Inflammation and hypoxia linked to renal injury by CCAAT/enhancer-binding protein δ
Authors: Junna Yamaguchi, Tetsuhiro Tanaka, Nobuaki Eto, Masaomi Nangaku
Journal: Kidney International (2015)

Tubulointerstitial hypoxia plays a critical role in the pathogenesis of kidney injury, and hypoxia-inducible factor (HIF)-1 is a master regulator of cellular adaptation to hypoxia. Aside from oxygen molecules, factors that modify HIF-1 expression and functional operation remain obscure. Therefore, we sought to identify novel HIF-1-regulating genes in kidney. A short-hairpin RNA library consisting of 150 hypoxia-inducible genes was derived from a microarray analysis of the rat renal artery stenosis model screened for the effect on HIF-1 response. We report that CCAAT/enhancer-binding protein δ (CEBPD), a transcription factor and inflammatory response gene, is a novel HIF-1 regulator in kidney. CEBPD was induced in the nuclei of tubular epithelial cells in both acute and chronic hypoxic kidneys. In turn, CEBPD induction augmented HIF-1α expression and its transcriptional activity. Mechanistically, CEBPD directly bound to the HIF-1α promoter and enhanced its transcription. Notably, CEBPD was rapidly induced by inflammatory cytokines, such as IL-1β in a nuclear factor-κB-dependent manner, which not only increased HIF-1α expression during hypoxia, but was also indispensable for the non-hypoxic induction of HIF-1α. Thus our study provides novel insight into HIF-1 regulation in tubular epithelial cells and offers a potential hypoxia and inflammation link relevant in both acute and chronic kidney diseases. Tubulointerstitial hypoxia plays a critical role in the pathogenesis of kidney injury, and hypoxia-inducible factor (HIF)-1 is a master regulator of cellular adaptation to hypoxia. Aside from oxygen molecules, factors that modify HIF-1 expression and functional operation remain obscure. Therefore, we sought to identify novel HIF-1-regulating genes in kidney. A short-hairpin RNA library consisting of 150 hypoxia-inducible genes was derived from a microarray analysis of the rat renal artery stenosis model screened for the effect on HIF-1 response. We report that CCAAT/enhancer-binding protein δ (CEBPD), a transcription factor and inflammatory response gene, is a novel HIF-1 regulator in kidney. CEBPD was induced in the nuclei of tubular epithelial cells in both acute and chronic hypoxic kidneys. In turn, CEBPD induction augmented HIF-1α expression and its transcriptional activity. Mechanistically, CEBPD directly bound to the HIF-1α promoter and enhanced its transcription. Notably, CEBPD was rapidly induced by inflammatory cytokines, such as IL-1β in a nuclear factor-κB-dependent manner, which not only increased HIF-1α expression during hypoxia, but was also indispensable for the non-hypoxic induction of HIF-1α. Thus our study provides novel insight into HIF-1 regulation in tubular epithelial cells and offers a potential hypoxia and inflammation link relevant in both acute and chronic kidney diseases. Oxygen homeostasis underlies many developmental and physiological processes, and, its disturbances have key roles in the pathogenesis of many human diseases, including kidney disease.1.Semenza G.L. Oxygen sensing, homeostasis, and disease.N Engl J Med. 2011; 365: 537-547Crossref PubMed Scopus (756) Google Scholar The kidney requires a complicated vasculature to maintain its structure and function and is thus highly susceptible to ischemic insults. Indeed, tubulointerstitial fibrosis is an end-stage feature in chronic kidney disease (CKD), which is frequently associated with the loss of peritubular capillary networks reminiscent of hypoxia.2.Bohle A. Mackensen-Haen S. Wehrmann M. Significance of postglomerular capillaries in the pathogenesis of chronic renal failure.Kidney Blood Press Res. 1996; 19: 191-195Crossref PubMed Scopus (175) Google Scholar To date, there is an increasing body of evidence showing that tubulointerstitial hypoxia is not only a key player in ischemic acute kidney injury (AKI) but is also a critical mediator toward end-stage renal disease in CKD settings.3.Fine L.G. Bandyopadhay D. Norman J.T. Is there a common mechanism for the progression of different types of renal diseases other than proteinuria? Towards the unifying theme of chronic hypoxia.Kidney Int. 2000; 57: S22-S26Abstract Full Text Full Text PDF Scopus (286) Google Scholar,4.Nangaku M. Chronic hypoxia and tubulointerstitial injury: a final common pathway to end-stage renal failure.J Am Soc Nephrol. 