Regulation of CXCL1 chemokine and CSF3 cytokine levels in myometrial cells by the MAFF transcription factor

Abstract Cytokines play key roles in a variety of reproductive processes including normal parturition as well as preterm birth. Our previous data have shown that MAFF, a member of the MAF family of bZIP transcription factors, is rapidly induced by pro‐inflammatory cytokines in PHM1‐31 myometrial cells. We performed loss‐of‐function studies in PHM1‐31 cells to identify MAFF dependent genes. We showed that knockdown of MAFF significantly decreased CXCL1 chemokine and CSF3 cytokine transcript and protein levels. Using chromatin immunoprecipitation analyzes, we confirmed CXCL1 and CSF3 genes as direct MAFF targets. We also demonstrated that MAFF function in PHM1‐31 myometrial cells is able to control cytokine and matrix metalloproteinase gene expression in THP‐1 monocytic cells in a paracrine fashion. Our studies provide valuable insights into the MAFF dependent transcriptional network governing myometrial cell function. The data suggest a role of MAFF in parturition and/or infection‐induced preterm labour through modulation of inflammatory processes in the microenvironment.

Small MAF proteins can form homodimers through which they may function as transcriptional repressors by acting as dominant negative factors 19 or heterodimerize with other bZIP proteins including the cap "n" collar (CNC) family members NFE2, 20 NFE2L1, 21 NFE2L2,22 NFE2L3, 23,24 BACH1 and BACH2. 25 The resulting homo-or heterodimers bind to palindromic Maf recognition element (MARE) sequences, NF-E2 binding motifs, antioxidant/electrophile response elements (ARE/EpRE) and CNC-sMAF binding elements (CsMBE). 13,17,26,27 The small MAFs play important roles in the control of mammalian gene expression, and have been linked to a variety of pathways and/or pathologies, 17,18,28 including cellular stress response and detoxification, 29 diabetes, 30 neuronal disease 31 and cancer. 32,33 Previously, we found that MAFF transcript and protein levels are induced by the pro-inflammatory cytokine IL1B and TNF alpha in PHM1-31 myometrial cells. 11,12 MAFF was the only small MAF protein to be induced by cytokines, suggesting a specific role for this protein in the inflammatory response in uterine smooth muscle cells. 12 An earlier report showed that MAFF transcripts are present in human term myometrium, but not in early gestation period or non-pregnant myometrium. 34 In the current study, we further explored the link between proinflammatory cytokines and myometrial cell function in PHM1-31 cells. We used knockdown approaches to dissect the cytokine- WISENT Inc., QC, Canada), 10% foetal bovine serum, 2 mmol/L L-glutamine and 2% antibiotic-antimycotic solution containing 5000 U/mL penicillin and 5000 U/mL streptomycin as previously described. 12 Cells were passaged using 0.05% trypsin-EDTA (25300-054; Invitrogen (qHsaCED0005290) and IL6 (qHsaCID0020314). Primer sequences used for CCL2, MMP2 and MMP9 have been described. 35 Additional custom primer sequences are listed in Table S1. The qPCR analysis was performed in a CFX96 Touch ™ Real Time PCR detection system (Bio-Rad). Data were analysed by the threshold cycle (Ct) comparative method. Samples were normalized to TBP and PPIA for PHM1-31 extracts and to Beta Actin and RPL37A for THP-1 extracts. 36

| Cell lysis and immunoblot analysis
Whole-cell extracts were prepared by scraping cells using 1x PBS and cells were lysed for 10 minutes in whole-cell lysis buffer (10 mmol/L Tris-HCl pH 8.0, 420 mmol/L NaCl, 250 mmol/L sucrose, 2 mmol/L MgCl 2 , 1 mmol/L CaCl 2 , 1% Triton-X100) supplemented with complete protease inhibitor cocktail (Roche, Mississauga, Canada, 04 693 116 001), and then centrifuged at 17 000 g for 10 minutes at 4°C. Supernatants were collected and protein concentrations were determined using a protein assay kit (Bio-Rad, 500-0006). Thirty microgram of the total protein lysate were separated by electrophoresis on a 15% sodium dodecyl sulphate polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Millipore). Blots were blocked using 5% milk in TBST (500 mmol/L Tris pH 7.6, 2 mol/L NaCl, 0.5% Tween) at room temperature for at least 1 hour and then incubated overnight at 4°C with primary antibodies specific for MAFF (1:1000) 12

| Chemokine and cytokine assays
ELISA assays were carried out using human CXCL1/GRO alpha (R&D Systems, DY275-05) and G-CSF (CSF3) DuoSet (R&D Systems, Inc., Minneapolis, MN, USA, DY214-05) ELISAs according to the manufacturer's instructions. Plates were prepared by coating the capture antibody (mouse anti-human GROα at 4 μg/mL and human G-CSF at 1 μg/mL) overnight at room temperature followed by three washes with PBS. Subsequently, plates were blocked for at least an hour with reagent diluent followed by another three washes. For the assay procedure, detection antibodies were used at 40 ng/mL for biotinylated goat anti-human GROα, at 300 ng/mL for the human G-CSF, and streptavidin-HRP was used at a 40-fold dilution into reagent diluent. The optical density of each well was calculated using a microplate reader (Fluostar Optima; BMG Labtech, Cary, NC, USA) by subtracting the absorbance reading measured at 600 nm (background) from the one obtained at 450 nm.

| Chromatin immunoprecipitation
ChIP assays were carried out using SimpleChIP ® Enzymatic Chro- per IP prep on ice for 2 minutes. This was followed by nuclei lysis with three sets of 20-second pulses at 15% intensity using a Fisher Scientific model 500 sonic dismembrator. The samples were then incubated overnight with ChIP-Grade Protein G Magnetic Beads (Cell Signaling, 9006) with an antibody specific for MAFF. 12 Flag rabbit IgG (DYKDDDDK Tag antibody; Cell Signaling, 2368S) was used as a negative control. After stringent washes, chromatin was eluted in ChIP Elution Buffer (Cell Signaling, 7009). Eluates were reverse cross-linked by adding 6 μL 5 mol/L NaCl and 2 μL Proteinase K (Cell Signaling, 10012), and incubated for 2 hours at 65°C. DNA was extracted and purified using Spin Columns (Cell Signaling, 10010) and eluted in DNA Elution Buffer (Cell Signaling, 10009). ChIP-qPCR analyses were performed in a CFX96 Touch ™ Real Time PCR detection system (Bio-Rad) and the primers used are listed in Table S1.

| Statistical analysis
Data are presented as mean ± standard error of the mean (SEM).
Differences between experiments were calculated with two-tailed Student t-test, for at least three independent experiments and statistically significant data are indicated. One asterisk indicates P < 0.05, two asterisks indicate P < 0.01 and three asterisks indicate 3.1 | CXCL1 and CSF3 levels are controlled by MAFF MAFF transcripts are highly expressed in term myometrium, but not during early gestation periods and in non-pregnant myometrium. 34 Our previous data showed that pro-inflammatory cytokines including IL1B rapidly induce MAFF transcript and protein levels in human PHM1-31 myometrial cells, 12 and that a larger network of inflammatory genes is equally modulated by IL1B. 11 We thus hypothesized that the MAFF transcription factor is involved in the regulation of uterine smooth muscle genes linked to inflammation at the onset of parturition. We chose to pursue our studies in PHM1-31 cells, a myometrial cell model we have analysed previously 11,12 and for which we optimized lentiviral based shRNA knockdown strategies for loss-of-function studies. We confirmed efficient shRNA mediated MAFF knockdown at the transcript and protein level in these cells with or without IL1B treatment ( Figure 1A,B). We also observed a prominent increase of MAFF mRNA and protein upon IL1B treatment, confirming our previous data. With respect to MAFF targets, we focused on genes coding for CXCL1 chemokine and CSF3 cytokine, as our earlier results had shown that they are also significantly induced by IL1B 11 and since they have been shown to be either directly or indirectly involved in the processes leading to parturition. [37][38][39][40][41] Indeed, the knockdown of MAFF resulted in a significant decrease of CXCL1 and CSF3 gene transcripts under basal conditions and following IL1B treatment for 1, 3 and 8 hours (Figure 2A,B), suggesting that MAFF is involved in the control of the expression of these genes. In the non-targeting control samples, both, CXCL1 and SALIBA ET AL.
| 2519 CSF3 mRNA levels are strongly increased upon IL1B treatment in a time dependent manner, emphasizing the role of MAFF in the regulation of these genes upon IL1B induction (Figure 2A).
In additional studies, we performed ELISA assays to confirm our data at the protein level. CXCL1 chemokine and CSF3 cytokine secretion are greatly reduced upon MAFF knockdown in PHM1-31 cells following 8 and 12 hours of IL1B treatment ( Figure 3A,B). Protein levels without IL1B treatment were below the sensitivity threshold of the ELISA kits used. Taken together, these data suggest that Data represent mean values ± SEM; unpaired t-test; *P < 0.05; **P < 0.01; ***P < 0.001, ΔΔΔ P < 0.001. Asteriks correspond to the significance with the control (NTC) at the same time point and triangles to the significance between controls F I G U R E 2 Control of CXCL1 and CSF3 transcript levels by MAFF in PHM1-31 myometrial cells. PHM1-31cells were transduced with shRNA (NTC, sh1 or sh2) and treated (or not) with 10 ng/mL IL1B for 1, 3 or 8 h. The regulation of CXCL1 (A) and CSF3 (B) mRNA expression upon MAFF knockdown was quantified by quantitative PCR. Data represent mean values ± SEM; unpaired ttest; *P < 0.05; **P < 0.01; ***P < 0.001, ΔΔΔ P < 0.001. Asterisks correspond to the significance with the control (NTC) at the same time point and triangles to the significance between controls F I G U R E 3 Regulation of CXCL1 and CSF3 protein levels by MAFF in PHM1-31 myometrial cells. PHM1-31cells were transduced with shRNA (NTC, sh1 or sh2) and treated with 10 ng/mL IL1B for 8 or 12 h. The regulation of CXCL1 chemokine secretion (A) and CSF3 cytokine secretion (B) upon MAFF knockdown was quantified by ELISA assays. Data represent mean values ± SEM; unpaired t-test; *P < 0.05; **P < 0.01; ***P < 0.001, ΔΔΔ P < 0.001. Asterisks correspond to the significance with the control (NTC) at the same time point and triangles to the significance between controls ( Figure 4A). For CSF3, two DNA regions were chosen, A and B.
While peak A is the most significant binding peak in HEPG2 cells, peak B is the second strongest and located in the proximity of the CSF3 loci ( Figure 5A). We performed ChIP assays examining those regions and found that MAFF binds to the analysed genomic regions in both the CXCL1 and CSF3 genes in a significant manner in PHM1-31 cells (Figures 4B and 5B Our data suggest that CXCL1 chemokine and CSF3 cytokine loci are direct targets of the MAFF transcription factor.

| Effect of MAFF activity in myometrial cells on monocyte gene expression
It has been proposed that labour resembles an inflammatory process, with the activation of leucocytes from maternal tissue as well as the with 10 ng/mL IL1B for 3 h and analysed by chromatin immunoprecipitation using antibodies against MAFF or FLAG to control for non-specific binding. Data represent mean values ± SEM; unpaired t-test; *P < 0.05; **P < 0.01; ***P < 0.001 F I G U R E 6 Effect of MAFF mediated regulation in PHM1-31 cells on THP-1 monocytes. THP 1 cells were cultured for 24 h with supernatant from PHM1-31 cells transduced with NTC, sh1 or sh2. The regulation of CCL2, IL6, MMP2 and MMP9 mRNA expression was quantified by quantitative PCR. Data represent mean values ± SEM; unpaired t-test; *P < 0.05; **P < 0.01; ***P < 0.001 SALIBA ET AL.
| 2521 increasing levels of cytokines and chemokines from gestational tissue. 43 Thus, we examined whether regulation of MAFF expression in PHM1-31 cells might have an impact on the leucocyte environment. As a study model, we chose human THP-1 monocytic cells. 44 We investigated the expression of CCL2 and IL6 cytokine genes that are highly up-regulated in vivo in labouring myometrium as well as the genes coding for two monocyte activation markers, MMP2 and MMP9 that contribute to cervical ripening. 35 We treated THP-1 cells for 24 hours with supernatant from PHM1-31 cells that have been transduced with control or MAFF shRNAs. MAFF knockdown leads to a significant decrease in CCL2, IL6, MMP2 and MMP9 transcript levels ( Figure 6A-D). Our results indicate that MAFF transcription factor activity in myometrial cells is able to control cytokine and activation marker gene expression levels in neighbouring monocytes in a paracrine fashion.

