Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2015 May 1.
Published in final edited form as: Mol Cancer Res. 2014 Feb 12;12(5):728–741. doi: 10.1158/1541-7786.MCR-13-0607

HIFs Enhance the Transcriptional Activation and Splicing of Adrenomedullin

Johnny A Sena 1,^, Liyi Wang 2,^, Matthew R Pawlus 1, Cheng-Jun Hu 1,2,*
PMCID: PMC4020993  NIHMSID: NIHMS566976  PMID: 24523299

Abstract

Adrenomedullin (ADM) is important for tumor angiogenesis, tumor cell growth and survival. Under normoxic conditions, the ADM gene was found to produce two alternative transcripts, a fully-spliced transcript that produces AM and PAMP peptides and a intron-3-retaining transcript that produces a less functionally significant PAMP peptide only. ADM is a well-established hypoxia inducible gene; however, it is not clear which ADM isoform is induced by hypoxia. In this study, it was determined that various cancer and normal cells express two predominant types of ADM transcripts, a AM/PAMP peptide producing FL transcript in which all introns are removed, and a non-protein producing I1-3 transcript in which all introns are retained. Interestingly, hypoxia preferentially induced the FL isoform. Moreover, HIFs, but not hypoxia per se are necessary and sufficient to increase splicing of ADM pre-mRNA. ADM splicing reporters confirmed that transcriptional activation by HIF or other transcription factors is sufficient to enhance splicing. However, HIFs are more potent in enhancing ADM pre-mRNA splicing than other transcriptional activators. Thus, ADM intron retention is not a consequence of abnormal splicing, but is an important mechanism to regulate ADM expression. These results demonstrate a novel function of HIFs in regulating ADM expression by enhancing its pre-mRNA splicing. Importantly, using endogenous and cloned ADM gene, further evidence is provided for the coupling of transcription and RNA splicing.

Keywords: Hypoxia, RNA splicing, HIF, adrenomedullin, intron retention

Introduction

Hypoxia is a common characteristic of many solid tumors. The hypoxic intratumoral microenvironment stabilizes hypoxia-inducible transcription factor 1α (HIF1α) and 2α (HIF2α) that are normally degraded by the 26S proteasome upon pVHL-mediated ubiquitination under normoxia. Stabilized HIF1α and HIF2α proteins translocate to the nucleus and heterodimerize with a constitutive nuclear protein, the aryl hydrocarbon receptor nuclear translocator (ARNT, also called HIF1β) to form HIF1α/ARNT (HIF1) and HIF2α/ARNT (HIF2) heterodimers. Then, HIF1 and HIF2 bind to HIF binding sites (HBS) on HIF target gene promoters and/or enhancers and activate genes involved in neovascularization, glycolysis, cellular proliferation, and metastasis. Thus, the HIF-mediated hypoxic transcriptional response is critical for tumor progression by allowing cancer cells to adapt to a low oxygen environment (1-4).

Adrenomedullin (ADM) is a well-established hypoxia induced gene. The ADM gene codes for a 185 amino acid propreadrenomedullin protein that is cleaved into a 52 amino acid AM peptide and a 20-amino acid peptide called “proadrenomedullin N-terminal 20 peptide” or PAMP (Fig. 1D). AM peptide plays important roles in tumorigenesis by inducing tumor angiogenesis, enhancing tumor cell proliferation, and reducing tumor cell apoptosis (5-13). However, PAMP appears to be less important in tumorigenesis because PAMP has no activity in tumor cell proliferation and survival, although PAMP is a stronger vasodilator and angiogenic factor than the AM peptide (14-16). Thus, hypoxia induced ADM gene expression is an important component of the hypoxia response that is crucial for tumor progression and metastasis.

Figure 1. Hypoxia increases the levels of fully-spliced ADM transcripts in cancer and normal cells.

Figure 1

Open in a new tab

RT-PCR (left panel) and RT-qPCR (right panel) analysis of ADM transcripts in normoxic or hypoxic Hep3B (A), HK2 (B), and HUVEC (C) cells. FL indicates ADM transcripts in which all introns are removed. I1 refers to ADM RNA containng intron 1. I1-3 refers to ADM RNA in which all three introns are retained. I2 or I3 refers to ADM RNA retaining intron 2 or 3. In the RT-qPCR panel, the numbers next to the sample labels represent the expression ratio of ADM FL to ADM I1-3 ± the standard deviation. D) Schematic diagram of the ADM gene including 4 exons (boxes) and three introns (lines). The start and stop codons for the propreADM protein were indicated. The exon regions that encode the PAMP and the AM peptides are indicated by black boxes and striped boxes respectively. E) Western blot analysis of HIF1α, HIF2α, Beta-Actin, and ADM proteins in normoxic and hypoxic Hep3B cell.

Interestingly, various cancer cells cultured under normoxia were found to produce two isoforms (17). One isoform is devoid of introns (full-length, FL) and produces both PAMP and AM peptides. A second isoform, in which the third intron is retained (I3), produces only the PAMP peptide due to a premature stop codon in intron 3 (17). Moreover, the relative ratio of I3/FL was increased by hypoxia, resulting in an increased PAMP/AM peptide ratio even though both isoforms were induced by hypoxia (17). This data suggested that hypoxia favors intron 3 retention and expression of PAMP peptide. The goal of our study is to clarify if hypoxia favors PAMP generation and to determine how hypoxia regulates the ADM isoform ratio change.

Materials and Methods

Cell culture

Hep3B cells were cultured in MEM/EBSS (Hyclone) containing 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100,000 units/L Penicillin/Streptomycin, 1.5 g/L sodium bicarbonate, and 1X non-essential amino acids (NEAA). Hela cells were grown in high-glucose DMEM (Hyclone) with 10% FBS, 2 mM L-glutamine, 100,000 units/L Penicillin/Streptomycin and 1X NEAA. HEK293T, RCC4, and RCC4T cells were grown in high-glucose Dulbecco modified Eagle medium (DMEM: Hyclone) with 10% FBS, 2 mM L-glutamine, 100,000 units/L Penicillin/Streptomycin and 1X NEAA. HK2 cells were grown in keratinocyte serum free medium (K-SFM) (GIBCO) with 0.05 mg/ml bovine pituitary extract (BPE), 5 ng/ml recombinant epidermal growth factor (EGF), 2 mM L-glutamine, 100,000 units/L Penicillin/Streptomycin and 1X NEAA. HUVEC cells were grown in F-12K medium (ATCC) containing 10% FBS with 0.1 mg/ml heparin, 0.04 mg/ml endothelial cell growth supplement (ECGS), 2 mM L-glutamine, 100,000 units/L Penicillin/Streptomycin and 1X NEAA. Prior to hypoxia treatment, 25 mM HEPES was added to growth media and cells were incubated under normoxia (21% O2) or hypoxia (1.5% O2) for 12-16 hrs. All parental cell lines were purchased from ATCC. After completing the experiments, the parental (Hep3B, HEK293T, RCC4, HK2 and HUVEC) and modified cell lines (RCC4T) were authenticated by DNA profiling or “fingerprinting” by the University of Colorado DNA Sequencing & Analysis Core.

Knockdown of endogenous mRNA using small interfering RNAs (siRNAs)

Control (Qiagen,1027281) or siRNAs specific for human ARNT (Qiagen, equal mix of SI00304220, SI00304234, and SI03020913), HIF1α (Qiagen, SI02664053) or HIF2α (Qiagen, SI00380212) mRNAs were transfected into Hep3B cells at 50% confluency using HiPerFect Transfection Reagent (Qiagen) according to the manufacturer’s protocol. 32hr post-transfection, cells were cultured at 21% or 1.5% O2 for 12-16hr and then collected to prepare mRNA or protein for analysis.

Plasmid constructs and viral transduction

The CA9P/ADM, PAI1P/ADM, and 2HRE/ADM splicing reporters were in the pcDNA3.1 (+) vector. The CA9 and PAI1 promoters were cloned from CA9P/Luc and PAI1P/Luc (18, 19) using Advantage GC cDNA polymerase (Clontech) and then inserted into the pcDNA3.1 plasmid, replacing the CMV promoter. 2 copies of HRE (hypoxia response element/HIF binding sites) from the PAI1 promoter were added upstream of SV40 minimal promoter in the pGL3/Luc vector to produced the 2HRE/Luc construct, from which the 2HRE/SV40 was PCR amplified and replaced the CMV promoter in the pcDNA3.1 vector. Next, the ADM gene (full-length gene) from exon 1 to exon 4 was amplified from human genomic DNA and inserted downstream of the CA9, PA11 and 2HRE promoters to yield the CA9P/ADM, PAI1P/ADM or 2HRE/ADM splicing reporter constructs. The CA9Pm2HRE/ADM, PAI1PmHRE/ADM, and ΔHRE/ADM constructs were synthesized from the CA9P/ADM, PAI1P/ADM, and the 2HRE/ADM constructs using Pfu Ultra II polymerase (Invitrogen)-mediated mutagenesis PCR, in which the HREs were mutated or deleted from the constructs. The G5P/ADM reporter was generated by replacing the CA9 promoter in CA9P/ADM with the 5 copies of Gal4 DNA binding elements from G5/Luc. The HIF1αTM-Flag, HIF2αTM-Flag, HIF1αDBD-Flag, HIF1αDBD/VP16TAD-Flag, and USF expression plasmids were described previously (18, 20). The HIF1αDBD/E2F1TAD-Flag construct was synthesized by adding the E2F transcription factor 1 transactivation domain from the PHKgalE2F 380-437 plasmid (gift from David Bentley) (21) to the HIF1αDBD construct using PCR. The Gal4DBD fusion constructs were synthesized by replacing the HIF1αDBD with the Gal4 DNA binding domain.

The HIF1α and HIF2α lentiviral constructs were synthesized by PCR cloning the HIF1α TM (triple mutation) and the HIF2α TM CDS, from the plasmids describe above, into the pLEX-MCS vector containing a C-terminal 2X Flag tag (Thermo Scientific). The GFP lentiviral construct was synthesized by cloning green fluorescent protein cDNA (CDS) into the pLEX-MCS vector containing a C-terminal 2X Flag tag. The GFP, HIF1αTM, or HIF2αTM plasmid were co-transfected with a psPAX2 packaging vector (Addgene) and a pMD2.G envelope vector (Addgene) into HEK293T cells, grown in 10 cm dishes (40% confluent), using TransIT LT-1 transfection reagent (Mirus). 24 hours after transfection, transfection media was replaced with complete Hep3B media. The following day, viral media was filtered and viral particles were concentrated using high-speed ultracentrifugation at 26,000 X G. The viral pellet was re-suspended in 1mL of PBS and added to Hep3B cells (30% confluency) for 6 hours. The following day, a second round of viral PBS was added to Hep3B cells. 24 hours after the second viral transduction, Hep3B cells were harvested for RNA and protein preparation.

RNA stability assay

Hep3B cells were grown to ~50% confluency in 6-well plates and place under hypoxia for 16 hours. After which, the cells were treated with 2 μg/ml of actinomycin D to inhibit de novo transcription and placed back under normoxia or hypoxia for 2, 4 or 8 hours. Following treatment, cells were collected for RNA isolation using SurePrep Nuclear or Cytoplasmic RNA Purification Kit (Fisher BioReagents, BP2805-25). cDNA synthesis was performed and ADM FL and ADM I1-3 mRNA stability was measured using RT-qPCR.

ADM splicing reporter assay

ADM splicing reporter assays were performed in Hep3B cells using Lipofectamine Reagent (invitrogen 18324-012) and PLUS Reagent (invitrogen 11514-015) to transfect plasmid DNA. Typically, 2 X105 cells/per well in 6-well plates were co-transfected with 0.2 μg of splicing report construct and 1.8 μg of transcription activator. 48 hours after transfection, cells were collected for mRNA and protein analysis.

Protein Analysis

Whole cell lysates were prepared and quantified for protein concentration. Western blot analysis was performed using standard protocols with the following primary antibodies: anti-ADM (C20) pAb (SC-16496, Santa Cruz), anti-Flag mAb (F3165, Sigma), anti-HIF1α mAb (610959, BD Bioscience), anti-HIF2α mAb (D9E3, Cell Signaling), anti-ARNT mAb (NB 100-124, Novus Biological), anti-Actin pAb (SC-1616, Santa Cruz), and anti-Gal4DBD pAb (SC-577, Santa Cruz).

RNA preparation and reverse transcription PCR or quantitative-PCR (RT-qPCR)

RNA was isolated from cells using the RNeasy Plus mini kit (Qiagen) which removes DNA, then was reverse transcribed using the iSCRIPT Advanced cDNA synthesis kit (Bio-Rad) containing oligo-dT and random hexamer. mRNA levels were semi quantified by RT-PCR or quantified by RT-qPCR using iQ Sybr Green supermix (Bio-Rad) in triplicate on the CFX384 Real-Time System (BioRad). All primer sets for RT-qPCR designed to measure mRNA levels were validated for their specificity and amplification efficiency (85%-110%) using melt curve analysis, RT-qPCR product sequencing, and standard dilution analysis. RT-qPCR results were normalized using the ΔΔCT method using18s rRNA and β-Actin as reference genes and untreated normoxia samples, GFP lentivirus, or empty vectors (His) as controls. At least three independent experiments were performed to generate the results presented in the figures.

Statistics

One-way analysis of variance was performed unless otherwise stated. Error bars in figures indicate standard deviation. Asterisks indicate statistical significance as follows: *, P < 0.05; **, P < 0.01. Controls for statistical analysis are specified in each figure. All experiments were performed at least three separate times.

Results

Hypoxia preferentially increases fully-spliced ADM transcript levels in various cell-lines

The ADM gene was reported to produce two isoforms, one isoform devoid of introns (ADM FL) and a second isoform in which the third intron is retained (ADM I3) (17). To determine if hypoxia differentially regulates the levels of these ADM transcripts, RNA prepared from normoxic and hypoxic Hep3B cells was used for RT-PCR. ADM transcripts were detected using forward and reverse primers located in the first exon (exon 1) and the last exon (exon 4), respectively, of the ADM gene. Interestingly, normoxic Hep3B cells, a hepatocellular carcinoma cell-line, expressed multiple ADM transcripts (Fig. 1A), a fully spliced transcript in which all introns were removed (Fig.1A, FL), a transcript in which all three introns were included (Fig.1A, I1-3), a transcript in which intron 1 was included (Fig. 1A, I1), and two minor isoforms in which intron 2 was included (Fig.1A, I2) and a previously reported intron 3-containing isoform (Fig. 1A, I3) (17). Interestingly, hypoxia induced the levels of ADM FL but not the other intron-containing isoforms (Fig. 1A). To better quantify the ADM isoforms, RT-qPCR was used to measure the relative ratios of the ADM FL and I1-3 transcripts since they were the dominantly expressed transcripts in Hep3B cells. Using transcript specific qPCR primers for ADM FL (E1-E2/E3-E2, detects both FL and I3) and ADM I1-3 (I1-E2/I2-E2, detects I1-3 only), it was found that Hep3B cells exhibited an ADM FL/I1-3 ratio of 3.28 and 15.95 under normoxia and hypoxia (Fig. 1A, bar graph), suggesting that hypoxia favors ADM FL expression even though both isoforms are induced. We also found that normoxic MDA-MB-231, Hela, and HEK293 cells expressed ADM FL, I1-3, and I1 isoforms and that hypoxia preferentially induced the levels of ADM FL in these cells (data not shown).

To assess ADM expression in relatively normal cells, HK2 cells, an HPV-16 transformed kidney cell-line was analyzed. Using RT-PCR, HK2 cells were found to express the FL, I1, and I1-3 transcripts with FL and I1-3 being the primarily expressed transcripts under normoxia. Interestingly, hypoxia increased the ADM FL, but not the I1-3 transcript levels (Fig. 1B). According to RT-qPCR, hypoxia increased the levels of ADM FL and I1-3 transcript by 5.98 and 1.16 fold (Fig. 1B, bar graph). As a result, the ADM FL/I1-3 ratio was increased from 11.7 under normoxia to 62.16 under hypoxia.

Similarly, HUVEC cells, a primary umbilical vein vascular endothelial cell-line, also exhibited splicing patterns similar to those observed in Hep3B and HK2 cells under normoxia (Fig. 1C). Furthermore, hypoxia induced the levels of ADM FL, but not I1-3 transcripts (Fig. 1C, right). Again, RT-qPCR confirmed that the levels of ADM FL transcript was increased by 6.52 fold but the levels of ADM I1-3 transcript was not induced (0.91 fold) (Fig. 1B, bar graph), thus hypoxia enhanced the FL/I1-3 ratio from 5.17 under normoxia to 37.62 under hypoxia. Since ADM FL and I1-3 transcripts are the dominantly expressed transcripts in all of the cell lines tested here; the FL and I1-3 transcripts are the focus for the remainder of this study.

The ADM FL transcript codes for a 182-amino acid pro-pre-adrenomedullin precursor protein that is cleaved to produce a 52-amino acid AM peptide and a 20-amino acid PAMP peptide (Fig. 1D). In contrast, the ADM I3 transcript only codes for the PAMP peptide due to a premature stop codon in intron 3, whereas the ADM I1, I2, and I1-3 isoforms do not code for either the AM or PAMP peptides due to a premature stop codon in introns 1 and 2 (Fig. 1D). After establishing that hypoxia increased the expression of the ADM FL transcript, western blots for the AM peptide were performed. As expected, high levels of HIF1α and HIF2α proteins were detected in hypoxic but not in normoxic Hep3B cells (Fig. 1E). Importantly, the ADM precursor (pro, 22 kDa) and the AM peptide (6 kDa, doublet likely due to post-translational modifications) derived from the ADM FL transcript were detected under normoxia. Interestingly, hypoxia significantly increased the levels of AM peptide (Fig.1E, anti-AM). However, the levels of the ADM precursor protein were not significantly different between normoxic and hypoxic Hep3B cells (Fig. 1E, Pro), likely due to its cleavage which generates the AM and PAMP peptides. These results indicated that hypoxia increases the levels of ADM FL transcript in cancer and normal cell lines.

The increased ADM FL/I1-3 ratio is due to RNA splicing but not due to differential RNA stability

Intron-retaining transcripts frequently contain premature termination codons and are targeted for nonsense-mediated decay (NMD) (22, 23). As stated above, ADM I1-3, I1, I2, and I3 transcripts contain premature stop codons in introns 1, 2, and 3. These intron-retaining ADM transcripts are predicted to undergo NMD when they are exported to cytoplasm, which might explain why they exhibit lower induction under hypoxia. To test this, Hep3B cells were placed under hypoxia for 16 hrs to increase the levels of both ADM FL and I1-3, followed by treatment with actinomycin D to inhibit de novo transcription. Cells were then placed back under normoxia or hypoxia for 0, 2, 4, or 8 hours and harvested for RNA isolation and cDNA synthesis. Using RT-qPCR, both ADM FL and I1-3 transcripts were found to be very unstable since both transcripts were reduced by 82-97% 2 hrs post actinomycin D treatment under normoxia or hypoxia (Fig. 2A). In addition, the ADM FL and I1-3 mRNA levels did not change significantly from 2-8 hrs following actinomycin D treatment (Fig. 2A). Interestingly, we found that the mRNA stability of ADM FL did not significantly differ between normoxia and hypoxia at 2, 4 and 8 hrs (Fig. 2A). However, hypoxia reduced the stability of ADM I1-3. For instance, ADM I1-3 was reduced by 82, 83, and 84% at 2, 4, and 8 hrs, respectively under normoxia but was reduced by 91-93% under hypoxia at 2, 4, and 8 hrs (Fig. 2A). It is not clear why ADM transcripts degrade rapidly although these results are consistent with a previous report, which demonstrates similar trends in ADM mRNA stability (24). Overall, ADM I1-3 was more stable than ADM FL both under normoxia and hypoxia; therefore, this data suggests that the hypoxia induced increase of the ADM FL/I1-3 ratio is not due to preferential de-stabilization of ADM I1-3 under hypoxia.

Figure 2. ADM FL and I1-3 transcript stability does not account for the hypoxia-mediated increase in the ADM FL/I1-3 ratio, and ADM FL and I1-3 transcripts are differentially localized in Hep3B cells.

Figure 2

Open in a new tab

A) RT-qPCR analysis ADM FL and I1-3 transcripts in normoxic and hypoxic Hep3B cells treated with actinomycin D for 2-8 hours. RT-qPCR analysis of beta-actin (B), ADM FL (C), ADM I1-3 (D) and ADM total (E) transcripts in nuclear and cytoplasmic fractions from normoxic and hypoxic Hep3B cells.

ADM I1-3 transcripts are primarily located in the nucleus

Intron-retaining transcripts frequently contain premature termination codons and are either targeted for nonsense mediated decay or their presence is restricted to the nucleus (22, 23). Next, the sub-cellular localization of ADM FL and I1-3 transcripts were determined in normoxia or hypoxia Hep3B cells by examining the levels of ADM FL and I1-3 transcripts in the nucleus or cytoplasm. First, we measured the expression of beta actin RNA (B-ACT) as a positive control for mRNA splicing and nuclear export, and as a negative control for hypoxia induction. As expected, B-ACT expression was not induced by hypoxia and a majority of the RNA was localized in the cytoplasm, with cytoplasmic to nuclear ratios of 3.05 and 3.87 under normoxia and hypoxia respectively (Fig. 2B). As expected of a mature transcript, ADM FL was found both in the nucleus and in the cytoplasmic fractions (Fig. 2C). However, more ADM FL transcript was found in the cytoplasmic fraction, with the cytoplasmic to nuclear ratios of ADM FL being 1.75 and 2.7 under normoxia and hypoxia respectively. In contrast, a majority of the ADM I1-3 transcript accumulated in the nucleus compared to the cytoplasm, with the cytoplasmic to nuclear ratios being 0.029 and 0.15 under normoxia and hypoxia respectively. This data suggested that ADM I1-3 is an unspliced ADM transcript that is restricted primarily to the nucleus (Fig. 2D). Additionally, we measured the total levels of ADM transcript (using primers located in exon 4) and found that more ADM transcripts were localized to the cytoplasm, with the cytoplasmic to nuclear ratio of total ADM being 1.33 under normoxia and 1.9 under hypoxia (Fig. 2E). In addition, we did not detect a signal by qPCR in our nuclear or cytoplasmic fractions in which we excluded reverse transcriptase from our cDNA synthesis (Fig.2B-E, no reverse transcriptase), suggesting that all of our transcripts including ADM I1-3 are in fact mRNAs and not DNA contamination.

HIF activity is required for increased splicing of ADM pre-mRNA

ADM is a HIF regulated gene; therefore we wanted to determine if HIF activity was necessary for the hypoxia induced ADM FL/I1-3 ratio increase. To test this, the levels of ARNT, HIF1α, or HIF2α mRNAs in Hep3B cells were reduced by 75-87% using siRNAs (Fig. 3A). ARNT knockdown inhibited hypoxic induction of HIF1α target genes, LDHA and PGK1, and HIF2α target genes, EPO and PAI1 (Fig. 3B). On the other hand, HIF1α knockdown only inhibited hypoxic induction of HIF1α target genes, whereas HIF2α knockdown only inhibited hypoxic induction of HIF2α target genes (Fig. 3B). Hypoxia preferentially increased the levels of the ADM FL transcript in Hep3B/control and HIF2α siRNA cells (Fig. 3C) while knockdown of ARNT and HIF1α dramatically inhibited the hypoxic induction of ADM FL mRNA (Fig. 3C). Using RT-qPCR, we found that hypoxia induced the expression of both the ADM FL and I1-3 transcripts but favored the induction of ADM FL transcript in Hep3B/control siRNA cells (Fig. 3D). Interestingly, ARNT knockdown reduced the hypoxic induction of both the ADM FL and I1-3 transcripts and also reduced the ADM FL/I1-3 ratio to 7.4 versus 11.4 observed in hypoxic Hep3B/control siRNA cells. In addition, HIF1α and HIF2α knockdown also decreased the hypoxia induction of ADM FL and I1-3 transcripts; however, individual knockdown of either HIF1 or HIF2 did not significantly alter the ADM FL/I1-3 ratio. For instance, HIF1 knockdown reduced the ADM FL/I1-3 ratio to 9.9 whereas HIF2 knockdown increased the ADM FL/I1-3 ratio to 12.9. This is likely because ADM is a common HIF1 and HIF2 target gene. This data suggested that HIF activity, but not hypoxia per se, is necessary for hypoxia-induced pre-mRNA splicing of ADM.