2006; 17: 17-25Crossref PubMed Scopus (877) Google Scholar Hypoxia-inducible factors (HIFs) regulate adaptive responses to hypoxia at the molecular, cellular, and tissue levels. HIF is a heterodimeric transcription factor, consisting of an oxygen-labile α subunit (HIF-1α, HIF-2α, and HIF-3α) and a constitutively expressed β subunit.5.Semenza G.L. Hypoxia-inducible factors in physiology and medicine.Cell. 2012; 148: 399-408Abstract Full Text Full Text PDF PubMed Scopus (2062) Google Scholar,6.Tanaka T. Wiesener M. Bernhardt W. et al.The human HIF (hypoxia-inducible factor)-3alpha gene is a HIF-1 target gene and may modulate hypoxic gene induction.Biochem J. 2009; 424: 143-151Crossref PubMed Scopus (79) Google Scholar Under normoxia, HIF-α is hydroxylated by prolyl hydroxylase domain-containing protein (PHDs) and undergoes proteasomal degradation assisted by the von Hippel-Lindau (VHL) tumor-suppressor protein. In hypoxia, the lack of oxygen inhibits PHD activity and disallows hydroxylation of HIF-α. Stabilized HIF-α translocates to the nucleus, dimerizes with HIF-1β, and transactivates its target genes. In response to hypoxia in kidney, HIF-1α is expressed in tubular epithelial cells, including proximal and distal tubules, connecting tubules, and collecting ducts.7.Rosenberger C. Mandriota S. Jürgensen J.S. et al.Expression of hypoxia-inducible factor-1α and -2α in hypoxic and ischemic rat kidneys.J Am Soc Nephrol. 2002; 13: 1721-1732Crossref PubMed Scopus (466) Google Scholar Functional studies both in vivo and in vitro show that HIF-1 appears to have protective roles in various acute and chronic kidney injuries.8.Matsumoto M. Makino Y. Tanaka T. et al.Induction of renoprotective gene expression by cobalt ameliorates ischemic injury of the kidney in rats.J Am Soc Nephrol. 2003; 14: 1825-1832Crossref PubMed Scopus (219) Google Scholar, 9.Tanaka T. Miyata T. Inagi R. et al.Hypoxia in renal disease with proteinuria and/or glomerular hypertension.Am J Pathol. 2004; 165: 1979-1992Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 10.Tanaka T. Kojima I. Ohse T. et al.Hypoxia-inducible factor modulates tubular cell survival in cisplatin nephrotoxicity.Am J Physiol Renal Physiol. 2005; 289: F1123-F1133Crossref PubMed Scopus (89) Google Scholar, 11.Tanaka T. Kojima I. Ohse T. et al.Cobalt promotes angiogenesis via hypoxia-inducible factor and protects tubulointerstitium in the remnant kidney model.Lab Invest. 2005; 85: 1292-1307Crossref PubMed Scopus (190) Google Scholar, 12.Tanaka T. Matsumoto M. Inagi R. et al.Induction of protective genes by cobalt ameliorates tubulointerstitial injury in the progressive Thy1 nephritis.Kidney Int. 2005; 68: 2714-2725Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 13.Bernhardt W.M. Câmpean V. Kany S. et al.Preconditional activation of hypoxia-inducible factors ameliorates ischemic acute renal failure.J Am Soc Nephrol. 2006; 17: 1970-1978Crossref PubMed Scopus (232) Google Scholar, 14.Bernhardt W.M. Gottmann U. Doyon F. et al.Donor treatment with a PHD-inhibitor activating HIFs prevents graft injury and prolongs survival in an allogenic kidney transplant model.Proc Natl Acad Sci USA. 2009; 106: 21276-21281Crossref PubMed Scopus (112) Google Scholar, 15.Hill P. Shukla D. Tran M.G. et al.Inhibition of hypoxia inducible factor hydroxylases protects against renal ischemia-reperfusion injury.J Am Soc Nephrol. 2008; 19: 39-46Crossref PubMed Scopus (221) Google Scholar In addition to its expression control by protein stabilization, HIF-1α undergoes transcriptional and translational regulation, particularly in tumor and inflammatory cells via the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin, nuclear factor (NF)-κB, and extracellular signal–regulated kinase mitogen-activated protein kinase pathways.16.Semenza G.L. Targeting HIF-1 for cancer therapy.Nat Rev Cancer. 