Limits...
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 nuclear factor (NF)-κB pathway regulates the CCAAT/enhancer-binding protein δ/hypoxia-inducible factor 1 (CEBPD/HIF-1) pathway under hypoxia or interleukin (IL)-1β treatment. (a) NF-κB reporter activity was measured under normoxia or hypoxia. The pRL-TK vector served as a control. Hypoxia (0.1% O2, 6 h) increased NF-κB activity in HK-2 cells. (b) Inhibition of the NF-κB pathway by p65 knockdown significantly decreased HREluc activity under hypoxia in HK-2 cells. (c) (Upper panel) HK-2 cells were pretreated with BAY11-7082 (IKK inhibitor, 10 μM) or dimethyl sulfoxide (for control) for 30 min and then treated with hypoxia (upper left panel) or IL-1β (1 ng/ml; upper right panel) for 6 h. CEBPD and HIF-1α protein induction under both conditions were inhibited. The same phenomena were observed using small interfering RNA (siRNA) against p65-mediated NF-κB inhibition (lower left panel for hypoxia and lower right panel for IL-1β). (d) Human CEBPD stable overexpression HK-2 clones were generated using a retroviral system. Upon NF-κB inhibition by BAY11-7082 (10 μM), HIF-1α protein induction under hypoxia (left panel) or IL-1β (1 ng/ml; right panel) was reduced in control HK-2 cells. In stable CEBPD-expressed HK-2 cells, the HIF-1α protein induction was restored under both conditions, indicating the necessity for HIF-1α protein regulation by CEBPD. The endogenous CEBPD protein is hardly detected, because exposure time was optimized for the overexpressed protein. Bar graph (mean±s.e.m. or representative of at least three independent experiments) statistics performed using two-way analysis of variance with Bonferroni's post-hoc tests. **P<0.01. (e) A scheme of a pathway proposed in this study. CEBPD is upregulated in tubular epithelial cells, either by hypoxia or IL-1β via the NF-κB-dependent pathway and regulates HIF-1 expression and its transcriptional activity.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4526770&req=5

fig7: The nuclear factor (NF)-κB pathway regulates the CCAAT/enhancer-binding protein δ/hypoxia-inducible factor 1 (CEBPD/HIF-1) pathway under hypoxia or interleukin (IL)-1β treatment. (a) NF-κB reporter activity was measured under normoxia or hypoxia. The pRL-TK vector served as a control. Hypoxia (0.1% O2, 6 h) increased NF-κB activity in HK-2 cells. (b) Inhibition of the NF-κB pathway by p65 knockdown significantly decreased HREluc activity under hypoxia in HK-2 cells. (c) (Upper panel) HK-2 cells were pretreated with BAY11-7082 (IKK inhibitor, 10 μM) or dimethyl sulfoxide (for control) for 30 min and then treated with hypoxia (upper left panel) or IL-1β (1 ng/ml; upper right panel) for 6 h. CEBPD and HIF-1α protein induction under both conditions were inhibited. The same phenomena were observed using small interfering RNA (siRNA) against p65-mediated NF-κB inhibition (lower left panel for hypoxia and lower right panel for IL-1β). (d) Human CEBPD stable overexpression HK-2 clones were generated using a retroviral system. Upon NF-κB inhibition by BAY11-7082 (10 μM), HIF-1α protein induction under hypoxia (left panel) or IL-1β (1 ng/ml; right panel) was reduced in control HK-2 cells. In stable CEBPD-expressed HK-2 cells, the HIF-1α protein induction was restored under both conditions, indicating the necessity for HIF-1α protein regulation by CEBPD. The endogenous CEBPD protein is hardly detected, because exposure time was optimized for the overexpressed protein. Bar graph (mean±s.e.m. or representative of at least three independent experiments) statistics performed using two-way analysis of variance with Bonferroni's post-hoc tests. **P<0.01. (e) A scheme of a pathway proposed in this study. CEBPD is upregulated in tubular epithelial cells, either by hypoxia or IL-1β via the NF-κB-dependent pathway and regulates HIF-1 expression and its transcriptional activity.

Mentions: 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, 35 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).


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 nuclear factor (NF)-κB pathway regulates the CCAAT/enhancer-binding protein δ/hypoxia-inducible factor 1 (CEBPD/HIF-1) pathway under hypoxia or interleukin (IL)-1β treatment. (a) NF-κB reporter activity was measured under normoxia or hypoxia. The pRL-TK vector served as a control. Hypoxia (0.1% O2, 6 h) increased NF-κB activity in HK-2 cells. (b) Inhibition of the NF-κB pathway by p65 knockdown significantly decreased HREluc activity under hypoxia in HK-2 cells. (c) (Upper panel) HK-2 cells were pretreated with BAY11-7082 (IKK inhibitor, 10 μM) or dimethyl sulfoxide (for control) for 30 min and then treated with hypoxia (upper left panel) or IL-1β (1 ng/ml; upper right panel) for 6 h. CEBPD and HIF-1α protein induction under both conditions were inhibited. The same phenomena were observed using small interfering RNA (siRNA) against p65-mediated NF-κB inhibition (lower left panel for hypoxia and lower right panel for IL-1β). (d) Human CEBPD stable overexpression HK-2 clones were generated using a retroviral system. Upon NF-κB inhibition by BAY11-7082 (10 μM), HIF-1α protein induction under hypoxia (left panel) or IL-1β (1 ng/ml; right panel) was reduced in control HK-2 cells. In stable CEBPD-expressed HK-2 cells, the HIF-1α protein induction was restored under both conditions, indicating the necessity for HIF-1α protein regulation by CEBPD. The endogenous CEBPD protein is hardly detected, because exposure time was optimized for the overexpressed protein. Bar graph (mean±s.e.m. or representative of at least three independent experiments) statistics performed using two-way analysis of variance with Bonferroni's post-hoc tests. **P<0.01. (e) A scheme of a pathway proposed in this study. CEBPD is upregulated in tubular epithelial cells, either by hypoxia or IL-1β via the NF-κB-dependent pathway and regulates HIF-1 expression and its transcriptional activity.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig7: The nuclear factor (NF)-κB pathway regulates the CCAAT/enhancer-binding protein δ/hypoxia-inducible factor 1 (CEBPD/HIF-1) pathway under hypoxia or interleukin (IL)-1β treatment. (a) NF-κB reporter activity was measured under normoxia or hypoxia. The pRL-TK vector served as a control. Hypoxia (0.1% O2, 6 h) increased NF-κB activity in HK-2 cells. (b) Inhibition of the NF-κB pathway by p65 knockdown significantly decreased HREluc activity under hypoxia in HK-2 cells. (c) (Upper panel) HK-2 cells were pretreated with BAY11-7082 (IKK inhibitor, 10 μM) or dimethyl sulfoxide (for control) for 30 min and then treated with hypoxia (upper left panel) or IL-1β (1 ng/ml; upper right panel) for 6 h. CEBPD and HIF-1α protein induction under both conditions were inhibited. The same phenomena were observed using small interfering RNA (siRNA) against p65-mediated NF-κB inhibition (lower left panel for hypoxia and lower right panel for IL-1β). (d) Human CEBPD stable overexpression HK-2 clones were generated using a retroviral system. Upon NF-κB inhibition by BAY11-7082 (10 μM), HIF-1α protein induction under hypoxia (left panel) or IL-1β (1 ng/ml; right panel) was reduced in control HK-2 cells. In stable CEBPD-expressed HK-2 cells, the HIF-1α protein induction was restored under both conditions, indicating the necessity for HIF-1α protein regulation by CEBPD. The endogenous CEBPD protein is hardly detected, because exposure time was optimized for the overexpressed protein. Bar graph (mean±s.e.m. or representative of at least three independent experiments) statistics performed using two-way analysis of variance with Bonferroni's post-hoc tests. **P<0.01. (e) A scheme of a pathway proposed in this study. CEBPD is upregulated in tubular epithelial cells, either by hypoxia or IL-1β via the NF-κB-dependent pathway and regulates HIF-1 expression and its transcriptional activity.
Mentions: 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, 35 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).

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