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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

The identification of hypoxia-inducible factor (HIF)-1-regulating genes. (a) Scheme of short hairpin RNA (shRNA) library screening. (b) HeLa cells transfected with small interfering RNA (siRNA) against the five candidate genes, CCAAT/enhancer-binding protein δ (CEBPD), transforming growth factor-beta-induced factor (TGIF), nuclear receptor superfamily 4A member 1 (NR4A1), paired mesoderm homeobox protein 2A (PHOX2A), or P300/CBP-associated factor (PCAF), were treated under hypoxia for 16 h to confirm the shRNA study results. The cells were processed for immunoblot analysis of HIF-1α and HIF-2α. Four genes except for PHOX2A showed a reduction in HIF-1α protein expression under hypoxia. The data are representative of three independent experiments, shown in duplicate. (c) Immunoblot analyses of HIF-1α (left panel) and HREluc activity (right panel) are shown representatively for siRNA-mediated CEBPD knockdown in HeLa cells. Knockdown of CEBPD decreased HIF-1α and hypoxic induction of HREluc activity in HeLa cells. (right panel) The ratio of luciferase reporter activity (firefly/TK-Renilla) to that of control siRNA under normoxia is indicated. Bar graph (mean±s.e.m. from three independent experiments) statistics performed using two-way analysis of variance (ANOVA) with Bonferroni post-hoc tests. *P<0.05. RAS, renal artery stenosis.
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fig1: The identification of hypoxia-inducible factor (HIF)-1-regulating genes. (a) Scheme of short hairpin RNA (shRNA) library screening. (b) HeLa cells transfected with small interfering RNA (siRNA) against the five candidate genes, CCAAT/enhancer-binding protein δ (CEBPD), transforming growth factor-beta-induced factor (TGIF), nuclear receptor superfamily 4A member 1 (NR4A1), paired mesoderm homeobox protein 2A (PHOX2A), or P300/CBP-associated factor (PCAF), were treated under hypoxia for 16 h to confirm the shRNA study results. The cells were processed for immunoblot analysis of HIF-1α and HIF-2α. Four genes except for PHOX2A showed a reduction in HIF-1α protein expression under hypoxia. The data are representative of three independent experiments, shown in duplicate. (c) Immunoblot analyses of HIF-1α (left panel) and HREluc activity (right panel) are shown representatively for siRNA-mediated CEBPD knockdown in HeLa cells. Knockdown of CEBPD decreased HIF-1α and hypoxic induction of HREluc activity in HeLa cells. (right panel) The ratio of luciferase reporter activity (firefly/TK-Renilla) to that of control siRNA under normoxia is indicated. Bar graph (mean±s.e.m. from three independent experiments) statistics performed using two-way analysis of variance (ANOVA) with Bonferroni post-hoc tests. *P<0.05. RAS, renal artery stenosis.

Mentions: 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 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.


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

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

The identification of hypoxia-inducible factor (HIF)-1-regulating genes. (a) Scheme of short hairpin RNA (shRNA) library screening. (b) HeLa cells transfected with small interfering RNA (siRNA) against the five candidate genes, CCAAT/enhancer-binding protein δ (CEBPD), transforming growth factor-beta-induced factor (TGIF), nuclear receptor superfamily 4A member 1 (NR4A1), paired mesoderm homeobox protein 2A (PHOX2A), or P300/CBP-associated factor (PCAF), were treated under hypoxia for 16 h to confirm the shRNA study results. The cells were processed for immunoblot analysis of HIF-1α and HIF-2α. Four genes except for PHOX2A showed a reduction in HIF-1α protein expression under hypoxia. The data are representative of three independent experiments, shown in duplicate. (c) Immunoblot analyses of HIF-1α (left panel) and HREluc activity (right panel) are shown representatively for siRNA-mediated CEBPD knockdown in HeLa cells. Knockdown of CEBPD decreased HIF-1α and hypoxic induction of HREluc activity in HeLa cells. (right panel) The ratio of luciferase reporter activity (firefly/TK-Renilla) to that of control siRNA under normoxia is indicated. Bar graph (mean±s.e.m. from three independent experiments) statistics performed using two-way analysis of variance (ANOVA) with Bonferroni post-hoc tests. *P<0.05. RAS, renal artery stenosis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig1: The identification of hypoxia-inducible factor (HIF)-1-regulating genes. (a) Scheme of short hairpin RNA (shRNA) library screening. (b) HeLa cells transfected with small interfering RNA (siRNA) against the five candidate genes, CCAAT/enhancer-binding protein δ (CEBPD), transforming growth factor-beta-induced factor (TGIF), nuclear receptor superfamily 4A member 1 (NR4A1), paired mesoderm homeobox protein 2A (PHOX2A), or P300/CBP-associated factor (PCAF), were treated under hypoxia for 16 h to confirm the shRNA study results. The cells were processed for immunoblot analysis of HIF-1α and HIF-2α. Four genes except for PHOX2A showed a reduction in HIF-1α protein expression under hypoxia. The data are representative of three independent experiments, shown in duplicate. (c) Immunoblot analyses of HIF-1α (left panel) and HREluc activity (right panel) are shown representatively for siRNA-mediated CEBPD knockdown in HeLa cells. Knockdown of CEBPD decreased HIF-1α and hypoxic induction of HREluc activity in HeLa cells. (right panel) The ratio of luciferase reporter activity (firefly/TK-Renilla) to that of control siRNA under normoxia is indicated. Bar graph (mean±s.e.m. from three independent experiments) statistics performed using two-way analysis of variance (ANOVA) with Bonferroni post-hoc tests. *P<0.05. RAS, renal artery stenosis.
Mentions: 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 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.

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