanism. Moreover, results from siRNA knockdown experiments confirmed the role of BRD4 in SSc fibrosis. This was further confirmed using BRD4 specific inhibitors. Functional studies showed that Ca2+ and Ca2+-related gene CAMK2A plays a critical role in the anti-fibrotic effects of JQ1, AZD5153, and ARV825. Together, these results suggest that BRD4 is critically involved in promoting fibrosis in SSc, and inhibition of BRD4, and perhaps targeting intracellular Ca2+, would be effective treatments for this disease.
JQ1, a first in-class BET inhibitor, competitively binds to the acetyl-lysine recognition areas of these proteins, and displaces them from acetylated chromatin, thereby repressing transcription of target genes. Early studies suggested that JQ1 exerted potent anti-proliferative properties in multiple myeloma via cell-growth arrest and senescence in a c-MYC-dependent manner (12). Since then, more studies haveshown the benefits of JQ1 in other proliferative disorders (5–8). In addition, this pan-BET inhibitor has demonstrated great efficacy in blocking fibrotic progression in a range of fibrosis models (5–8). In a lung fibrosis model, JQ1 significantly reduced collagen deposition in bleomycin-treated mice compared to control mice (8). This drug also ameliorated the phenotypic changes of lung fibroblasts from idiopathic pulmonary fibrosis patients. We showed in this study that JQ1 inhibits proliferation of dcSSc dermal fibroblasts through inducing cell cycle arrest and apoptosis. It also exhibits potent anti-fibrotic effects in patient-derived cells and in the bleomycin skin fibrosis model. The functional changes in cell proliferation are also confirmed from the RNA-seq results, as genes enriched in cell cycle, mitosis, and ferroptosis pathways are highlighted in the pathway analysis (Figure 3E). Our results are consistent with the findings from Shin et al, where they showed that JQ1 repressed collagen expression in SSc skin organ culture, likely due to the increase in MMP1 (13).
BET proteins are located in the nucleus. Because of their involvement in various cellular processes, BETs have been shown to participate in tumor development, autoimmunity, infections, and inflammation (14–17). Specifically, BRD4 has been studied extensively for its role in gene transcription, including regulation (through acetylated histones), initiation (via engaging RNA polymerase II), and elongation (by interaction with P-TEFb)(18). Indeed, BRD4 is reported to control various fibrosis-related genes, including ACTA2 and COL1A1, shown from BRD4 knockdown studies or ChIP analyses (5, 7, 8, 19). This is not surprising, as both genes are reported to possess acetylated histones in their promoter region in fibroblasts (Supplemental Figure 9). In dcSSc fibroblasts, inhibition of BETs by JQ1 downregulated both ACTA2and COL1A1, and this appears to be mediated through blockade of BRD4, as BRD4 knockdown or inhibition led to decreased ACTA2 and COL1A1 levels, while knockdown of BRD2 or BRD3 did not. Interestingly, TGFB1, which was significantly upregulated by JQ1, was downregulated in BRD4 knocked down cells. Similar results were seen in MMP1 expression. This discrepancy suggests that JQ1, as a pan-BET inhibitor, has off-target effects compared to BRD4 knockdown, especially at the higher concentrations. This could potentially explain the U-shaped dose-response curve of JQ1 in the gel contraction assay. Of note, our results showing that BRD2 knockdown leads to COL1A1 upregulation are consistent with previous reports (19). Since JQ1 dose-dependently increased BRD2 expression in dcSSc fibroblasts, it is possible that the anti-fibrotic effect of JQ1 is partly mediated by enhancing the anti-fibrotic potential of BRD2. However, BRD2 inhibition, although trending to induction of fibroblast migration and pro-fibrotic gene expression (Figure 7A-C), had no significant effect on enhancing myofibroblast functions. Furthermore, BIC1 significantly increased intracellular Ca2+ levels in dcSSc fibroblasts, in contrast to BRD4 inhibition.
