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Research Article |
Lady Davis Institute for Medical Research3
and Department of Medicine,4 McGill University, Montreal, Quebec, Canada H3T 1E2
Departments of Biochemistry and Molecular Biology,5 University of Texas Medical School, Houston, Texas 77030
Biomedical Sciences,6 Colorado State University, Fort Collins, Colorado 80523
ABSTRACT
The MAF (proto-)oncogene family of basic-leucine zipper transcription factors plays crucial roles in the control of mammalian gene expression and development. Here we analyzed the regulation of the human MAFF gene, coding for a small MAF transcription factor, in uterine smooth muscle cells. We found that MAFF transcript levels are induced by proinflammatory cytokines in PHM1–31 myometrial cells. We observed an important induction by interleukin 1 beta (IL1B) and a weaker upregulation by tumor necrosis factor (TNF), whereas interleukin 6 (IL6) treatment had no effect. Time course experiments revealed a rapid induction of MAFF transcripts within 30 min following IL1B treatment. The presence of actinomycin D inhibited the upregulation, suggesting that regulation of MAFF mRNA levels occurs at the transcriptional level. We generated a MAFF-specific antiserum and determined that MAFF protein was also induced by TNF and IL1B in PHM1–31 cells. In contrast, it was particularly interesting that the transcript and protein levels of the highly homologous MAFG and MAFK genes are not modulated by these cytokines. Our results suggest a possible specific role for MAFF in proinflammatory cytokine-mediated control of myometrial gene expression and provide the first link between a small MAF transcription factor and the inflammatory response.
CNC, cytokines, gene regulation, IL1B, MAF, parturition, signal transduction, TNF, transcription factor, uterus
INTRODUCTION
MAF (v-maf musculoaponeurotic fibrosarcoma oncogene homolog [avian]) basic-leucine zipper (bZIP) transcription factors play crucial roles in gene regulation, differentiation, oncogenesis, and development in many organisms. The founding member, the v-maf oncogene, was isolated as the transforming agent of an avian retrovirus [1–3]; v- maf and its human cellular counterpart MAF (previously known as c-Maf) have been implicated in tumorigenesis [4]. There are two subgroups of MAF proteins, the large and the small MAF. The large MAF, containing an activation domain, include MAF, MAFA, MAFB, and NRL proteins [5–9]. Large MAF play important roles in the differentiation and development of a variety of hematopoietic and neural tissues [1, 3]. The small MAF, devoid of an obvious transactivation domain, include MAFF, MAFG, and MAFK factors [10, 11]. A unique domain located adjacent to the basic domain, called the extended homology region, is common to all MAF transcription factors and has been shown to be important for their DNA binding specificity [12]. Small MAF proteins can form heterodimers with the CNC (cap'n' collar) proteins, which also belong to the bZIP family of transcription factors. The resulting CNC/small MAF complexes have been shown to recognize NFE2 (nuclear factor erythroid derived 2)-, MARE (MAF recognition element)-, and ARE (antioxidant response element)-type of DNA binding motifs [1, 3].
The small MAF are widely expressed, but transcript levels vary considerably in different tissues [1]. Small MAF homodimers have been reported to function as transcriptional repressors [13–15]. In contrast, it was reported that CNC/small MAF heterodimers serve as transcriptional activators of gene expression [1, 16–18]. Small MAF play a role in the cellular response to hydrogen peroxide, heavy metals, and electrophiles [19–21]. Data from several studies suggest that CNC/small MAF heterodimers are involved in the regulation of detoxifying enzyme genes and in the response to oxidative stress [14, 15, 17, 18, 22, 23]. Targeted disruption of the MafK and MafF loci yielded null mice that were indistinguishable from their wild-type littermates, indicating that these small MAF factors are dispensable in vivo and are replaced by other small MAF [24, 25]. MafG null mice are also viable but suffer from a mild thrombocytopenia [26]. Thus, functional redundancy has made it difficult to assign specific in vivo roles to each of the small MAF transcription factors.
