Methyltransferase SETD4 mediates macrophages proliferation through EGFR signaling

doi:10.21203/rs.3.rs-1958184/v2

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Methyltransferase SETD4 mediates macrophages proliferation through EGFR signaling

https://doi.org/10.21203/rs.3.rs-1958184/v2

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Purpose: Epigenetic modifications have important impacts on the biological functions of cells. This study explored the effects of SET domain-containing protein (SETD)4, a histone lysine methyltransferase, on the general biological functions of bone marrow-derived macrophages (BMMs).

Materials and Methods: We employed a CRISPR/Cas9 technology-edited mouse model. After isolated from SETD4knockout (KO) and Wild-type (WT) mice, BMMs were maintained and identified. Then, serials in vitro experiments were performed to test the levels of methylation of histone lysine sites and the abilities in proliferation, cell cycle, migration, inducing vascular tube-formation and polarity. The phosphorylation pathway profiling arrays focused on the Mitogen-Activated Protein Kinase (MAPK), AKT, Janus Kinase/signal Transducer and Activator of Transcription(JAK/STAT), Nuclear Factor-κB (NF-κB), and Transforming Growth Factor β (TGF-β) signaling pathways were performed. After the Differentially Expressed Proteins (DEPs) were screened, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were adopted on those DEPs. Finally, CI-1033, an inhibitor for pan Epidermal Growth Factor Receptor (EGFR) was administrated to treat the BMMs, the cell viability and expression of phosphorylated(p)-EGFR(Ser1070) were assessed.

Results: BMMs from SETD4 KO mice exhibited significant inhibition of monomethylation, dimethylation or trimethylation of histones H3K4, H3K36, H3K79 and H4K20; significant enhancement of the proliferation, EdU-positive cell ratio, Ki67 mRNA levels, and ability to form vascular structures; and attenuation of cell migration ability. After induction by lipopolysaccharide + interferon-γ or interleukin (IL)-4, a majority of the markers that were tested, including pro-inflammatory Interleukin-1β, Interleukin-6, Inducible nitric oxide synthase and tumor necrosis factor-α and anti-inflammatory Fizz-1, Arginase 1 and Mannose receptor C-type 1 were all downregulated, while TGF-β1 was upregulated. The phosphorylation pathway profiling arrays revealed 32 DEPs, six that were upregulated and 26 that were downregulated in BMMs from SETD4 KO mice versus WT mice. These DEPs were involved in 1365 terms from the GO and 137 signals from the KEGG database. Inhibition of the most significantly upregulated protein, EGFR(Ser1070), by CI-1033 significantly reduced p-EGFR(Ser1070), and was accompanied by a decrease in the cell viability of BMMs, particularly in the SETD4 KO cells.

Conclusion: This is the first study to implicate the involvement of SETD4-mediated EGFR signaling in the proliferation of BMMs. SETD4 mediates the methylation of multiple histone lysine sites in BMMs, in turn affecting cell biology.

SETD4

Bone marrow-derived macrophages

Histone lysine methylation

Cell biology

Phosphorylation Pathway Profiling Array

Epigenetic regulation of gene expression is persistent, heritable, and does not involve changes in DNA sequence.¹ The regulatory mechanisms of epigenetics mainly involve DNA methylation, histone post-translational modification, chromosome remodeling, and microRNA regulation. Methyltransferases are the key enzymes of histone modification, and are broadly categorized into two major families: histone arginine methyltransferases and histone lysine methyltransferases. As a histone lysine methyltransferase, SET domain-containing protein 4 (SETD4) is a member of the SETD family. It was first reported to function in breast cancer cell proliferation.² Recent reports have begun to uncover other functions of SETD4, suggesting that it plays certain roles in tumors,³ organ development and regeneration,^4-7 and inflammatory responses,⁸ but its exact biological functions have not yet been fully elucidated.

In recent years, studies on the biology, immune escape, tumor escape, and tissue damage repair of macrophages have attracted much attention.^9,10 Bone marrow-derived macrophages (BMMs) in particular are widely involved in treating infection and sepsis in vitro,^11,12 attenuating atherosclerosis,^13,14 promoting pancreatic cancer progression,¹⁵ and protecting against lung cancer and glioma progression^16,17 and cardiovascular diseases.^18,19 Our previous investigation reported for the first time that a SETD4 gene knockout (KO) in mice affected the genomic methylation of bone marrow-derived mesenchymal stem cells, which was accompanied by changes in cell proliferation, migration, paracrine and differentiation functions,²⁰ suggesting that SETD4 plays a specific role in bone marrow-derived cells. In this study, we expanded our characterization of SETD4 and explored possible mechanisms of SETD4 in the biology of BMMs. Our results may provide new ideas for epigenetic regulation of diseases of inflammation and immune response.

Animals, Ethics, and BMM Culture, Identification and Polarization

All animal procedures were conducted in accordance with protocols approved by the Experimental Animal Ethics Committee of Guangdong Medical University (no. GDY2003014), and conformed to the Helsinki Declaration of 1975 (as revised in 2008) concerning animal rights. Heterozygous SETD4 KO C57BL/6J mice were bred, mated and genotyped as described previously.²⁰ Wild type (WT) and SETD4^−/− mice (male, 8 weeks old, 18–23 g) were subjected to BMM isolation. BMMs were maintained in Dulbecco’s modified Eagle's medium (DMEM; ThermoFisher Scientific, Shanghai, China) supplemented with L929 cell supernatant (25%, v/v), 20% fetal bovine serum (FBS; ThermoFisher Scientific), 100 U/mL penicillin and 0.1 μg/mL streptomycin, and were incubated in 5% CO₂ saturated humidity at 37°C. The culture medium was changed every 3 days. The supernatant of L929 cells was obtained by continuously culturing L929 cells (kindly provided by Cell Bank, Chinese Academy of Sciences, Shanghai, China) in L929 cell special medium (Cat: SCSP-5039, Cell Bank) for 7 days. Undifferentiated BMMs (M0) were subjected to immunofluorescence staining of F4/80 (Cat: 11-4801-82, ThermoFisher Scientific), and their purity was calculated. M0 macrophages treated with 100 ng/mL lipopolysaccharide (LPS; Cat: L2630, Sigma-Aldrich, Shanghai, China) + 20 ng/mL interferon-γ (IFN-γ; Cat: 315-05, PeproTech, Cranbury, NJ, USA) were induced to differentiate into a pro-inflammatory type (M1 polarization), while those treated with 20 ng/mL murine interleukin-4 (IL-4; Cat: 214-4, PeproTech) for 24 h were induced into an anti-inflammatory type (M2 polarization). The M0, M1 and M2 cells were subjected to the following experiments.

Cell Counting Kit (CCK)-8 and EdU Incorporation Assays

For CCK-8 assays, M0 cells were seeded onto 96-well plates (8000 cells/well) and allowed to recover overnight. At 0-, 1-, 2- and 3-day time points, 10 μL of CCK-8 reagents (Cat: SPDA-D010, Beyotime Institute of Biotechnology, Nanjing, China) was added, cells were maintained for additional 3 h, and optical density (OD) at 450 nm was measured using a microplate reader (ThermoFisher Scientific). Each group was duplicated in 4–6 wells. For EdU incorporation assays, cells were added to 96-well plates (10,000 cells/well) and cultured in a 37°C incubator for 24 h before adding EdU DNA proliferation in vitro detection reagents (Cat: C10310, RiboBio, Guangzhou, China), following the instruction of the manufacturer. Fluorescence microscopy was used to image and record the rate of positive cells. The numbers of cells with red fluorescence and Hoechst staining (blue fluorescence) in each field were used to calculate the cell proliferation ratio. Assays of each group were repeated in three wells.

Cell Cycle Determination by Flow Cytometry (FCM)

Serum-starved M0 cells were seeded in 6-well plates at a concentration of 5 × 10⁵ cells/well and incubated in L929 supernatant-enriched medium for 12 h, 24 h, 48 h and 72 h. Cells were collected and suspended in 70% ethanol for 24 h at 4°C. After washing with cold phosphate-buffered saline, cells were incubated with RNase A and propidium iodide at 37°C for 30 min. FCM was performed to assess the cell cycle distribution, and data were analyzed using FlowJo 7.6 software (Treestar, Ashland, OR, USA). Each time point was repeated in three wells.

Cell Migration Assay

M0 cells were serum-starved for 12 h and trypsinized; 4 × 10⁴ cells were collected in a total volume of 200 μL DMEM medium, which was added to the upper Transwell chambers; and 500 μL DMEM medium containing 15% FBS was added to the lower Transwell chambers. A membrane with an 8-μm pore size separated the upper and lower Transwell chambers. After 24 h, the chambers were disassembled. The membranes were fixed in 4% paraformaldehyde for 20 min, stained with 0.1% crystal violet for 15 min, and washed with ddH₂O for observation and photography with a microscope. Each cell sample was repeated in three chambers.

Tube-Formation Assay

Considering that M2 cells are beneficial for tube formation during tissue repair, we tested the contribution of SETD4 to the tube-formation ability of M2 cells. The supernatant was collected from M2 cells derived from WT and SETD4 KO macrophages induced by 20 ng/mL IL-4 for 24 h. Aliquots of 100 μL diluted Matrigel (Corning, NY, USA) were added 48-well plates and incubated at 37°C for 30 min, followed by the addition of 4 × 10⁴ serum-starved human umbilical vein endothelial cells (HUVECs; provided by Dr. XJ Zhang, Guangdong Medical University) and 200 μL of the M2 cell supernatants. After a 4-h incubation, the cells were subjected to microscopic observation and photography, and the formation of tube-like structures by the HUVECs was analyzed by ImageJ software (Version 7, https://imagej. nih.gov/ij/).

RNA Isolation, Reverse Transcription (RT) and PCR

Total cellular RNAs were extracted using TRIZOL reagent (Cat: 15596-026, ThermoFisher Scientific) and quantified by SimpliNano (Biochrom, Germany). Totally 500 ng RNA was used to generate cDNA using a RT kit (Cat: RR047, TaKaRa Beijing, China). Quantitative RT-PCR (qRT-PCR) was performed using a LightCycler 480 II machine (Roche, Basel, Switzerland). The 20-μL reaction mixture comprised 10 µL SYBR Green I PCR Master Mix (#QPK-201, TOYOBO Guangzhou Branch, China), 0.4 µL forward primer (10 µM), 0.4 µL reverse primer (10 µM), 2 µL cDNA, and 7.2 µL ddH₂O. PCR amplification was performed as follows: 95°C for 1 min, and 45 cycles of 95°C for 5 s and 60°C for 20 s. The 2⁻^△△CT method was used to determine the levels of mRNAs. Primers spanning the exon–exon junction are listed in Table S1.

Western Blotting

Cells were lysed with RIPA buffer (Cat.P0013B, Beyotime Institute of Biotechnology). Samples of total protein (35 µg) were subjected to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes (Merck China, Shanghai). After washing twice with Tris-buffered saline containing 0.1% Tween 20 (TBST), the membranes were incubated with 5% skim milk powder in TBST at 37°C for 1 h and then incubated with primary antibodies at 4°C overnight. The primary antibodies are listed in Table S2. After washing with TBST twice, the membranes were incubated with horseradish peroxidase (HRP)-conjugated IgGs (1:5000; Cat. SA00001-10, Cat. SA00001-2; ProteinTech, Wuhan, China) for 1 h at 37°C. Bands were visualized using enhanced chemiluminescence (ECL) reagents (ThermoFisher Scientific) and analyzed with a gel analysis system (Tanon, Shanghai, China).

Phosphorylation Pathway Profiling Array

The RayBio® Phosphorylation Pathway Profiling Array (Cat.AAH-PPP-1-2, RayBiotech, Guangzhou, China) was used as instructed by the manufacturer to detect the relative levels of phosphorylation of 55 unique proteins from five well-known signaling pathways, including mitogen-associated protein kinase (MAPK), AKT, Janus kinase/signal transducer and activator of transcription (JAK/STAT), nuclear factor (NF)-κB and transforming growth factor (TGF)-β. Briefly, total protein was extracted from M0 cells from WT and SETD4 KO mice. The protein chip membranes were incubated with Blocking Buffer at room temperature for 1 h, and then incubated with 1 mL of a 1:20 dilution of cell lysate overnight at 4°C. The membranes were washed twice with Wash Buffer I & II and then incubated with Detection Antibody Cocktail at 25°C for 2 h. After washing twice with Wash Buffer, the membranes were incubated with HRP-labeled anti-rabbit secondary antibody at 25°C for 2 h. The signals were collected by an ImageQuant LAS4000 Scanner (GE Healthcare Corporation, Pittsburgh, PA, USA) after incubation with an ECL reagent. The original raw data obtained by chip scanning were processed by Raybiotech software for chip background removal and normalization. Differentially expressed proteins (DEPs) were identified by screening for P < 0.05 and a fold-change (FC) in expression of ≤ 0.83, or P < 0.05 and FC ≥ 1.2.²¹

Gene Ontology (GO) Terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Enrichment

Protein function annotation for GO and KEGG pathways were analyzed by the R package “clusterProfiler”, with the standard number of DEPs for a certain GO term/pathway set at ≥ 5 and P < 0.05 (Fisher's exact test). KEGG was used to enrich the DEP-related pathways.

Epidermal Growth Factor Receptor (EGFR) Inhibitor Treatment of BMMs

The EGFR inhibitor CI-1033 (also known as Canertinib or PD183805, Cat. S1019; Selleck China, Shanghai) was used to treat BMMs. BMMs isolated from WT and SETD4 KO mice were seeded onto 96-well plates and 6-cm dishes and allowed to recover overnight. BMMs were incubated with complete medium supplemented with various concentrations of CI-1033 for 24–48 h. Cells in 96-well plates were subjected to a CCK-8 assay for cell viability, while cells in 6-cm dishes were used for protein extraction and western blotting analysis of phosphorylated (p)-EGFR (Ser1070) expression. We used the change in p-EGFR(Ser1070) levels to indicate the activation of ERFR signaling.

Statistical Analysis

Statistical analyses were conducted using SPSS software (version 26.0, IBM Corporation, Armonk, NY, USA) and GraphPad Prism (Version 9.0, GraphPad Software, San Diego, CA, USA). QQ plot was used to assess data distributions, the measurement data conforming to normal distribution were expressed by mean ± standard deviation (SD), and analyzed by t-test. Dunnett test in one-way analysis of variance (ANOVA) was used for comparison between multiple groups. P values < 0.05 were considered statistically significant.

SETD4 KO Changes Histone Methylation Status in BMMs

There were no apparent differences in morphology between M0 BMMs from WT and SETD4 KO mice, with both exhibiting high expression of the F4/80 macrophage marker. In SETD4 KO M0s, expression of SETD4 was undetectable at both the mRNA and protein levels. These results confirmed that BMMs derived from WT and SETD4 KO mice had been successfully isolated and identified (Figure 1). Through western blotting, we found that the SETD4 KO mainly affected the methylation of multiple sites of histone 3 (H3) and histone 4 (H4) in BMMs. As shown in Figure 2, compared with WT, the SETD4 KO had a specific inhibitory effect on the epigenetic signatures of H3K4, H3K36, H3K79 and H4K20: namely, H3K4me1, H3K4me2, H3K36me3, H3K79me1, H3K79me2, H3K79me3, H4K20me1 and H4K20me2.

SETD4 KO Facilitates BMM Proliferation

The effect of the SETD4 KO on cell proliferation ability was assessed using multiple methods. The CCK-8 assay indicated that the SETD4 KO increased cellular viability of BMMs (Figure 3a). Ki67, a marker of cell proliferation, exhibited upregulated mRNA levels in SETD4 KO BMMs compared with those in WT BMMs (Figure 3b). We also performed an EdU incorporation assay, which revealed that SETD4 KO BMMs processed a high rate of cells positive for EdU staining (Figure 3c). Finally, western blotting results revealed enhanced PCNA protein expression in SETD4 KO BMMs compared with that in WT BMMs (Figure 3d). Taken together, these results demonstrated that the KO of SETD4 enhanced the proliferation ability of BMMs, and implied that the expression level of SETD4 may be negatively correlated with the proliferation of BMMs.

SETD4 KO Advances the Cell Cycle of BMMs

Considering that the cell cycle is closely related to cell proliferation, we used western blotting to detect changes in proteins involved in cell cycle distribution. As shown in Figure 4a, compared with that in WT BMMs, the expression of CyclinA2, CyclinB1, CyclinD1, CDK2 and CDK4 was significantly upregulated in SETD4 KO BMMs between 24 and 48 h post seeding. At 72 h, the levels of these proteins declined to levels similar to those in WT cells. Next, we used FCM to examine cell cycle distribution. As shown in Figure 4b–c, there were significant differences between the two groups at 24 h after cell seeding: compared with the WT group, the SETD4 KO group had a lower proportion of G1 phase (P < 0.05) and a significantly increased proportion of G2/M phase (P < 0.01), with no significant change in the proportion of S phase. At 48 h after cell seeding, the trend in cell cycle distribution in the two groups was exactly opposite to that at the 24 h time point, and when the time was extended to 72 h, there was no difference in the cell cycle distributions of the two groups of BMMs. These results suggested that SETD4 may help to advance the cell cycle in BMMs.

SETD4 KO Impairs BMM Migration

Because changes in the levels of matrix metalloproteinases (MMPs) are critical for cell migration, we examined the expression of MMP-2 and MMP-9 in BMMs. As shown in Figure 5a and b, compared with WT BMMs, SETD4 KO BMMs showed downregulated expression of MMP-2 and MMP-9 at the protein level as well as the mRNA level. A Transwell migration experiment provided further indication that the SETD4 KO impaired cell migration ability (Figure 5c).

Supernatant from SETD4 KO BMM Cultures Benefits Angiogenesis

Vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis. Using qRT-PCR, we found that the levels of VEGF-A mRNA were dramatically higher in SETD4 KO M2 cells compared with those in WT M2 cells (Figure 5e). Next, we performed a Matrigel tube-formation experiment to functionally analyze the ability of M2 cells to induce angiogenesis in HUVECs. As shown in Figure 5f–g, the supernatant from SETD4 KO M2 cells enhanced the potential in HUVECs to induce the formation of tubular structures.

Effects of SETD4 KO on the Expression of Inflammatory Factors in BMMs

To induce polarization, we treated BMMs with LPS + IFN-γ or IL4, and examined them for the release of inflammation-related factors using qRT-PCR. At baseline, the mRNA levels of most of the M1 and M2 polarity-related factors were lower in the SETD4 KO group than in the WT group (Figure 6). After induction for 24 h, the levels of M1 and M2 polarity-related factors increased significantly in the WT group, indicating that the induction method was feasible. By contrast, in the SETD4 KO group, except for TGF-β1, most of the other factors, including IL-1β, IL-6, Inducible nitric oxide synthase (iNOS), Tumor necrosis factor (TNF)-α, Fizz1, Arginare 1 and Mannose receptor C-type 1(MRC1), were downregulated post 24 h induction (Figure 6). These results suggested that the absence of SETD4 led to generally inhibited expression of major polarity-related inflammatory factors.

SETD4-Related DEPs and Bioinformatics in BMMs

We screened for DEPs from the JAK/STAT, MAPK, AKT, NF-κB and TGF-β signaling pathways between WT and SETD4 KO BMMs using Phosphorylation Pathway Profiling Arrays, and these arrays include total 55 unique proteins (Figure S1). In total, 32 DEPs were identified, of which 26 were downregulated and 6 were upregulated in SETD4 KO cells (Figure 7a). Details on the DEPs can be found in Table S3. Among these DEPs, EGFR(Ser1070), P27, AKT, Smad2 and AMPKa were the top 5 most significantly upregulated, while Mek1, P53, JNK, P38 and SHP1 (Ser591) were the top 5 most significantly downregulated in SETD4 KO cells. Next, we used the clusterProfiler data package of R/bioconductor to perform GO term enrichment analysis on the two sets of DEPs. As shown in Figure 7b, the enriched biological processes (BPs) were gland development, cellular response to chemical stress, and peptidyl-tyrosine phosphorylation. The enriched molecular functions (MFs) were protein serine/threonine kinase activity, phosphatase binding, and protein phosphatase binding. The enriched cellular components (CCs) were transcription regulator complex, nuclear chromatin, and RNA polymerase II transcription regulator complex. In total, 1365 GO terms were enriched, and are described in detail in Table S4. As shown in Figure 7c, the enriched KEGG pathways mainly involve inflammation and immune response, viral infection, PD-L1 expression and the PD-1 checkpoint pathway in cancer, the Toll-like receptor signaling pathway, and Th17 cell differentiation. In total, 137 KEGG signals were enriched, and are described in detail in Table S5.

SETD4-Mediated EGFR Signaling Promotes the Viability of BMMs

In view of the finding that the most significantly upregulated DEP in SETD4 KO BMMs was EGFR(Ser1070), we treated BMMs with the EGFR broad-spectrum inhibitor CI-1033 to observe the effects of EGFR inhibition on cell viability. As shown in Figure 8a, CI-1033 inhibited BMM viability in a concentration-dependent manner, suggesting that EGFR signaling plays a positive role in the proliferation of BMMs. Additionally, in the early stage (24 h) of CI-1033 stimulation, the WT and SETD4 KO groups had different responses: the WT showed no response to CI-1033, while the KO group showed concentration-dependent inhibition even at 3 μM CI-1033. Prior to the addition of CI-1033(CI-1033, 0 μM), the cell viability of the SETD4 KO group was higher than that of the WT group. This observation suggested that, in the absence of CI-1033, there was more obvious proliferation in the SETD4 KO group than in the WT group, which was consistent with the results of Figure 3a-b. Next, western blotting was used to examine the levels of p-EGFR(Ser1070) under CI-1033 treatment. CI-1033 inhibited the expression of p-EGFR(Ser1070) much more obvious in SETD4 KO cells than in WT cells (Figure 8b).

To undertake this exploration of the biological effects of SETD4 on macrophages, we first sought to obtain qualified BMMs from bone marrow cells derived from our previously reported C57BL/6J mice carrying the SETD4 KO. Successful culture of macrophages in vitro reportedly requires a special cytokine microenvironment of factors that mainly include macrophage colony-stimulating factor (M-CSF) and granulocyte–macrophage CSF.²² Because the supernatant of L929 cell cultures contains M-CSF,²³ we used the L929 supernatant as conditioned medium, which has been reported previously.^24-26 We found that, morphologically, there was no significant difference between BMMs isolated from the SETD4 KO and WT groups. Immunofluorescence staining confirmed that both groups of cells expressed the macrophage marker gene F4/80, at positivity rates > 95%. Further FCM results also confirmed that the BMMs exhibited high purity. We concluded that the BMMs sorted in this study met the qualification requirement, laying a solid foundation for subsequent biological function research.

Considering that the SET family are histone methyltransferases,²⁷ we tested our KO for multiple site-specific methylation changes to H3 and H4. The SETD4 KO showed significant changes in the levels of certain histone lysine methyltransferases, mainly H3K4, H3K36, H3K79 and H4K20. These sites included H3K4me1, H3K4me2, H3K36me3, H3K79me1, H3K79me2, H3K79me3, H4K20me1 and H4K20me2. These results are largely consistent with our previous results of SETD4 on bone marrow mesenchymal stem cells.²⁰ It is speculated that even cells derived from bone marrow have some differences in their histone methylation sites, suggesting that SETD4 may have cell specificity for methylation changes to histone sites. It has been reported that the histone methylation sites H3K9me2, H3K27me3, H3K79me3 and H4K20me3 are related to gene transcription inhibition, while the methylation sites H3K4me2 and H3K36me3 are related to gene transcription activation.^{28, 29} Methylation changes to a large number of histone-specific sites would undoubtedly affect the transcription of many genes, and may have been related to the general downregulation of major polarity-related factors in BMMs from the SETD4 KO in this study. However, more studies are needed to uncover the effects of special histone methylation patterns on gene expression.

During the processes of inflammatory and immune response and regulation, the proliferation and migration of macrophages are among the most basic biological functions.^{30, 31} Thus, we integrated the results of CCK-8 assays, EdU staining, qPCR and western blotting to analyze the effects of SETD4 on the proliferation of BMMs. The results confirmed that insufficient expression of SETD4 promotes the proliferation of BMMs. Because the cell cycle is precisely regulated by the activities of a variety of cyclins, cyclin-dependent kinases (CDKs) and cyclin-dependent kinase inhibitors (CDKIs),³² we looked for changes in the expression of proteins involved in cell cycle regulation. Unsurprisingly, compared with WT BMMs, SETD4 KO BMMs exhibited significant upregulation of CyclinA2, CyclinB1, CyclinD1, CDK2 and CDK4. Therefore, we speculated that the absence of SETD4 would regulate the cell cycle phases, manifesting in cells as a tendency to accelerate proliferation. To verify this conjecture, we employed FCM to detect specific changes in the cell cycle at different time points. At the 24-h time point after seeding of SETD4 KO BMMs, cells in G1 phase decreased and the ratio of cells in G2/M increased. At the 48-h time point, these patterns in the G1 and G2/M phases were opposite to those at the 24-h time point. At 72 h, there were no significant differences between the two groups in the distribution of cells in each phase. These results suggested that the SETD4 KO led to cells entering the cycle earlier than those in the WT group.

In the inflammatory response, the gradual increase in inflammatory cells in lesion tissue is related not only to cell proliferation, but also to the chemotactic movement of inflammatory cells.³³ For this reason, we also analyzed the absence of SETD4 expression on the migration ability of BMMs. Given that MMPs play important roles in cell migration,³⁴ we used western blotting and RT-PCR to demonstrate that the protein and mRNA expression of MMP-2 and MMP-9 were both significantly reduced in the SETD4 KO group compared with those in the WT group. This suggested that, in the absence of SETD4, it is possible to inhibit cell migration by downregulating the expression of MMPs. Functionally, the Transwell experiments showed that the number of cells that passed through the 8-μm pore size polycarbonate membrane in 24 h in the SETD4 KO group was significantly reduced compared with that in the WT group. This result was consistent with our previous observation that SETD4 KO is detrimental to BMSCs migration.²⁰ We also observed that the change in MMP-2 expression was more dramatic than that in MMP-9, suggesting that MMP-2 is the main functional MMP in this system.

As we continued to explore the effect of the SETD4 KO on the polarization of BMMs, the previously reported mature protocol ^35,36yielded a general upward-regulated expression trend in M1 and M2 marker genes in WT cells, confirming that the induction programs used in this study were successful. Interestingly, in the SETD4 KO group, except for the significant increase in TGF-β1 expression, most of the other factors, including IL-1β, IL-6, iNOS, TNF-α, Fizz-1, Arginase1 and MRC1, were downregulated. Although this result demonstrated that SETD4 had a general inhibitory effect on the M1 and M2 polar differentiation-related factors of BMMs, the specific reason is unclear. An early report has shown that SETD4 positively regulates the expression of IL-6 and TNF-α in macrophages by activating H3K4 methylation.⁸ It is possible that the KO of SETD4 affects the methylation of such a large group of histone sites that the expression of genes related to inflammatory factors are ultimately inhibited.

M2 macrophages have anti-inflammatory abilities and participate in the repair processes of late tissue damage, such as the promotion of angiogenesis and the progression of fibrosis.^37,38 We observed that, although the expression of TGF-β1 was upregulated in the SETD4 KO, the level of upregulation was weak. Therefore, we focused on the expression of pro-angiogenic factors secreted by M2 macrophages. Given that VEGF is the core factor in the promotion of angiogenesis, ^39,40 we tested the mRNA levels of VEGF-A in M2 type BMMs was significantly higher in the SETD4 KO group than the WT group. Consistently, the consequent tube-forming experiment showed that the supernatant from M2-type SETD4 KO BMMs promoted the obvious formation of vascular tube structures in HUVEC cultures. Furthermore, a 2021 report by Xing et al. proposed that SETD4 negatively impacts the angiogenesis of c-Kit+ cells,⁵ which is supported by our current study.

To clarify the molecular mechanism underlying the differences in general biological behavior of BMMs with and without SETD4, we screened for DEPs, with a focus on the MAPK, AKT, JAK/STAT, NF-κB and TGF-β signaling pathways. Compared with the WT group, the SETD4 KO group had 26 downregulated proteins, of which the top 5 were Mek1, p53, JNK, P38 and SHP1 (Ser591), and 6 upregulated proteins, of which the top 5 were EGFR(Ser1070), p27, AKT, Smad2 and AMPKa. Using R language software, totally 1365 GO terms and 137 KEGG signals were found to be enriched. The SETD4 KO mainly affected the following: BPs of gland development, cellular response to chemical stress, and peptidyl-tyrosine phosphorylation; MFs of protein serine/threonine kinase activity, phosphatase binding, and protein phosphatase binding; and CCs of transcription regulator complex, nuclear chromatin, and RNA polymerase II transcription regulator complex. The above GO function annotations suggested that SETD4 mainly affects the cellular response to chemical and oxidative stress, amino acid phosphorylation modification, protein kinase activity regulation, and transcription regulation. In terms of KEGG signal enrichment, SETD4 KO appears to mainly affect signal pathways involving immune regulation, inflammatory cell differentiation, and viral infection. These bioinformatics analyses suggest that SETD4 plays an important role in the immune and inflammatory responses, making it a potential target for inflammatory response intervention.

We targeted EGFR(Ser1070), the most significantly upregulated protein in the SETD4 KO, for intervention to explore its influence on macrophage viability. EGFR signal activation can be achieved through phosphorylation, and this signal activation has been reported in macrophages, in which it is related to proliferation, migration, polarization and other functions.^41,42 In this study, we used the broad-spectrum EGFR signaling inhibitor CI-1033, also known as canertinib, a pan-ErbB inhibitor that targets EGFR and ErbB2.⁴³Studies found that CI-1033 inhibits the expression of p-EGFR(Ser1070) in a concentration-dependent manner, suggesting that treatment of BMMs with CI-1033 might inhibit the activation of EGFR signaling. Functionally, we found that CI-1033 inhibition of EGFR signaling showed differences between BMMs from the WT and KO groups in the early stage of cell culture. Inhibition of EGFR had no obvious effect on the proliferation of BMMs in the WT group, but for the SETD4 KO group, 3 μM CI-1033 had a significant inhibitory effect at 24 h. This is likely attributable to the significantly higher expression of p-EGFR(Ser1070) in SETD4 KO BMMs compared with that in WT BMMs; i.e. the activation of EGFR was stronger, so the targeted inhibitory effect of CI-1033 was stronger. This study is the first to identify potential roles for SETD4 in EGFR signaling and the regulation of macrophage proliferation.

There are some limitations to this study. First, because the SEDT4 KO will be expected to causes changes in the lysine methylation of multiple histones, the identification of specific target genes of these histone methylases would undoubtedly help to explain the changes in polarity-related factors in the KO. Second, this study was focused on cytological research. In the future, these results will need to be replicated in animal models of inflammation to verify in vivo functions. Third, among the 26 DEPs and related KEGG signals that were identified in this study, specific signals or transcription factors of interest will need to be selected for targeted intervention studies to clarify the exact role of SETD4 in macrophage biology.

This study provided a detailed exploration of the relationships between the histone methylase SETD4 and the biological functions of macrophages. SETD4 was found to be associated with proliferation, cell cycle, migration, and paracrine and polarization differentiation of BMMs. Changes in the degree of methylation of histone-specific sites regulated by SETD4 in macrophages were preliminarily analyzed, and SETD4-mediated EGFR(Ser1070) signaling was found to play a role in BMM proliferation. These results provide new clues for the elucidation of the inflammation and immune responses from the perspective of epigenetic methylation modification by SETD4.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising, or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Acknowledgments

We thank Michelle Kahmeyer-Gabbe, PhD, from Edanz Group (https://en-author-services. Edanzgroup.com/) for editing a draft of this manuscript

Funding

This work was supported by the grant from the Key Laboratory of Tropical Cardiovascular Disease Research of Hainan Province (No. KLTCDR-202101) and National Nature Cultivation Project of Tongji Hospital (No. GJPY2142). The funders had no role in the design of the study, collection, analysis or interpretation of data, nor were they involved in writing the manuscript.

Supporting Files

Additional file_1: Table S1. Primers used for PCR.

Additional file_2: Table S2. Antibodies used.

Additional file_3: Table S3: Full list of DEPs.

Additional file_4: Figure S1. Images of scanned arrays.

Additional file_5: Table S4. Enriched GO terms, including GO-MF, GO-CC and GO-BP.

Additional file_6: Table S5. Enriched KEGG signals.

Disclosure

The authors have no conflicts of interest related to this work to report.

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No competing interests reported.

Additionalfile1TableS1.docx
Additional file_1: Table S1. Primers used for PCR.
Additionalfile2TableS2.docx
Additional file_2: Table S2. Antibodies used.
Additionalfile3TableS3.docx
Additional file_3: Table S3: Full list of DEPs.
Additionalfile4FigureS1.pdf
Additional file_4: Figure S1. Images of scanned arrays.
Additionalfile5TableS4.xls
Additional file_5: Table S4. Enriched GO terms, including GO-MF, GO-CC and GO-BP.
Additionalfile6TableS5.xls
Additional file_6: Table S5. Enriched KEGG signals.

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Methyltransferase SETD4 mediates macrophages proliferation through EGFR signaling

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