To gain mechanistic insight into the anti-fibrotic effect of BET inhibition in SSc, we performed mRNA-seq in JQ1-treated dcSSc fibroblasts. In addition to pathways in cell cycle regulation, ECM, and intracellular signaling, our analysis also revealed many novel pathways, including calcium signaling, metabolism, ferroptosis, and prostaglandin synthesis and regulation (Figure 3E). As these pathways have been implicated in various fibrotic conditions, it is possible that they are involved in the anti-fibrotic effect of BET inhibition in dcSSc fibroblasts. Specifically, the calcium signaling pathway, which is the most enriched pathway in our analysis (Figure 3C), has been shown to be critical invarious fibrotic conditions (10, 20, 21). Intracellular Ca2+ levels are controlled by Ca2+-permeable channels in the plasma membrane (voltage-operated channels and receptor-operated channels), Ca2+ release from the endoplasmic/sarcoplasmic reticulum, as well as Ca2+ extrusion pumps. The increase in intracellular Ca2+ levels can activate various pathways that are involved in many physiological functions (22–25). Studies show that interference of intracellular Ca2+ levels through blockade of Ca2+ channels or receptors has potential anti-fibrotic effects in various models (26–29). In dcSSc fibroblasts, JQ1 treatment significantly reduced Ca2+ levels in these cells (Figure 4C). This is possibly mediated by significant downregulation of calcium channels and receptors on the plasma membrane, including P2RX1 and P2RX5 (encoding for purinergic receptor P2X1 and 5), CACNA1D (calcium voltage-gated channel subunit alpha1D), and TPCN1 (voltage-dependent calcium channel protein TPC1), as well as ones on the endoplasmic/sarcoplasmic reticulum, such as RYR1 (Ryanodine receptor 1) (Figure 4B). Interestingly, certain Ca2+ channels, Ca2+ extrusion pumps, and Ca2+-release transporters that increase intracellular Ca2+ were affected by JQ1. It is possible that these events represent a compensatory mechanism that attempt to antagonize the decreased Ca2+ levels in these fibroblasts. The identification of Ca2+ pumps/carriers/ channels involved in Ca2+ entry, as well as their involvement in mediating the fibrotic effect of BET proteins in SSc requires further investigation.
In this study we identified CaMKII, specifically the α isoform, as a critical mediator for the fibrotic effect of BETs and BRD4. CaMKII, comprised of 4 isoforms, is a downstream messenger of the Ca2+ signaling pathway. CaMKII activation augmented collagen production in cardiac fibroblasts, while inhibition of CaMKII blockedproliferation, collagen and profibrotic cytokine production (22, 30, 31). We showed similar findings in dcSSc fibroblasts in this study. In addition to cardiac fibrosis, CaMKII is also involved in pulmonary fibrosis (32), ureteral scar formation (33), and renal fibrosis (34). CaMKII has been shown to be associated with FGFR3/FGF9 in SSc fibroblasts, functioning as a downstream mediator for the pro-fibrotic effect of FGFR3 (25). Interestingly, we found that overexpression of CAMK2A alone led to significant increase in fibrotic gene expression and cell proliferation in dcSSc fibroblasts, suggesting that this gene itself could play a critical role in SSc fibrosis.
Although JQ1 has been shown to possess desirable qualities of a small-molecular inhibitor, including high target potency and well-characterized selectivity, it is known for its inhibitory effect on lymphoid and hematopoietic tissues (35). JQ1 exhibits linear pharmacokinetics in mice with an oral bioavailability of approximately 50% and a half-life of 1 hour (35, 36). Due to the short half-life and potential off-target effects, this compound is only used in preclinical studies. Currently there are several BET inhibitors with better pharmacokinetic properties in clinical trials for various types of cancer (9). Recent trials have also focused on targeting specific BETs, including BRD4 inhibitors AZD5153 (NCT03205176), which was used in this study, and ABBC-744 (NCT03360006). Based on our results, BETs, in particular BRD4, regulate essential processes involved in SSc fibrosis. We not only revealed a novel anti-fibrotic mechanism involving calcium signaling by BET inhibition, but also highlighted the potential of epigenetic therapeutic strategies targeting BRD4 for SSc patients. These data, along with future studies focusing on additional molecular mechanisms underlyingthe role of BRD4 in SSc, should provide the framework that supports the use of more selective BRD4 inhibitors as a therapeutic option for SSc.
Methods
Bleomycin-induced skin fibrosis
The procedure to induce fibrosis by bleomycin in mice was published previously (2, 37). 100 μl of bleomycin (1 mg/ml) or PBS was injected subcutaneously into a single location on the shaved back of C57BL/6 mice once every day for 2 weeks. JQ1 (50 mg/kg, MedChemExpress) or vehicle control (20% DMSO/50% PBS/30% PEG) was given daily by oral gavage. In a separate experiment, BRD4 inhibitors AZD5153 (5mg/kg, Cayman), ARV825 (5mg/kg, MedChemExpress), or vehicle control (22% DMSO/48% PBS/30% PEG) were given daily intraperitonially. At the end of the experiment, fixed skin sections were stained with Masson’s trichrome. Dermal thickness was measured by analyzing the distance between the epidermal–dermal junction and the dermal–fat junction in three fields in two or more sections from each animal. Immunofluorescence was performed on sections using anti-αSMA (Abcam) or anti-F4/80 (Invitrogen) antibodies after antigen-retrieval. Collagen content in the skin was measured using the Hydroxyproline Kit (Abcam). All animal protocols were approved by the Institutional Animal Care & Use Committee at the University of Michigan.
Patients and Controls
All patients recruited in this study met the ACR/EULAR criteria for the classification of SSc (38). Punch biopsies from the distal forearm of healthy volunteers and diffuse cutaneous (dc)SSc patients were obtained for fibroblast isolation. This study was approved by the University of Michigan Institutional Review Board. The demographics and clinical characteristics of the enrolled individuals are summarized in Supplemental Table 2.
Cell culture
Punch biopsies obtained from healthy subjects and SSc patients were digested as previously described (2, 39). Briefly, biopsies were placed in 2.4 U/ml dispase overnight at 4°C. After removing the epidermal layers, the biopsies were transferred to 0.2% collagenase solution and incubated at 37°C for 45 min. The dissociated cells were then collected and cultured. After separating out the endothelial cells by magnetic bead selection, the resultant fibroblasts were maintained in RPMI supplemented with 10% fetal bovine serum (FBS) and antibiotics. Cells between passage 3 and 6 were used in all experiments.
Cell treatment and transfection
Dermal fibroblasts from dcSSc patients were treated with 0.01-22 µM of JQ1 (Cayman), a pan-BET inhibitor, for 48 hours. Normal dermal fibroblasts were treated with TGFβ (10 ng/ml, Cell Signaling) and/or JQ1 (1 µM) for 72 hours to induce a myofibroblast phenotype. In a separate experiment, BRD2 inhibitor BIC1 (Cayman), BRD4 inhibitors AZD5153 or ARV825 (0.1-10 µM, Cayman), were used to treat dcSSc fibroblasts for 48 to 72 hours. BRD2, BRD3, or BRD4 knockdown in dcSSc dermal fibroblasts was performed using BRD2 (200nM), BRD3 (100nM), or BRD4 (200nM) siRNA (ON-TARGETplus siRNA, Dharmacon). Scrambled siRNA (Dharmacon) was used as a control. The cells were transfected for 72 hours before downstream experiments were performed. CAMK2A in dcSSc fibroblasts was overexpressed using CAMK2A vectors from Origene. 0.1 μg/ml of CAMK2A vector was mixed with Lipofectamine 2000, then added to culture media. Inhibitors were added 24 hours after the transfection. PCMV6-XL6 vector (Origene) was used as a negativecontrol. To determine the effect of Ca2+ on myofibroblast transformation, dcSSc dermal fibroblasts were cultured in Ca2+-containing or Ca2+ -free DMEM media. In a separate experiment, dcSSc fibroblasts cultured in Ca2+-containing RPMI media were treated with BAPTA-AM (20 μM, Thermo Fisher) in the presence or absence of 1 μM JQ1 for 2 days.
Western blots
Western blotting was performed following the protocol as described previously (39). Equal amount of protein was separate by SDS-PAGE, followed by transferring to a nitrocellulose membrane. After blocking, the blots were probed with antibodies against BRD2 (Abcam), BRD3 (Santa Cruz), BRD4 (Abcam), collagen I (Abcam), or αSMA (Abcam). For loading control, the blots were immunoblotted with antibodies against β-actin (Sigma) or GAPDH (Cell Signaling) as controls. Band quantification was performed using ImageJ (40).
mRNA extraction and qRT-PCR
Total RNA was extracted using Direct-zol™ RNA MiniPrep Kit (Zymo Research) before converted to cDNA. Quantitative PCR was performed in a ViiA™ 7 Real-Time PCR System.
Cell proliferation assays
Proliferation of dermal fibroblasts was measured using the BrdU Cell Proliferation Assay Kit (BioVision). After 4 hours of BrdU incubation, cells were fixed before adding antibodies and substrate. The absorbance at 450 nm was measured. In a separate experiment, the IncuCyte® Live-Cell Imaging System was used to monitor cell proliferation. Cells were seeded and allowed to grow overnight.After adding different treatments cells were monitored by IncuCyte® up to 5 days. Cell counts were analyzed by the IncuCyte® S3 Analysis software.
Gel contraction and cell migration assays
The Cell Contraction Assay kit (Cell Biolabs) was used for gel contraction (39). After treatment, cells were suspended and mixed with collagen solution. Solidified gels were lifted after 24 hours and the areas of the gels were quantified using ImageJ (40). To evaluate the effect of JQ1 on cell migration, we performed a scratch wound assay using the IncuCyte® Live-Cell Imaging System. Cell migration was monitored by IncuCyte® up to 7 days.
Detection of apoptosis
To assess the effect of JQ1 on cell apoptosis, cells were plated in 96 well plates and treated with JQ1 in the presence of IncuCyte® Caspase-3/7 Apoptosis Reagent. The apoptotic cells were quantified by measuring cells with green fluorescent staining. The data were presented as green fluorescent cell number normalized to total cell count.
Cell cycle analysis
Fibroblasts were treated with 1 µM JQ1 for 48 hours, while the non-treated dcSSc fibroblasts were used as control. Both the JQ1- and non-treated cells were first synchronized using the double thymidine block method before treatment (41). Cells were then harvested and stained with 7-AAD viability dye and Vybrant DyeCycle Violet (Invitrogen) according to the manufacture’s protocol. The stained cells were analyzed using a BD FACSCanto II flow cytometer. Cell cycle analysis was performed on gated 7-AAD-live cells using Flowjo 7.0.
RNA sequencing and analysis
RNA-seq was performed on mRNA extracted from control- or JQ1 (1 μM)-treated dcSSc fibroblasts. Samples with an RNA-integrity number of more than 7 were subjected to library preparation and sequencing to 151 paired-end cycles on the NovaSeq-6000 platform (Illumina), resulting in approximately 35 million reads/sample. Differential gene expression analysis was performed using DESeq2 (42), using a negative binomial generalized linear model (thresholds: linear fold change > 1.5 or < -1.5, Benjamini-Hochberg FDR (Padj) <0.05). Plots were generated using variations of DESeq2 plotting functions and other packages with R version 3.3.3. Functional analysis, including candidate pathways activated or inhibited in comparison(s) and GO-term enrichments, was performed using iPathway Guide (Advaita)(43). Additional functional enrichment analyses to generate networks for visualization were performed using ClueGO (v2.5.7)/CluePedia (v1.5.7)(44, 45) and Cytoscape (v3.8.0)(46).
Intracellular Ca2+ measurement
Dermal fibroblasts from dcSSc patients were treated with or without 1 µM JQ1, AZD5153, ARV825, or BIC1 for 48 hours before being collected for Ca2+ measurement by flow cytometry. Detached cells (1×107/mL) were labeled in loading buffer (HBSS with 1% FBS, 1 mM CaCl2 and 1 mM MgCl2) containing 1 μM Fluo-4 AM (Thermo Fisher) with 0.02% Pluronic F-127 at 37 °C for 30 min with gentle agitation every 10 min. The labeled cells were washed twice with loading buffer containing 0.1 mM sulfinpyrazone, and resuspended in the same loading buffer with sulfinpyrazone at 1×106/mL, incubated at 37°C for 30 min before basal fluorescence ofFluo-4 were measured on a BD FACSCanto II flowcytometer. Mean fluorescent intensity of unlabeled cells was subtracted from the mean fluorescent intensity of the labeled cells.
Statistics
Normality test was conducted to determine whether the data is normally distributed or skewed. To determine the differences between groups, unpaired t test, Mann–Whitney U test, paired t-test, Wilcoxon test, one-way ANOVA with Sidak test, Kruskal–Wallis test with Dunn’s test, or two-way ANOVA with Dunnett’s test were performed using GraphPad Prism version 8 (GraphPad Software, Inc). P values of less than 0.05 were considered statistically significant. Results were expressed as mean ± SD.
Study Approval
This study was approved by the University of Michigan Institutional Review Board. Written informed consent was received from participants prior to inclusion in the study. All animal protocols were approved for use from the University of Michigan the Institutional Animal Care & Use Committee.
Competing interests
The authors declare no conflicts of interest.
Author contributions
All authors participated in the interpretation of study results, and in the drafting, critical revision and approval of the final version of the manuscript. PT, AHS, DF, YMD, and DK contributed to study conception and/or design. SV, MG, MNM, WDB, SM, PJP, MA, PLC, MAA, JHR, QW, EM, DMR, JLH, and PT contributed to the acquisition of study results. SV, PJP, MA, QW, and PT contributed to the analysis of study results.
Data availability
All data relevant to the study are included in the article. The RNA-sequencing data is deposited to GEO (GSE186961).
Acknowledgements
This work was supported by the funds from the American Autoimmune Related Disease Foundation and the Edward T. and Ellen K. Dryer Early Career Professorship (Tsou), Chugai, PCORI, Novartis, Sanofi-Genzyme, and Genentech (Mao-Draayer), National Institute of Arthritis and Musculoskeletal and Skin Diseases grant number K24AR063120 (Khanna) and R01AR070148 (Sawalha), and National Institute of Allergy and Infectious Diseases grant number UM1-AI110557-05 and UM1-AI144298-01 (Mao-Draayer), and R01AI097134 (Sawalha).
Footnotes
Funding info: This work was supported by the funds from the American Autoimmune Related Disease Foundation and the Edward T. and Ellen K. Dryer Early Career Professorship (Tsou), Chugai, PCORI, Novartis, Sanofi-Genzyme, and Genentech (Mao-Draayer), National Institute of Arthritis and Musculoskeletal and Skin Diseases grant number K24AR063120 (Khanna) and R01AR070148 (Sawalha), and National Institute of Allergy and Infectious Diseases grant number UM1-AI110557-05 and UM1-AI144298-01 (Fox, Khanna, Mao-Draayer), and R01AI097134 (Sawalha).
Conflict of interest: None.