Involvement of Circadian Clock Gene BMAL1 in Doxorubicin-Induced Inflammation in Vascular Smooth Muscle Cells

The molecular clock component Brain and Muscle Arnt-Like protein-1 (BMAL-1) affects various biologic processes, including cell survival, in numerous cell types. We previously demonstrated that BMAL1 positively regulates cell proliferation in Vascular Smooth Muscle Cells (VSMCs). However, its role in VSMC inflammation remains unelucidated. Because doxorubicin causes phlebitis associated with vascular inflammation, the present study used cultured VSMCs to investigate whether BMAL1 affected doxorubicin-induced vascular inflammation. Doxorubicin treatment led to Increased Interleukin (IL)-6 mRNA expression with an increase in BMAL1 expression in VSMCs. BMAL1 knockdown significantly increased IL-6 mRNA and further enhanced doxorubicin-induced IL-6 mRNA expression in VSMCs. BMAL1 knockdown also significantly decreased cell viability and affected the expression of other clock genes, including Per1 and Clock. Furthermore, the levels of nuclear factor erythroid 2-related factor 2, which has anti-inflammatory effects, increased in VSMCs with BMAL1 knockdown. Finally, BMAL1 knockdown increased NADPH oxidase 4 mRNA, p38α mRNA, and p38β mRNA levels, leading to increased total p38 Mito-gen-Activated Protein Kinase (MAPK) and phosphorylated p38 MAPK. IL-6 mRNA induction caused by BMAL1 knockdown was significantly inhibited in VSMCs following pretreatment with SB203580, a p38 MAPK inhibitor. Our findings demonstrated that decreased BMAL1 expression caused VSMC inflammation via p38 MAPK activation. Moreover, doxorubicin-induced inflammation in VSMCs was further enhanced when BMAL1 expression levels were low. Thus, BMAL1 may be a novel therapeutic target to treat inflammatory disease, including doxorubicin-induced phlebitis.


Introduction
Doxorubicin is a highly potent, effective broad-spectrum anticancer chemotherapeutic drug used to treat various cancers, including solid and hematologic tumors. However, because it elicits dose-dependent toxic side effects associated with cardiotoxicity, leading to dilated cardiomyopathy and heart failure [1] [2], its clinical use is limited. Additionally, although the frequency of side effects is relatively low compared with that of cardiotoxicity, doxorubicin also causes systematic inflammation, including phlebitis [3] [4]. Phlebitis is characterized by an inflammatory response associated with elevated interleukin (IL)-6 expression, causing various forms of tissue damage and symptoms of pain and warmth [5] [6].
Clock genes form transcription-translational feedback loops that maintain circadian rhythms [7] [8] [9]. Brain and Muscle Arnt-Like protein-1 (BMAL1) is a crucial circadian clock gene and the only Clock gene where deletion completely ablates all mammalian rhythms [10]. BMAL1 forms a heterodimer with a clock circadian regulator (CLOCK) and its heterodimers, which bind to the E-box element, increasing the expression of genes such as PER and CRY, which code for the Period Circadian Regulator (PER) and Cryptochrome Circadian Regulator (CRY), respectively. PER/CRY heterodimers inhibit BMAL1/CLOCK-dependent gene transcription. Additionally, the heterodimeric CLOCK: BMAL1 complex accelerates transcription of the DNA-binding orphan nuclear receptor reverse erythroblastosis virus (REV-ERB)-α/β and retinoid-related orphan receptor (ROR)-α, -β, -γ. RORs activate the transcriptional expression of BMAL1, whereas REV-ERBs repress it by binding to the ROR element [11] [12].
Circadian rhythm disruption increases the risk of various diseases, including metabolic disorders and cardiovascular diseases [13] [14]. BMAL1 was recently reported to affect a diverse range of biologic processes, including signal transduction associated with apoptosis, cell proliferation, and cell differentiation, in many cell types. Furthermore, accumulating evidence suggests that BMAL1 is crucial in the pathophysiology of vascular disorders, including hypertension and abdominal aortic aneurysms [15] [16] [17]. We recently reported that BMAL1 positively regulates Vascular Smooth Muscle Cell (VSMC) proliferation [18]. Thus, although BMAL1 regulates a variety of vascular functions, its role in VSMC inflammation remains largely unelucidated. Therefore, this study aimed to determine whether BMAL1 affects inflammation associated with doxorubicin-induced phlebitis in VSMCs.