Figure 3. HIF activity is required for hypoxia induced splicing of ADM pre-mRNA.

Figure 3

Open in a new tab

A) RT-qPCR analysis of ARNT, HIF1α, and HIF2α mRNA levels in normoxic and hypoxic Hep3B cells targeted with control, ARNT, HIF1α, or HIF2α siRNAs. B) RT-qPCR analysis of the levels of HIF1 target genes, LDHA and PGK1, and HIF2 target genes, EPO and PAI1, in normoxic and hypoxic Hep3B cells targeted with control, ARNT, HIF1α, or HIF2α siRNAs. C) RT-PCR analysis of ADM transcripts in normoxic and hypoxic Hep3B cells targeted with control, ARNT, HIF1α, or HIF2α siRNAs. D) RT-qPCR analysis of ADM FL, I1-3 and total transcripts in normoxic and hypoxic Hep3B cells targeted with control, ARNT, HIF1α, or HIF2α siRNAs.

HIF activity is sufficient for increased splicing of ADM pre-mRNA

Next, we determined if HIF activity is sufficient for increased splicing of ADM pre-mRNA. To test this, Hep3B cells were transduced with lentiviruses expressing normoxia active, flag-tagged HIF1α, HIF2α, or GFP. Western blot analysis using an anti-flag antibody detected expression of HIF1α, HIF2α, and GFP proteins in virus infected Hep3B cells under normoxia (Fig. 4A). In addition, HIF1α and HIF2α proteins were functional under normoxia since HIF1α target genes, LDHA and PGK1, and HIF2α target genes, EPO and PAI1, were induced in HIF1α or HIF2α transduced cells compared to the GFP transduced cells (Fig. 4B). Importantly, both HIF1α and HIF2α increased the levels of ADM FL, but not I1-3 as determined by RT-PCR (Fig. 4C). RT-qPCR also determined that both HIF1α and HIF2α induced the expression of ADM FL 4.8 and 6.2 fold, respectively, and also induced the expression of ADM I1-3 2.3 and 2.35 fold, respectively (Fig. 4C left). Thus, HIF1α or HIF2α transduction increased the ADM FL/I1-3 ratio to 19.36 or 25.19 versus 4.19 for the GFP transduced cells.

Figure 4. HIF activity is sufficient to promote splicing of ADM pre-mRNA.

Figure 4

Open in a new tab

A) Western Blot analysis of Flag-tagged HIF1α, HIF2α, and GFP proteins in Hep3B cells transduced with lenti-virus expressing normoxia active Flag tagged HIF1αTM (TM, triple mutation), or normoxia active HIF2αTM or GFP proteins under normoxia. B) RT-qPCR analysis of HIF1 target genes, LDHA and PGK1, and HIF2 target genes, EPO and PAI1 in the above-described cells. C) RT-PCR (left) and RT-qPCR (right) analysis of ADM FL and I1-3 transcripts in the above-described cells. The ADM FL/I1-3 ratio is indicated next to the graph legend. D-E) RT-PCR (D) and RT-qPCR (E) analysis of ADM transcripts in normoxic and hypoxic RCC4 or RCC4T cells with ADM FL/I1-3 ratio indicated next to the graph legend.

To further validate that HIFs are sufficient to promote splicing of ADM pre-mRNA, we used RCC4 cells, a renal cell carcinoma cell-line that expresses constitutively active HIF1α and HIF2α protein even under normoxia due to mutation of the VHL gene. In addition, we used RCC4T cells in which functional pVHL is re-introduced into RCC4 cells and therefore HIF proteins are only active under hypoxia (25). RCC4T cells expressed both ADM FL and I1-3 transcripts (Fig. 4D); moreover, hypoxia preferentially induced the levels of the ADM FL transcript (Fig. 4D). However, RCC4 cells mainly expressed the ADM FL transcript under both normoxia and hypoxia (Fig. 4D). Using RT-qPCR, hypoxia was found to induce the expression of ADM FL and I1-3, 10 and 1.4 fold above normoxic RCC4T cells respectively and also increased the ADM FL/I1-3 ratio to 54.16 versus 8.72 in normoxic RCC4T cells (Fig. 4E). However, hypoxia only weakly increased the ADM FL/I1-3 ratio in RCC4 cells since ADM FL was already favored even under normoxia. For instance, the ADM FL/I1-3 ratio was 53.41 under normoxia and 67.11 under hypoxia. These findings supported the idea that HIFs are sufficient to increase splicing of ADM pre-mRNA.

ADM splicing reporters recapitulate splicing changes observed for the endogenous ADM gene

After determining the relationship between ADM gene activation and increased intron-removal using the endogenous ADM gene, we wanted to see if HIF-mediated transcription activation of an ADM splicing reporter would result in increased ADM splicing. To test this idea, the full-length ADM gene (exons1-4 including introns) was cloned and placed downstream of the CA9 promoter, a HIF1 target gene promoter, or the PAI1 promoter, a HIF2 target gene promoter, or an artificial hypoxia/HIF induced promoter containing two hypoxia-responsive-elements (2HRE) and the SV40 minimal promoter (Fig. 5A). Additionally, splicing reporters in which the HREs were mutated (mHRE) or deleted (ΔHRE) were generated. These ADM splicing reporters were then co-transfected into Hep3B cells with empty vector expressing a His tag (His) or with normoxia active HIF1α or HIF2α expression plasmids. All of the splicing reporters expressed two major transcripts, a transcript in which introns 1-3 were removed (intron skipping or IS, to distinguish from the endogenous FL transcript) and a second transcript in which introns 1-3 were retained (intron retaining, IR) (Fig.5B-C). The ADM IS and IR transcripts corresponded to endogenous ADM FL and ADM I1-3 respectively, demonstrating that the splicing reporters recapitulated the expression patterns of the endogenous ADM gene.

Figure 5. HIFs regulate splicing of ADM splicing reporters.

Figure 5

Open in a new tab

A) Diagram of ADM splicing reporters. Arrows represent primers used for RT-PCR and RT-qPCR. Forward primer in the promoter region (CA9P, PAI1P, or 2HRE) and ADM RT-PCR primer (in E4) were used in RT-PCR. CA9P, PAI1P, or 2HRE primers in conjunction with ADMIS, ADMIR or ADM total were used in RT-qPCR. B) RT-PCR (upper panel) and RT-qPCR (lower panel) detection of ADM transcripts in normoxic Hep3B cells co-transfected with CA9P/ADM or CA9Pm2HRE/ADM and with His tagged empty vector control (His), HIF1αTM or HIF2αTM expression plasmids. C) RT-PCR (top panel) and RT-qPCR (middle and bottom) analysis of ADM transcripts in normoxic Hep3B cells transfected with PAI1P/ADM, PAI1PmHRE/ADM, 2HRE/ADM, or ΔHRE/ADM splicing reporters and empty vector (His), HIF1αTM, or HIF2αTM expression plasmids.

Using reporter specific primers and RT-PCR, we found that co-transfected HIF1α activated the CA9P/ADM reporter and increased the levels of the ADM IS transcript but not the ADM IR transcript (Fig. 5B). Co-transfected HIF2α also increased the levels of the ADM IS transcript although to a lesser extent than HIF1 (Fig. 5B). In contrast, the CA9Pm2HRE/ADM reporter was not activated by HIF1 or HIF2 nor were the ADM IS and IR transcripts induced (Fig.5B). RT-qPCR confirmed that HIF1α induced the expression of ADM IS and IR by 16 and 2.6 fold respectively. Similarly, HIF2α induced the expression of the ADM IS and IR transcripts by 4 and 1.7 fold respectively. Moreover, HIF1α or HIF2α increased the ADM IS/IR ratio to 4.22 or 2.23 respectively versus 0.93 for the empty vector (His) (Fig.5B, bar graph). In contrast, the CA9Pm2HRE/ADM reporter was not induced by HIF1α or HIF2α, nor did HIF1α or HIF2α alter the ADM IS/IR ratio.