2003; 3: 721-732Crossref PubMed Scopus (5295) Google Scholar, 17.Blouin C.C. Pagé E.L. Soucy G.M. et al.Hypoxic gene activation by lipopolysaccharide in macrophages: implication of hypoxia-inducible factor 1alpha.Blood. 2004; 103: 1124-1130Crossref PubMed Scopus (361) Google Scholar, 18.Rius J. Guma M. Schachtrup C. et al.NF-kappaB links innate immunity to the hypoxic response through transcriptional regulation of HIF-1alpha.Nature. 2008; 453: 807-811Crossref PubMed Scopus (1160) Google Scholar However, it remains unknown whether the transcriptional and translational control of HIF-1α is operative in cells of noncancerous tissues, such as renal tubular cells, and which, if any, genes are responsible for the HIF-1α upstream signaling. After taking into account the accumulating knowledge of HIF-1 on the pathogenesis of both AKI and CKD, we designed this study to identify novel HIF-1 regulators through RNAi library screening. To identify which genes are relevant in HIF-1 regulation, we created a shRNA library against the top 150 hypoxia-inducible genes screened from a microarray analysis of the rat renal artery stenosis (RAS) model, a representative chronic hypoxic kidney injury model (Supplementary Table S1 online).19.Kojima I. Tanaka T. Inagi R. et al.Metallothionein is upregulated by hypoxia and stabilizes hypoxia-inducible factor in the kidney.Kidney Int. 2009; 75: 268-277Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar For the initial screening, the impact of candidate genes on HIF-1 was evaluated in vitro through a hypoxia-responsive element (HRE)-driven luciferase (HREluc) reporter assay (Figure 1a); shRNA transfection against a bona fide HIF-1-regulating gene would affect HREluc activity. The hypoxic induction (defined as (luciferase activity in 1% O2)/(luciferase activity in 21% O2)) was also calculated to exclude the nonspecific effect. The shRNA-transfected clones showing hypoxic changes within 50 percentile compared with the positive control (shRNA anti-HIF-1α) were extracted as hit clones. Eighteen hit genes proceeded to the second screening. Download .xls (.04 MB) Help with xls files Supplementary Table S1 Stable HeLa cells transfected with shRNA against candidate genes were further assessed for the hypoxic induction of HREluc, HIF-1α protein, and supernatant vascular endothelial growth factor (VEGF, an endogenous HIF-1 target) expression (Supplementary Figure S1 online). At this stage, five genes were selected as novel, potential HIF-1-regulating genes: CCAAT/enhancer-binding protein δ (CEBPD); transforming growth factor-beta-induced factor (TGIF); paired mesoderm homeobox protein 2A (PHOX2A); nuclear receptor superfamily 4A member 1 (NR4A1); and P300/CBP-associated factor (PCAF). Intercellular adhesion molecule 1 (ICAM1); leucine-rich alpha-2-glycoprotein 1 (LRG1); cytochrome P-450, family 4, subfamily B, polypeptide 1 (CYP4B1); and renin (RENIN) were extracted as hit genes by HREluc activity criteria but did not have positive effects on HIF-1α or VEGF and were therefore not selected as candidates (data not shown). Download .jpg (.03 MB) Help with files Supplementary Figure S1 Finally, small interfering RNAs (siRNAs) against these five candidates were designed and transfected into HeLa cells, and the above experiments were repeated. PHOX2A was excluded because of a failure to reduce HIF-1α expression by siRNA, whereas the remaining four matched the criteria. As a result, we identified four novel genes––TGIF, NR4A1, PCAF, and CEBPD––that functionally regulated HIF-1α protein expression and its transcriptional activity (Figure 1b). None of them had an effect on HIF-2α (Figure 1b). Of these genes, CEBPD had the most pronounced effect on HIF-1α expression, which we investigated further (Figure 1c). CEBPD is a member of the highly conserved C/EBP family of basic region leucine zipper transcription factors. Although its basic expression level is low in most normal tissues and cells, CEBPD is rapidly upregulated by a variety of stimuli, including inflammatory signals.20.Takiguchi M. The C/EBP family of transcription factors in the liver and other organs.Int J Exp Pathol. 1998; 79: 369-391Crossref PubMed Scopus (97) Google Scholar,21.Ramji D.P. Foka P. CCAAT/enhancer-binding proteins: structure, function and regulation.Biochem J. 2002; 365: 561-575Crossref PubMed Google Scholar We investigated whether CEBPD expression in vivo correlates with hypoxia in the kidney. Rodent kidneys were subjected to a series of hypoxic stimuli and tested for CEPBD expression. CEBPD was induced in kidneys exposed to systemic hypoxia (8% O2, 6 h), as well as in models of kidney injuries (ischemia–reperfusion (I/R) injury, cisplatin nephrotoxicity, RAS, and 5/6 nephrectomy (remnant kidney (RK)); Figure 2a and b). Although the pathogenesis of these models is multifactorial, they serve as models of acute and chronic ischemic kidney injury. Cisplatin nephrotoxicity is hypoxic in its outer medulla because of the reduced blood flow caused by the toxin,10.Tanaka T. Kojima I. Ohse T. et al.Hypoxia-inducible factor modulates tubular cell survival in cisplatin nephrotoxicity.Am J Physiol Renal Physiol. 2005; 289: F1123-F1133Crossref PubMed Scopus (89) Google Scholar and activation of the renin–angiotensin system results in decreased peritubular capillary flow with subsequent hypoxia in RK.22.Manotham K. Tanaka T. Matsumoto M. et al.Evidence of tubular hypoxia in the early phase in the remnant kidney model.J Am Soc Nephrol. 2004; 15: 1277-1288Crossref PubMed Scopus (194) Google Scholar Immunofluorescence studies of kidney sections using anti-CEBPD antibody and Phaseolus vulgaris Erythroagglutinin (PHA-E), a cell-specific marker for proximal tubular cells, demonstrated that in the control kidney CEBPD was most evidently expressed in the S3 segment of proximal tubules in the outer stripe of the outer medulla, physiologically hypoxic and the most susceptible portion of kidney to hypoxia (Figure 2c).7.Rosenberger C. Mandriota S. Jürgensen J.S. et al.Expression of hypoxia-inducible factor-1α and -2α in hypoxic and ischemic rat kidneys.J Am Soc Nephrol. 2002; 13: 1721-1732Crossref PubMed Scopus (466) Google Scholar Upon hypoxic stimulation, CEBPD was robustly induced predominantly in the nuclei of proximal tubular cells (Figure 2d). CEBPD co-localized with PHA-E in the hypoxic models and did not co-localize with Tamm–Horsfall protein (THP, a marker for thick ascending limbs) or with calcium-binding protein (CaBP, a marker for ascending limb or distal tubules; data not shown). These data demonstrate that CEBPD is induced by hypoxia predominantly in proximal tubular cells. CEBPD induction in proximal tubules following a series of hypoxic injury conditions prompted us to investigate whether these events occur in a cell-autonomous manner. In HK-2 cells, hypoxia increased CEBPD expression (Figure 3a). CEBPD mRNA expression was increased by threefold, which was not blunted by HIF-1α siRNA (Supplementary Figure S2A online). This suggested that the hypoxic induction of CEBPD was mediated independently of HIF-1. Conversely, CEBPD knockdown by siRNA resulted in reduced HIF-1α protein expression under hypoxia (Figure 3b), which was accompanied by a subsequent reduction in HIF transcriptional activity (Figure 3c) and reduced expression of the endogenous HIF-1 target genes glucose transporter 1 (GLUT1) and VEGF (Figure 3d). We further investigated whether other members of the C/EBP family might similarly regulate HIF-1. CCAAT/enhancer-binding protein β (CEBPB), a known homologue to CEBPD, was not induced by hypoxia nor did it regulate HIF-1α expression (Supplementary Figure S2B online). In contrast, CEBPD overexpression significantly increased HIF-1α protein levels (Supplementary Figure S2C online). Interestingly, HIF-1α protein upregulation by CEBPD overexpression was more pronounced in 1% hypoxia but was evident at near anoxic conditions (0.1% hypoxia). These data collectively demonstrate that CEBPD regulates endogenous HIF-1 in tubular cells, which is consistent with previous report in cancer cell line,23.Min Y. Ghose S. Boelte K. et al.C/EBP-δ regulates VEGF-C autocrine signaling in lymphangiogenesis and metastasis of lung cancer through HIF-1α.Oncogene. 2011; 30: 4901-4909Crossref PubMed Scopus (56) Google Scholar and this effect is strictly retained within the context of the CEBPD–HIF-1 axis. Download .jpg (.04 MB) Help with files Supplementary Figure S2 CEBPD is an inflammatory response gene, which contributes to both the stimulation and the resolution of inflammation.20.Takiguchi M. The C/EBP family of transcription factors in the liver and other organs.Int J Exp Pathol. 1998; 79: 369-391Crossref PubMed Scopus (97) Google Scholar,24.Alam T. An M.R. Papaconstantinou J. Differential expression of three C/EBP isoforms in multiple tissues during the acute phase response.J Biol Chem. 1992; 267: 5021-5024Abstract Full Text PDF PubMed Google Scholar CEBPD is induced by bacterial lipopolysaccharide (LPS), interferon-γ, interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6 in myeloid cells.24.Alam T. An M.R. Papaconstantinou J. Differential expression of three C/EBP isoforms in multiple tissues during the acute phase response.J Biol Chem. 1992; 267: 5021-5024Abstract Full Text PDF PubMed Google Scholar, 25.Juan T.S. Wilson D.R. Wilde M.D. et al.Participation of the transcription factor C/EBP delta in the acute-phase regulation of the human gene for complement component C3.Proc Natl Acad Sci USA. 1993; 90: 2584-2588Crossref PubMed Scopus (125) Google Scholar, 26.Balamurugan K. Sharan S. Klarmann K.D. et al.FBXW7α attenuates inflammatory signalling by downregulating C/EBPδ and its target gene Tlr4.Nat Commun. 2013; 4: 1662Crossref PubMed Scopus (66) Google Scholar On the other hand, HIF-1α is induced under normoxic condition by LPS, growth factors such as epidermal growth factor or insulin, and inflammatory cytokines such as IL-1β or TNF-α,27.Treins C. Giorgetti-Peraldi S. Murdaca J. et al.Insulin stimulates hypoxia-inducible factor 1 through a phosphatidylinositol 3-kinase/target of rapamycin-dependent signaling pathway.J Biol Chem. 2002; 277: 27975-27981Crossref PubMed Scopus (469) Google Scholar, 28.Zhong H. Chiles K. Feldser D. et al.Modulation of hypoxia-inducible factor 1alpha expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/AKT/FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics.Cancer Res. 2000; 60: 1541-1545PubMed Google Scholar, 29.Stiehl D.P. Jelkmann W. Wenger R.H. et al.Normoxic induction of the hypoxia-inducible factor 1alpha by insulin and interleukin-1beta involves the phosphatidylinositol 3-kinase pathway.FEBS Lett. 2002; 512: 157-162Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar depending on the cell type, as well as the pathophysiologial contexts. These facts led us to hypothesize that tissue inflammation might fine-tune HIF-1 through CEBPD expression. In the kidney, inflammation is mediated by resident tubular cells and inflammatory cells, most commonly by macrophages.30.Ricardo S.D. van Goor H. Eddy A.A. Macrophage diversity in renal injury and repair.J Clin Invest. 2008; 118: 3522-3530Crossref PubMed Scopus (562) Google Scholar,31.Chung A.C. Lan H.Y. Chemokines in renal injury.J Am Soc Nephrol. 2011; 22: 802-809Crossref PubMed Scopus (193) Google Scholar Macrophages are present in the tubulointerstitium of all of the above-indicated hypoxic kidney models (Figure 4a). Macrophages and tubular cells under inflammatory conditions produce various cytokines and chemokines, including IL-1β, TNF-α, and monocyte chemotactic protein-1. These were confirmed in all the above hypoxic kidney models by real-time quantitative (q)RT-PCR of the diseased renal cortex (Figure 4b). Endogenous HIF-1 target genes, including ANPLT4 (angiopoietin-like 4), VEGF, SOD1 (superoxidase dismutase 1), and HO-1 (hemeoxygenase-1), were overall upregulated except for the RK model, in which the insufficient expression of HIF-target genes is reported (Figure 4b).32.Tanaka T. Yamaguchi J. Higashijima Y. et al.Indoxyl sulfate signals for rapid mRNA stabilization of Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (CITED2) and suppresses the expression of hypoxia-inducible genes in experimental CKD and uremia.FASEB J. 2013; 27: 4059-4075Crossref PubMed Scopus (34) Google Scholar This confirmed that inflammation and hypoxia coexist and may have interactions in kidney diseases. HK-2 cells were accordingly treated with various inflammatory stimuli, including LPS, TNF-α, and IL-1β (Supplementary Figure S3A and B online, Figure 5a and b). Whereas neither LPS nor TNF-α influenced the expression of CEBPD or HIF-1α (Supplementary Figures 3A and B online), IL-1β induced CEBPD and HIF-1α protein expression in a dose- and time-dependent manner under normoxia (Figure 5a and b). The parallel measurement of IL-6 or monocyte chemotactic protein-1 confirmed the efficacy of each treatment (Supplementary Figure S3A online, Figure 5a). A time-lapse study revealed that the induction of CEBPD preceded that of HIF-1α (Figure 5b), suggesting the existence of a CEBPD/HIF-1 pathway; indeed, HIF-1α induction by IL-1β was shown to be CEBPD dependent through siRNA knockdown and overexpression experiments (Figure 5c and Supplementary Figure S3C online). This regulation was also observed in A549 cells, a human lung carcinoma cell line (data not shown). These results indicate that CEBPD regulates the expression of HIF-1α protein not only under hypoxia but also under inflammatory conditions. Download .jpg (.03 MB) Help with files Supplementary Figure S3 HIF-α expression is tightly regulated at multiple steps. While the posttranslational ubiquitination subsequent to oxygen-dependent hydroxylation by PHD is the dominant step,16.Semenza G.L. Targeting HIF-1 for cancer therapy.Nat Rev Cancer. 2003; 3: 721-732Crossref PubMed Scopus (5295) Google Scholar the quantity of HIF-α is also influenced through changes in the transcriptional and translational rates, particularly in tumor and inflammatory cells.18.Rius J. Guma M. Schachtrup C. et al.NF-kappaB links innate immunity to the hypoxic response through transcriptional regulation of HIF-1alpha.Nature. 2008; 453: 807-811Crossref PubMed Scopus (1160) Google Scholar Because CEBPD regulated HIF-1α expression under various O2 concentrations, including those under which the PHD activity is already severely impaired, we hypothesized that the latter mechanisms may be involved in the control of HIF-1α by CEBPD. In HK-2 cells, treatment with actinomycin, a transcription inhibitor, decreased HIF-1α expression under hypoxia or IL-1β treatment (Figure 6a). HIF-1α mRNA showed a CEBPD-dependent increase under both conditions (Figure 6b). Cycloheximide, a protein synthesis inhibitor, did not show differences in HIF-1α protein half-lives between control and CEBPD siRNA groups, whereas MG132, a proteasome inhibitor, clearly demonstrated a decrease in the HIF-1α synthesis in the CEBPD siRNA group (Supplementary Figure S4A and B online), which indicated that CEBPD regulates HIF-1α at the transcription or the translation level. This was also confirmed by an experiment using a 2-oxoglutarate analog, DMOG, which disallows PHD enzymatic activity and thus prevents HIF-α degradation (Supplementary Figure S4C online). Furthermore, PI3K/AKT or mammalian target of rapamycin signaling has been implicated in the transcriptional or translational regulation of HIF-1α in several tumor cells. However, pretreatment with specific inhibitors, PI3K inhibitor (LY294002) and rapamycin, had no effect on the expression of HIF-1α, suggesting that these pathways were not involved in our study (Supplementary Figure S4D online).27.Treins C. Giorgetti-Peraldi S. Murdaca J. et al.Insulin stimulates hypoxia-inducible factor 1 through a phosphatidylinositol 3-kinase/target of rapamycin-dependent signaling pathway.J Biol Chem. 2002; 277: 27975-27981Crossref PubMed Scopus (469) Google Scholar, 29.Stiehl D.P. Jelkmann W. Wenger R.H. et al.Normoxic induction of the hypoxia-inducible factor 1alpha by insulin and interleukin-1beta involves the phosphatidylinositol 3-kinase pathway.FEBS Lett. 2002; 512: 157-162Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 