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Inflammation and hypoxia linked to renal injury by CCAAT/enhancer-binding protein δ.

Yamaguchi J, Tanaka T, Eto N, Nangaku M - Kidney Int. (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Nephrology and Endocrinology, The University of Tokyo Graduate School of Medicine, Tokyo, Japan.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

CCAAT/enhancer-binding protein δ (CEBPD) mechanistically regulates hypoxia-inducible factor (HIF)-1α at the transcription level. (a) Transcription inhibition reduces the induction of HIF-1α protein both under hypoxic and interleukin (IL)-1β-treated conditions. HK-2 cells pretreated with actinomycin for 20 min were either exposed to hypoxia (0.1% O2; left panel) or treated with IL-1β (1 ng/ml; right panel) for 6 h and assessed for HIF-1α and CEBPD protein expression levels through immunoblot analysis. (b) Small interfering RNA (siRNA)-mediated CEBPD knockdown cancelled the increase in HIF-1α mRNA under hypoxia (left panel) or IL-1β (right panel). Data are the mean±s.e.m. of three independent experiments. *P<0.05 and **P<0.01 (two-way analysis of variance (ANOVA) with Bonferroni's post-hoc tests). (c) Position and nucleotide sequence for the binding of the C/EBP transcription factors are shown as motifs 1–4. The hHIF-1α1.3K promoter activity transfected either with siRNA against control or CEBPD together with TK-Renilla vector was measured under hypoxia. The activity of the pGL3 basic control vector was not influenced by the treatment. (d) Site-directed mutagenesis against the hHIF-1α1.3K promoter was performed to identify the responsible site. Out of the five candidate sites, a mutation at −227/−222 bp site decreased HIF-1α promoter activity. This result signified that this site −227/−222 was functionally responsible for the transcriptional activation of HIF-1α. (e) Chromatin immunoprecipitation–PCR revealed CEBPD–HIF-1α interaction following hypoxia or IL-1β treatment. CEBPD on HIF-1α promoter (−227/−222 bp site) was significantly enriched under hypoxia or IL-1β compared with normoxia. Cross-linked DNA–protein complexes in the normoxia, hypoxia, and IL-1β groups were captured by anti-CEBPD antibody, and enriched nucleotide fragments were quantitatively amplified by PCR using promoters specific to HIF-1α, IL-6 (positive control under IL-1β treatment), and HBB (negative control). This result indicates that CEBPD is recruited to the HIF-1α promoter under both conditions. (c–e) The data are the mean±s.d. of a representative of three independent experiments. *P<0.05 and **P<0.01 (two-way ANOVA with Bonferroni's post-hoc tests).
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fig6: CCAAT/enhancer-binding protein δ (CEBPD) mechanistically regulates hypoxia-inducible factor (HIF)-1α at the transcription level. (a) Transcription inhibition reduces the induction of HIF-1α protein both under hypoxic and interleukin (IL)-1β-treated conditions. HK-2 cells pretreated with actinomycin for 20 min were either exposed to hypoxia (0.1% O2; left panel) or treated with IL-1β (1 ng/ml; right panel) for 6 h and assessed for HIF-1α and CEBPD protein expression levels through immunoblot analysis. (b) Small interfering RNA (siRNA)-mediated CEBPD knockdown cancelled the increase in HIF-1α mRNA under hypoxia (left panel) or IL-1β (right panel). Data are the mean±s.e.m. of three independent experiments. *P<0.05 and **P<0.01 (two-way analysis of variance (ANOVA) with Bonferroni's post-hoc tests). (c) Position and nucleotide sequence for the binding of the C/EBP transcription factors are shown as motifs 1–4. The hHIF-1α1.3K promoter activity transfected either with siRNA against control or CEBPD together with TK-Renilla vector was measured under hypoxia. The activity of the pGL3 basic control vector was not influenced by the treatment. (d) Site-directed mutagenesis against the hHIF-1α1.3K promoter was performed to identify the responsible site. Out of the five candidate sites, a mutation at −227/−222 bp site decreased HIF-1α promoter activity. This result signified that this site −227/−222 was functionally responsible for the transcriptional activation of HIF-1α. (e) Chromatin immunoprecipitation–PCR revealed CEBPD–HIF-1α interaction following hypoxia or IL-1β treatment. CEBPD on HIF-1α promoter (−227/−222 bp site) was significantly enriched under hypoxia or IL-1β compared with normoxia. Cross-linked DNA–protein complexes in the normoxia, hypoxia, and IL-1β groups were captured by anti-CEBPD antibody, and enriched nucleotide fragments were quantitatively amplified by PCR using promoters specific to HIF-1α, IL-6 (positive control under IL-1β treatment), and HBB (negative control). This result indicates that CEBPD is recruited to the HIF-1α promoter under both conditions. (c–e) The data are the mean±s.d. of a representative of three independent experiments. *P<0.05 and **P<0.01 (two-way ANOVA with Bonferroni's post-hoc tests).

Mentions: 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 the quantity of HIF-α is also influenced through changes in the transcriptional and translational rates, particularly in tumor and inflammatory cells.18 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, 29, 33


Inflammation and hypoxia linked to renal injury by CCAAT/enhancer-binding protein δ.

Yamaguchi J, Tanaka T, Eto N, Nangaku M - Kidney Int. (2015)

CCAAT/enhancer-binding protein δ (CEBPD) mechanistically regulates hypoxia-inducible factor (HIF)-1α at the transcription level. (a) Transcription inhibition reduces the induction of HIF-1α protein both under hypoxic and interleukin (IL)-1β-treated conditions. HK-2 cells pretreated with actinomycin for 20 min were either exposed to hypoxia (0.1% O2; left panel) or treated with IL-1β (1 ng/ml; right panel) for 6 h and assessed for HIF-1α and CEBPD protein expression levels through immunoblot analysis. (b) Small interfering RNA (siRNA)-mediated CEBPD knockdown cancelled the increase in HIF-1α mRNA under hypoxia (left panel) or IL-1β (right panel). Data are the mean±s.e.m. of three independent experiments. *P<0.05 and **P<0.01 (two-way analysis of variance (ANOVA) with Bonferroni's post-hoc tests). (c) Position and nucleotide sequence for the binding of the C/EBP transcription factors are shown as motifs 1–4. The hHIF-1α1.3K promoter activity transfected either with siRNA against control or CEBPD together with TK-Renilla vector was measured under hypoxia. The activity of the pGL3 basic control vector was not influenced by the treatment. (d) Site-directed mutagenesis against the hHIF-1α1.3K promoter was performed to identify the responsible site. Out of the five candidate sites, a mutation at −227/−222 bp site decreased HIF-1α promoter activity. This result signified that this site −227/−222 was functionally responsible for the transcriptional activation of HIF-1α. (e) Chromatin immunoprecipitation–PCR revealed CEBPD–HIF-1α interaction following hypoxia or IL-1β treatment. CEBPD on HIF-1α promoter (−227/−222 bp site) was significantly enriched under hypoxia or IL-1β compared with normoxia. Cross-linked DNA–protein complexes in the normoxia, hypoxia, and IL-1β groups were captured by anti-CEBPD antibody, and enriched nucleotide fragments were quantitatively amplified by PCR using promoters specific to HIF-1α, IL-6 (positive control under IL-1β treatment), and HBB (negative control). This result indicates that CEBPD is recruited to the HIF-1α promoter under both conditions. (c–e) The data are the mean±s.d. of a representative of three independent experiments. *P<0.05 and **P<0.01 (two-way ANOVA with Bonferroni's post-hoc tests).
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fig6: CCAAT/enhancer-binding protein δ (CEBPD) mechanistically regulates hypoxia-inducible factor (HIF)-1α at the transcription level. (a) Transcription inhibition reduces the induction of HIF-1α protein both under hypoxic and interleukin (IL)-1β-treated conditions. HK-2 cells pretreated with actinomycin for 20 min were either exposed to hypoxia (0.1% O2; left panel) or treated with IL-1β (1 ng/ml; right panel) for 6 h and assessed for HIF-1α and CEBPD protein expression levels through immunoblot analysis. (b) Small interfering RNA (siRNA)-mediated CEBPD knockdown cancelled the increase in HIF-1α mRNA under hypoxia (left panel) or IL-1β (right panel). Data are the mean±s.e.m. of three independent experiments. *P<0.05 and **P<0.01 (two-way analysis of variance (ANOVA) with Bonferroni's post-hoc tests). (c) Position and nucleotide sequence for the binding of the C/EBP transcription factors are shown as motifs 1–4. The hHIF-1α1.3K promoter activity transfected either with siRNA against control or CEBPD together with TK-Renilla vector was measured under hypoxia. The activity of the pGL3 basic control vector was not influenced by the treatment. (d) Site-directed mutagenesis against the hHIF-1α1.3K promoter was performed to identify the responsible site. Out of the five candidate sites, a mutation at −227/−222 bp site decreased HIF-1α promoter activity. This result signified that this site −227/−222 was functionally responsible for the transcriptional activation of HIF-1α. (e) Chromatin immunoprecipitation–PCR revealed CEBPD–HIF-1α interaction following hypoxia or IL-1β treatment. CEBPD on HIF-1α promoter (−227/−222 bp site) was significantly enriched under hypoxia or IL-1β compared with normoxia. Cross-linked DNA–protein complexes in the normoxia, hypoxia, and IL-1β groups were captured by anti-CEBPD antibody, and enriched nucleotide fragments were quantitatively amplified by PCR using promoters specific to HIF-1α, IL-6 (positive control under IL-1β treatment), and HBB (negative control). This result indicates that CEBPD is recruited to the HIF-1α promoter under both conditions. (c–e) The data are the mean±s.d. of a representative of three independent experiments. *P<0.05 and **P<0.01 (two-way ANOVA with Bonferroni's post-hoc tests).
Mentions: 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 the quantity of HIF-α is also influenced through changes in the transcriptional and translational rates, particularly in tumor and inflammatory cells.18 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, 29, 33

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Nephrology and Endocrinology, The University of Tokyo Graduate School of Medicine, Tokyo, Japan.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus