The ETS Factor Myeloid Elf-1-Like Factor (MEF)/Elf4 Is Transcriptionally and Functionally Activated by Hypoxia

Abstract

Hypoxia-inducible factor (HIF)-1α is a transcription factor belonging to the HIF family that is activated in mammalian cells during conditions of low oxygen tension or hypoxia to induce an adaptive response and promote cell survival. Some of the genes targeted by HIF-1α are important for angiogenesis and proliferation. Here, we found that the E26 transformation-specific (ETS) transcription factor myeloid elf-1-like factor (MEF)/Elf4 is activated by HIF-1α. MEF induces genes such as human beta-defensin 2 (HβD2) and perforin (PRF1), and is known to affect the cell cycle. Treatment with hypoxia mimetic CoCl₂ or low O₂ incubation up-regulated MEF mRNA and protein levels in various cell lines. HIF-1α overexpression in HEK293 cells also increased MEF mRNA and protein levels. In contrast, HIF-1α knockdown by small interfering RNA (siRNA) suppressed the induction of MEF in response to hypoxia. HIF-1α binds to the hypoxia response element in the MEF promoter region (−200 bp) and activates MEF promoter under hypoxia condition. The induction of MEF by hypoxia/HIF-1α correlated with the increase of MEF target genes HβD2 and PRF1. Intriguingly, the hypoxia-induced expression of HIF-1α target gene vascular endothelial growth factor (VEGF) was enhanced by the exogenous addition of MEF. Overall, these data indicate that hypoxia or HIF-1α positively regulates MEF expression and function.

Oxygen homeostasis is a requirement for eukaryotic organisms to maintain proper cellular functions. During conditions of reduced O₂ availability or hypoxia, cells respond through changes in gene expression that are mainly mediated by hypoxia-inducible factor alpha (HIFα).^1,2) The master transcriptional regulator HIF-1α is a transcription factor that is activated by hypoxia and associates with other co-factors to transactivate genes that ultimately trigger adaptive responses to the lack of cellular oxygen.³⁾ HIF-1α binds to hypoxia response element (HRE) having a consensus sequence 5′-A/GCG TG-3′ in the proximal promoters of hypoxia-responsive genes. The genes that are regulated by HIF/hypoxia control important cellular processes such as stimulation of oxygen delivery through erythropoiesis and angiogenesis.⁴⁾ HIF-1α is known to activate the transcription of several growth factors including vascular endothelial growth factor (VEGF), which is a critical factor for angiogenesis. HIF-1α knockout mice mainly showed abnormal vascular development.⁵⁾ It was also reported that HIF-1α induces the expression of GATA1, a hematopoietic transcription factor that promotes erythroid differentiation under hypoxic condition.⁶⁾ Due to the cellular need to adapt to an environment of low oxygen tension, it is unsurprising that HIF-1α targets genes that are involved in vascular formation, red blood cell production and cellular proliferation. HIF-1α has also been shown to activate E26 transformation-specific (ETS)1, the founding member of the ETS transcription factor family that is mainly involved in cell growth and angiogenesis.⁷⁾

Myeloid elf-1-like factor (MEF)/Elf4, is a member of the ETS transcription factor family, and was shown to transactivate a number of genes such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3,⁸⁾ Sox-2,⁹⁾ MDM2,¹⁰⁾ and the innate immunity-related genes lysozyme, human beta-defensin 2 (HβD2) and perforin (PRF1).^11–13) MEF regulates the entry of hematopoietic stem cells from quiescent to G1 phase¹⁴⁾ and is involved in promoting the progression of the cell cycle from G1 to S phase.¹⁵⁾ We previously reported that MEF is transcriptionally activated by Sp1 and E2F1, with the activity of the latter being modulated by the tumor suppressor p53.^16,17) Recently, we have shown that MEF is degraded by the proteasome via the E3 ligase MDM2, its own target gene, revealing both an auto-regulatory loop and a mutual inhibitory relationship with p53 protein.¹⁸⁾ In our continuous investigations on how MEF is regulated in the cells, we explored the possibility that MEF could be affected by hypoxia. Here, we found that MEF is a transcriptional target of HIF-1α. The increase in MEF mRNA and protein levels under hypoxic conditions or in the presence of HIF-1α related to the increase of MEF transactivating potential. Interestingly, the data revealed a hitherto unknown potential of MEF to up-regulate VEGF and enhancing VEGF transcription in hypoxic condition.

MATERIALS AND METHODS

Cell Culture, Treatment and Transfection

The cell lines HEK293, K562, Jurkat and A549 were obtained from the American Type Culture Collection. Maintenance of HEK293 and A549 cells was described previously.¹⁸⁾ K562 and Jurkat cells were maintained in RPMI 1640 medium supplemented with 10% inactivated fetal bovine serum (FBS) and 2% antibiotics. MEF-stable A549 cells (clone71) were generated previously.¹¹⁾ For MEF-stable HEK293 cells, MEF cDNA plasmid¹¹⁾ was sub-cloned into pEBMulti-Neo vector at the XhoI–BamHI site, and transfected in HEK293 cells. Clones were selected using geneticin sulfate (G418; Sigma-Aldrich, St. Louis, MO, U.S.A.). G418-resistant single colonies were cultured and passaged in G418-containing Dulbecco’s modified Eagle’s medium (DMEM). The clones that highly expressed MEF (clone8 and clone17) were used.

For hypoxia culture, cells were kept at 37°C in 1% O₂ incubator (APM-30D, ASTEC Co., Ltd., Fukuoka, Japan) for the indicated time. Anhydrous cobalt(II) chloride (CoCl₂) was purchased from Nacalai Tesque (Kyoto, Japan). A stock solution was made using dimethyl sulfoxide (DMSO) solvent. Cells were treated with CoCl₂ for the indicated time.

Transient transfection of DNAs for promoter assay was performed using TransIT-LT1 reagent (Mirus Bio LLC, Madison, WI, U.S.A.) according to the protocol reported previously.¹⁸⁾ Transfection of HIF-1 small-interfering RNA (si-RNA) or GL2 si-RNA was carried out using TransIT-TKO reagent (Mirus Bio LLC) as described in ref. 18. The sequence of si-HIF-1α is as follows: forward, 5′-ugugaguucgcaucuugau-3′ and reverse, 5′-aucaagaugcgaacucaca-3′. The sequence of si-GL2 was published in ref. 18.

Plasmids and Luciferase Assay

HIF-1α in pcDNA3.1 plasmid¹⁹⁾ was obtained from Addgene (plasmid 18949). Dominant-negative HIF-1α plasmid was a generous gift from Kizaka-Kondoh and colleagues²⁰⁾ (Tokyo Institute of Technology, Japan). MEF deletion promoters in pGL3b vector were constructed previously.¹⁶⁾ Point mutations in MEF promoter (−200 bp) at the HRE consensus sites were performed using KOD Plus Neo kit (Toyobo Co., Ltd., Osaka, Japan) according to the manufacturer’s instructions. The sequences of mutagenesis primers used are shown in Table 1. Polymerase chain reaction (PCR) products were digested with DpnI, and transformed in E. coli. Positive clones were sequenced for confirmation. MEF promoter constructs were transfected in HEK293 cells together with HIF-1α or empty vector and phRG-TK Renilla luciferase plasmid (Promega, Madison, WI, U.S.A.). Luciferase activity was assayed according to the protocol performed previously.¹⁸⁾

Table 1. Primers Used for Mutagenesis of HRE in MEF Promoter

Primer	Sequence
HRE mutant 1 (MT1) Forward	5′-cttccgaggggagactatgacctggcggggctg-3′
HRE mutant 1 (MT1) Reverse	5′-cagccccgccaggtcatagtctcccctcggaag-3′
HRE mutant 2 (MT2) Forward	5′-gcgccgaggtgagtgattgatgcggggtggcgg-3′
HRE mutant 2 (MT2) Reverse	5′-ccgccaccccgcatcaatcactcacctcggcgc-3′

The mutated bases are in italics.

Real-Time Quantitative PCR Analysis

Total RNA extraction was performed as previously described.¹⁸⁾ Equal amount of RNA samples were reacted with PrimeScript RT master mix as indicated in the recommended protocol. RT products were subjected to real-time quantitative PCR using SYBR Premix Ex Taq II (TaKaRa Bio Inc., Shiga, Japan) according to the manufacturer’s instructions. Amplification and quantification were performed using Bio-Rad CFX Real Time System (Bio-Rad, CA, U.S.A.) and as described previously.¹⁸⁾ The oligonucleotide primers used in the real-time quantitative PCR are listed in Table 2.

Table 2. Primers Used for Real-Time Quantitative RT-PCR Analysis

Gene (for q-PCR)	Forward	Reverse
MEF	5′-tggaaggcagttttttgctga-3′	5′-gacttccgcggttgacatg-3′
Perforin	5′-cgcctacctcaggcttatctc-3′	5′-cctcgacagtcaggcagtc-3′
Human β-defensin	5′-atcagccatgagggtcttgt-3′	5′-gagaccacaggtgccaattt-3′
VEGF	5′-atgacgagggcctggagtgtg-3′	5′-cctatgtgctggccttggtgag-3′
18S rRNA	5′-cggctaccacatccaaggaa-3′	5′-gctggaattaccgcggct-3′

Western Blotting, Antibodies, and Chromatin Immunoprecipitation (ChIP) Assay

Nuclear extracts were isolated using the protocol reported previously, and subjected to immunoblotting.¹⁸⁾ Blots were probed with the appropriate antibodies and visualized by chemiluminescence. The antibodies used were from Santa Cruz Biotechnologies (Santa Cruz, CA, U.S.A.): rabbit anti-Elf4 (M20), goat anti-γ-tubulin (C-20), goat anti-actin (I-19) and normal mouse immunoglobulin G (IgG) (sc-2025), and from BD Transduction Laboratories: mouse anti-HIF-1α (#610958) antibody. For ChIP experiments, nuclear extracts of HEK293 cells were cross-linked using 1% formaldehyde. ChIP assay was performed following the protocol described previously.²¹⁾ The sequences of the primers for MEF and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoters are reported in ref. 17.

RESULTS AND DISCUSSION

Hypoxia Increases MEF mRNA Expression Level

To determine the effect of hypoxia, we mimicked hypoxia condition by treating HEK293 cells with increasing dose of CoCl₂. MEF mRNA level was significantly increased at 500 to 1000 µM CoCl₂ treatment (Fig. 1A). Treatment with CoCl₂ for 12 or 24 h up-regulated MEF mRNA expression (Fig. 1B). HEK293 cells exposed for 24 h to low O₂ resulted in an increased expression of MEF mRNA (Fig. 1C). To investigate whether the hypoxia-induced increase of MEF was mediated by HIF-1α, we transfected dominant-negative (dn) HIF-1α construct into HEK293 cells. The hypoxia-induced increase of MEF mRNA was dose-dependently suppressed by dn-HIF1α (Fig. 1D). Exogenous addition of HIF-1α plasmid resulted in a statistically significant increase of MEF gene (Fig. 1E), and conversely, transfection of HIF-1α si-RNA inhibited the CoCl₂-induced increase of MEF mRNA level (Fig. 1F; CoCl₂). HIF-1α si-RNA did not down-regulate basal MEF transcription (Fig. 1F; DMSO), suggesting that low level of HIF-1α does not contribute to the regulation of basal MEF expression. Leukemia cell lines K562 and Jurkat likewise exhibited an increase in MEF mRNA expression in hypoxia conditions induced by low O₂ incubation (Figs. 1G, H) or treatment with CoCl₂ (Figs. 1I, J). These results showed that hypoxia positively affects MEF transcription.

Fig. 1. Hypoxia Increases MEF mRNA Expression

Quantitative RT-PCR analysis of MEF expression was performed on total RNA extracted from HEK293 cells: (A) treated with DMSO (control) or the indicated concentration of CoCl₂ for 24 h; (B) treated with 1000 µM CoCl₂ for the indicated time; (C) exposed to low O₂ for 24 h; (D) transfected with dominant negative (dn) HIF-1α plasmid or empty vector and 24 h post-transfection, cells were incubated in low O₂ incubator for 24 h; (E) transfected with HIF-1α plasmid for 48 h; (F) transfected with si-GL2 (control) or si-HIF-1α, and 24 h after, cells were treated with DMSO or 1000 µM CoCl₂. (G, H) MEF mRNA was determined by quantitative PCR analysis in K562 cells (G) and Jurkat cells (H) incubated in low O₂ for 24 h. (I, J) K562 cells (I) and Jurkat cells (J) were treated with the indicated concentration of CoCl₂ or DMSO for 12 h, and MEF mRNA level was determined by quantitative PCR analysis. Data shown are the mean±S.D. (n=3). * p<0.05, ** p<0.01 vs. non-treated and/or non-transfected control, assessed by ANOVA with Dunnett’s multiple comparison test (for A, I, J), ANOVA with Tukey–Kramer test (for D, F) or t-test (for B, C, E, G, H).