Next, similar experiments were performed on ADM splicing reporters driven by the PAI1 promoter (PAI1P/ADM and PAI1PmHRE/ADM). Using reporter specific primers and RT-PCR, we found that HIF1α activated the PAI1P/ADM reporter and increased the levels of the ADM IS, but not IR transcript (Fig. 5C top). Similarly, HIF2α also increased the levels of ADM IS but not IR (Fig.5C top). In contrast, the PAI1PmHRE/ADM reporter was not activated by HIF1α or HIF2α and nor did HIF change the levels of ADM IS and IR transcripts (Fig.5C top). Using RT-qPCR, we found that HIF1α induced the levels of ADM IS and IR by 2.8 and 1.3 fold respectively (Fig.5C, middle) while HIF2α increased the levels of the ADM IS and IR transcripts by 18 and 3.3 fold respectively. As a result, HIF1α or HIF2α increased the ADM IS/IR ratio to 9.85 or 19.73 respectively versus 3.31 for the His vector (Fig.5C, middle). In contrast, the PAI1PmHRE/ADM reporter was not activated by HIF1α or HIF2α, nor did HIF1α or HIF2α significantly alter the ADM IS/IR expression ratio (Fig.5C, middle left).

Finally, we utilized ADM splicing reporters driven by the SV40 minimal promoter in which two HREs from the PAI1 promoter were added to produce an artificial HIF/hypoxia responsive promoter (2HRE/ADM and ΔHRE/ADM). Using reporter specific primers and RT-PCR, we found that both HIF1α and HIF2α activated the 2HRE/ADM reporter and increased the expression of the ADM IS transcript (Fig. 5C, top left). In contrast, the ΔHRE/ADM reporter was not activated by HIF1α or HIF2α and therefore the ADM IS and IR transcripts were not induced (Fig. 5C, top left). Again, RT-qPCR confirmed that HIF1α induced the expression of ADM IS 4 fold but reduced the expression of ADM IR 1.4 fold (Fig. 5C, bottom right). Moreover, HIF2α induced the expression of the ADM IS and IR transcripts 17 and 1.2 fold respectively (Fig. 5C, bottom right). HIF1α and HIF2α increased the ADM IS/IR ratio to 4.55 and 11.28 respectively versus 0.5 for the His vector (Fig. 5C, bottom right). In contrast, the ΔHRE/ADM reporter was not activated by HIF1α or HIF2α, nor did HIF1α or HIF2α significantly alter the ADM IS/IR ratio (Fig. 5C, bottom left). Taken together, this data showed that splicing of the ADM splicing reporters can recapitulate the HIF dependent splicing changes observed for the endogenous ADM gene. This data further supported our conclusion that HIF activity is sufficient to promote splicing of ADM pre-mRNA. In addition, this data suggested that transcription activation of ADM is necessary to promote splicing of ADM pre-mRNA since mutation or deletion of HREs in the reporters prevented transcription activation and also the increase in the ADM IS/IR ratio.

The transactivation domain of HIFα protein is not required for increased RNA splicing of the ADM splicing reporter

Some transcription factors have dual roles in RNA splicing and gene transcription (26-28). To determine if the activation domain of HIFα protein is important for ADM pre-mRNA splicing, we constructed hybrid proteins that contained the HIF1α DNA binding domain and ARNT dimerization domain (DBD), fused to the transactivation domains from the E2F1 or VP16 transcription factors (Fig. 6A). To facilitate the detection of these hybrid proteins, 2xFlag tag was added at the C-terminuses of these constructs (Fig.6A). As determined by anti-Flag western blot, the HIF1αDBD, HIF1αDBD/E2F1TAD, and HIF1αDBD/VP16TAD expression plasmids expressed higher levels of proteins than the normoxia active HIF1α and HIF2α expression plasmids even though the same amount of plasmid DNAs were transfected (Fig. 6B). However, the CA9P/ADM reporter was activated similarly by HIF1α (2.2 fold) and HIF1αDBD/E2F1 (2.1 fold) while the HIF2α and HIF1αDBD/VP16 weakly activated the CA9P/ADM reporter (Fig. 6C, ADM total). Interestingly, HIF1α was more potent in increasing the ADM IS/IR ratio than the HIF1αDBD/E2F1, increasing the ADM IS/IR ratio to 4.37 whereas HIF1αDBD/E2F1 increased the ADM IS/IR ratio to 2.47 versus 0.94 for the His control, although the constructs were able to activate the reporter similarly (Fig. 6C). In addition, HIF2α and HIF1αDBD/VP16 only increased the ADM IS/IR ratio slightly (Fig. 6C).

Figure 6. Activation of ADM splicing reporter is sufficient to increase splicing of ADM pre-mRNA.

Figure 6

Open in a new tab

A) Schematic of HIF1αTM, HIF2αTM, and HIF1α DNA binding and PAS domains (HIF1αDBD) fused with the activation domain of VP16 or E2F1. All of these constructs are tagged with a 2XFlag epitope at their C-terminus. B) Western blot analysis of Flag tagged HIF1αTM, HIF2αTM, HIF1αDBD, HIF1αDBD/VP16TAD, and HIF1αDBD/E2F1TAD expression constructs. C-D) RT-qPCR analysis of ADM transcript levels in normoxic Hep3B cells co-transfected with the CA9P/ADM reporter (C) or the PAI1P/ADM reporter (D) and the above described expression constructs. E) RT-qPCR analysis of HIF2α and USF2 mRNA levels (top panel) and splicing reporter specific ADM transcripts (bottom panels) in Hep3B cells co-transfected with the PAI1P/ADM reporter and control (His), HIF2αTM or USF2 expression plasmids.

In addition, the above-described fusion constructs were able to activate the PAI1P/ADM reporter in Hep3B cells (Fig. 6D, ADM total). HIF1α activated the reporter 1.96 fold whereas HIF2α activated the reporter more than any other expression plasmid, 8.84 fold. Interestingly, even the HIF1αDBD slightly activated this reporter, 1.67 fold. In addition, the HIF1αDBD/E2F1 and the HIF1αDBD/VP16 activated the reporter 2.17 and 2.35 fold respectively (Fig. 6D, ADM total). In addition, HIF1α and HIF2α were able to increase the ADM IS/IR ratio to 15.09 and 23.4 versus 7.5 for the His control (Fig. 6D). Although HIF1αDBD activated the reporter slightly, it did not significantly change the ADM IS/IR splicing ratio (7.7) compared to the His vector (7.5). On the other hand, HIF1αDBD/E2F1 and HIF1αDBD/VP16 increased the ADM IS/IR splicing ratio to 13.3 and 9.85 respectively versus 7.5 for the His control (Fig.6D). Furthermore, the fusion constructs were also able to activate and promote splicing of endogenous ADM even under normoxia (data not shown). In summary, this data suggested that other transcription factors are also able to promote splicing of the ADM pre-mRNA and the transactivation domain of the HIFα protein is replaceable for increased RNA splicing of the ADM splicing reporter.

Activation of endogenous HIF target genes is not absolutely required for increased splicing of the ADM splicing reporter

As stated above, transcription activation by HIF1 or HIFDBD hybrid constructs is sufficient to promote splicing of the ADM pre-mRNA. However, both HIF and the fusion constructs can activate endogenous HIF target genes; therefore it is possible that HIF target genes may be responsible for promoting splicing of ADM pre-mRNA. To rule out or confirm this possibility, we utilized an ADM splicing reporter driven by the PAI1 promoter (PAI1P/ADM), which contains an HRE and also binding sites for the upstream stimulatory factor 2 (USF2) (18). In contrast to HIFs and the HIF fusion constructs, USF2 does not activate endogenous HIF target genes. To test this, normoxic Hep3B cells were co-transfected with the PAI1/ADM splicing reporter and either HIF2α or USF2 expression plasmid. As determined by RT-qPCR using primers that detect endogenous and transfected HIF2α or USF2 RNA, we found that HIF2α transfection increased HIF2α mRNAs levels 18.31 fold and USF2 transfection increased USF2 levels 105.94 fold above the His vector (Fig. 6E, top). As determined by RT-qPCR using reporter specific primers that detect total reporter specific ADM transcripts, we found that HIF2α activated the PAI1P/ADM reporter 3 fold whereas USF2 activated the reporter 2 fold compared to the His vector (Fig. 6E, bottom, ADM total). Also, HIF2α induced the expression of ADM IS and IR 11.4 and 1.7 fold respectively while USF2 induced the expression of ADM IS and IR 5.2 and 1.9 fold respectively. Thus, HIF2α or USF2 increased the ADM IS/IR ratio to 9.13 or 3.71 versus 1.42 for the His control (Fig. 6E, lower panel).

