BMAL1/REV-ERBα loop as a novel inflammatory sensor to drive NF-κB-mediated inflammation in vascular smooth muscle cells by modulating oxidative stress

doi:10.21203/rs.3.rs-4353009/v1

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Research Article

BMAL1/REV-ERBα loop as a novel inflammatory sensor to drive NF-κB-mediated inflammation in vascular smooth muscle cells by modulating oxidative stress

https://doi.org/10.21203/rs.3.rs-4353009/v1

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Objectives and design:

As a pleiotropic inflammatory cytokine, TNF-α could act as a kind of zeitgeber mediator to integrate with circadian clock and modulate inflammatory signaling. We aimed to investigate how circadian gene Bmal1 regulating inflammation in vascular smooth muscle cells (VSMCs) upon TNF-α stimulation.

Methods

Circadian rhythmicity of Bmal1 expression was detected in the mouse VSMCs challenged with TNF-α, and then Bmal1 was knocked down or overexpressed by adenovirus transfection to investigate the effects and machines of Bmal1 on inflammatory signaling.

Results

1) TNF-α stimulated Bmal1 transcription and disrupted its circadian expression in VSMCs. 2) Transcriptional activation of Bmal1 furtherly activated TNF-α-induced- NF-κB signaling and exacerbated VSMCs inflammation by triggering oxidative stress. 3) TNF-α-activated JNK signaling enhanced REV-ERBα phosphorylation and degradation, and thus promoted Bmal1 transcription in VSMCs.

Conclusion

Our work identified a specific pathway by which the transcriptional activation of Bmal1, mediated by the TNF-α-induced REV-ERBα phosphorylation, triggered oxidative stress to exacerbate inflammatory response in VSMCs, which represents a new opportunity for clock gene Bmal1 being a potential diagnostic marker and therapeutic target for TNF-α mediated vascular inflammation.

Tumor necrosis factor (TNF-α) is one of the most important pro-inflammatory mediators which exert both homeostatic and pathogenic bioactivities in the development of many diseases^{1, 2}. Multiple pathological or sub-healthy conditions, including infections³, autoimmune diseases⁴, aging⁵ and long-term stress⁶, could induce enhanced and prolonged production of TNF-α, causing immune imbalance and exacerbating pathological changes. Generally, TNF-α initiates signaling transduction through binding to the receptors of TNFR1 and TNFR2, and subsequently activates the pathways of NF-κB, apoptosis, extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38 MAPK), and c-Jun N-terminal kinase (JNK), to exert biological effects in regulating immunity, oxidative stress, cell death and differentiation^{7, 8}. As the major constituents of medium- and large-sized arteries, vascular smooth muscle cells (VSMCs) show remarkable plasticity in response to vascular injury, inflammation, and lipoprotein accumulation, and the inflammatory phenotype transition of VSMCs are usually detrimental, which could release cytokines to stimulate adjacent ECs and enhance monocyte/macrophages infiltration, exacerbating diseases progression^{9, 10}. TNF-α as a pleiotropic cytokine has extensive effects on VSMCs and is critically implicated in the pathogenesis of many cardiovascular diseases (CVDs)¹¹.

The circadian clock is an endogenous timing system, which consist of transcriptional-translational feedback loops (TTFLs) to drive the periodic expression of clock gene products, and maintain overall 24 h circadian rhythmicity of mammalian behavioral and physiological processes¹². It is generally reported that the immune parameters change with time of day and the dysfunction of circadian clock genes is linked to many inflammatory pathologies^{13, 14}. Besides, recent evidences reveal a bidirectional communication existing between immune system and circadian clock, in which TNF-α functions as a bridging element to modulate inflammatory response through interfering with circadian clock system¹⁵. As reported, TNF-α could induce CREB1 phosphorylation and promote BMAL1 transcription in a calcium-dependent manner¹⁶. TNF-α also could activate p38 MAPK to repressing PER and CRY gene transcription, causing prolonged rest periods in the dark¹⁷. Besides, there is a negative feed-back loop between melatonin and TNF-α, in which TNF-α-induced suppression of melatonin synthesis prolongs rest period, while melatonin-induced TNF-α suppression induces sleep period^18–20. These studies suggested that the pro-inflammatory cytokine TNF-α could affect circadian clock system in both positive and negative way. But at the cellular level, TNF-α regulates genes expression and signaling transduction usually in a cell-type-specific manner⁸, and the effects of TNF-α on the VSMCs circadian clock and inflammation are rarely studied.