Hypoxia Up-Regulates MEF Promoter Activity in HEK293 Cells

Reporter assays revealed that CoCl₂ treatment increased the MEF promoter activity at the proximal (−200 bp) region of MEF promoter (Fig. 2A). Low O₂ incubation also yielded similar results (Fig. 2B). Transfection of HIF-1α cDNA plasmid dose-dependently up-regulated MEF promoter activity (Fig. 2C). The −200 bp segment of MEF promoter contains 2 HREs, assessed by in silico analysis. To clarify the importance of these sites for HIF response, we generated point mutant constructs, MT1, MT2 and dMT that carry mutations in HRE1, HRE2 and both, respectively (Fig. 2D, inset). Mutations in either of the HREs resulted in the suppression of MEF promoter’s response to HIF-1α overexpression. Mutations in both sites further down-regulated the activity of MEF promoter, indicating that both HREs are important for the effect of HIF-1α (Fig. 2D). Consistent with these results, we observed that HIF-1α binds to the promoter of MEF as determined by ChIP assay using nuclear extracts from HEK293 cells treated with CoCl₂ (Fig. 2E) or transfected with HIF-1α cDNA plasmid in the presence of CoCl₂ (Fig. 2F). GAPDH was used as positive control for HIF-1α binding.²²⁾

Fig. 2. Hypoxia Increases MEF Promoter Activity

(A) MEF promoter (−200, −400, −800 bp) in luciferase reporter plasmid, or empty vector (pGL3b) was transfected in HEK293 cells. Forty-eight hours post-transfection, cells were treated with 1000 µM CoCl₂ for 24 h. (B) HEK293 cells transfected with the indicated MEF promoter constructs were exposed to hypoxia (1% O₂) for 6, 12 or 24 h. (C) HEK293 cells were transfected with −200 bp MEF promoter and/or the indicated amount of HIF-1α cDNA. (D) Empty vector, −200 bp MEF wild-type (WT) and MEF HRE mutant (MT) promoters were transfected in HEK293 cells. Inset, two HREs are contained in −200 bp MEF promoter. These are singly (MT1 or MT2) or doubly (dMT) mutated. (A–D) Promoter activity was assayed, normalized to Renilla luciferase and expressed as fold activation over pGL3b vector. Data are the mean±S.E. (n=3). * p<0.05, ** p<0.01 vs. the indicated control, assessed using ANOVA with Tukey–Kramer test (A, B, D) or with Dunnett’s test (C). (E, F) HIF-1α binding to MEF promoter (−200 bp) was assessed by ChIP assay using nuclear extracts of HEK293 cells untreated or treated with the indicated concentration of CoCl₂ for 24 h (E) or cells transfected with HIF-1α and treated with 1000 µM CoCl₂ for 6 h (F). GAPDH promoter was used as positive control for HIF-1α binding. Data shown are representative of 2 independent experiments.

Hypoxia Increases MEF Protein Expression Level

HEK293 cells treated with CoCl₂ showed an increase of endogenous MEF protein expression level (Fig. 3A), as did the exposure of cells to low O₂ for 24 h (Fig. 3B). Transfection of HIF-1α and/or treatment with CoCl₂ also up-regulated MEF protein level (Fig. 3C), while transfection of HIF-1α si-RNA suppressed the CoCl₂-induced increase of endogenous MEF protein in a statistically significant manner (Fig. 3D). This was not specific to HEK293 because K562 cells and Jurkat cells incubated under hypoxia condition showed an up-regulation of MEF protein (Figs. 3E, F). Moreover, CoCl₂ dose-dependently increased MEF protein expression in K562 and Jurkat cells (Figs. 3G, H). These data indicate that hypoxia positively affects MEF protein expression. We detected two bands of activated HIF-1α protein (such as in Fig. 3H). It has been shown that HIF-1α is expressed as a doublet that represents post-translational modification of this protein.²³⁾

Fig. 3. Hypoxia Increases MEF Protein Expression

(A–D) HEK293 cells were (A) treated with the indicated concentration of CoCl₂ for 24 h; (B) incubated at low oxygen (1% O₂); (C) transfected with HIF-1α or pcDNA3.1 and treated with CoCl₂ for 24 h; (D) transfected with the indicated si-HIF-1α or si-GL2 and treated with CoCl₂ for 24 h. Right panel, Immunoblots were analyzed by Image Gauge software (ver. 4.23, FUJIFILM, Japan), and normalized to Actin. Graphs are the mean±S.E. (n=3). * p<0.05 vs. control (DMSO, 0 nM si-HIF-1α); ^† p<0.05 vs. CoCl₂, 0 nM si-HIF-1α. (E, F) K562 and Jurkat cells were incubated for 12 and 24 h, respectively, under normoxic (control) or hypoxic (1% O₂) condition. (G, H) K562 and Jurkat cells were treated with the indicated concentration of CoCl₂ for 24 h. (A–H) Nuclear extracts were isolated and analyzed by Western blotting for the expression of MEF protein. Data shown are representative of 2–3 independent experiments.