To further validate that transcription activation of ADM was sufficient to promote splicing of ADM pre-mRNA, ADM splicing reporter was placed under the control of a promoter containing 5 copies of the Gal4 DNA binding element (Fig. 7A). In addition, fusion constructs containing the Gal4 DNA binding domain fused to the transactivation domains of normoixa active HIF1α, normoixa active HIF2α, VP16, or E2F1 were generated (Fig.7A). Therefore, the Gal4 DBD fusion constructs are expected to activate the G5P/ADM splicing reporter but not endogenous HIF target genes. Next, we co-transfected Hep3B cells with the G5P/ADM splicing reporter and the Gal4 DBD fusion constructs. An anti-gal4 western blot indicated that the fusion constructs expressed proteins as expected (Fig. 7B). Using RT-qPCR to assess activation of the G5P/ADM reporter, we found that the Gal4 DBD did not significantly activate the reporter above the His control (Fig. 7C, ADM total). In contrast, Gal4/HIF1αTAD, HIF2αTAD, VP16TAD, and E2F1TAD were able to activate the reporter 16.4, 4.05, 71.4 and 66.75 fold respectively (Fig. 7C, ADM total). Moreover, the Gal4 DBD expression plasmid did not alter the expression of ADM IS and IR transcripts and did not change the ADM IS/IR ratio (Fig. 7C). However, the Gal4/HIF1αTAD expression plasmid increased the expression of ADM IS and IR by 80.95 and 3 fold and also increased the ADM IS/IR ratio to 45.98 from 2.26 for the His vector (Fig.7C). Similarly, the Gal4/HIF2αTAD expression plasmid increased the expression of ADM IS and IR by 12.3 and 2.35 fold and increased the ADM IS/IR expression ratio to 8.58 (Fig.7C). Also, the Gal4/VP16TAD increased the expression of ADM IS and IR by 284.8 and 14.1 fold and thus increased the ADM IS/IR ratio to 39.4 (Fig. 7C). Moreover, the Gal4/E2F1TAD expression plasmid increased the expression of ADM IS and IR by 255.13 and 8.03 fold and increased the ADM IS/IR ratio to 52.12 (Fig. 7C). Taken together, this data further confirmed that transcriptional activation of ADM is sufficient to enhance intron removal in the absence of endogenous HIF target gene activation.

Figure 7. Activation of endogenous HIF target genes is not required for increased splicing of an ADM splicing reporter.

Figure 7

Open in a new tab

A) Schematic of the G5P/ADM splicing reporter and hybrid constructs containing the Gal4 DNA binding domain (Gal4) fussed to the transactivation domain of HIF1αTM, HIF2αTM, VP16, or E2F1. B) Western blot analysis of the Gal4 DBD expression constructs using antibody specific for the Gal4 DNA binding domain. C) RT-qPCR analysis of ADM transcript levels in normoxic Hep3B cells co-transfected with the G5P/ADM reporter and the above described expression constructs.

Discussion

Martinez et al reported that the ADM gene expresses FL and I3-containing isoforms in normoxic cell lines (17). By analyzing ADM RNA transcripts using RT-PCR, we found that normoxic cancer cells express fully-spliced (FL) and several intron-retaining RNA transcripts including I1, I2, I3, and I1-3 isoforms (Fig.1). Furthermore, the FL and I1-3 isoforms, but not the I3 isoform, are the predominate isoforms expressed in normoxic cells (Figs 1 and 4). To our knowledge, this study is the first to report that the ADM gene expresses I1-3, I1, and I2 isoforms in normoxic cells. Importantly, in addition to cancer cells, we found that intron-retaining ADM isoforms are expressed in normal human tissues (data not shown) and normal HUVEC cells, suggesting that expression of intron-retaining isoforms is not a consequence of abnormal splicing, but a normal control mechanism of gene expression.

ADM is a well-established HIF target gene (25, 29). However, it was reported that the I3 but not the FL isoform was preferentially induced by hypoxia (17). Because the ADM FL RNA codes for a more functionally important AM peptide than the ADM I3 RNA, it is puzzling that I3 is preferentially induced by hypoxia. Thus, we investigated ADM isoform expression in hypoxic cells. Interestingly, we found that hypoxia preferentially induces the levels of ADM FL over intron-containing ADM transcripts. It is not clear if this discrepancy is due to different cell lines used or other differences.

To address the molecular mechanism concerning preferential induction of ADM FL RNA under hypoxia, we first tested the hypothesis that ADM I1-3 transcripts are selectively degraded during hypoxia. However, we found that ADM FL is even less stable than the ADM I1-3 isoform, suggesting that differential transcript stability does not account for the hypoxia induced ADM FL/I1-3 ratio increase. Furthermore, we found that the both ADM I1-3 and FL transcripts are very unstable both under normoxia and hypoxia (Fig.2A), a result consistent with a previous report that demonstrates similar patterns of ADM mRNA degradation (24). Since there are significant amounts of ADM FL and I1-3 in the nucleus, one possibility is that the ADM FL and ADM I1-3 transcripts undergo Traf4/Trf5-Air1/Air2-Mtr4 (TRAMP) mediated polyadenylation and RNA degradation by the nuclear exosome complex (30-32). In the future, it will be interesting to address why ADM transcripts are so unstable.

After establishing that increased splicing of ADM pre-mRNA is responsible for the increased ADM FL/I1-3 ratio under hypoxia, using the endogenous ADM gene (Figs. 3 and 4) as well as ADM splicing reporters (Fig.5), we determined that HIF activity, but not hypoxia per se, is necessary and sufficient to regulate hypoxia induced splicing of ADM pre-mRNA. Furthermore, we determined that transcription activation of ADM is necessary and sufficient to regulate splicing of ADM pre-mRNA independent of endogenous HIF target gene activation (Fig 6E and 7). Thus, our data demonstrated that ADM gene transcription and pre-mRNA splicing is coupled. Although it is well established that transcription and splicing are functionally coupled (33-35), these conclusions are mainly derived from studies using chimeric splicing reporters in which an alternatively spliced exon is placed between two constitutive exons of a different gene. To our knowledge, this is the first report that utilizes endogenous and cloned genes in the same study as models to demonstrate functional coupling of transcription and RNA splicing.

Additionally, our data indicated that transcription activation strength contributes to splicing of ADM pre-mRNA. For instance, HUVEC cells exhibit an ADM FL/I1-3 splicing ratio of 5.17 (Fig. 1C) under normoxia. In contrast, Hep3B cells exhibit an ADM FL/I1-3 ratio of 3.28 under normoxia (Fig. 1A). Interestingly, basal level expression of the ADM FL and the ADM I1-3 isoforms are 2.62 and 2.89 fold higher in HUVECs than in Hep3B cells (data not shown). In addition, the HIF2α virus is a stronger inducer of ADM gene transcription and increases the ADM FL/I1-3 ratio more than the HIF1α virus (Fig. 4C). Further support is garnered from our ADM splicing reporter studies. For instance the ADM IS/IR ratio is 0.5 under basal conditions (His) for the 2HRE/ADM reporter while the ADM IS/IR ratio is 3.31 for the PAI1P/ADM reporter under basal conditions (His) (Fig. 5C). Interestingly, the basal expression of PAI1P/ADM reporter is 2.8 fold higher than the basal expression of the 2HRE/ADM reporter (data not shown). Moreover, higher ADM IS/IR expression ratios correlate with higher levels of reporter activation. For instance, if HIF1α activates the reporter more than HIF2α, HIF1α also increases the ADM IS/IR ratio more than HIF2α (Fig. 5B) or if HIF2α activates the reporter more than HIF1α, HIF2α also increases the ADM IS/IR ratio more than HIF1α (Fig. 5C). In summary, these findings indicate that transcription activation strength regulates pre-mRNA splicing efficiency of ADM independent of promoter differences. These findings are novel and in contrast to what have been observed for alternatively spliced genes. For instance, previous studies suggest that differences in promoter structure lead to differences in alternative splicing of particular transcripts, however transcription activation strength is not responsible for this effect (35-37). It is not clear why our results differ from previous studies. We speculate that these differences may be due to the fact that we studied intron-removal whereas previous studies assessed alternative exon inclusion. Moreover, while previous studies compared different transcription factors in regulating alternative splicing we compared basal level expression of ADM transcripts in different cell lines. In fact, if we compared HIF with other transcription factors in regulating ADM splicing (Fig. 6C-D and Fig. 7C), our data is in agreement with previous studies and suggests that activation strength does not determine the efficiency of ADM splicing (see below).