| DISCUSSION
Our previous data linked the MAFF transcription factor to proinflammatory cytokine signaling in uterine smooth muscle cells. 11,12 It has been established that at parturition, leucocytes invade the uterus and together with the local cells secrete cytokines, thus creating an inflammatory microenvironment in the reproductive tissues. 3 This process is essential for membrane rupture, cervical dilation, myometrial contraction and expulsion of the foetus. 1 In earlier studies, it has been reported that MAFF is present at high levels in pregnant myometrium at term. 34 Using PHM1-31 uterine smooth muscle cells as well as primary myometrial cells as models, we previously showed that MAFF transcript and protein levels are up-regulated by IL1B and TNF, 11,12 both of which have been linked to the onset of labour. 45 The MAFF gene is highly sensitive to IL1B and the induction is very rapid, as MAFF mRNA increases are observed at as low as 0.05 ng/mL of the cytokine and as early as 30 minutes following the treatment. 12 We also found that MAFF is the only member of the small MAF family, to be regulated by IL1B and TNF in this cell type. 12 Together, these studies suggested a specific role for MAFF in controlling inflammatory processes in myometrial cells. In the present study, we identified the genes coding for CXCL1 chemokine and CSF3 cytokine as MAFF targets use a loss-of-function approach. One important question was whether MAFF, as a transcription factor, directly controls the expression of the identified targets. We consulted UCSC browser data of genome wide MAFF binding analyses in HEPG2 cells and identified a series of significant MAFF DNA binding peaks present in the CXCL1 and CSF3 loci. Our data showed that MAFF is binding to these regions in PHM1-31 cells, and that the binding preference varies depending on the peak region analysed. As we based our analysis on ChIP-sequencing data generated in hepatic cells, it is conceivable that in our myometrial cell model MAFF may also bind to other sequences in these genes. We conclude that MAFF acts as a direct upstream regulator of the transcription of these genes.
As CXCL1 and CSF3 are both secreted proteins, we hypothesized that regulation of myometrial gene expression by MAFF may contribute to inflammatory processes in the microenvironment.
Indeed, we found that MAFF knockdown in PHM1-31 cells can also affect the gene expression pattern in THP-1 monocyte cells in a paracrine manner. MAFF levels in myometrial cells are able to mediate the regulation of CCL2 chemokine, IL6 cytokine and activation makers such as MMP2 and MMP9 in monocytes. This is of interest, as leucocyte infiltration of the uterine cervix, predominantly consisting of neutrophils and macrophages, coincides with the onset of labour. 53 Others have observed phenotypic and metabolic changes in maternal neutrophils and granulocytes in preterm labour. 54 Of note, CXCL1 has been shown to be involved in monocyte recruitment. 55 Also, CSF3 functions as an important maturation factor for monocytes as well as granulocytes. [56][57][58] In conclusion, our data further support the involvement of CXCL1 and CSF3 in monocyte and granulocyte recruitment and maturation, and establishes MAFF as a key upstream regulator of these cytokine-mediated signaling pathways in myometrial cells, including the recruitment of leuuocytes, a critical step for the labour process.
Based on our data, we propose a model (Figure 7), in which the MAFF transcription factor functions as a key regulator in the labour process, acting as a link between initiation of the process and the following activation of leucocytes and subsequent uterine activation. 40,48,[59][60][61] In future studies, it will be important to further dissect the MAFF-mediated regulatory network in myometrial cells, including signaling pathways and interacting proteins that control the activity of the MAFF transcription factor as well as coordinated regulation of the labour process together with other transcriptional regulators. A better understanding of the MAFF regulatory network in parturition may provide means to prevent preterm labour.

ACKNOWLEDGEMENTS
We wish to thank Joo Yeoun (Sophie) Park, Audrey Burban and Jennie Jackson for critical reading of the manuscript and/or fruitful discussions. We also would like to thank Barbara Sanborn for providing PHM1-31 myometrial cells and Andrew Mouland for providing THP-1 cells, and Anna Derjuga for expert technical assistance. We thank François Lefebvre for his help with the bioinformatics analysis of RNA sequencing data at an earlier stage. We would like to acknowledge the ENCODE project consortium and the Michael Snyder laboratory, Stanford for the MAFF ChIP-seq data. This work was

CONFLI CT OF INTEREST
The authors declare no conflict of interest.