In this study, we found that the treatment of TNF-α up-regulated the expression of Bmal1 and disrupted its rhythm in VSMCs through phosphorylating REV-ERBα. And the overexpression of Bmal1 furtherly enhanced the oxidative stress and NF-κB signaling, which aggravated the TNF-α-induced inflammatory response in VSMCs, and inhibition of oxidative stress could significantly alleviate the Bmal1 overexpression-exacerbated inflammation by TNF-α. In conclusion, our present work confirmed the pathogenic relationship between TNF-α and the circadian clock gene Bmal1 in VSMCs, and explored a specific pathway by which the transcriptional activation of Bmal1, mediated by the TNF-α-induced REV-ERBα phosphorylation, triggered oxidative stress to exacerbate inflammatory response in VSMCs. Thus, the clock gene Bmal1 may be a potential diagnostic marker and therapeutic target for TNF-α mediated vascular inflammation.

Cell Culture and Treatment

Mice Vascular smooth muscle cells (VSMCs) were cultured in RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% FBS and 1% penicillin-streptomycin solution at 37°C in a humidified atmosphere containing of 5% CO₂. VSMCs were treated with or without TNF-α (50ng/mL) for 6 h.

Cell Transfection

Recombinant adenovirus carrying shRNA for Bmal1 knockdown (Ad-shBmal1) and overexpression (Ad-Bmal1) were designed by Genechem Company. The VSMCs were starved with no serum media for 6–8 h, then transfected with the corresponding adenoviruses for 12 h, followed by standard or HGHP m9edia for another 48 h.

Western blotting (WB)

Protein concentration was measured using a BCA reagent kit (Thermo Fisher Scientific, USA). Protein samples were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (10% gel), and transferred onto polyvinylidene fluoride membrane. After blocking with 5% skim milk, the membrane was incubated with a primary antibody (anti-p65, anti-p-p65, anti-SOD2, anti-IL-6, anti-MCP-1, anti-REV-ERBα or anti-RORα) overnight at 4°C, followed by incubation with a horseradish peroxidase-conjugated secondary antibody. Protein bands were detected using the BioRad ChemiDoc XRS + Imager (BioRad). Quantification of detected protein bands was performed with the ImageLab 6.0.1 software (Life Science, Waltham, MA). All antibody were purchased from Proteintech (Wuhan, China).

Immunoprecipitation (IP) for REV-ERBα phosphorylation

The IP experiment was performed using Protein A/G magnetic beads (Beyotime Institute of Biotechnology) according to the manufacturer’s instructions. In brief, lysis buffer (Beyotime Institute of Biotechnology) supplemented with 1% protease inhibitor cocktail (Sigma) was used to lyse cells on ice for 15 min to obtain protein. The antibody was prepared into 20ug/ml antibody working solution, and 20 µl Protein A/G magnetic beads was added to 500 µl antibody working solution, incubated in a rotator at room temperature for 1h. Then, 500ug protein were added into each reaction, and incubated in rotator at 4°C overnight. After incubation, the beads were washed three times in cold lysis buffer and centrifuged 1000 rpm at 4°C for 5 min. Before the WB assay for detecting REV-ERBα phosphorylation, 1× sodium dodecyl sulfate (SDS)-containing buffer was added into beads and boiled for 10 min to denature the protein.