Although transcription activation and activation strength influence ADM pre-mRNA splicing, HIFs are more potent than other transcriptional activators in promoting intron removal under condition in which both endogenous HIF target genes are activated or not activated. For example, HIF1α is more potent than HIF1αDBD/E2F1 in inducing ADM IS from the CA9P/ADM splicing reporter despite similar activation of the reporter by the two activators (Fig. 6C). Furthermore, Gal4/HIF1αTAD is also a stronger inducer of ADM IS than Gal4/VP16TAD or Gal4/E2F1TAD although Gal4/VP16TAD or E2F1TAD activate the G5/ADM reporter more than Gal4/HIF1αTAD (Fig. 7C). It is not clear why HIFs are more efficient than other transcription factors in increasing ADM pre-mRNA splicing. Based on the recruitment model of RNA splicing (38-40), we speculate that HIF may have stronger activity than other transcription factors in recruiting splicing factors to HIF target gene promoters. So far, HIFs have not been reported to interact with splicing factors such as serine-arginine rich (SR) proteins or heterogeneous nuclear ribonucleoproteins (hnRNPs) or with components of the spliceosome. However, HIFs have been shown to recruit co-transcription factors that can promote splicing. For example, studies indicated that BRM, a component of the SWI/SNF chromatin remodeling complex, can act as a co-activator for HIF target genes (41). Moreover, a separate study showed that BRM could regulate alternative splicing by slowing the rate of RNA Pol II elongation (42). Therefore, it is possible that HIFs are more efficient than other transcription factors in recruiting BRM to the ADM promoter and regulate splicing by altering the elongation rate of RNA Pol II (38-40, 43).

In summary, we demonstrate for the first time that HIFs enhance pre-mRNA splicing of ADM in addition to regulating ADM transcription. In the future, it will be interesting to assess whether other HIF target genes also express intron-retaining or exon-skipping isoforms under normoxia and whether HIF promotes RNA splicing of additional HIF target genes. In addition, it will be interesting to know if activation of other HIF target genes will lead to increased RNA splicing independent of endogenous HIF target gene activation.

Implications.

Here, a novel function of HIFs in regulating ADM gene expression is identified by enhancing ADM pre-mRNA splicing.

Acknowledgments

This work was supported by grants from the National Cancer Institute (RO1CA134687, Hu) and Cancer League of Colorado (Hu). Johnny Sena was supported by “Research Supplemental to Promote Diversity” (NCI) from June 1, 2010 to May 30, 2012. Dr. David Bentley generously provided the Gal4/E2F1TAD plasmid used in this study.

Financial supports

National Cancer Institute (RO1CA134687, Hu) and Cancer League of Colorado (Hu). Johnny Sena was supported by “Research Supplemental to Promote Diversity” (RO1CA134687-S3 and RO1CA134687-S4)

Footnotes

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

References

  • 1.Finger EC, Giaccia AJ. Hypoxia, inflammation, and the tumor microenvironment in metastatic disease. Cancer metastasis reviews. 2010;29:285–93. doi: 10.1007/s10555-010-9224-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Kaelin WG., Jr Cancer and altered metabolism: potential importance of hypoxia-inducible factor and 2-oxoglutarate-dependent dioxygenases. Cold Spring Harbor symposia on quantitative biology. 2011;76:335–45. doi: 10.1101/sqb.2011.76.010975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Keith B, Johnson RS, Simon MC. HIF1alpha and HIF2alpha: sibling rivalry in hypoxic tumour growth and progression. Nature reviews Cancer. 2012;12:9–22. doi: 10.1038/nrc3183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Semenza GL. Hypoxia-inducible factors in physiology and medicine. Cell. 2012;148:399–408. doi: 10.1016/j.cell.2012.01.021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Park SJ, Kim JG, Son TG, Yi JM, Kim ND, Yang K, et al. The histone demethylase JMJD1A regulates adrenomedullin-mediated cell proliferation in hepatocellular carcinoma under hypoxia. Biochemical and biophysical research communications. 2013;434:722–7. doi: 10.1016/j.bbrc.2013.03.091. [DOI] [PubMed] [Google Scholar]
  • 6.Ouafik L, Sauze S, Boudouresque F, Chinot O, Delfino C, Fina F, et al. Neutralization of adrenomedullin inhibits the growth of human glioblastoma cell lines in vitro and suppresses tumor xenograft growth in vivo. The American journal of pathology. 2002;160:1279–92. doi: 10.1016/S0002-9440(10)62555-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Martinez A. Biology of adrenomedullin. Introduction. Microscopy research and technique. 2002;57:1–2. doi: 10.1002/jemt.10045. [DOI] [PubMed] [Google Scholar]
  • 8.Kawakami Y. Adrenomedullin antagonist suppresses in vivo proliferation of cancer cells in SCID mice via angiogenesis inhibition. [Hokkaido igaku zasshi] The Hokkaido journal of medical science. 2005;80:575–83. [PubMed] [Google Scholar]
  • 9.Tsuchiya K, Hida K, Hida Y, Muraki C, Ohga N, Akino T, et al. Adrenomedullin antagonist suppresses tumor formation in renal cell carcinoma through inhibitory effects on tumor endothelial cells and endothelial progenitor mobilization. International journal of oncology. 2010;36:1379–86. doi: 10.3892/ijo_00000622. [DOI] [PubMed] [Google Scholar]
  • 10.Deville JL, Bartoli C, Berenguer C, Fernandez-Sauze S, Kaafarani I, Delfino C, et al. Expression and role of adrenomedullin in renal tumors and value of its mRNA levels as prognostic factor in clear-cell renal carcinoma. International journal of cancer Journal international du cancer. 2009;125:2307–15. doi: 10.1002/ijc.24568. [DOI] [PubMed] [Google Scholar]
  • 11.Oehler MK, Norbury C, Hague S, Rees MC, Bicknell R. Adrenomedullin inhibits hypoxic cell death by upregulation of Bcl-2 in endometrial cancer cells: a possible promotion mechanism for tumour growth. Oncogene. 2001;20:2937–45. doi: 10.1038/sj.onc.1204422. [DOI] [PubMed] [Google Scholar]
  • 12.Oehler MK, Hague S, Rees MC, Bicknell R. Adrenomedullin promotes formation of xenografted endometrial tumors by stimulation of autocrine growth and angiogenesis. Oncogene. 2002;21:2815–21. doi: 10.1038/sj.onc.1205374. [DOI] [PubMed] [Google Scholar]
  • 13.Nikitenko LL, MacKenzie IZ, Rees MC, Bicknell R. Adrenomedullin is an autocrine regulator of endothelial growth in human endometrium. Molecular human reproduction. 2000;6:811–9. doi: 10.1093/molehr/6.9.811. [DOI] [PubMed] [Google Scholar]
  • 14.Eto T, Samson WK. Adrenomedullin and proadrenomedullin N-terminal 20 peptide: vasodilatory peptides with multiple cardiovascular and endocrine actions. Trends in endocrinology and metabolism: TEM. 2001;12:91–3. doi: 10.1016/s1043-2760(01)00374-5. [DOI] [PubMed] [Google Scholar]
  • 15.Nakamura M, Yoshida H, Hiramori K. Comparison of vasodilator potency of adrenomedulling and proadrenomedullin N-terminal 20 peptide in human. Life sciences. 1999;65:2151–6. doi: 10.1016/s0024-3205(99)00480-4. [DOI] [PubMed] [Google Scholar]
  • 16.Martinez A, Zudaire E, Portal-Nunez S, Guedez L, Libutti SK, Stetler-Stevenson WG, et al. Proadrenomedullin NH2-terminal 20 peptide is a potent angiogenic factor, and its inhibition results in reduction of tumor growth. Cancer research. 2004;64:6489–94. doi: 10.1158/0008-5472.CAN-04-0103. [DOI] [PubMed] [Google Scholar]
  • 17.Martinez A, Hodge DL, Garayoa M, Young HA, Cuttitta F. Alternative splicing of the proadrenomedullin gene results in differential expression of gene products. Journal of molecular endocrinology. 2001;27:31–41. doi: 10.1677/jme.0.0270031. [DOI] [PubMed] [Google Scholar]
  • 18.Pawlus MR, Wang L, Ware K, Hu CJ. Upstream stimulatory factor 2 and hypoxia-inducible factor 2alpha (HIF2alpha) cooperatively activate HIF2 target genes during hypoxia. Molecular and cellular biology. 2012;32:4595–610. doi: 10.1128/MCB.00724-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Pawlus MR, Wang L, Murakami A, Dai G, Hu CJ. STAT3 or USF2 contributes to HIF target gene specificity. PloS one. 8:e72358. doi: 10.1371/journal.pone.0072358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Hu CJ, Sataur A, Wang L, Chen H, Simon MC. The N-terminal transactivation domain confers target gene specificity of hypoxia-inducible factors HIF-1alpha and HIF-2alpha. Molecular biology of the cell. 2007;18:4528–42. doi: 10.1091/mbc.E06-05-0419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Blau J, Xiao H, McCracken S, O’Hare P, Greenblatt J, Bentley D. Three functional classes of transcriptional activation domain. Molecular and cellular biology. 1996;16:2044–55. doi: 10.1128/mcb.16.5.2044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Lejeune F, Maquat LE. Mechanistic links between nonsense-mediated mRNA decay and pre-mRNA splicing in mammalian cells. Current opinion in cell biology. 2005;17:309–15. doi: 10.1016/j.ceb.2005.03.002. [DOI] [PubMed] [Google Scholar]
  • 23.Wong JJ, Ritchie W, Ebner OA, Selbach M, Wong JW, Huang Y, et al. Orchestrated intron retention regulates normal granulocyte differentiation. Cell. 2013;154:583–95. doi: 10.1016/j.cell.2013.06.052. [DOI] [PubMed] [Google Scholar]
  • 24.Kitamuro T, Takahashi K, Nakayama M, Murakami O, Hida W, Shirato K, et al. Induction of adrenomedullin during hypoxia in cultured human glioblastoma cells. Journal of neurochemistry. 2000;75:1826–33. doi: 10.1046/j.1471-4159.2000.0751826.x. [DOI] [PubMed] [Google Scholar]
  • 25.Hu CJ, Wang LY, Chodosh LA, Keith B, Simon MC. Differential roles of hypoxia-inducible factor 1alpha (HIF-1alpha) and HIF-2alpha in hypoxic gene regulation. Molecular and cellular biology. 2003;23:9361–74. doi: 10.1128/MCB.23.24.9361-9374.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Auboeuf D, Dowhan DH, Kang YK, Larkin K, Lee JW, Berget SM, et al. Differential recruitment of nuclear receptor coactivators may determine alternative RNA splice site choice in target genes. Proceedings of the National Academy of Sciences of the United States of America. 2004;101:2270–4. doi: 10.1073/pnas.0308133100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Auboeuf D, Dowhan DH, Li X, Larkin K, Ko L, Berget SM, et al. CoAA, a nuclear receptor coactivator protein at the interface of transcriptional coactivation and RNA splicing. Molecular and cellular biology. 2004;24:442–53. doi: 10.1128/MCB.24.1.442-453.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Cheng D, Cote J, Shaaban S, Bedford MT. The arginine methyltransferase CARM1 regulates the coupling of transcription and mRNA processing. Molecular cell. 2007;25:71–83. doi: 10.1016/j.molcel.2006.11.019. [DOI] [PubMed] [Google Scholar]
  • 29.Yang BC, Lippton H, Gumusel B, Hyman A, Mehta JL. Adrenomedullin dilates rat pulmonary artery rings during hypoxia: role of nitric oxide and vasodilator prostaglandins. Journal of cardiovascular pharmacology. 1996;28:458–62. doi: 10.1097/00005344-199609000-00016. [DOI] [PubMed] [Google Scholar]
  • 30.LaCava J, Houseley J, Saveanu C, Petfalski E, Thompson E, Jacquier A, et al. RNA degradation by the exosome is promoted by a nuclear polyadenylation complex. Cell. 2005;121:713–24. doi: 10.1016/j.cell.2005.04.029. [DOI] [PubMed] [Google Scholar]
  • 31.Vanacova S, Wolf J, Martin G, Blank D, Dettwiler S, Friedlein A, et al. A new yeast poly(A) polymerase complex involved in RNA quality control. PLoS biology. 2005;3:e189. doi: 10.1371/journal.pbio.0030189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Wyers F, Rougemaille M, Badis G, Rousselle JC, Dufour ME, Boulay J, et al. Cryptic pol II transcripts are degraded by a nuclear quality control pathway involving a new poly(A) polymerase. Cell. 2005;121:725–37. doi: 10.1016/j.cell.2005.04.030. [DOI] [PubMed] [Google Scholar]
  • 33.Rosonina E, Ip JY, Calarco JA, Bakowski MA, Emili A, McCracken S, et al. Role for PSF in mediating transcriptional activator-dependent stimulation of pre-mRNA processing in vivo. Molecular and cellular biology. 2005;25:6734–46. doi: 10.1128/MCB.25.15.6734-6746.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Nogues G, Kadener S, Cramer P, Bentley D, Kornblihtt AR. Transcriptional activators differ in their abilities to control alternative splicing. The Journal of biological chemistry. 2002;277:43110–4. doi: 10.1074/jbc.M208418200. [DOI] [PubMed] [Google Scholar]
  • 35.Cramer P, Pesce CG, Baralle FE, Kornblihtt AR. Functional association between promoter structure and transcript alternative splicing. Proceedings of the National Academy of Sciences of the United States of America. 1997;94:11456–60. doi: 10.1073/pnas.94.21.11456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Cramer P, Caceres JF, Cazalla D, Kadener S, Muro AF, Baralle FE, et al. Coupling of transcription with alternative splicing: RNA pol II promoters modulate SF2/ASF and 9G8 effects on an exonic splicing enhancer. Molecular cell. 1999;4:251–8. doi: 10.1016/s1097-2765(00)80372-x. [DOI] [PubMed] [Google Scholar]
  • 37.Auboeuf D, Honig A, Berget SM, O’Malley BW. Coordinate regulation of transcription and splicing by steroid receptor coregulators. Science. 2002;298:416–9. doi: 10.1126/science.1073734. [DOI] [PubMed] [Google Scholar]
  • 38.Kornblihtt AR. Coupling transcription and alternative splicing. Advances in experimental medicine and biology. 2007;623:175–89. doi: 10.1007/978-0-387-77374-2_11. [DOI] [PubMed] [Google Scholar]
  • 39.Kornblihtt AR, de la Mata M, Fededa JP, Munoz MJ, Nogues G. Multiple links between transcription and splicing. RNA. 2004;10:1489–98. doi: 10.1261/rna.7100104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Perales R, Bentley D. “Cotranscriptionality”: the transcription elongation complex as a nexus for nuclear transactions. Molecular cell. 2009;36:178–91. doi: 10.1016/j.molcel.2009.09.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Sena JA, Wang L, Hu CJ. BRG1 and BRM chromatin-remodeling complexes regulate the hypoxia response by acting as coactivators for a subset of hypoxia-inducible transcription factor target genes. Molecular and cellular biology. 2013;33:3849–63. doi: 10.1128/MCB.00731-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Batsche E, Yaniv M, Muchardt C. The human SWI/SNF subunit Brm is a regulator of alternative splicing. Nature structural & molecular biology. 2006;13:22–9. doi: 10.1038/nsmb1030. [DOI] [PubMed] [Google Scholar]
  • 43.Braunschweig U, Gueroussov S, Plocik AM, Graveley BR, Blencowe BJ. Dynamic integration of splicing within gene regulatory pathways. Cell. 2013;152:1252–69. doi: 10.1016/j.cell.2013.02.034. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES