TNFα induces matrix metalloproteinase-9 expression in monocytic cells through ACSL1/JNK/NF- kB signaling pathways

doi:10.21203/rs.3.rs-2503035/v1

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TNFα induces matrix metalloproteinase-9 expression in monocytic cells through ACSL1/JNK/NF- kB signaling pathways

https://doi.org/10.21203/rs.3.rs-2503035/v1

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published 01 Sep, 2023

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Background

Studies have established the association between increased plasma levels of matrix metalloproteinase (MMP)-9 and adipose tissue inflammation. Tumor necrosis factor α (TNFα) was elevated in obesity and is involved in the induction of MMP-9 in monocytic cells. However, the underlying molecular mechanism was incompletely understood. As per our recent report, TNFα mediates inflammatory responses through long-chain acyl-CoA synthetase 1 (ACSL1). Therefore, we further investigated the role of ACSL1 in TNFα-mediated MMP-9 secretion in monocytic cells and macrophages.

Methods

Monocytic THP-1 cells and macrophages were used to study MMP-9 expression. mRNA and protein levels of MMP-9 were determined by qRT-PCR and ELISA, respectively, and its biological activity was determined by zymography. Signaling pathways were studied using Western blotting, inhibitors, and NF-kB/AP1 reporter cells.

Results

We found that THP-1 monocytic cells and macrophages displayed increased MMP-9 mRNA expression, as well as biologically active protein secretion after incubation with TNFα. Inhibition of ACSL1 in the cells with triacsin C significantly reduced MMP-9 secretion. However, inhibition of β-oxidation and ceramide biosynthesis was not affected by TNFα-induced MMP-9 production. Using small interfering RNA-mediated knockdown, we further confirmed that TNFα-induced MMP-9 secretion was significantly reduced in ACSL1-deficient cells. Moreover, TNFα-mediated MMP-9 expression was significantly reduced by inhibition of ERK1/ERK2, JNK, and NF-kB signaling pathways. We further observed TNFα-induced phosphorylation of JNK, ERK1/ERK2, and NF-kB. On the other hand, inhibition of ACSL1 reduced TNFα-mediated phosphorylation of JNK, c-Jun, ERK1/2, and NF-kB in THP-1 monocytic cells. In addition, increased NF-κB/AP-1 activity was inhibited in triacsin C-treated cells.

Conclusion

Altogether, our findings suggest that ACSL1/JNK/ ERK/NF-kB axis plays an important role in the regulation of MMP-9 induced by TNFα in monocytic THP-1 cells and macrophages.

Biological sciences/Immunology

Biological sciences/Molecular biology

ACSL1

TNFα

MMP-9

monocytes/macrophages

Matrix metalloproteinases (MMPs) are factors involved in various biological events, including angiogenesis, embryogenesis, inflammation, and wound healing. Their role in healthy tissue remodeling has been well-established¹. Abnormalities in the expression of MMPs result in the development of various inflammatory disorders, such as heart diseases, arthritis, cancer metastasis, and atherosclerosis. Among MMPs, MMP-9 is predominantly involved in denaturing native type IV, which is a common component of the basement membrane. MMP-9 plays a role in the breakdown of various non-extracellular matrix (ECM) molecules, such as substance P, IL-1β, and myelin basic protein². The overexpression of MMP-9 results in cells migrating to inflammation sites, sustaining the survival of target cells, along with promoting macrophages renewal, thereby contributing toward the development and progression of chronic inflammatory diseases³. MMP-9 is mainly secreted by monocytes or macrophages, and contributes toward the pathogenesis of obesity-induced inflammation, insulin resistance, and cancer metastasis^4–6. The expression of MMP-2 and MMP-9 is augmented in inflamed tissues during chronic inflammatory conditions, such as obesity, arthritis, atherosclerosis, and periodontal disease. These infiltrated immune cells overexpress MMP-9, causing degradation of connective tissue and triggering pathogenesis ^7–10. MMP-9 also induces the degradation of the basement membrane and ECM components, which facilitates the trans-endothelial migration of monocytes/macrophages ¹¹.

MMP-9 expression is increased by lipopolysaccharide and proinflammatory cytokines such as interleukin-1 (IL-1) ¹², IL-6 ¹³, and tumor necrosis factor α (TNFα) ¹⁴. Notably, elevated circulating concentrations of TNFα and MMP-9 have been found in inflammatory disorders including obesity, cancer, atherosclerosis, and diabetes ¹⁵. However, the mechanism by which TNFα induces MMP-9 is incompletely understood. Herein, we determined TNFα-induced MMP-9 production in monocytic cells, monocytes/macrophages, and the signal transduction pathway(s) that were involved in this induction. Since TNFα-mediated immune responses, in part, have been induced by Acyl-CoA synthetase 1 (ACSL1) ¹⁶, we also investigated the role of ACSL1 in TNFα-mediated MMP-9 secretion. We found that pharmacologic and genetic inhibition of ACSL1 repressed TNFα-stimulated MMP-9 expression in monocytic cells, along with the activation of JNK, c-Jun, ERK and NF-kB.

THP-1 Monocytic Cell Culture and Stimulation

THP-1 cells were obtained from the American Type Culture Collection (ATCC) and cultured according to their recommendation ^17–19. In brief, cells were maintained in RPMI-1640 culture medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 100 ug/ml Normocin, 50 U/ ml penicillin, and 50 µg/ml streptomycin (Gibco, Life Technologies, Grand Island, NY, USA). For experimentation, cells were plated in 12-well plates (Costar, Corning Incorporated, Corning, NY, USA) at 1 × 10⁶ cells/well (unless indicated otherwise). Cells were then stimulated for 24 hours with 10ng/ml TNFα (R&D Systems, Minneapolis, MN, USA) or 0.1% BSA as vehicle control. All cultures were incubated under recommended cell culture conditions at 37°C (with humidity) in 5% CO₂. At the endpoint of the experiment, cells were harvested for RNA isolation, and the conditioned medium was used for the determination of MMP-9 secreted protein. For NF-kB/AP-1 reporter cells, cells were cultured in complete RPMI medium with the addition of zeocin (200 µg/ml) as a selective factor (InvivoGen, San Diego, CA, USA).

THP-1-Derived Macrophages

THP-1 monocytic cell-derived macrophages were generated as previously described by stimulating them with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) ²⁰. The treated cells were then incubated at 37°C (under humidity) in 5% CO₂ for 48 hours, and the morphological presentation of the cells was monitored throughout. Upon cells' transformation and adherence, the culture medium was removed and replaced by fresh media. Transformed macrophages were treated with TNF-α or vehicle as mentioned previously.

Real-Time Quantitative Polymerase Chain Reaction (PCR)

Total RNA was isolated from cultured cells using RNeasy Mini Kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions. cDNA synthesis was carried out using 1 µg of the total RNA isolated through the use of a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). 500 ng cDNA was then amplified, and the gene expression of (MMP-9, Hs00234579_m1 ; ACSL1, Hs00960561; and GAPDH, Hs03929097_g1) was conducted through the use of TaqMan® Gene Expression Master Mix (Applied Biosystems, Foster City, CA, USA) according to manufacturer’s instructions ^20–23. The threshold cycle (Ct) was normalized to the house-keeping gene GAPDH, and the expression of the target gene was calculated relatively to control using the ΔΔCt-method ^24–27. Relative mRNA expression was visualized as fold expression over the average of control gene expression, with the control treatment assumed to be 1²⁸. The data is presented as mean standard error of the mean (± SEM), and statistical analyses were deemed significant at p < 0.05.

MMP-9 Determination

Sandwich ELISA was used according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA) to find MMP-9 protein in the supernatants of treated cells.

Gelatin Zymography

Gelatin zymography was used to detect the gelatinase activity of MMP-9 as previously described and in accordance with BioRad technology ²⁹. In brief, the collected conditioned medium from treated cells was mixed with zymogram sample buffer containing (62.5mM Tris-HCl, pH.6.8, 25% glycerol, 4% SDS and 0.01% bromophenol blue) and wad subjected to electrophoresis on 10% polyacrylamide gel with gelatin (10% Ready Gel® Zymogram Gel, Biorad) as the substrate. After electrophoresis was completed, the gel was incubated for 1 h at room temperature in a 2.5% Triton X-100 solution and treated with a zymogram developing buffer containing 50mM Tris-HCl, pH 7.5, 200mM NaCl, and 5mM CaCl2 at 37°C for 24 hours. The gels were stained with 0.5% Coomassie Brilliant Blue R-250, 40% Methanol, 10% Acetic Acid solution for 2 hours, followed by destaining step in 40% Methanol, and 10% Acetic Acid solution to visualize the bands. Proteolytic activity was indicated by the presence of clear bands against the blue background of the stained gel.

siRNA Transfections

We performed small interfering RNA (siRNA) transfection, as previously described by Al-Roub et al ³⁰. Briefly, we washed THP-1 monocytic cells and resuspended them in nucleofector solution (100 µl; Amaxa Nucleofector Kit V). We transfected the cells separately with siRNA against ACSL1 (30 nM; OriGene Technologies, Inc., city, MD, USA), scramble siRNA (30 nM; OriGene Technologies, Inc., city, MD, USA), and pmaxGFP (0.5 ug; Amaxa Nucleofector Kit V for THP-1cells, Lonza, city, country). We performed all transfection experiments with an Amaxa Cell Line Nucleofector Kit V for monocytic cells (Lonza, city, Germany) using an Amaxa Electroporation System (Amaxa Inc., city, Germany)¹⁷. After 36 hours, we treated the siRNA transfected cells with TNFα. Next, after 24 hours, we harvested the monocytic cells and conditioned media. Lastly, we assessed the gene knockdown level of ACSL1 using real-time PCR.

Western Blotting

We performed Western blotting, as described earlier³¹. We first harvested treated and untreated THP-1 monocytic cells. Then, we treated the cells with lysis buffer (10X Lysis Buffer, Cell Signaling, USA) for 30 minutes. We resolved the lysates by 12% SDS-PAGE, as described earlier, ³¹ and transferred the cellular proteins to an Immuno-Blot PVDF membrane (Bio-Rad Laboratories, USA) by electroblotting. We blocked the Immuno-Blot PVDF membranes with 5% non-fat milk in phosphate buffered saline (PBS) for 1 hour. We then incubated the membranes with primary antibodies against p-44/42 mitogen-activated protein kinases (MAPK; ERK1/2), p-SAPK/JNK, p-c-Jun, p-NF-κB, and the respective unphosphorylated antibodies in 1:1000 dilution overnight at 4°C. We procured all primary antibodies from Cell Signaling (Cell Signaling Technology Inc., Danvers, MA, USA). We then washed the blots and incubated them for 1 hour with horseradish peroxidase-conjugated secondary antibody (Promega, Madison, WI, USA). We developed immunoreactive bands using an Amersham ECL Plus Western Blotting Detection System (GE Health Care, city, UK) and visualized them by Molecular Imager® VersaDocTM MP Imaging Systems (Bio-Rad Laboratories, Hercules, CA, USA).

Statistical Analysis

We performed statistical analyses on the GraphPad Prism software ( La Jolla, CA, USA). Data are presented as mean ± standard error of the mean (SEM). We used unpaired Student’s t-test and one-way ANOVA to compare means between groups. P-value < 0.05 was considered significant (*P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001).

TNFα Induced MMP-9 Gene Expression in Human Monocytes and Macrophages

We treated monocytic cells with TNFα for 24 hours and investigated the impact of TNFα on MMP-9 gene expression regulation in these cells. Our results demonstrate that the mRNA expression levels of MMP-9 were significantly elevated (6.621-fold; p < 0.0014) in TNFα-treated THP-1 monocytic cells, as compared to controls, i.e., cells treated with vehicle alone (Fig. 1A). In concordance, the protein levels of MMP-9 were also significantly elevated in the supernatant of cells stimulated with TNFα (26073 pg/ml; P < 0.0025; Fig. 1B). To ensure that MMP-9 produced by TNFα or LPS was biologically active, we performed gelatin zymography on the conditioned media obtained from cells treated with TNFα. Zymography showed high level of MMP-9 activity in the condition media obtained from TNFα-treated cells, as compared to controls (Fig. 1C).

Since macrophages play a key role in producing inflammation in peripheral tissues, we also examined the effect of TNFα on macrophages in regard to MMP-9 production. We found that TNFα upregulates the gene expression of MMP-9 (44.48-fold, P = 0.0003) and protein production in macrophages (32321 pg/mL, P = 0.0026) (Fig. 1D and 1E). Zymography showed high level of MMP-9 activity in the condition media obtained from TNFα-treated macrophages, as compared to controls (Fig. 1F).

TNFα-Induced MMP-9 Production is Suppressed by the Inhibition of ACSL1

Accumulating evidence suggests that ACSL1 participates in TNFα-mediated immune regulation ^16,32. We, therefore, investigated whether ACSL1 was involved in TNFα- mediated MMP-9 production by THP-1 cells. ACSL1 activity was inhibited in THP-1 monocytic cells using triacsin C. As per our results, pretreatment of the monocytic cells with triacsin C followed by exposure to TNFα resulted in a significant decrease in MMP-9 expression (Fig. 2A and 2B; p < 0.05). Zymography showed that MMP-9 activity was inhibited in the condition media obtained from the cells pretreated with triacsin C followed by TNFα exposure (Fig. 2C).

We observed similar results in macrophages (Fig. 2D–F). Since the gene expression of MMP-9 is activated by TNFα via ACSL1, which steer fatty acids towards β-oxidation ³³ and ceramide production ³⁴, we next aimed to elucidate whether these factors play a role in TNFα-mediated MMP-9 production. We therefore incubated monocytes and macrophages with inhibitors of fatty acid oxidation (etomoxir) or ceramide synthesis (myriocin) prior to TNFα exposure. Interestingly, etomoxir and myriocin did not reduce the expression of MMP-9 (Monocytes Fig. 2A–C; Macrophages Fig. 2D–F).

ACSL1 Deficiency Suppresses TNFα-Induced MMP-9

To further verify if TNFα-induced MMP-9 in the THP-1 monocytic cells was dependent on ACSL1, we transfected cells with ACSL1 siRNA that achieved > 50% reduction in ACSL1 mRNA levels, as compared to scramble (control) siRNA (Fig. 3A). As expected, the MMP-9 gene expression was significantly reduced in ACSL1 siRNA-transfected monocytic cells after stimulation with TNFα, when compared to scramble siRNA-transfected monocytic cells (Fig. 3B). Similarly, the protein expression of MMP-9 was significantly suppressed in TNFα-activated ACSL1-deficient cells (Fig. 3C). Altogether, the results demonstrate that ACSL1 is a key effector in TNFα-mediated MMP-9 production in THP-1 monocytic cells.

ACSL1 is Involved in TNFα-Activated MAPKs and NF-kB Signaling Pathways in THP-1 Monocytic Cells

MAPK signaling (ERK1/2, p38, JNK) and NF-kB signaling pathways are activated by TNFα ^35,36. We next investigated if these molecules play a role in the regulation of TNFα-stimulated MMP-9 expression in THP-1 monocytic cells. First, we incubated the cells with inhibitors of JNK (SP600125), ERK1/2 (PD98059, U0126), or p38 MAPK (SB203580), as appropriate, prior to treatment with TNFα. We found that TNFα-mediated MMP-9 mRNA expression was reduced (Fig. 4A) after treatment with either JNK (SP600125) or ERK1/2 (PD98059, U0126) inhibitor. However, p38 MAPK (SB203580) did not suppress the gene expression of MMP-9. Consistent with quantitative reverse transcription (qRT)-PCR results, we found a significant reduction in MMP-9 levels in culture supernatants of THP-1 monocytic cells (Fig. 4B). Secondly, we preincubated the cells with NF-κB inhibitors (Bay11-7085, Triptolide or resveratrol) before TNFα treatment. We found that TNFα-mediated MMP-9 mRNA and protein expression was reduced by inhibition of NF-κB signaling (Fig. 4C and 4D).

To gain further insight into the role of ACSL1 in TNFα-induced activation of MAPK and NF-kB signaling proteins in monocytic cells, we treated THP-1 monocytic cells with inhibitors of ACSL1 prior to TNFα exposure. ACSL1 inhibition significantly decreased TNFα-induced phosphorylation of JNK, c-Jun, ERK1/2, and NF-kB (Fig. 4E and 4F). These findings suggest that JNK, c-Jun, ERK1/2, and NF-kB were downstream of TNFα/ACSL1 signaling in monocytic cells. Given that the MMP-9 gene promoter has NF-κB/AP-1 binding sites, the loss of NF-κB/AP-1 activation is expected to result in the suppression of MMP-9 gene expression^37,38. We used NF-κB/AP-1 reporter cells and treated them with TNFα. We found that TNFα induced NF-kB/AP-1 activity in the reporter cells (Fig. 5A). Consistent with NF-κB /AP-1 activity, MMP-9 gene expression and protein production were increased in the reporter monocytic cells (Fig. 5B and 5C).

Next, we used NF-kB/AP-1 reporter monocytic cells to confirm the role of ACSL1 in TNFα-induced NF-kB/AP-1 activity on MMP-9 gene expression and protein production. We observed that ACSL1 inhibition suppressed the NF-kB/AP-1 activation induced by TNFα in the reporter cells (Fig. 5D). Consistent with NF-κB /AP-1 activity, MMP-9 gene expression and protein production were increased in the reporter monocytic cells (Fig. 5E and 5F).

It is already known that MMP-9 overproduction is involved in the pathogenesis of inflammatory diseases and obesity-associated chronic low-grade inflammation^6,39. In regard to this, our results demonstrated elevated MMP-9 levels with increasing duration of diabetes. This supports the previous statement of increased risk of diabetes incidence in individuals with high MMP-9 levels ³⁹. We also found that TNFα increases MMP-9 production; however, the underlying molecular mechanism associated with MMP-9 overexpression by TNFα in monocytic cells is yet to be elucidated.

In this study, we investigated a specific mechanism by which TNFα promotes MMP-9 expression in monocytic cells. We found that TNFα stimulated MMP-9 expression in monocytic cells and macrophages. Results from previous in vitro studies support the fact that TNFα plays a functional role in MMP-9 regulation in various cell lines^40,41. Therefore, our results strengthen the hypothesis that TNFα plays an active role in MMP-9 induction in monocytic cells. MMP-9 is increased in the skin and sera of patients with vitiligo, and MMP-9 is produced by keratinocytes in response to IFN-γ and TNFα ⁴². MMP-9 is also causal in the establishment of colitis in mice ⁴³. Plasma MMP-8, MMP-9, and TNFα, and highly sensitive C-reactive protein were substantially higher in cases of metabolic syndrome ⁴⁴. Moreover, elevated MMP-9 and TNFα levels are correlated with the progression of inflammatory disease.

To explore the underlying mechanisms and contexts by which TNFα stimulated the MMP-9 induction, it was central to determine whether ACSL1-mediated signaling is involved in TNFα-mediated MMP-9 gene expression regulation in monocytic cells. We previously showed that ACSL1 deficiency prevents TNFα-mediated induction of cell surface and secretory inflammatory markers in monocytic cells ^18,45. Herein, we used pharmacological and genetic approaches to support the evidence that TNFα-induced gene expression and protein production of MMP-9 depends on ACSL1. First, we found that MMP-9 production by TNFα is repressed by the pharmacologic inhibition of ACSL1 with triacsin C. Next, our results demonstrated that ACSL1-deficient monocytic cells do not support TNFα-stimulated gene expression and secretion of MMP-9. In a recent study, it was shown that monocytic cells require ACSL1 to facilitate inflammatory marker expression induced by TNFα ¹⁸. ACSL1 is a key enzyme that directs fatty acids towards β-oxidation ³³ and ceramide production ³⁴, and its deficiency prevents TNFα-mediated induction of IL-1β and MCP-1¹⁸. In addition to these results, when THP-1 cells were treated with inhibitors of fatty acid oxidation (etomoxir) or ceramide synthesis (myriocin) prior to TNFα treatment, we found that etomoxir and myriocin did not block TNFα-induced MMP-9 production. Interestingly, our results suggest that TNFα induces the gene and protein expression of MMP-9 via the involvement of ACSL1, without significant impact on β-oxidation and ceramide formation, which is consistent with a previous report ⁴⁶.

