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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

Hypoxia-inducible factor (HIF)-1 regulation by CCAAT/enhancer-binding protein δ (CEBPD) in tubular epithelial cells. (a) CEBPD protein is upregulated by hypoxia (0.1% O2) in HK-2 cells. (b) Knockdown of CEBPD decreased HIF-1α protein expression. Right panel shows densitometrical quantification of HIF-1α protein under hypoxic condition of three independent experiments. (c) Knockdown of CEBPD significantly decreased HREluc activity under hypoxia. The ratio of luciferase reporter activity (firefly/CMV-Renilla) to that of control small interfering RNA (siRNA) under normoxia is indicated. (d) Real-time qRT-PCR for HIF-1 target genes, glucose transporter 1 (GLUT1) and vascular endothelial growth factor (VEGF), also demonstrated that their induction under hypoxia is dependent on CEBPD. Bar graph (mean±s.e.m. or representative of three independent experiments) statistics performed using two-way analysis of variance (ANOVA) with Bonferroni post-hoc tests. *P<0.05 and **P<0.01.
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fig3: Hypoxia-inducible factor (HIF)-1 regulation by CCAAT/enhancer-binding protein δ (CEBPD) in tubular epithelial cells. (a) CEBPD protein is upregulated by hypoxia (0.1% O2) in HK-2 cells. (b) Knockdown of CEBPD decreased HIF-1α protein expression. Right panel shows densitometrical quantification of HIF-1α protein under hypoxic condition of three independent experiments. (c) Knockdown of CEBPD significantly decreased HREluc activity under hypoxia. The ratio of luciferase reporter activity (firefly/CMV-Renilla) to that of control small interfering RNA (siRNA) under normoxia is indicated. (d) Real-time qRT-PCR for HIF-1 target genes, glucose transporter 1 (GLUT1) and vascular endothelial growth factor (VEGF), also demonstrated that their induction under hypoxia is dependent on CEBPD. Bar graph (mean±s.e.m. or representative of three independent experiments) statistics performed using two-way analysis of variance (ANOVA) with Bonferroni post-hoc tests. *P<0.05 and **P<0.01.

Mentions: 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 and this effect is strictly retained within the context of the CEBPD–HIF-1 axis.


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

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

Hypoxia-inducible factor (HIF)-1 regulation by CCAAT/enhancer-binding protein δ (CEBPD) in tubular epithelial cells. (a) CEBPD protein is upregulated by hypoxia (0.1% O2) in HK-2 cells. (b) Knockdown of CEBPD decreased HIF-1α protein expression. Right panel shows densitometrical quantification of HIF-1α protein under hypoxic condition of three independent experiments. (c) Knockdown of CEBPD significantly decreased HREluc activity under hypoxia. The ratio of luciferase reporter activity (firefly/CMV-Renilla) to that of control small interfering RNA (siRNA) under normoxia is indicated. (d) Real-time qRT-PCR for HIF-1 target genes, glucose transporter 1 (GLUT1) and vascular endothelial growth factor (VEGF), also demonstrated that their induction under hypoxia is dependent on CEBPD. Bar graph (mean±s.e.m. or representative of three independent experiments) statistics performed using two-way analysis of variance (ANOVA) with Bonferroni post-hoc tests. *P<0.05 and **P<0.01.
© Copyright Policy - open-access
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Show All Figures
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fig3: Hypoxia-inducible factor (HIF)-1 regulation by CCAAT/enhancer-binding protein δ (CEBPD) in tubular epithelial cells. (a) CEBPD protein is upregulated by hypoxia (0.1% O2) in HK-2 cells. (b) Knockdown of CEBPD decreased HIF-1α protein expression. Right panel shows densitometrical quantification of HIF-1α protein under hypoxic condition of three independent experiments. (c) Knockdown of CEBPD significantly decreased HREluc activity under hypoxia. The ratio of luciferase reporter activity (firefly/CMV-Renilla) to that of control small interfering RNA (siRNA) under normoxia is indicated. (d) Real-time qRT-PCR for HIF-1 target genes, glucose transporter 1 (GLUT1) and vascular endothelial growth factor (VEGF), also demonstrated that their induction under hypoxia is dependent on CEBPD. Bar graph (mean±s.e.m. or representative of three independent experiments) statistics performed using two-way analysis of variance (ANOVA) with Bonferroni post-hoc tests. *P<0.05 and **P<0.01.
Mentions: 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 and this effect is strictly retained within the context of the CEBPD–HIF-1 axis.

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