Transcription Factors Sp1 and Hif2α Mediate Induction of the Copper-transporting ATPase (Atp7a) Gene in Intestinal Epithelial Cells during Hypoxia*

Background: A hypoxia-inducible transcription factor (Hif2α) mediates induction of intestinal iron and copper transporters during iron deficiency. Results: Specificity factor 1 (Sp1) is required for transcriptional induction of an intestinal copper transporter (Atp7a) by Hif2α. Conclusion: Sp1 and Hif2α may synergistically mediate the genetic response to iron deficiency. Significance: Understanding molecular mechanisms governing iron absorption may allow modulation of this process during disease states. Genes with G/C-rich promoters were up-regulated in the duodenal epithelium of iron-deficient rats including those encoding iron (e.g. Dmt1 and Dcytb) and copper (e.g. Atp7a and Mt1) metabolism-related proteins. It was shown previously that an intestinal copper transporter (Atp7a) was co-regulated with iron transport-related genes by a hypoxia-inducible transcription factor, Hif2α. In the current study, we sought to test the role of Sp1 in transcriptional regulation of Atp7a expression during iron deprivation/hypoxia. Initial studies in IEC-6 cells showed that mithramycin, an Sp1 inhibitor, reduced expression of Atp7a and iron transport-related genes (Dmt1, Dcytb, and Fpn1) and blocked their induction by CoCl2, a hypoxia mimetic. Consistent with this, overexpression of Sp1 increased endogenous Atp7a mRNA and protein expression and stimulated Atp7a, Dmt1, and Dcytb promoter activity. Site-directed mutagenesis and functional analysis of a basal Atp7a promoter construct revealed four functional Sp1 binding sites that were necessary for Hif2α-mediated induction of promoter activity. Furthermore, chromatin immunoprecipitation (ChIP) assays confirmed that Sp1 specifically interacts with the Atp7a promoter in IEC-6 cells and in rat duodenal enterocytes. This investigation has thus revealed a novel aspect of Atp7a gene regulation in which Sp1 may be necessary for the HIF-mediated induction of gene transcription during iron deficiency/hypoxia. Understanding regulation of Atp7a expression may help further clarify the physiological role of copper in the maintenance of iron homeostasis. Furthermore, this Sp1/Hif2α regulatory mechanism may have broader implications for understanding the genetic response of the intestinal epithelium to maintain whole-body iron homeostasis during states of deficiency.

mice lacking intestinal Hif2␣, whereas induction of iron absorption during iron deprivation was maintained in mice lacking Hif1␣. It was further shown that the Dmt1, Dcytb, and Fpn1 promoters contained functional hypoxia-responsive elements (HREs) that specifically interacted with Hif2␣, explaining their induction during iron deficiency (and tissue hypoxia) (9 -11). Hif2␣ is thus critical to maintain intestinal iron homeostasis in mice.
Interestingly, our previous studies in iron-deficient rats showed that Atp7a was up-regulated in the duodenal epithelium similarly to Dmt1, Dcytb, and Fpn1 (2), and we thus hypothesized that Atp7a was coordinately regulated with these iron transport-related genes. Subsequently, it was demonstrated that the Atp7a promoter was indeed a direct Hif2␣ target in rat intestinal epithelial (IEC-6) cells (12). Furthermore, in a previous investigation, it was noted that promoters of genes induced in the duodenal epithelium of iron-deficient rats contained a statistical overrepresentation of G/C-rich sequences (13). It was also shown that an abundance of genes up-regulated in differentiated Caco-2 cells (human intestinal adenocarcinoma cells) in response to iron chelation contained G/C-rich promoter sequences as well as putative HREs (14). Importantly, many of the iron and copper homeostasis-related genes induced in both models of intestinal iron transport contained G/C-rich promoters and putative HREs. These observations led us to hypothesize that a G/C-binding protein (e.g. specificity factor 1 (Sp1) or a related trans-acting factor) was important for the transcriptional response of the intestinal epithelium to iron deprivation (14). To test this hypothesis, in the current investigation, we performed a series of experiments to determine whether Sp1 is important for the Hif2␣-mediated transactivation of Atp7a gene expression using an in vitro model of the intestinal epithelium (IEC-6 cells) and iron-deprived rats. Results of this investigation showed that Sp1 specifically interacts with cis-elements in the Atp7a promoter and furthermore that Sp1 binding is necessary for Hif2␣-mediated induction of Atp7a transcription during hypoxia.