The human MAFF protein has previously been identified in a one-hybrid assay as a factor binding to regulatory sequences of the oxytocin receptor gene [27]. MAFF transcripts are highly expressed in term myometrium but are not present in early gestation (14 wk) and nonpregnant myometrial tissue [27]. It was hypothesized that the MAFF transcription factor may be involved in uterine gene regulation at the onset of parturition. Recent evidence suggests that cytokines may play an important role in the events that lead to preterm labor and delivery, causing morbidity and mortality of the fetus and serious health complications for the mother [28, 29]. Increased cytokine levels due to uterine infections, such as in chorioamnionitis, may cause the early onset of contractions [29]. The proinflammatory cytokines interleukin 1 beta (IL1B), interleukin 6 (IL6), and tumor necrosis factor (TNF) and the chemotactic factor IL8 have been implicated as mediators in both preterm and term labor, particularly in association with intrauterine infection. Elevated levels of these inflammatory cytokines have been observed in the amniotic fluid following infections ascending from the vagina and cervix [28]. Furthermore, it has been reported that expression of the oxytocin receptor is regulated by IL1B, IL6, and TNF in uterine smooth muscle cells [29–31]. Hence, there appears to be a link between the presence of inflammatory cytokines in the uterus or amnion and the induction of labor.
Based on these previous results, we investigated the regulation of the MAFF transcription factor by proinflammatory cytokines. We found that both IL1B and TNF can rapidly induce MAFF transcript and protein levels in myometrial cells, whereas the expression of the highly homologous MAFG and MAFK genes is not modulated. Inhibitor studies suggest that regulation of the MAFF gene takes place at the transcriptional level. Since proinflammatory cytokines have been implicated as mediators in both preterm and term labor, we speculate that MAFF might participate in the control of these processes. Our results suggest that transcriptional complexes comprising MAFF may play a role in the regulation of uterine gene expression, linking for the first time small MAF transcription factor-mediated gene regulation to cytokine signaling and inflammation.
MATERIALS AND METHODS
Cell Culture
Human embryonic kidney 293T (HEK293T, ATCC) cells were cultured in minimal essential medium alpha, 10% bovine serum, plus penicillin and streptomycin antibiotics.
Derivation of PHM1–31 Cells
PHM1–31 myometrial cells were derived from the immortalization mixture used to derive PHM1–41 cells [32]. Briefly, myometrium was removed from the upper edge of the uterine segment at the time of caesarian section in a term pregnant patient who was not in labor, using a protocol that had received prior institutional approval (University of Texas Health Sciences Center, Houston). Myometrial cells were isolated by enzymatic digestion, infected at passage 2 with replication-defective adenovirus vector pLXSN16E6E7 (J.K. McDougal, University of Washington, Seattle) expressing the E6/E7 proteins of human papilloma virus 16 and the neomycin resistance gene, and selected with the neomycin analog Geneticin (Invitrogen) [32]. PHM1–31 cells were recovered by subcloning foci and were maintained at 37°C in high-glucose DMEM containing 0.1 mg/ml Geneticin, 10% fetal bovine serum, 2 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin. The cells reach confluency 3–4 days after being cultured at 2 x 105 cells/100-mm dish and have been used in this study up to passage 25. The cells maintain smooth muscle 
-actin expression at passage 27 and retain the ability to respond to oxytocin and thapsigargin with an increase in intracellular calcium.
Cytokine and Actinomycin D Treatment
Cells reaching 80%–90% confluency were FBS starved for 16 to 24 h. They were then treated with TNF (100 ng/ml; Medicorp), IL1B (0.005–100 ng/ml; Research Diagnostics), or IL6 (100ng/ml; RDI) or combinations of these cytokines. Induction time was as indicated in the figures. For the inhibition experiments, the cells were starved overnight, and then actinomycin D (5 µg/ml) dissolved in dimethyl sulfoxide (DMSO) (0.5%) or DMSO alone (0.5%) was added to the cells 1 h before beginning the 3-h treatment with IL1B (1 ng/ml).
Cloning and Northern Analysis
The human MAFF and MAFK cDNAs, comprising the entire coding region, were recovered by RT-PCR using the following primer pairs: MAFF-F2 (5'-GGGCACCTTCTGCAAACATGT-3') and MAFF-R2 (5'-GAGGCGGCGCTCAGGCACTTT-3') and hMAFK-F1 (5'-CGTGCCCGGGTTATGACGACT-3') and hMAFK-R1 (5'-GGCCGGCACTAGGATGCAGC-3'). The resulting PCR fragments were cloned into the pCR-BluntII-TOPO vector (Invitrogen). To obtain MAFF- and MAFK-specific probes for Northern analysis, we performed an EcoRI digestion of the pCR-BluntII-MAFF and pCR-BluntII-MAFK plasmids, respectively. For MAFG, we amplified a 407-base-pair cDNA fragment with the primers MAFG611F (5'-CCGATCGTAGGGACGCGCGT-3') and MAFG1018R (5'-CCACTCGGGAGTGGAGGGAA-3') using the pMT2 MAFG vector as template [33] and cloned it into the pCR-BluntII-TOPO vector. The resulting plasmid was digested with EcoRI to obtain a MAFG -specific probe. For Northern analysis, total RNA was isolated using the protocol described for Trizol (Invitrogen). After subjecting RNA to gel electrophoresis, the gel was transferred overnight using standard techniques. The membrane was hybridized as described [34].