Accumulating evidence states that MAPKs and NF-κB signaling pathways are involved in TNFα stimulation of several inflammatory cytokines that contribute to the pathogenesis of various inflammatory conditions ^35,36. Additionally, the expression of MMP-9 seems to be highly controlled through MAPKs and NF-κB in numerous cell types ^47,48. It is important to look at the function of ACSL1 in TNFα-induced activation of MAPK and NF-κB signaling events. In the present study, we found that the inhibition of ACSL1 suppresses TNFα-mediated ERK1/2, JNK, and NF-kB phosphorylation, but not p38. Furthermore, inhibition of MAPKs and NF-kB decreased MMP-9 gene expression and protein production induced by TNFα. Interestingly inhibition of p38 MAPK had no effect on TNFα-induced MMP-9 expression. Cohen et al. reported that TNFα-induced MMP-9 expression, secretion, and activity were completely blocked by the inhibition of JNK and ERK, but not p38 MAPK ⁴⁹. A study suggests that TNFα-induced MMP-9 expression by osteoblast-like MC3T3-E1 cells was partially blocked by the inhibitor of ERK, JNK, p38 MAPK, or NF-kB ⁵⁰. In this context, another study showed that p38 MAPK regulates TNFα-induced RANTES and MMP-9 production in human bronchial epithelial cells ⁵¹. In addition, it is of interest that the MMP-9 gene is regulated by MAPK pathways which are dependent on AP-1 and NF-κB for transcription ^52,53. In human vascular smooth muscle cells, the transcription factors NF-κB and AP-1 involved in the ERK1/2-mediated MMP-9 expression in response to TNFα have been investigated ⁵³. Consistent with our findings that inhibition of ACSL1 reduces MAPKs and NF-κB phosphorylation, ACSL1 inhibition has a significant impact on the TNFα induced activation of AP-1 and NF-kB. Overall, our findings suggest that ACSL1 acts upstream of MAPK and NF-κB signaling pathways.

Altogether, our results indicate that in monocytic cells, TNFα likely activates two different pathways (MAPKs and NF-κB) leading to MMP-9 expression through the involvement of ACSL1. Our results, therefore, provide novel insights into the mechanisms of action of TNFα, stating that ACSL1-dependent MAPKs and NF-κB may be associated with the upregulation of MMP-9 in monocytic cells.

Acknowledgements

This work was supported by Kuwait Foundation for the Advancement of Sciences (KFAS) grant RA AM 2016- 007.

Conflict of interest

The authors declare no conflict of interests and no permission is required for publication.

Author contributions

AA, NA, FAR, AW performed experiments, analyzed data and participated in writing manuscript. FAR, FA, FAM, SS participated in designing, planning experiments and in critical review and editing manuscript, RA planned, designed experimental work, interpreted data and wrote the manuscript.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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No competing interests reported.

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TNFα induces matrix metalloproteinase-9 expression in monocytic cells through ACSL1/JNK/NF- kB signaling pathways

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Journal Publication

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Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Material And Methods

THP-1 Monocytic Cell Culture and Stimulation

THP-1-Derived Macrophages

Real-Time Quantitative Polymerase Chain Reaction (PCR)

MMP-9 Determination

Gelatin Zymography

siRNA Transfections

Western Blotting

Statistical Analysis

Results

TNFα Induced MMP-9 Gene Expression in Human Monocytes and Macrophages

TNFα-Induced MMP-9 Production is Suppressed by the Inhibition of ACSL1

ACSL1 Deficiency Suppresses TNFα-Induced MMP-9

ACSL1 is Involved in TNFα-Activated MAPKs and NF-kB Signaling Pathways in THP-1 Monocytic Cells

Discussion

Declarations

References

Additional Declarations

Status:

Journal Publication

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