EXPERIMENTAL PROCEDURES
Cell Culture-Rat intestinal epithelial (IEC-6) cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured as described previously (12,15). For hypoxia experiments, IEC-6 cells at ϳ85% confluence were cultured in a hypoxia chamber with 1% O 2 and 5% CO 2 (with the balance being nitrogen). To mimic hypoxia, 200 M CoCl 2 was added to the culture medium when the cells were ϳ85% confluent, and cells were then cultured for 60 h. To interrupt Sp1 binding, fully differentiated IEC-6 cells (i.e. 7 days postconfluence) were treated with mithramycin (a G/C base pair-specific, DNA-binding antibiotic) (16,17)

at various concentrations for 24 h.
Animals and Diets-Weanling Sprague-Dawley rats (male) were purchased from Harlan (n ϭ 12); raised in overhanging, wire mesh-bottomed cages in a room with 12-h light/dark cycles; and sacrificed at 10 a.m. Rats were fed custom AIN93Gbased diets (Dyets, Bethlehem, PA) that varied only in iron content for 5 weeks; the control diet contained 198 ppm iron, whereas the iron-deficient diet contained 3 ppm iron. Animals were weighed weekly. Subsequently, rats were anesthetized by CO 2 exposure and killed by cervical dislocation. Blood was col-lected by cardiac puncture, and hemoglobin and hematocrit were measured by routine methods (8). The duodenum was excised and inverted on a wooden stick after which enterocytes were isolated using a well established, previously published method (8,18). Duodenal enterocytes were used for mRNA isolation, Western blot analysis, and chromatin immunoprecipitation experiments. All animal studies were approved by the Institutional Animal Care and Use Committee of the University of Florida.
RNA Isolation and Real Time Quantitative RT-PCR-Total RNA was isolated from IEC-6 cells or duodenal enterocytes using TRIzol reagent (Invitrogen) following a standard protocol. RNA was reverse transcribed using the iScript cDNA Synthesis kit (Bio-Rad), and the resulting cDNA was utilized for qRT-PCRs with SYBR Green PCR Master Mix (Bio-Rad). Primers (listed in supplemental Table S1) were designed to span large introns to avoid amplification from genomic DNA. Standard curve reactions and melt curves were routinely run to validate primer pairs and PCRs. Experimental genes were normalized to 18 S rRNA, and relative gene expression was quantified using routine methods.
Plasmid Construction-The rat Sp1 open reading frame (ORF) (GenBank accession number D12768) was cloned by PCR from cDNA derived from IEC-6 cells using Phusion High Fidelity DNA Polymerase (Thermo Scientific, Pittsburgh, PA). The forward primer contained the translational start codon, and the reverse primer ended just 5Ј of the stop codon. Primers were designed with overhanging KpnI (forward) and EcoRV (reverse) restriction enzyme cutting sites. The PCR-amplified Sp1 ORF amplicon and pcDNA3.1 expression vector (Invitrogen) were double digested with KpnI and EcoRV followed by column purification. Sp1 ORF was then subcloned into double digested pcDNA3.1 with the LigaFast TM Rapid DNA Ligation System (Promega, Madison, WI). An HA tag was inserted into the 3Ј-end of the Sp1 ORF by PCR amplifying the entire pcDNA-Sp1 plasmid with primers containing overhanging sequences containing an HA sequence tag and a stop codon. Primers were designed with the forward primer at the 3Ј-end of the Sp1 ORF and the reverse primer at the 5Ј-end of the EcoRV site on the pcDNA3.1 vector. Each primer was phosphorylated at the 5Ј-end, which allowed ligation of the PCR amplicons to reform the intact plasmid. Primer sequences are provided in supplemental Table S1.
Mutant Atp7a promoter constructs were prepared by PCR amplifying the entire wild-type (WT) promoter fragment (Ϫ224/ϩ88) in the pGL4.18 vector (Promega) with the QuikChange Lightning Site-Directed Mutagenesis kit (Agilent Technologies, Santa Clara, CA). Primers contained mutations in putative Sp1 binding sites with amplification reactions proceeding in opposite directions. PCR products were digested with DpnI restriction enzyme (Agilent Technologies) to remove the template DNA (which was methylated during replication in bacteria). All DNA constructs were sequenced to confirm that promoter amplicons did not contain random mutations. Primer sequences are listed in supplemental Table S1.
Transfection and Luciferase Assays-WT or mutated Atp7a promoter constructs in the pGL4.18 vector (1 g) were transiently transfected into IEC-6 cells at ϳ60% confluence and cultured in 24-well plates. For Sp1 and Hif2␣ overexpression experiments, 1 g of Atp7a promoter construct (WT or mutated) was co-transfected with 1 g of either Sp1 (described above) or Hif2␣ overexpression vector (described previously) (12). Other constructs used were pGL4.18 plasmids containing ϳ1-kb mouse Dcytb and Dmt1 promoter fragments (kindly provided by Dr. Yatrik Shah, University of Michigan). Co-transfected pRL-CMV plasmid (Invitrogen) expressing Renilla luciferase was used to normalize expression of firefly luciferase driven by experimental promoters. 36 h after transfection, luciferase activity was measured with the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions.
Stable Sp1 Overexpression-IEC-6 cells were grown in 6-well plates and transfected with pcDNA3.1 (empty vector) or pcDNA-Sp1-HA vector with TurboFect in Vitro Transfection kit (Thermo Scientific). 60 h after transfection, cells were treated with G418 (at a predetermined concentration) to kill non-transfected cells, allowing transfected cells expressing the neomycin gene in the pcDNA3.1 or pcDNA-Sp1-HA vectors to survive. IEC-6 cells with stable overexpression of Sp1 were used to analyze Atp7a and Sp1 mRNA expression using qRT-PCR and protein expression by Western blotting and to determine the effect of forced Sp1 expression on Atp7a promoter activity.
Protein Isolation and Western Blot Analysis-Cytosolic and nuclear proteins were isolated from IEC-6 cells cultured in 10-cm cell culture dishes using a kit from Active Motif as described previously (15). Protein concentrations were determined by BCA Protein Assay (Pierce). 30 g of cytosolic or 50 g of nuclear proteins were resolved by 7.5% SDS-PAGE followed by transfer to PVDF membranes. The membranes were blocked with 5% (w/v) nonfat milk and then incubated with one of the following primary antibodies: Atp7a (called 54-10) (3), Sp1 (catalog number 07-645, Millipore), phosphorylated Sp1 (catalog number ab37707, Abcam, Cambridge, MA), Hif1␣ (catalog number NB100-105, Novus Biologicals, Littleton, CO), or Hif2␣ (catalog number NB100-122, Novus Biologicals). Subsequently, membranes were incubated with an anti-rabbit IgG secondary antibody. Antibody binding was visualized using homemade ECL reagent (8) followed by exposure to x-ray film. For quantification, protein expression was normalized to total proteins on stained blots as this method does not rely on the expression level of any individual protein that may or may not be affected by various treatments (and as used extensively by us in the past).
Chromatin Immunoprecipitation (ChIP) Assay-Assays were performed as described previously (12). Briefly, IEC-6 cells or rat duodenal enterocytes were cross-linked with 1.1% (v/v) chloroform for 10 min followed by quenching with 0.3 M glycine. Cells were subsequently lysed with hypotonic buffer (Active Motif) and homogenized. Nuclei were collected and resuspended in nuclear lysis buffer followed by sonication with a BioRuptor (Diagenode, Liege, Belgium) for 30 cycles with 30 s on and 30 s off. Target DNA with bound protein was pulled down with anti-Sp1 (Millipore) or anti-Hif2␣ (Novus Biologicals) antibody. After removing cross-links, DNA samples were analyzed by PCR with primer sets listed in supplemental Table S1. Primers were designed to amplify regions of the Atp7a promoter containing putative Sp1 or Hif2␣ binding sites or other up-or downstream regions that did not contain predicted Sp-or HIF-like sites.
Statistical Analysis-One-way analysis of variance (with Tukey's post hoc test) and paired Student's t test (GraphPad, La Jolla, CA) were used to statistically compare data across groups. p Ͻ 0.05 was considered statistically significant.

Mithramycin Inhibits Expression of Iron and Copper
Transport-related Genes-Expression of Atp7a and other genes was analyzed by qRT-PCR after mithramycin treatment of differentiated IEC-6 cells (Table 1). Mithramycin reduced expression of all experimental genes tested (Atp7a, Dmt1, Dcytb, and Fpn1) as well as positive control genes including ankyrin repeat domain 37 (Ankrd37), Hif2␣, and Sp1. The inhibition was most significant for all tested genes with 500 nM mithramycin; higher concentrations were without additional effect (data not shown), although cellular toxicity was not noted with concentrations up to 1 M. Ankrd37, which was strongly induced by iron deprivation (2), is a known Sp1 target gene (19) as is Hif2␣ (20). Interestingly, Sp1 is self-regulated via a positive feedback loop (21). Sp6 and transferrin receptor 1 (Tfr1) were selected as negative controls as neither gene is known to be regulated by Sp-like factors. Expression of Sp6 was unaffected by mithramycin treatment, whereas for unknown reasons, Tfr1 expression was induced.
Inhibition of Sp1 Binding Blocks Hypoxia-mediated Gene Expression-Under normoxic conditions, the Hif␣ subunits are hydroxylated on conserved proline residues and subsequently targeted for proteasomal degradation. Hypoxia stabilizes the Hif␣ subunits by inhibiting the HIF prolyl hydroxylase enzymes that mediate this hydroxylation reaction (22). Hypoxia can be mimicked by treating cells with cobalt chloride, which effectively inhibits proteasomal degradation of the HIF␣ subunits under normoxic conditions (23,24). Here, CoCl 2 was utilized to mimic hypoxia in IEC-6 cells. Results showed that expression of experimental (Atp7a, Dcytb, Dmt1, and Fpn1) and positive control (Ankrd37 and vascular endothelial growth factor (Vegf)) genes was increased by CoCl 2 exposure (Fig.  1). The Ankrd37 and Vegf genes are known Sp1 targets (19). Moreover, mithramycin decreased basal expression of all tested genes, and it inhibited the induction of Atp7a, Dcytb, Dmt1, and Fpn1 by CoCl 2 . Conversely, however, mithramycin did not affect the induction of Ankrd37 or Vegf expression by CoCl 2 .

Sp1-like cis-Elements Are Required for Basal Atp7a
Promoter Activity-Phylogenetic footprinting analysis showed that multiple G/C-rich sequences in the basal Atp7a promoter region  (Ϫ224/ϩ88) were conserved among rats, mice, and humans (data not shown). Moreover, TFSEARCH was utilized to predict putative Sp1 binding sites; nine potential sites were identified. Initially, all nine sites were individually mutated in the basal Atp7a promoter, and promoter activity was assessed in IEC-6 cells. These experiments (n ϭ 3) showed that four putative Sp1 binding sites had the most significant effects on basal promoter activity (data not shown), so these four sites (called S1-S4) were selected for further analysis. Mutation of each site individually significantly reduced basal promoter activity (Fig.  3). Combinatorial mutations (i.e. triple and quadruple mutations), however, had little additional effect on basal promoter activity. Additionally, to consider the functional role of the putative Sp1 binding sites, the effect of Sp1 overexpression on Atp7a promoter activity was assessed. Sp1 overexpression induced activity of the WT promoter (ϳ2.5-fold), whereas individual and combinatorial mutations had varying effects on promoter activity, with some mutations (e.g. S1-S3 and S2-S4) abolishing the increase caused by Sp1 overexpression (Fig. 3).

Sp1
Physically Interacts with the Atp7a Promoter-To assess potential interactions between Sp1 and the Atp7a promoter, ChIP assays were conducted. Chromosomal DNA containing cross-linked proteins was isolated from IEC-6 cells and sheared to ϳ200 bp, and then DNA samples were pulled down with a ChIP-grade anti-Sp1 antibody. After reversing cross-links and purifying DNA, PCR analysis was utilized to determine whether specific regions of the Atp7a promoter were present in the immunoprecipitated samples. Results showed that all four putative Sp1 binding site regions were present, whereas up-or downstream Atp7a promoter regions lacking putative Sp1-like binding sites were not detected (Fig. 4). It was further shown that mithramycin significantly reduced the amount of Atp7a promoter DNA pulled down (containing all four putative Sp1 binding sites). In this experiment (and others), there was no apparent difference in the amount of input DNA among different reactions.
Sp1 Binding Is Required for Hif2␣-mediated Up-regulation of Atp7a Expression-We next sought to determine whether the putative Sp1 binding sites were required for Hif2␣-mediated  ϩ88). The 5Ј-most transcriptional start site identified previously is marked as ϩ1. Also shown are the previously identified HREs and the putative Sp1 binding sites (designated as S1-S4) with the mutated bases shown below the line. These putative Sp1 binding sites were mutated individually or in combination in the basal Atp7a promoter construct and subsequently transfected into IEC-6 cells. B shows the activity of mutated promoters in relation to the activity of the WT promoter. C shows the effect of forced Sp1 expression on WT and mutated Atp7a promoter activity. In B and C, each bar represents the mean value ϮS.D. Different letters above bars indicate significant differences among groups (unpaired Student's t test; n ϭ 3-4). AUGUST 16, 2013 • VOLUME 288 • NUMBER 33 induction of Atp7a promoter activity. As shown previously (12), Hif2␣ overexpression induced Atp7a promoter activity ϳ5-fold in IEC-6 cells (Fig. 5). This induction was blunted by mutation of each Sp1 binding site individually, and combinatorial mutations abolished transactivation by Hif2␣.

Sp1 and Hif2␣ Regulate Atp7a Transcription during Hypoxia
Sp1 and Hif2␣ Interact with the Atp7a Promoter in Vivo-As all experiments reported so far were from an in vitro model of the mammalian intestinal epithelium, it was important to confirm these observations in vivo. Accordingly, studies were performed in rats that were deprived of dietary iron for 5 weeks after weaning. The intent was to determine whether Sp1 and Hif2␣ bound to the Atp7a promoter in rat intestine and whether iron deprivation altered DNA-protein interactions. Iron-deprived rats had significantly decreased hemoglobin and hematocrit levels (both reduced Ͼ75%), which are indicative of iron deficiency anemia, consistent with previous observations (2, 8) (data not shown). Body weights were also lower (ϳ19%) in the rats fed the low iron diet. Dcytb, Dmt1, and Atp7a mRNA expression increased in duodenal enterocytes isolated from the iron-deficient rats as expected, and serum ceruloplasmin protein expression increased, consistent with previous observations (Fig. 6) (8). Moreover, Atp7a protein expression increased, and although inconsistent between animals, Hif2␣ protein levels were higher in the iron-deficient rats. Lack of Atp7a detection in one sample and Hif2␣ in two samples may relate to delays in sample processing (and subsequent partial protein degradation) because RNA purification was undertaken first. However, degradation was not observed upon visual inspection of the stained blots. Furthermore, Hif1␣ was undetectable under these conditions (but the antibody had been validated by us using other nuclear protein samples).
ChIP experiments confirmed that Sp1 and Hif2␣ specifically interacted with the Atp7a promoter in vivo (Fig. 6). There was a trend toward increased Hif2␣ binding in samples derived from iron-deficient rats, whereas conversely, no differences in Sp1 binding were noted among groups (data not shown). The detection of Hif2 binding in control rat samples was unexpected as the protein is normally degraded during normoxia. However, the intestinal epithelium exists in a natural state of mild hypoxia (25), particularly in epithelial cells at the villus tip, which is furthest from the blood supply (and where iron and copper transporters are expressed). This may explain the stabilization of the Hif2 protein in control duodenum.
Sp1 and Hif2␣ Synergistically Activate the Atp7a Promoter-Forced expression of Sp1 or Hif2␣ activates the Atp7a promoter in IEC-6 cells. To determine whether these two transacting factors can further enhance the promoter response when FIGURE 4. ChIP analysis of Sp1 binding to the Atp7a promoter. Crosslinked chromosomal DNA was immunoprecipitated from IEC-6 cell nuclear extracts using a ChIP-grade Sp1 antibody. Subsequent PCR analysis was utilized to determine whether certain regions of the Atp7a promoter were pulled down by the antibody. A shows the typical size of DNA fragments after sonication. B depicts PCR analysis of recovered DNA. Results showed that all four putative Sp1 binding sites were present in the immunoprecipitated sample (Atp7a), but other regions of the promoter not containing putative Sp1 binding sites (Ϫ) were not detected. Also shown is amplification of the Sp1 binding site regions from the input DNA. ChIP analysis was also performed with nuclear extracts derived from control or mithramycin (Mith)-treated IEC-6 cells (C and D). C shows the effect of 500 nM mithramycin on the activity of the WT Atp7a promoter-transfected into cells. Each bar represents the mean value ϮS.D. (n ϭ 3; ***, p Ͻ 0.005; paired Student's t test). Mithramycin also decreased the amount of Atp7a promoter DNA containing the putative Sp1 binding sites detected by PCR after ChIP (Atp7a) (D). Amplification from input DNA samples was similar, indicating that equal amounts of starting material were used. Again, other unrelated promoter regions were not detected (Ϫ). ChIP experiments depicted here are typical of three independent experiments performed with similar results. Ctrl, control. FIGURE 5. Effect of Hif2␣ overexpression on Atp7a promoter activity. Hif2␣ expression vector was co-transfected into IEC-6 cells along with WT or mutant Atp7a promoter constructs, and luciferase assays were performed. The effect of Hif2␣ overexpression is shown relative to activity of the WT promoter without Hif2␣ overexpression. The specific Sp1 sites (S) mutated are indicated below each bar, and Hif2␣ overexpression is indicated further below. Each bar represents the mean value ϮS.D. Different letters above bars indicate statistical differences between groups (p Ͻ 0.05; paired Student's t test; n ϭ 3-4).
co-overexpressed, IEC-6 cells were transfected with Sp1 and Hif2␣ expression vectors individually or together along with the basal Atp7a promoter construct, and reporter gene assays were performed. Forced Sp1 expression increased activity ϳ3-fold, whereas Hif2␣ overexpression increased activity ϳ5-fold (Fig. 7). When both were overexpressed together, Atp7a promoter activity was further transactivated to ϳ8-fold over control (empty vector-transfected) cells.
CoCl 2 and Low Oxygen Enhance Phosphorylation of Sp1-Sp1 transactivation properties are regulated by phosphorylation. We thus sought to determine whether the level of phospho-Sp1 was altered by hypoxia. Accordingly, IEC-6 cells were treated with CoCl 2 to mimic hypoxia or grown in a hypoxia chamber (12), and Sp1/phospho-Sp1 proteins levels were determined by immunoblot analysis. Results showed significantly higher levels of phospho-Sp1 in treated cells, whereas total immunoreactive Sp1 levels were relatively constant when comparing the treatment groups with control (untreated) cells (Fig. 8).

DISCUSSION
During iron deficiency, hemoglobin levels fall, decreasing oxygen delivery to tissues and cells, which leads to a hypoxic response. At the molecular level, this causes stabilization of the HIF␣ subunits that promotes nuclear localization and interaction with a constitutively expressed HIF␤ subunit followed by FIGURE 6. Molecular analysis of control and iron-deficient rats. Weanling rats consumed control or low iron diets for 5 weeks and then were sacrificed. Expression of known iron-responsive genes was analyzed in isolated duodenal enterocytes by qRT-PCR (A-C). Each bar represents the mean value ϮS.D. **, p Ͻ 0.01; ***, p Ͻ 0.005 (paired Student's t test; n ϭ 6). Ceruloplasmin (Cp; in serum; D), Atp7a (in enterocytes; E), and Hif1/2␣ (in enterocytes; F) protein expression was assessed by Western blotting. Shown below the blots are total stained proteins exemplifying equal loading of the gels and efficient transfer of proteins to membranes. ChIP experiments were also performed using cross-linked chromosomal DNA isolated from duodenal enterocytes and ChIP-grade Hif2a (G) or Sp1 (H) antibodies. For Hif2a ChIP (G), primers that covered the region containing the three HREs on the Atp7a promoter were used. In H, the primers encompassed the Sp1 binding sites on the promoter. In G and H, (Ϫ) indicates the use of primers from unrelated up-or downstream sites within the Atp7a promoter, and "Input" indicates amplification from the DNA samples prior to immunoprecipitation. Ctrl, control; FeD, iron-deficient. FIGURE 7. Effect of Hif2␣ and/or Sp1 overexpression on Atp7a promoter activity. The basal Atp7a promoter construct (Ϫ224/ϩ88) was co-transfected along with Hif2␣ and/or Sp1 expression plasmids into IEC-6 cells. Subsequent luciferase assays indicated promoter activity, which is shown in relation to promoter activity in the absence of Sp1 or Hif2␣ overexpression (Ctrl). Each bar represents the mean Ϯ S.D. Different letters above each bar (a, b, c, and d) indicate significant differences (p Ͻ 0.05; one-way analysis of variance; n ϭ 3-4). DNA binding and activation of genes related to energy metabolism (glycolysis), angiogenesis, and iron homeostasis. In the intestinal mucosa, during iron deficiency/hypoxia, a Hif2␣-specific transcriptional response enhances absorption of dietary iron by transactivating genes encoding proteins that mediate iron transport. Interestingly, Hif2␣ may also modulate intestinal copper absorption as reflected by induction of Atp7a and metallothionein in duodenal enterocytes during iron deprivation. The co-regulation of iron and copper transport during iron deficiency supports the concept that copper plays an important physiologic role in the maintenance of iron homeostasis. The current studies aimed to further evaluate mechanistic aspects of the Hif2␣ transcriptional response.
It was noted previously that many genes induced by iron deprivation in the rat intestine and in Caco-2 cells have G/Crich promoters (14), suggesting regulation by a trans-acting factor with an affinity for G-C base pairs. The classic example of such a transcription factor is Sp1. This widely expressed protein is a member of the Sp1-like and Krüppel-like factor family of DNA-binding proteins, which are integral parts of the transcriptional machinery of eukaryotic cells (26,27). Sp1/Krüppellike factors have highly conserved carboxyl-terminal DNAbinding domains containing three tandem zinc finger motifs. The amino-terminal regions are variable and contain transcription regulatory domains that interact with co-regulators. By regulating the expression of a large number of genes containing G/C-rich promoters, Sp1/Krüppel-like factor proteins are involved in many biological processes including cell proliferation, differentiation, apoptosis, and neoplastic transformation (26). Few studies to date have investigated Sp1-like factors in the regulation of iron homeostasis-related genes. One recent study suggested that the age-related decline in hepatic transferrin gene expression may relate in part to Sp1-like DNA binding activity (28). The hepatic Hfe gene, which is mutated in some types of hereditary hemochromatosis in humans, also has apparent Sp1-like binding sites (29). Sp1 and Sp3 were also shown to bind to an enhancer in the ferritin H gene and activate expression in fibroblast and liver cell lines (30). Additionally, it was reported previously that the Dmt1 gene has three predicted Sp1-like binding sites, but these were not experimentally verified (31). Moreover, although a role for Sp1-like factors in mediating the transcriptional response of intestinal epithelial cells to iron deprivation was postulated previously (13), this possibility has not been experimentally tested to date.
To elucidate a potential role for Sp1 in the Hif2␣-mediated transcriptional response to iron deprivation, we performed initial studies on the Atp7a gene, which is coordinately regulated with iron transport-related genes during iron deprivation. Atp7a-mediated regulation of copper absorption may play an important physiologic role in the maintenance of intestinal iron transport possibly by enhancing activity of a multicopper ferroxidase (hephaestin), which couples iron oxidation to efflux via Fpn1 (32,33). We previously evaluated the rat Atp7a promoter (12,34) including mapping the transcriptional start site and defining the basal promoter region (Ϫ224/ϩ88). The role of Sp1 in basal and Hif2␣-stimulated Atp7a transcription, however, has not been examined. This investigation was thus undertaken to test the hypothesis that Sp1 (or an Sp1-like factor) is necessary for the Hif2␣-mediated induction of gene expression in the duodenal mucosa during iron deficiency. Whenever possible, whether Atp7a-specific regulatory mechanisms were conserved among iron homeostasis-related genes (e.g. Dmt1, Dctyb, and Fpn1) was assessed to broaden the scope of this experimental analysis.
Initial experiments utilized a drug that blocks Sp1 binding to DNA (mithramycin) to assess a possible role for Sp1 in Atp7a gene transcription in IEC-6 cells. Mithramycin is a DNA-binding antibiotic that binds to the minor groove of G-C base pairs (35,36). This interaction with DNA blocks trans-acting factor binding to G/C-rich regions. Although initial studies showed specific inhibition of Sp1 binding (16,17,37), mithramycin could theoretically block binding of any protein with an affinity for G-C base pairs. In the current study, mithramycin was utilized to show that Atp7a and other iron homeostasis-related genes were potentially regulated by Sp1 as mRNA expression was significantly inhibited. However, a G/C-binding protein was not absolutely required for basal transcriptional activation of these genes as expression was not abolished. These observations provide preliminary evidence that intestinal genes induced by Hif2␣ during iron deprivation/hypoxia may be regulated by Sp1.
CoCl 2 chemically mimics hypoxia (under normoxic conditions) by stabilizing the HIF␣ subunits; Hif1␣ and Hif2␣ are both stabilized via inhibition of oxygen-dependent degradation. Expression of Atp7a and iron transport-related genes increased with CoCl 2 treatment, consistent with their known regulation by Hif2␣. Ankrd37 and Vegf were also up-regulated, likely reflecting regulation by Hif1␣ (19,38). Interestingly, mithramycin had differing effects on the induction of mRNA expression by CoCl 2 ; it blocked the increase of some genes, whereas other genes were unaffected. This exemplified two modes of regulation: one in which Sp1 (or a related G/C-binding protein) is necessary for the HIF response (e.g. for Atp7a and Dmt1) and another in which Sp1 is not required (e.g. for Ankrd37 and Vegf). These opposing regulatory mechanisms may relate to distinct transactivation properties of the different HIF␣ subunits. A trans-acting factor with affinity for G/C-rich DNA regions may thus be required for the Hif2␣-mediated increase in gene expression, which ultimately promotes iron absorption during hypoxia.
Several experimental observations presented herein suggest that Atp7a gene transcription is regulated by Sp1 including the FIGURE 8. Immunoblot analysis of phosphorylated Sp1 protein expression. IEC-6 cells at 85% confluence were either untreated and grown under control conditions (Ctrl), treated with 200 M CoCl 2 , or cultured in a hypoxia chamber (with 1% O 2 ) for 60 h. Nuclear proteins were then isolated, and immunoblots were run for detection of Sp1 and phosphorylated Sp1 (pSp1). The pSp1 band was detected at ϳ120 kDa, whereas the total Sp1 protein band was detected at ϳ108 kDa. The blots shown are representative of three independent experiments with comparable results.
following. 1) Sp1 overexpression increased endogenous Atp7a mRNA and protein expression in IEC-6 cells and stimulated the exogenously expressed Atp7a promoter. 2) Putative Sp1 binding sites were shown to be required for full transactivation of Atp7a gene expression. 3) ChIP assays showed that Sp1 directly interacts with the Atp7a gene in IEC-6 cells and in rat duodenal enterocytes. 4) Mithramycin significantly decreased pulldown of Atp7a promoter DNA containing the putative Sp1 binding sites from IEC-6 cells, consistent with the documented decrease in Atp7a promoter activity in the presence of mithramycin. Furthermore, in the current investigation, the previously reported binding of Hif2␣ to the Atp7a promoter in IEC-6 cells (12) was confirmed in rat duodenal enterocytes. Atp7a is thus a bona fide Sp1 and Hif2␣ target gene.
A final series of experiments was designed to determine whether putative Sp1 binding sites were necessary for Hif2␣mediated induction of Atp7a promoter activity. Forced Hif2␣ expression increased promoter activity ϳ5-fold, whereas individual Sp1 binding site mutations attenuated this increase to ϳ3-fold. Combinatorial Sp1 site mutations abolished transactivation by Hif2␣ overexpression. Interestingly, Hif2␣ overexpression maintained basal Atp7a promoter activity at WT levels even when multiple Sp1 sites were mutated (in contrast to decreases in basal activity without forced Hif2␣ expression). Putative Sp1 binding sites are thus necessary for transactivation of the Atp7a gene by Hif2␣.
Data presented here show that the HIF-mediated induction of Atp7a expression during iron deficiency/hypoxia involves Sp1. Sp1-dependent Hif2␣ transactivation of gene expression has not been reported in the scientific literature (to our knowledge), suggesting that this is a novel regulatory mechanism. Hif2␣ is preferentially stabilized during iron deprivation in the intestine of mice (9, 10) and rats (this study) and in Caco-2 cells (14). Hif2␣ protein levels likely increase due to tissue hypoxia in iron-deprived mice and rats and as a result of inhibition of the iron-dependent HIF prolyl hydroxylases in Caco-2 cells treated with deferoxamine (an iron chelator). What is not clear is the specific molecular mechanism by which Sp1 potentiates the HIF-mediated induction of Atp7a gene transcription. Sp1 is known to be regulated by phosphorylation (39 -41), which alters its DNA binding affinity and/or transactivation capabilities. As such, we quantified Sp1/phospho-Sp1 levels in CoCl 2treated IEC-6 cells and in cells grown in 1% O 2 . Phosphorylation of Sp1 increased dramatically in treated cells, suggesting that posttranslational modification of the protein may play a role in induction of Atp7a expression during iron deprivation/ hypoxia. Because ChIP assays showed no difference in the amount of Atp7a promoter DNA pulled down with Sp1 antibody from enterocytes isolated from control or iron-deficient rats, we speculate that Sp1 phosphorylation increases transactivation of Atp7a gene expression.
This investigation focused on the gene encoding the primary enterocyte copper exporter, Atp7a. Lack of fully functional Atp7a is the underlying cause of Menkes disease in humans, a Mendelian disorder in which inefficient absorption of dietary copper leads to systemic copper deficiency and the dire physiologic consequences of copper depletion (e.g. neurological damage, hypopigmentation, etc.) (42,43). During iron deficiency/ hypoxia, Atp7a expression increases dramatically, implicating copper in control of iron homeostasis. In fact, copper increases in tissues and cells important for homeostatic control of iron homeostasis (e.g. enterocytes and hepatocytes) during iron deficiency (3,8). Given that Atp7a represents the rate-limiting step in acquisition of dietary copper, it may then play a key role in the compensatory response to iron deficiency. Thus, a detailed mechanistic understanding of Atp7a gene regulation may increase knowledge of regulatory aspects of whole-body iron homeostasis.
In summary, Sp1 binding is necessary for the hypoxia-mediated induction of Atp7a promoter activity in IEC-6 cells. Whether this mechanism is also true of in vivo regulation of Atp7a gene expression during iron deprivation is unknown, but we provide evidence that the Atp7a gene is a direct Hif2␣ and Sp1 target in rat duodenal enterocytes. Three lines of evidence suggest that these observations may have importance beyond understanding Atp7a gene regulation. 1) Atp7a is coordinately regulated by Hif2␣ along with genes encoding proteins required for iron absorption (Dcytb, Dmt1, and Fpn1). 2) Many genes up-regulated by iron deficiency in the mammalian duodenum have G/C-rich promoters and evolutionarily conserved HREs. 3) Hypoxia resulted in increased phosphorylation of Sp1, likely altering its transactivation properties. Sp1-dependent, Hif2␣-mediated induction of gene expression may thus have broader implications for understanding additional mechanistic aspects of intestinal iron homeostasis.