RNA extraction and qRT-PCR

Total RNA was extracted from the cell samples using TRIzol reagent (Technologies, USA) following the manufacturer’s protocol. The mRNA was transcribed into cDNA using the PrimeScript RT Master Mix Kit (TaKaRa, Japan), and the cDNAs were amplified using the FastStart Universal SYBR Green Master Kit (TaKaRa, Japan), according to the manufacturer's protocol. The relative expression of mRNA was normalized with the expression of β-actin.

Mitochondrial Reactive Oxygen Species (ROS) staining

MitoSOX Red (Thermo Fisher Scientific) was used to detect mitochondrial ROS. Cells were stained with 10 µM MitoSOX Red in PBS for 15 minutes at 37°C and 5% CO2, Then, the cells were washed with PBS twice. Images were acquired with a laser scanning confocal microscopy system (LSM 710, Carl Zeiss) and mean fluorescence intensities were calculated with ImageJ software.

Measurement of Malondialdehyde (MDA) level

MDA, a reliable marker of lipid peroxidation, was determined with thiobarbituric acid (TBA) according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, China). MDA reacts with thiobarbituric acid (TBA) at 90–100°C and acidic condition. The reaction yields a pink MDA-TBA conjugate, which was measured at 532 nm using a Multi-Mode Microplate Readers (SpectramMax M5). The cellular MDA was expressed nmol/mg cellular protein.

Transwell Assay

VSMCs at density of 1 × 10⁶ cells with or without treatment of TNF-α in supernatant medium was added to the lower chamber of the transwell chamber, while RAW264.7 macrophage cell was added to the upper chamber. After 12 h of incubation, non-migrated cells in the upper chamber were removed via cotton swab, and those migrated into the lower chamber were fixed, stained with 10% crystal violet, and then quantified from 3 random fields under the inverted microscope (CX71, Olympus, Japan).

Luciferase Reporter Assay

To detect whether TNF-α activated NF-κB signaling, a dual-luciferase reporter assay was applied. NF-κB luciferase reporter vector was transfected into VSMCs using Lipofectamine 2000 (Invitrogen), with the pRL-TK control vector (Promega, Madison, WI, USA) transfected as control. The luciferase activity was analyzed with the Dual-Luciferase Reporter Assay System (E1910; Promega) after transfecting for 48 hours.

ELISA Assay

The supernatant concentrations of IL-6, MCP-1 in the TNF-α treated VSMCs were detected using enzyme-linked immunosorbent assay (ELISA) assay with commercial kits (R&D Systems, USA). In brief, VSMCs at a density of 0.5 × 10⁶ cells/mL were induced with TNF-α (50 ng/mL) for 6 h. Then the cell culture supernatants were collected by centrifugation and subjected to the quantification of secreted proinflammatory cytokines and chemokines of IL-6 and MCP-1 ELISA kits according to manufacturer’s instructions.

Statistical Analysis

Data were evaluated on GraphPad Prism 8.0. Differences were compared by using analysis of variance (ANOVA) followed by Tukey’s posttest analysis for comparison of multiple independent groups or unpaired Student’s t test for direct analysis of two independent groups. Results are presented as mean ± SEM from at least three independent experiments. Differences were considered significant at the values of *P < 0.05, **P < 0.01, ***P < 0.001 and **** P < 0.0001.

1. TNF-a induced oxidative stress and activated NF-κB-mediated inflammatory signaling in VSMCs.

Oxidative stress was usually triggered in the TNF-α-induced inflammation²¹. To investigate the oxidative stress in VSMCs upon TNF-α stimulation, we added 50 ng/ml TNF-α in the cultured VSMC for 6h, and then evaluated the cellular levels of mitochondrial reactive oxygen species (ROS), malonaldehyde (MDA) and superoxide dismutase (SOD2) through fluorescence staining, TBA reaction and Western blotting analysis, respectively. As shown in Fig. 1A-1C, stimulation with TNF-α significantly increased the mitochondrial ROS, MDA and SOD2 levels, suggesting the induction of oxidative stress by TNF-α in VSMCs. Next, we constructed and transfected NF-κB responsive firefly luciferase plasmid into VSMCs and found the addition of TNF-α significantly activated the firefly luciferase activity (Fig. 1D). Western blotting and RT-qPCR analyses also shown the protein levels of phosphorylated-p65 (p-p65)/p65 (Fig. 1E), and the expression levels of inflammatory cytokines (IL6 and MCP-1) (Fig. 1F and 1G) were significantly enhanced in the VSMCs upon TNF-α stimulation. ELISA and the co-culture with RAW264.7 macrophages transwell assays furtherly indicated that the production and release of IL6 and MCP-1 (Fig. 1H), and the inflammatory cells migration into VSMCs were both activated by TNF-α stimulation (Fig. 1I). These results suggested TNF-α triggered oxidative stress and NF-κB signaling, promoting cytokines production and exacerbating inflammatory response in VSMCs.