33.Balamurugan K. Wang J.M. Tsai H.H. et al.The tumour suppressor C/EBPδ inhibits FBXW7 expression and promotes mammary tumour metastasis.EMBO J. 2010; 29: 4106-4117Crossref PubMed Scopus (83) Google Scholar Download .jpg (.04 MB) Help with files Supplementary Figure S4 These results altogether indicated that HIF-1α expression levels under these conditions were regulated at the transcriptional and/or the translational level. Consistently, the activity of 1.3k human HIF-1α promoter was dependent on CEBPD (Figure 6c). siRNA knockdown of CEBPD significantly decreased HIF-1α promoter activity. The HIF-1α promoter contains six potential binding motifs for C/EBP (motif 1–4) within its 1.3-kb region (Figure 6c);21.Ramji D.P. Foka P. CCAAT/enhancer-binding proteins: structure, function and regulation.Biochem J. 2002; 365: 561-575Crossref PubMed Google Scholar,34.Osada S. Yamamoto H. Nishihara T. et al.DNA binding specificity of the CCAAT/enhancer-binding protein transcription factor family.J Biol Chem. 1996; 271: 3891-3896Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar therefore, we tested for potential binding of CEBPD to the promoter. Because a pilot transfection of the minimal promoter in close proximity to the HIF-1α transcriptional start site did not reveal significant changes following CEBPD stimulation (data not shown), we speculated that the potential CEBPD binding sites might be one (or more) of the five C/EBP motifs indicated in Figure 6c. Mutations were introduced against each of the candidate C/EBP motifs, and the mutant promoters were tested for the CEBPD response. A mutation at the -227/-222 bp site cancelled the CEBPD-dependent increase in promoter activity (Figure 6d). Chromatin immunoprecipitation (ChIP) –PCR analysis confirmed CEBPD recruitment to this HIF-1α promoter (-227/-222 bp) under hypoxia or IL-1β treatment (Figure 6e). Collectively, these results indicate that CEBPD binds to the HIF-1α promoter and transcriptionally upregulates its expression in response to hypoxia or inflammatory stimulation. The expression levels of both CEBPD and HIF-1α are regulated by NF-κB signaling in certain cell types, although the detailed pathways of the latter remain largely unknown.19.Kojima I. Tanaka T. Inagi R. et al.Metallothionein is upregulated by hypoxia and stabilizes hypoxia-inducible factor in the kidney.Kidney Int. 2009; 75: 268-277Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar,35.Litvak V. Ramsey S.A. Rust A.G. et al.Function of C/EBPdelta in a regulatory circuit that discriminates between transient and persistent TLR4-induced signals.Nat Immunol. 2009; 10: 437-443Crossref PubMed Scopus (231) Google Scholar In HK-2 cells, NF-κB activity was increased by hypoxia (Figure 7a); conversely, siRNA knockdown of p65, a component of NF-κB, led to a decrease in HREluc, indicating that the HIF activity was partially mediated by NF-κB (Figure 7b). On the basis of our previous observation that CEBPD augments HIF-1α expression in hypoxia and allows its normoxic, IL-1β-dependent accumulation, we hypothesized that NF-κB signaling might underlie hypoxic- or IL-1β-stimulated CEBPD expression, which is responsible for the induction of HIF-1α. Upon NF-κB inhibition by the IκB-α phosphorylation and degradation inhibitor (BAY11-7082) or p65 siRNA, the expression levels of both CEBPD and HIF-1α significantly decreased (Figure 7c). Conversely, stable CEBPD overexpression partially rescued the reduced HIF-1α expression caused by NF-κB inhibition (Figure 7d). In summary, these results signify a novel NF-κB/CEBPD/HIF-1 pathway in which CEBPD was primarily responsible for the NF-κB-mediated fine-tuning of HIF-1α (Figure 7e). In this study, we identified novel regulators of HIF-1α expression in kidney using shRNA library screening. Out of the four identified genes, CEBPD had the most striking effect, which was induced by hypoxia or inflammation in tubular epithelial cells via HIF-1-independent, NF-κB-dependent pathways, and CEBPD regulated HIF-1α expression through transcriptional upregulation. CEBPD is a transcription facto