Transfection
For transfection studies, the MAFF and MAFK cDNA fragments, obtained by EcoRI digestion of the pCR-BluntII-MAFF and pCR-BluntII-MAFK plasmids, were cloned into the EcoRI site of the pMT2 expression vector. The pMT2 MAFG plasmid has been described previously [33]. Transient transfections of HEK293T cells were performed using the standard calcium-phosphate coprecipitation procedure. We used 10 µg of the pMT2 MAFF, pMT2 MAFG, or pMT2 MAFK expression vectors to transfect a 100-mm dish of 30%–50% confluent HEK293T cells.
Generation of MAFF Antiserum
To construct the GST-MAFF fusion protein vector, we generated a human MAFF cDNA using the primer pairs Bam-MAFF (5'-GTCGGATCCATGTCTGTGGATCCCCTATCC-3') and MAFF-Eco (5'-GCAGAATTCTAGGAGCAGGAGGCCGGGCC-3') and digested the amplicon with EcoRI and BamHI. Subsequently, the product was cloned into the EcoRI and BamHI sites of the pGEX-2TK fusion vector (Amersham). Rabbits at Pocono Rabbit Farm & Laboratory, Inc., were immunized with the purified GST fusion protein comprising the entire 164-amino-acid coding region of human MAFF. Preimmune serum was used to confirm the specificity of the generated antiserum to MAFF.
Immunoblot Analysis
To prepare whole-cell extracts, the cells were scraped using 1x PBS and centrifuged, and the pellets were then resuspended in NB buffer (250 mM sucrose, 420 mM NaCl, 10 mM Tris/HCl, 2 mM MgCl2, 1 mM CaCl2, 1% Triton X-100) and protease inhibitors. After incubation on ice for 10 min, samples were briefly centrifuged, and the supernatant was collected. Cytoplasmic extracts were generated using the same procedure as for whole-cell extracts except that NaCl in buffer was omitted. Nuclear extracts were prepared by resuspending the pellet from the cytoplasmic extract preparation in NB buffer with 420 mM NaCl, centrifuging, and collecting the supernatant. The protein concentrations were determined using a protein assay kit (Bio Rad, Hercules, CA); 20–30 µg of the lysates were electrophoresed in 4%–12% NuPage Novex Bis-Tris (Invitrogen) (Fig. 6) or 12% SDS-polyacrylamide gels (Fig. 7). Resolved proteins were transferred electrophoretically to PVDF membranes (Millipore). After transfer, the membrane was blocked for 1 h at room temperature and was subsequently incubated overnight with polyclonal sera specific for either ACTB (actin, beta) (1:10000) (Sigma #A-5441), human MAFF (1:200), human MAFG (1:200) [33], or human MAFK (1 µg/ml) (Santa Cruz). Secondary HRP-conjugated antibodies (Pierce) were applied for 1 h, and subsequently the proteins were visualized using chemiluminescence (Pierce).
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Quantification and Statistical Analysis
Quantification of Northern experiments was performed using a phosphoimager (Molecular Dynamics) and Image Quant software (version 5.2) or densitometry using NIH image software (version 1.63). All data are expressed as the mean ± SEM and were analyzed using one-way analysis of variance or, where stated, nonlinear regression analysis. Values of P < 0.05 (*) or P < 0.01 (**) were used as criteria for declaring significance.
RESULTS
Induction of MAFF Transcripts by Cytokines in PHM1–31 Myometrial Cells
Proinflammatory cytokines such as IL1B, TNF, and IL6 are likely to play a role in regular and preterm labor [28, 29] as well as in the regulation of oxytocin receptor expression in myometrial cells [29, 30]. We hypothesized that MAFF expression may be modulated by cytokines. Our preliminary results in primary myometrial cells showed that MAFF mRNA levels were induced by IL1B and TNF (data not shown). To minimize the need for primary myometrial tissue obtained from patients undergoing cesarian section, we used myometrial PHM1–31 cells in our subsequent studies. These cells were derived from primary myometrial cells by immortalization through overexpression of the human papilloma virus E6/E7 proteins [32]. The appearance of PHM1–31 cells was determined by phase contrast light microscopy (Fig. 1). The morphology of the cells is similar to that of regular proliferating smooth muscle cells. They appear long and spindle shaped with a central nucleus and a sheet-like growth pattern at confluency. We examined MAFF mRNA expression in PHM1–31 cells treated with proinflammatory cytokines (Fig. 2). We found a significant upregulation of MAFF mRNA levels by TNF and an even stronger induction by IL1B. In contrast, treatment with IL6 did not result in a significant change of MAFF mRNA levels (Fig. 2A). Combination of TNF and IL1B did not increase MAFF mRNA levels further than IL1B treatment alone (Fig. 2A). This result suggests that PHM1–31 cells, although immortalized, have conserved their myometrial phenotype with respect to cytokine responses. In contrast to MAFF, the transcript level of the highly homologous MAFG and MAFK genes were not induced by IL1B or TNF, suggesting that stimulation by these cytokines is specific for the MAFF gene (Fig. 2, B and C).
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Regulation of MAFF Expression at the Transcriptional Level
Dose-response experiments revealed that 0.005 ng/ml of IL1B upregulates MAFF gene expression, and maximal induction is reached with 0.5 ng/ml of IL1B (Fig. 3A). Experiments using PHM1–31 cells revealed induction of MAFF gene transcription by 30 min, reaching maximal levels by 1 h. Elevated MAFF transcript levels were sustained over a 24-h period (Fig. 3, B and C). In addition, actinomycin D inhibited induction of MAFF mRNA by IL1B (Fig. 4), suggesting regulation at the transcriptional level.
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Analysis of MAFF Protein Levels in Myometrial Cells
To characterize MAFF biochemically, we generated an antiserum specific for this protein. We showed that the antiserum specifically recognizes MAFF, an 18-kDa protein, in cell extracts from human embryonic kidney 293T (HEK293T) cells transiently transfected with an expression vector coding for human MAFF, using immunoblot analysis (Fig. 5). The MAFF-specific antiserum does not cross-react with the homologous small MAF transcription factors MAFG and MAFK in immunoblot assays (Fig. 5). We investigated whether MAFF protein is expressed in myometrial cells analyzing whole-cell extracts, nuclear and cytoplasmic extracts prepared from PHM1–31 cells (Fig. 6). In accordance with its function as a transcription factor, MAFF is found in the nucleus of PHM1–31 cells. As expected, MAFG and MAFK are equally found in the nucleus (Fig. 6, B and C). In addition, we examined whether the upregulation of MAFF transcripts also leads to an increase in MAFF protein levels. Analysis of nuclear extracts of cytokine-induced PHM1–31 cells showed that both TNF and IL1B induce MAFF protein expression (Fig. 7A). In contrast, expression of MAFG and MAFK protein was not affected by cytokine treatment (Fig. 7, B and C). Hence, cytokine regulation of MAFF mRNA and protein is coordinate (Fig. 2).
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DISCUSSION
Members of the MAF and CNC families of bZIP transcription factors play diverse roles in mammalian gene regulation, differentiation, oncogenesis, and development [1, 3]. In the present study, we found an intriguing new link between proinflammatory cytokine signaling and MAF transcription factor gene regulation in uterine smooth muscle cells. In our studies, we examined MAFF expression in the myometrial cell line PHM1–31. We found that PHM1–31 cells mimic primary myometrial cells with respect to morphology and upregulation of MAFF in response to cytokines (Figs. 2, 3, and 7; data not shown) [35, 36]. We conclude that PHM1–31 cells provide a valuable model to study myometrial gene expression and function. Most important, our novel data show that MAFF mRNA is rapidly induced by IL1B and TNF in primary myometrial and PHM1–31 cells (Figs. 2 and 3; data not shown). The presence of actinomycin D inhibits this induction (Fig. 4), suggesting regulation at the transcriptional level. The induction of MAFF also occurs at the protein level in the PHM1–31 cell line (Fig. 7). It is of interest to note that IL6 does not modulate MAFF transcript and protein levels, suggesting that the observed regulation is specific to a subset of cytokines. Moreover, the expression of the highly homologous MAFG and MAFK transcripts and proteins is not modulated by these cytokines (Figs. 2 and 7), suggesting a specific function of MAFF in the inflammatory response. This is of interest, as overlapping functions and functional redundancy in studies using in vitro, transgenic, and gene-targeting approaches have made it difficult to assign specific roles to each of the small MAF [13, 24–26, 33, 37–39]. Examining MAFF as well as double small MAF-deficient mice, using, for example, bacterial and viral induced inflammation models, may thus reveal novel functions for these transcription factors.
The identification of novel targets of cytokine signaling in myometrial cells is important, as elevated levels of proinflammatory cytokines such as IL1B and TNF in the uterus or amnion are likely to function as mediators in both preterm and normal term labor, in particular in association with cervical and intrauterine infection in the mother [29, 40]. Elevated levels of IL1B are observed in the amniotic fluid only late in pregnancy, during the third trimester. With infection, the levels of IL1B, IL6, TNF, and IL8 rise even further, and this increase is linked to the early onset of labor [29, 41]. About one-third of preterm births are associated with infective processes, accounting for a high number of neonatal deaths and a higher incidence of brain, lung, and metabolic disorders [42]. Thus, a better understanding of proinflammatory cytokine signaling in gestational tissues is crucial. Based on these previous studies implicating cytokines in normal and preterm labor, we hypothesize that MAFF may participate in these processes. This is further supported by our finding that prostaglandin-endoperoxide synthase 2 (PTGS2, previously known as cyclooxygenase-2) mRNA, proposed to be a major regulator of gestation and parturition [36], is stimulated by IL1B and TNF but not IL6 in the PHM1–31 cell line (data not shown). Earlier reports have shown that PTGS2 expression is upregulated by IL1B in primary myometrial cells [43, 44].
The stimulation of MAFF expression by IL1B and TNF may also be important with respect to the regulation of the oxytocin receptor gene, a putative MAFF target [27], by cytokines. The oxytocin-signaling pathway may play an important role in the control of parturition, and its abnormal regulation may be implicated in preterm labor [45]. There have been conflicting reports as to whether oxytocin receptor gene expression is increased, decreased, or not changed following treatment of myometrial cells with IL1B and IL6 [30, 31, 46, 47]. Thus, it is not clear how MAFF may be implicated in the control of the oxytocin pathway by cytokines. Transfection experiments using MAFF cDNA alone failed to modulate oxytocin receptor gene transcription [27]. This result may be explained by the absence of appropriate CNC factors, which function as binding partners of MAFF. Further studies using a dominant-negative MAFF factor or cotransfection experiments, expressing both MAFF and CNC proteins, should clarify whether the oxytocin receptor gene is regulated by MAFF.
Previous reports showed that MAFF is able to interact with members of the CNC transcription factor family, including NFE2, NFE2-like 1, and NFE2-like 2 [13, 23]. We propose that CNC/MAFF heterodimers may be involved in the regulation of myometrial gene expression by proinflammatory cytokines. CNC/small MAF heterodimers have been shown to bind a variety of AP1-like binding motifs, including the NFE2, MARE, and ARE recognition sites [1, 48]. It has been proposed earlier that small MAF factors, which do not contain an activation domain, increase the binding site specificity of the complex, whereas the large CNC subunit confers the transcriptional activation properties to the heterodimer [11]. Thus, cytokine stimulation leading to increased MAFF levels may result in gene expression changes of a specific set of genes in myometrial cells.
In conclusion, our studies suggest that the MAFF transcription factor may play a role in inflammatory responses in uterine smooth muscle cells. This is important because of the possible role that proinflammatory cytokines may play in preterm and normal term labor [29]. In future studies, it will be critical to identify the MAFF target genes in myometrial cells. This research should eventually lead to a better understanding of the molecular mechanisms governing small MAF factor-mediated regulation of gene expression and help to uncover the possible functions of MAFF in the inflammatory response in uterine smooth muscle cells.
ACKNOWLEDGMENTS
We would like to thank Benoît Chénais for helpful discussions and critical reading of the manuscript and Zaynab Nouhi for assistance with revision experiments.
FOOTNOTES
1 Supported by McGill Cancer Consortium and J.W. McConnell McGill Major Research studentships to W.M. Also supported by NIH award (HD09618) to B.M.S. V.B. supported by a Chercheur Boursier Fonds de la Recherche en Santé du Quebec and a McGill University Charles O. Monat award. Also supported by grants from the Hospital for Sick Children Foundation/Institute for Human Development, Child and Youth Health-CIHR and from the Cancer Research Society Inc. to V.B. 
2 Correspondence: Volker Blank, Lady Davis Institute for Medical Research, Department of Medicine, McGill University, 3755 Cote Sainte-Catherine Road, Montreal, Quebec, Canada H3T 1E2. FAX: 514 340 7573; volker.blank{at}mcgill.ca
Received: 15 July 2005.
First decision: 19 August 2005.
Accepted: 12 December 2005.
REFERENCES
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