1. TNF-a triggered oxidative stress and activated NF-κB-mediated inflammatory response in cultured VSMCs.

A-I, Immortalized mouse cerebral smooth muscle cells were treated with TNF-a (50 ng/mL) for 6h. A, Representative images and quantification of mitochondrial ROS staining, by confocal microscopy (n = 3); Scale bar = 10µm. B, Relative level of MDA, by TBA reaction (n = 3). C, Relative protein level of SOD2, by Western blotting analysis (n = 3). D, NF-κB responsive firefly luciferase plasmid was transfected into cultured VSMCs and then the fluorescence intensity of firefly luciferase activity was detected upon TNF-a stimulation (n = 3). E, Relative protein levels of phosphorylated-p65 (p-p65)/p65, by Western blotting analysis (n = 3). F and G, Relative mRNA expression (F) and protein (G) levels of inflammatory cytokines (IL6 and MCP-1), by RT-qPCR analysis and Western blotting analysis (n = 3). H, Protein levels of inflammatory cytokines (IL6 and MCP-1) in the supernatant, by ELISA assay (n = 3). I, VSMCs were co-cultured with RAW264.7 macrophages upon TNF-α stimulation and then counts of macrophages migration were quantified, by transwell assay (n = 3). A-I, Data represented as mean ± SEM. *P < 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001. Statistical significance was determined by unpaired Student’s 2-tailed t test with Welch's correction.

2. TNF-α activated but disrupted the circadian expression of Bmal1 in VSMCs.

It is recently reported that TNF-α interfered with circadian clock to modulate inflammatory response^{16, 22, 23}. To assess the effect of TNF-α on the circadian gene Bmal1, VSMCs were treated with TNF-α in different concentrations (0, 20, 50,100 and 200 ng/ml) (Fig. 2A) and for different time times (0, 0.5, 2, 6, 12 and 24 h) (Fig. 2B), and RT-qPCR analysis found the mRNA expression level of Bmal1 were significantly increased, and the up-regulation peaked at the stimulation of 50 ng/ml TNF-α for 6 h. Considering Bmal1 has stable period and amplitude as a circadian clock gene, next we aimed to identify whether TNF-α disturbed the circadian rhythmicity of Bmal1 expression. The cultured VSMCs were treated with 100 nM dexamethasone for 2 h to reset circadian time in vitro, and subsequently stimulated with 50 ng/ml TNF-α at CT0 (the end of dexamethasone shock). As shown in Fig. 2C-2F, the mRNA and protein expression of Bmal1 both exhibited robust circadian rhythmicity after dexamethasone shock; but upon the stimulation with TNF-α, the Bmal1 peak expression and the horizontal baseline both were raised in most phases, with the amplitude being attenuated, suggesting a disruption of Bmal1 circadian expression. These results indicated the enhanced but rhythmicity-disrupted expression of circadian gene Bmal1 in VSMCs upon TNF-α stimulation.