HIF-1α Enhances MEF-Induced Activation of MEF Target Genes

To confirm the functional relevance of the up-regulation of MEF expression induced by hypoxia, we assessed the effect of HIF-1α or CoCl₂ on the target genes of MEF. While HIF-1α did not increase HβD2 and PRF1, MEF induced the transcription of these genes^12,13) (Figs. 4A, B). Importantly, the addition of HIF-1α enhanced the activation of HβD2 and PRF1 (Figs. 4A, B). Interestingly, VEGF, which is regulated by HIF-1α, was also induced by MEF alone and was more up-regulated in the presence of HIF-1α and MEF (Fig. 1C). Treatment with CoCl₂ enhanced the MEF-induced expression of HβD2, PRF1 and VEGF. The up-regulation resulting from the combination of MEF and CoCl₂ was statistically significant compared with either MEF or CoCl₂ alone (Figs. 4D, F). Taken together, these data indicate that HIF-1α or hypoxia condition increases MEF expression and MEF transactivating potential resulting in the enhancement of the transcription of MEF target genes. Because VEGF has not been previously proven to be a MEF target and it is an important factor in many physiological and pathological processes, we further verified the combined effect of MEF and hypoxia on VEGF transcription. HEK293 cells stably expressing MEF, clone8 and clone17 and A549 cells stably expressing MEF, clone71 (Figs. 4H, J, L) were untreated or treated with CoCl₂. VEGF was slightly elevated in MEF stable cell lines (Figs. 4G, I, K). Treatment of MEF stable cells with CoCl₂ enhanced the MEF-induced increase of VEGF, suggesting that MEF and hypoxia have additive effect on VEGF transcription. We can speculate that this additive effect is due to MEF being able to independently activate VEGF directly, thus providing a cooperative activation of VEGF together with hypoxia/HIF-1α. Alternatively, the enhanced VEGF expression is due to the positive regulation of MEF expression and function by HIF-1α/hypoxia. Whether VEGF is a bona fide target gene of MEF remains to be clarified by further studies.

Fig. 4. HIF-1α Enhances MEF-Induced Activation of MEF Target Genes

(A–C) HEK293 cells were transfected with empty vectors or MEF and/or HIF-1α. Forty-eight hours post-transfection, cells were harvested for RNA extraction. (D–F) HEK293 cells were transfected with empty vector or MEF. Twenty-four hours post-transfection, cells were treated with DMSO or 1000 µM CoCl₂, and total RNA was extracted. (G–J) Parental HEK293 cells or stably transfected MEF cells (G, H) clone8 and (I, J) clone17, and (K, L) parental A549 cells or stably transfected MEF cells (clone71) were treated for 24 h with 1000 µM CoCl₂, then total RNA was isolated. (A–L) The mRNA expression of the indicated genes was analyzed by quantitative RT-PCR. Data shown are the mean±S.D. (n=3). * p<0.05, ** p<0.01 vs. control (mock-transfected and/or DMSO-treated). For D–F, ^# p<0.05, ^## p<0.01 vs. mock-transfected, CoCl₂-treated group. ^† p<0.05, ^†† p<0.01 vs. MEF-transfected, DMSO-treated group. For G, I and K, ^†† p<0.01 vs. CoCl₂-treated HEK293 or A549 parental cells. n.s., not significant. Statistical differences were assessed using ANOVA with Tukey–Kramer test except for H, J and L, which was assessed using t-test.

Taken together, we show here that hypoxia up-regulates the mRNA and protein expression levels of MEF via the activation of MEF promoter by HIF-1α. Although we cannot completely rule out the possibility that HIF-2α can also activate MEF, the si-RNA specific to HIF-1α was able to inhibit the CoCl₂-induced increase of MEF (Figs. 1F, 3D). In addition, Park et al., demonstrated that hypoxia-induced gene activation is solely mediated by HIF-1α. HIF-2α is primarily localized in the cytoplasm and remains transcriptionally inactive under hypoxic conditions.²⁴⁾ Considering that MEF is constitutively localized in the nucleus and is degraded in the nucleus as what we reported previously,^18,25) it is unlikely that HIF-2α mediates the effect of hypoxia on MEF. The contribution of MEF in hypoxia in tumor cells or normal cells remains to be elucidated, but the findings presented here that MEF is activated by HIF-1α adds to the growing possibilities of the functions of MEF in health and disease.

Acknowledgments

The dominant negative HIF-1α construct was a generous gift from Shinae Kizaka-Kondoh from the Tokyo Institute of Technology. The authors acknowledge the technical assistance provided by Ms. Masako Shimamoto. This work was supported by Grants-in-Aid for Science Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan KAKENHI (#22390015 to H.K. and #23590082 to M.S.).

Conflict of Interest

The authors declare no conflict of interest.

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