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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/hypoxia-inducible factor 1 (CEBPD/HIF-1) pathway activation under normoxia by interleukin (IL)-1β. (a) HK-2 cells were treated with IL-1β (0–1 ng/ml) under 21% O2 for 6 h and assessed for CEBPD and monocyte chemotactic protein-1 (MCP-1) mRNA by real-time qRT-PCR. CEBPD and MCP-1 mRNA levels were increased in a dose-dependent manner. (b) Immunoblot analysis of CEBPD and HIF-1α expression levels in HK-2 cells under IL-1β treatment. CEBPD and HIF-1α protein levels increased in a dose- and time-dependent manner. (Upper panel) HK-2 cells were treated with IL-1β (0–1 ng/ml) under 21% O2 or 1% O2 for 6 h. (Lower panel) HK-2 cells were treated with IL-1β (1 ng/ml) for 0–8 h under normoxia. (c) HK-2 cells transfected with small interfering RNA (siRNA) against CEBPD were treated with IL-1β for 6 h under 21% O2 or 1% O2 and assessed for HIF-1α expression through immunoblot analysis. Knockdown of CEBPD reduced the HIF-1α induction by IL-1β both under normoxia and hypoxia. Bar graph (mean±s.e.m. of three independent experiments) statistics performed using one-way analysis of variance (ANOVA) with Dunnett's post-hoc tests. *P<0.05 and **P<0.01.
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fig5: CCAAT/enhancer-binding protein δ/hypoxia-inducible factor 1 (CEBPD/HIF-1) pathway activation under normoxia by interleukin (IL)-1β. (a) HK-2 cells were treated with IL-1β (0–1 ng/ml) under 21% O2 for 6 h and assessed for CEBPD and monocyte chemotactic protein-1 (MCP-1) mRNA by real-time qRT-PCR. CEBPD and MCP-1 mRNA levels were increased in a dose-dependent manner. (b) Immunoblot analysis of CEBPD and HIF-1α expression levels in HK-2 cells under IL-1β treatment. CEBPD and HIF-1α protein levels increased in a dose- and time-dependent manner. (Upper panel) HK-2 cells were treated with IL-1β (0–1 ng/ml) under 21% O2 or 1% O2 for 6 h. (Lower panel) HK-2 cells were treated with IL-1β (1 ng/ml) for 0–8 h under normoxia. (c) HK-2 cells transfected with small interfering RNA (siRNA) against CEBPD were treated with IL-1β for 6 h under 21% O2 or 1% O2 and assessed for HIF-1α expression through immunoblot analysis. Knockdown of CEBPD reduced the HIF-1α induction by IL-1β both under normoxia and hypoxia. Bar graph (mean±s.e.m. of three independent experiments) statistics performed using one-way analysis of variance (ANOVA) with Dunnett's post-hoc tests. *P<0.05 and **P<0.01.

Mentions: 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.


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/hypoxia-inducible factor 1 (CEBPD/HIF-1) pathway activation under normoxia by interleukin (IL)-1β. (a) HK-2 cells were treated with IL-1β (0–1 ng/ml) under 21% O2 for 6 h and assessed for CEBPD and monocyte chemotactic protein-1 (MCP-1) mRNA by real-time qRT-PCR. CEBPD and MCP-1 mRNA levels were increased in a dose-dependent manner. (b) Immunoblot analysis of CEBPD and HIF-1α expression levels in HK-2 cells under IL-1β treatment. CEBPD and HIF-1α protein levels increased in a dose- and time-dependent manner. (Upper panel) HK-2 cells were treated with IL-1β (0–1 ng/ml) under 21% O2 or 1% O2 for 6 h. (Lower panel) HK-2 cells were treated with IL-1β (1 ng/ml) for 0–8 h under normoxia. (c) HK-2 cells transfected with small interfering RNA (siRNA) against CEBPD were treated with IL-1β for 6 h under 21% O2 or 1% O2 and assessed for HIF-1α expression through immunoblot analysis. Knockdown of CEBPD reduced the HIF-1α induction by IL-1β both under normoxia and hypoxia. Bar graph (mean±s.e.m. of three independent experiments) statistics performed using one-way analysis of variance (ANOVA) with Dunnett's post-hoc tests. *P<0.05 and **P<0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526770&req=5

fig5: CCAAT/enhancer-binding protein δ/hypoxia-inducible factor 1 (CEBPD/HIF-1) pathway activation under normoxia by interleukin (IL)-1β. (a) HK-2 cells were treated with IL-1β (0–1 ng/ml) under 21% O2 for 6 h and assessed for CEBPD and monocyte chemotactic protein-1 (MCP-1) mRNA by real-time qRT-PCR. CEBPD and MCP-1 mRNA levels were increased in a dose-dependent manner. (b) Immunoblot analysis of CEBPD and HIF-1α expression levels in HK-2 cells under IL-1β treatment. CEBPD and HIF-1α protein levels increased in a dose- and time-dependent manner. (Upper panel) HK-2 cells were treated with IL-1β (0–1 ng/ml) under 21% O2 or 1% O2 for 6 h. (Lower panel) HK-2 cells were treated with IL-1β (1 ng/ml) for 0–8 h under normoxia. (c) HK-2 cells transfected with small interfering RNA (siRNA) against CEBPD were treated with IL-1β for 6 h under 21% O2 or 1% O2 and assessed for HIF-1α expression through immunoblot analysis. Knockdown of CEBPD reduced the HIF-1α induction by IL-1β both under normoxia and hypoxia. Bar graph (mean±s.e.m. of three independent experiments) statistics performed using one-way analysis of variance (ANOVA) with Dunnett's post-hoc tests. *P<0.05 and **P<0.01.
Mentions: 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.

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