A and B, Relative mRNA expression of circadian gene Bmal1 in cultured VSMCs upon TNF-α stimulation in different concentrations (0, 20, 50,100 and 200 ng/ml) (A) and for different time times (0, 0.5, 2, 6, 12 and 24 h) (B), by RT-qPCR analysis (n = 3). C-E, Immortalized mouse cerebral smooth muscle cells were reset circadian time in vitro by dexamethasone shock and subsequently they were treated with 50 ng/ml TNF-α at CT0. C and D, Relative mRNA expression of Bmal1 was detected at 0, 4, 8, 12, 16, 20 and 24h after the TNF-α stimulation without dexamethasone shock (C) and with dexamethasone shock (D), by RT-qPCR analysis (n = 3). E and F, Relative protein level of BMAL1 was detected at 0, 4, 8, 12, 16, 20 and 24h after the TNF-α stimulation without dexamethasone shock (E) and with dexamethasone shock (F), by Western blotting analysis (n = 3). A-F, Data represented as mean ± SEM.

3. Transcriptional activation of Bmal1 exacerbated TNF-a-induced oxidative stress and inflammatory response in VSMCs

Circadian clock is critically implicated in the inflammatory response¹³. To evaluate the role of Bmal1 in the TNF-α-induced inflammation, we applied plasmid or siRNA transfection to overexpress or inhibit Bmal1 expression in the TNF-α-stimulated VSMCs respectively, and then detected the levels of oxidative stress, NF-κB signaling, and inflammatory cytokines. Results indicated that overexpression of Bmal1 not only induced but also furtherly aggregated the TNF-α-increased mitochondrial ROS and MDA production (Fig. 3A and 3B), NF-κB signaling activation (Fig. 3C), and inflammatory cytokines expression (Fig. 3D and 3E); while inhibition of Bmal1 alleviated the activation of oxidative stress and inflammatory signaling upon TNF-α stimulation (Fig. 3A-3E). These results suggested that the TNF-α-stimulated transcriptional activation of Bmal1 exacerbated oxidative stress and inflammatory response in VSMCs.

A-E, cultured VSMCs were transfected with plasmid or siRNA targeting Bmal1 to overexpress or inhibit Bmal1 expression respectively and then stimulated with 50 ng/mL TNF-α for 6h. A, Representative images and quantification of mitochondrial ROS staining, by confocal microscopy; Scale bar = 10µm. B, Relative level of MDA, by TBA reaction (n = 3). C, Relative protein levels of phosphorylated-p65 (p-p65)/p65, by Western blotting analysis (n = 3). D and E, Relative mRNA expression (D) and protein (E) levels of inflammatory cytokines (IL6 and MCP-1), by RT-qPCR analysis and Western blotting analysis (n = 3). A-E, Data represented as mean ± SEM. *P < 0.05, **P < 0.01, *** P < 0.001 and **** P < 0.0001 vs. Control. #P < 0.05, ##P < 0.01, ### P < 0.001 and #### P < 0.0001 vs. TNF-α. Statistical significance was determined by one-way ANOVA.

4. Inhibition of oxidative stress alleviated Bmal1 activation-exacerbated inflammatory response upon TNF-a stimulation.

It is generally reported that oxidative stress is involved in the circadian disorders and inflammatory pathologies²⁴. In the present work, in the TNF-α-stimulated VSMCs, we added the mitochondria-targeted antioxidant MitoQ to attenuate oxidative stress²⁵, and combined with Bmal1 overexpression/ inhibition to detect the role of oxidative stress in the Bmal1 overexpression-exacerbated inflammation. As shown by mitochondrial ROS staining (Fig. 4A) and MDA evaluation (Fig. 4B), inhibiting oxidative stress significantly attenuated the TNF-α-induced and the Bmal1 overexpression-exacerbated oxidative stress in VSMCs. RT-qPCR (Fig. 4C) and Western blotting (Fig. 4D) analyses furtherly indicated the TNF-α-induced and Bmal1 overexpression-exacerbated inflammatory response also were significantly alleviated with oxidative stress inhibition. These results suggested that in the TNF-α-induced VSMCs inflammation, the transcriptional activation of Bmal1 exacerbated inflammatory signaling by triggering oxidative stress and clearance of oxidative stress could efficiently prevent the circadian disruption-mediated VSMCs inflammation.