Cell Culture and siRNA Transfection
The experimental plan was approved by the president of Hokkaido University of Inc., Tokyo, Japan) and 5.0 mg/kg butorphanol (Vetorphale®; Meiji Seika Pharma Co., Ltd., Tokyo, Japan) before dissection. VSMCs isolated from thoracic aortas using an enzymatic digestion method were cultured in DMEM containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin, as previously described [18]. When confluency of approximately 80% -90% was reached, the cells were transfected with 50 nM siRNA using Lipofectamine RNAi-MAX Transfection Reagent, according to the manufacturer's protocol.

Cell Viability Assay
After treating the VSMCs with doxorubicin at the indicated concentrations or with siRNA targeting BMAL1, cell viability was assessed using cell counting kit-8 (Dojindo Molecular Technologies, Kumamoto, Japan).

Western Blot
After treatment, cell lysis and subsequent western blots were performed, as previously described [18].

Reverse Transcription-Quantitative Polymerase Chain Reaction (qPCR) Analysis
Total RNA extraction, cDNA synthesis, and subsequent qPCR were performed, as previously described [18]. The primers used are presented in Table 1. GAPDH was used as the housekeeping (control) gene.
GAPDH was used as the housekeeping (control) gene.

Statistical Analysis of Data
Data were expressed as means ± Standard Error of the Mean (SEM). Differences between the groups were evaluated using the Student t-test. GraphPad Prism, version 6 (GraphPad Software, San Diego, CA, USA) was used for all statistical analyses. P values < 0.05 were considered statistically significant.

Doxorubicin Increases IL-6 Induction and BMAL1 Expression in VSMCs
We tested whether doxorubicin affected cell viability and IL-6 mRNA expression in VSMCs. Doxorubicin treatment significantly increased IL-6 mRNA expres-

BMAL1 Knockdown Further Increases Doxorubicin-Induced IL-6 Induction in VSMCs
We assessed doxorubicin-induced IL-6 mRNA expression in VSMCs with BMAL1 knockdown to determine the role of BMAL1 in doxorubicin-induced inflammation. The VSMCs treated with 50 nM siRNA targeting BMAL1 showed significantly decreased BMAL1 mRNA (Figure 2(a)) and BMAL1 protein (Figure 2(b)) expression. Interestingly, BMAL1 knockdown significantly reduced cell viability   (Figure 2(h)). On the other hand, the BMAL1 knockdown did not alter Per2 mRNA expression in VSMCs (Figure 2(g)). These findings suggested that other clock genes might be involved in increased IL-6 expression in VSMCs with BMAL1 knockdown.

p38 Mitogen-Activated Protein Kinase (MAPK) is Involved in the Induction of IL-6 mRNA Expression in VSMCs with BMAL1 Knockdown
The Reactive Oxygen Species (ROS)/p38 MAPK pathway is crucial for the induction of IL-6 expression in VSMCs. Therefore, we first examined the effect of BMAL1 knockdown on NADPH Oxidase (Nox) 1 and Nox4, which are involved in ROS production and p38 MAPK activation. BMAL1 knockdown significantly increased Nox4 but not Nox1 mRNA expression in VSMCs (Figure 4(a) and Figure 4(b)). Additionally, BMAL1 knockdown significantly increased the phosphorylated p38 MAPK in VSMCs Figure 4(c)). Interestingly, BMAL1 knockdown also markedly increased total p38 MAPK protein expression and p38 MAPK mRNA (Figures 4(c)-(e)), indicating that BMAL1 knockdown increased p38 MAPK activation accompanied by p38α and p38β mRNA upregulation in VSMCs. Increased IL-6 mRNA expression due to BMAL1 knockdown was significantly inhibited following pretreatment with SB203580, a p38 MAPK inhibitor ( Figure 4(f)). These results suggest that p38 MAPK activation played a critical role in increased IL-6 mRNA expression VSMCs with BMAL1 knockdown.

Discussion
Although clock gene proteins were recently recognized as signal molecules involved in a variety of cell responses, including proliferation and apoptosis [18] [19] [20], the role of BMAL1 in vascular inflammation is unclear. In the present study, we showed that doxorubicin increased BMAL1 mRNA and protein expression with elevated IL-6 mRNA expression in VSMCs. We also demonstrated that BMAL1 knockdown further increased doxorubicin-induced IL-6 mRNA expression and that IL-6 induction caused by BMAL1 knockdown was partially mediated by p38 MAPK activation in VSMCs. Vascular inflammation is caused by various inflammatory cytokines produced by VSMCs and macrophage expressed with alkaline phosphatase [21]. Among these cytokines, IL-6 plays a crucial role in vascular inflammation, leading to various cardiovascular diseases, including phlebitis and atherosclerosis [22] [23] [24]. IL-6 induction in VSMCs is triggered by a variety of stimuli, such as angiotensin II and superoxide [25] [26]. Our results revealed that doxorubicin increased IL-6 mRNA and protein expression, suggesting that doxorubicin induces inflammation, at least in part, through the induction of IL-6 in VSMCs. Consistent with our observation that doxorubicin increases BMAL1 expression, the anticancer drug paclitaxel upregulates BMAL1 expression in mouse kidney and cultured human HK-2 renal cells [27], suggesting it affects the expression of clock gene BMAL1 in a variety of cell types. Further studies are necessary to elucidate the mechanisms whereby doxorubicin affects BMAL1 expression in VSMCs.
It remains unclear whether BMAL1 affects doxorubicin-induced inflammation in VSMCs. We demonstrated that the BMAL1 knockdown increased doxorubicin-induced IL-6 mRNA expression, suggesting that doxorubicin-induced BMAL1 negatively regulates doxorubicin-induced inflammation in VSMCs. In support of our findings, cardiomyocyte-specific BMAL1 deletion in mice was reported to induce proinflammatory cytokines [28], and BMAL1 deletion in macrophages was found to further increase lipopolysaccharide-induced IL-1β mRNA expression [29]. Additionally, given that BMAL1 expression is relatively low at midnight compared with daytime [30], it should be noted that doxorubicin might cause vascular pain and phlebitis more easily when BMAL1 levels are low. BMAL1 deletion affects the expression of other clock genes and causes clock dysfunction [10]. We found that BMAL1 knockdown reduced Per1 and increased CLOCK in VSMCs. Per1 has been reported to increase IL-6 mRNA expression in primary spinal astrocytes [31], suggesting that decreased Per1 expression caused by BMAL1 knockdown partially contributes to the induction of IL-6 mRNA expression in VSMCs. Further investigations are necessary to determine whether other clock genes are involved in the mechanism of VSMC inflammation.
Nrf2 is a transcriptional factor that protects against inflammation and oxidative stress through the induction of HO-1 and directly binds to the promoter region of IL-6 to suppress IL-6 mRNA expression in bone marrow-derived macrophages [32]. Therefore, we investigated whether BMAL1 regulates inflammation by controlling Nrf2 expression in VSMCs. We found that BMAL1 knockdown significantly increased Nrf2 mRNA and protein expression, followed by increased HO-1 expression, the target gene of Nrf2 in VSMCs. In contrast, BMAL1 deletion was reported to decrease Nrf2 expression, which induces proinflammatory cytokines in macrophages [29], suggesting that the role of BMAL1 in Nrf2 expression differs among cell types.
p38 MAPK belongs to the MAPK kinase family, which regulates a variety of cellular responses, including apoptosis and inflammation [33] [34]. P38 MAPK is activated by ROS-dependent mechanisms in VSMCs and other cells [35] [36]. The increased expression of Nox family proteins, which are catalytic subunits of NAD(P)H oxidases, leads to ROS production in VSMCs. Among the Nox family members, Nox1 and Nox4 are expressed at relatively higher levels than Nox2 and Nox3 in VSMCs [37]. We found that Nox4, but not Nox 1, was significantly increased and that p38 MAPK phosphorylation was also significantly increased following BMAL1 knockdown in VSMCs, indicating that increased Nox4 activates p38 MAPK in VSMCs. Upregulated Nox4 expression was reported as associated with increased p38 MAPK phosphorylation in inflammation sites in atherosclerotic regions [38].
Increased IL-6 mRNA induced by BMAL1 knockdown was inhibited by pretreatment with a p38 MAPK inhibitor, indicating that p38 MAPK activation is crucial in the induction of IL-6 by BMAL1 knockdown in VSMCs. Interestingly, total p38 MAPK was also increased by BMAL1 knockdown through the upregulation of p38 MAPK mRNA expression in VSMCs. In support of our results, MAPK activity was reported to exhibit a circadian rhythm peak in the mid to late subjective night in various clock structures [39] [40]. This suggests that circadian clock genes affect MAPK expression and functions. In fact, several circadian clock genes exert a variety of biologic functions through the p38 MAPK signaling pathway [31] [41] [42].

Conclusion
Doxorubicin induces inflammation with increased BMAL1 expression in VSMCs.
BMAL1 knockdown increases IL-6 induction, which is partially mediated by p38 MAPK activations in VSMCs. Our findings suggest that decreased BMAL1 expression strengthens inflammation induced by doxorubicin.

Funding
This study was supported, in part, by JSPS KAKENHI [grant number 18K06703].