A-D, cultured VSMCs transfected with Bmal1 overexpression plasmid or siRNA were treated with MitoQ (a kind of oxidative stress inhibitor) for 2h and then stimulated with 50 ng/mL TNF-α for 6h. A, Representative images and quantification of mitochondrial ROS staining, by confocal microscopy (n = 3); Scale bar = 20µm. B, Relative level of MDA, by TBA reaction (n = 3). C and D, Relative mRNA expression (C) and protein (D) levels of inflammatory cytokines (IL6 and MCP-1), by RT-qPCR analysis and Western blotting analysis (n = 3). A-D, Data represented as mean ± SEM. *P < 0.05, **P < 0.01, *** P < 0.001 and **** P < 0.0001 vs. TNF-α. #P < 0.05, ##P < 0.01, ### P < 0.001 and #### P < 0.0001 vs. TNF-α + MitoQ, Statistical significance was determined by one-way ANOVA.

5. TNF-a promoted Bmal1 transcription by enhancing REV-ERBα phosphorylation and degradation in a JNK-dependent manner.

REV-ERBα and RORα are two crucial regulators within TTFLs to maintain the rhythmic expression of Bmal1²⁶. In order to clarify the molecular mechanism of Bmal1 transcriptional activation by TNF-a, we detected the protein levels of REV-ERBα and RORα in the TNF-α-stimulated VSMCs, and found RORα expression had no significant change but the expression of REV-ERBα was significantly decreased (Fig. 5A). Immunoprecipitated (IP) combined with immunoblot assay furtherly found TNF-α significantly enhanced the phosphorylated-serine/threonine level and decreased the REV-ERBα protein level (Fig. 5B). Recent studies indicated the phosphorylation of REV- ERBα could promoted the REV- ERBα protein degradation²⁷, and TNF-a usually exerts biological effects through activating the MAPK pathway of p38 and JNK signaling to phosphorylate downstream molecules⁷. In order to clarify which signaling accounted for the TNF-α-induced REV- ERBα phosphorylation, the cultured VSMCs were treated with p38 inhibitor and JNK inhibitor respectively, and results showed that the TNF-α-induced phosphorylation and degradation of REV- ERBα were significantly inhibited by blocking JNK signaling (Fig. 5C). These results suggested that TNF-α activated JNK signaling to phosphorylate REV- ERBα and promote its degradation, which finally enhanced the transcription of Bmal1.

A-C, cultured VSMCs were stimulated with TNF -α (50 ng/mL) for 6 h. A, Relative protein level of REV-ERBα and RORα, by Western blotting analysis (n = 3). B, REV-ERBα protein were immunoprecipitated (IP) and separated by SDS-PAGE, and then the relative phosphorylated-serine/threonine level of REV-ERBα was detected by immunoblot assay. C, cultured VSMCs were treated with p38 inhibitor (left) and JNK inhibitor (right) for 1h upon TNF-α stimulation, respectively. Next, REV-ERBα protein were immunoprecipitated to detect the relative phosphorylated-serine/threonine level in REV-ERB by immunoblot assay. A, Data represented as mean ± SEM. **P < 0.01. Statistical significance was determined by unpaired Student’s 2-tailed t test with Welch's correction.

The major and novel findings of this study are as follows: 1) TNF-α stimulated Bmal1 transcription and disrupted its circadian expression in VSMCs. 2) Transcriptional activation of Bmal1 furtherly activated TNF-α-induced- NF-κB signaling and exacerbated VSMCs inflammation by triggering oxidative stress. 3) TNF-α-activated JNK signaling enhanced REV-ERBα phosphorylation and degradation, and thus promoted Bmal1 transcription in VSMCs.

TNF-α activated oxidative stress and inflammatory signaling in VSMCs.

As a kind of highly plastic cells, VSMCs could undergo inflammatory phenotype in the stimulation of pro-inflammatory mediators, and the transition of inflammatory phenotype in VSMCs played crucial roles in the occurrence and development of many cardiovascular diseases (CVDs), such as atherosclerosis, arterial aneurysm and arteritis^{9, 10, 28}. TNF-α is one of the most important pro-inflammatory mediators, which is produced by macrophages and lymphocytes and could directly activate at least 5 different cellular signaling, including NF-κB, apoptosis, extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38 MAPK), and c-Jun N-terminal kinase (JNK MAPK)²⁹. Besides, the reciprocal interactions between oxidative stress and inflammatory response, in which the activation of inflammatory cells can secrete oxidative stress mediators, while oxidative stress mediators can also activate and enhance inflammatory molecules and inflammation, have been established in many studies^{21, 30}. Here, we found in the stimulation of pro-inflammatory mediator TNF-α, the oxidative stress (Fig. 1A-1C) and NF-κB signaling (Fig. 1D-1E) were activated in VSMCs, accompanied by the enhanced cytokines production and inflammatory cells migration (Fig. 1F-1I), suggesting TNF-α induced VSMCs inflammation via activating oxidative stress and NF-κB signaling. In the patients with CVDs, circulating levels of TNF-α are significantly increased and drove the inflammation within vessels wall, which is an important inducement of atherosclerotic vascular disease³¹.

Transcriptional activation of Bmal1 exacerbated TNF-α-induced VSMCs inflammation by triggering oxidative stress

Recent advances have unraveled novel oxidative stress and inflammatory mechanisms of vascular dysfunction in many cardiovascular diseases. And many inflammatory conditions associated with dysfunctional molecular clocks, have been reported were mediated by the oxidative stress activating NF-κB signaling^{32, 33}. For instance, the core clock regulator BMAL1 could act on the oxidative stress pathways by modulating Nrf2 expression, and thus regulate the production of proinflammatory cytokines in various tissue types³⁴. In our present study, we observed that in the stimulation of TNF-α, the Bmal1 transcription was significantly enhanced and furtherly triggered oxidative stress and exacerbate the inflammatory response in VSMCs (Fig. 3); while eliminating oxidative stress by adding MitoQ, could significantly relieve the Bmal1 activation-exacerbated VSMCs inflammation (Fig. 4). Actually, the regulatory role of Bmal1 within different cell types in inflammatory signaling is disparate and discrepant. It is reported that in the macrophages³⁴, myeloid cells³⁵ and skin keratinocytes³⁶, Bmal1 suppress inflammatory signaling; while in the gastric epithelial progenitor-derived cells³⁷, synovial cells²³ and myeloid cells³⁸, Bmal1 promote inflammatory response. Here, we determined that in the VSMCs, Bmal1 transcriptional activation indeed exacerbated the TNF-α-induced inflammatory response, in which oxidative stress played a crucial role and clearance of oxidative stress could efficiently prevent the circadian disruption-mediated VSMCs inflammation. suggesting a potential role of oxidative stress as the therapeutic target for the TNF-α-induced circadian disruption and cardiovascular disease.

TNF-α disrupted circadian expression of Bmal1 through phosphorylating REV-ERBα.

In addition to a pro-inflammatory cytokine, TNF-α also could act as a kind of zeitgeber mediator to integrate with circadian clock and modulate circadian rhythm by regulating clock genes transcription¹⁵. The operation of circadian clock is generally maintained by the transcriptional-translational feedback loops (TTFL), in which BMAL1/ CLOCK-heterodimer activate the transcription of clock-controlled output genes, as well as the repressors of PER and CRY, and the transcriptional regulators of retinoid-related orphan receptors (ROR α, β, γ) and REV-ERBα/REV-ERBβ, to inhibit further transcriptional activation and finally produce robust 24h rhythms of gene expression¹². Rhythmic changes in Bmal1 transcription are driven by the orphan nuclear-receptor genes (REV-ERBα and RORα), in which REV-ERB represses while RORα promotes the transcription of Bmal1²⁶. In our present study, we found in the stimulation of TNF-α, the peak expression and horizontal baseline of Bmal1 expression both were significantly increased in VSMCs, suggesting a consistent transcriptional activation of Bmal1 (Fig. 2A-2E). Previous research reported that the phosphorylation of REV-ERBα could promote its degradation, and finally disinhibit Bmal1 transcription²⁷. And here, we indeed found the phosphorylation and degradation of REV-ERBα were significantly enhanced in the TNF-α-stimulated VSMCs (Fig. 5A and 5B). There are two important phosphorylation signaling initiated by TNF-α: p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK MAPK)⁷. In order to clarify which signaling accounting for the phosphorylation degradation of REV-ERBα, we applied p38 inhibitor and JNK inhibitor respectively, and found it was the JNK pathway, rather than the p38 pathway, exerted functions in the TNF-α-phosphorylated REV-ERBα (Fig. 5C). Therefore, our present study firstly reported that TNF-α could enhance and disrupt the circadian rhythmicity of Bmal1 expression in VSMCs; and we also clarified that it was the increased phosphorylation degradation of REV-ERBα via JNK pathway that accounts for the TNF-α-enhanced Bmal1 transcription.

Practical implications and limitations

TNF-α as a pleiotropic cytokine plays an important role in the vascular inflammatory diseases. And it is also known that the dysfunction of molecular clock is critically involved in many inflammatory pathologies. In addition to the traditional role of TNF-α as a pro-inflammatory cytokine, our present work found that TNF-α also could act as a kind of zeitgeber mediator to induce the transcriptional activation of circadian clock gene Bmal1 via JNK- REV-ERBα phosphorylation pathway in VSMCs, which activate oxidative stress and NF-κB signaling, and finally exacerbate TNF-α-induced VSMCs inflammation. Therefore, it is thought that therapeutic manipulations targeting circadian rhythm, and oxidative stress in VSMCs may represent an opportunity to treat the TNF-α-induced vascular inflammation. However, considering that most of our present experiments were conducted in vitro, hence in the future, we are to be focusing on verifying our present findings in various in vivo models.

In summary, under the stimulation of TNF-α, the transcription of circadian gene Bmal1 and its circadian expression in VSMCs were significantly activated but rhythmicity-disrupted, which was mediated by the enhanced phosphorylation and degradation of REV-ERBα in a JNK-dependent manner. The transcriptional activation of Bmal1 furtherly triggered oxidative stress and thus activated NF-κB signaling to exacerbated the TNF-α-induced VSMCs inflammation. Our present work clarified the specific signaling pathway of Bmal1 activation and its deteriorating effects in the TNF-α-induced VSMCs inflammation, which imply that the therapeutic manipulations targeting circadian rhythm and oxidative stress in VSMCs may represent an opportunity to treat the TNF-α-induced vascular inflammation (Fig. 6).

The authors have no conflicts of interest to declare.

Funding

This study was supported by grants from the National Natural Science Foundation of China (82072101) and the National Key Research and Development Program of China (2020YFA0803603 and 2021YFA1301402).

Author Contribution

M-J.X., X-L.Y., Y-L.G. and P-J.L. designed and performed the experiments, prepared the ﬁgures, and wrote the manuscript. Y-R.B., B.Z., J.X., S-Y.H., Y-G.B., and Q-L.C. contributed to the performance of the experiments., J.M., and L.Z. supervised the work and wrote the manuscript.

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No competing interests reported.

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BMAL1/REV-ERBα loop as a novel inflammatory sensor to drive NF-κB-mediated inflammation in vascular smooth muscle cells by modulating oxidative stress

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Abstract

Objectives and design:

Methods

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Conclusion

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Introduction

Materials and methods

Cell Culture and Treatment

Cell Transfection

Western blotting (WB)

Immunoprecipitation (IP) for REV-ERBα phosphorylation

RNA extraction and qRT-PCR

Mitochondrial Reactive Oxygen Species (ROS) staining

Measurement of Malondialdehyde (MDA) level

Transwell Assay

Luciferase Reporter Assay

ELISA Assay

Statistical Analysis

Results

Discussion

Practical implications and limitations

Conclusions

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Funding

Author Contribution

References

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