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Inhibition of NF-κB by deoxycholic acid induces miR-21/PDCD4-dependent hepatocelular apoptosis.

M Rodrigues P, B Afonso M, L Simão A, M Borralho P, M P Rodrigues C, E Castro R - Sci Rep (2015)

Bottom Line: In fact, NF-κB overexpression or constitutive activation halted miR-21-dependent apoptosis by DCA while opposite results were observed upon NF-κB inhibition.In turn, DCA-induced oxidative stress resulted in caspase-2 activation and NF-κB/miR-21 inhibition, in a PIDD-dependent manner.These signalling circuits may constitute appealing targets for bile acid-associated liver pathologies.

View Article: PubMed Central - PubMed

Affiliation: Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, 1649-003 Lisboa, Portugal.

ABSTRACT
MicroRNAs (miRNAs/miRs) are key regulators of liver metabolism, while toxic bile acids participate in the development of several liver diseases. We previously demonstrated that deoxycholic acid (DCA), a cytotoxic bile acid implicated in the pathogenesis of non-alcoholic fatty liver disease, inhibits miR-21 expression in hepatocytes. Here, we investigated the mechanisms by which DCA modulates miR-21 and whether miR-21 contributes for DCA-induced cytotoxicity. DCA inhibited miR-21 expression in primary rat hepatocytes in a dose-dependent manner, and increased miR-21 pro-apoptotic target programmed cell death 4 (PDCD4) and apoptosis. Both miR-21 overexpression and PDCD4 silencing hampered DCA-induced cell death. Further, DCA decreased NF-κB activity, shown to represent an upstream mechanism leading to modulation of the miR-21/PDCD4 pathway. In fact, NF-κB overexpression or constitutive activation halted miR-21-dependent apoptosis by DCA while opposite results were observed upon NF-κB inhibition. In turn, DCA-induced oxidative stress resulted in caspase-2 activation and NF-κB/miR-21 inhibition, in a PIDD-dependent manner. Finally, modulation of the NF-κB/miR-21/PDCD4 pro-apoptotic pathway by DCA was also shown to occur in the rat liver in vivo. These signalling circuits may constitute appealing targets for bile acid-associated liver pathologies.

No MeSH data available.


Related in: MedlinePlus

Modulation of the NF-κB pathway halts DCA-modulation of miR-21/PDCD4 and cell viability.Cells were transfected with a plasmid encoding a constitutively active form of IKK (CA-IKK), NF-κB or an empty vector (pcDNA 3.1) or incubated with 10 μM BAY 11-7085 and treated with 100 μM DCA or no addition as described in Materials and Methods. (A) NF-κB transcriptional activity (n = 6). Cells were transfected with a mixture of an inducible NF-κB responsive construct, encoding the firefly luciferase reporter gene, and a constitutively expressing Renilla luciferase construct, as an internal standard control. (B) Real-Time RT-PCR analysis of miR-21 (top; n = 5) and immunoblotting of PDCD4 (bottom; n = 5). Representative blots are shown. Blots were normalized to endogenous β-actin. (C) Cell viability, measured by the ApoTox-GloTM Triplex assay (n = 5). (D) NF-κB transcriptional activity (n = 4). (E) Real-time RT-PCR analysis of miR-21 (top; n = 6) and immunoblotting of PDCD4 (bottom; n = 8). Representative blots are shown. Blots were normalized to endogenous β-actin. (F) Cell death, measured by LDH assay (top; n = 8) and caspase-3/7 activity measured by the ApoTox-GloTM Triplex assay (bottom; n = 6). Results are expressed as mean ± SEM fold change.
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f5: Modulation of the NF-κB pathway halts DCA-modulation of miR-21/PDCD4 and cell viability.Cells were transfected with a plasmid encoding a constitutively active form of IKK (CA-IKK), NF-κB or an empty vector (pcDNA 3.1) or incubated with 10 μM BAY 11-7085 and treated with 100 μM DCA or no addition as described in Materials and Methods. (A) NF-κB transcriptional activity (n = 6). Cells were transfected with a mixture of an inducible NF-κB responsive construct, encoding the firefly luciferase reporter gene, and a constitutively expressing Renilla luciferase construct, as an internal standard control. (B) Real-Time RT-PCR analysis of miR-21 (top; n = 5) and immunoblotting of PDCD4 (bottom; n = 5). Representative blots are shown. Blots were normalized to endogenous β-actin. (C) Cell viability, measured by the ApoTox-GloTM Triplex assay (n = 5). (D) NF-κB transcriptional activity (n = 4). (E) Real-time RT-PCR analysis of miR-21 (top; n = 6) and immunoblotting of PDCD4 (bottom; n = 8). Representative blots are shown. Blots were normalized to endogenous β-actin. (F) Cell death, measured by LDH assay (top; n = 8) and caspase-3/7 activity measured by the ApoTox-GloTM Triplex assay (bottom; n = 6). Results are expressed as mean ± SEM fold change.

Mentions: To elucidate the role of NF-κB in our model, we next differently modulated the NF-κB pathway by using a constitutively active form of IKK (CA-IKK), that overactivates the NF-κB pathway by inducing IκB degradation36, as well as an NF-κB overexpression construct37. Transfection of cells with the CA-IKK and NF-κB plasmids increased NF-κB transcriptional activity by ~2- and 9-fold, respectively (p < 0.01), while co-incubation of cells with DCA significantly hampered NF-κB activation (p < 0.05) (Fig. 5A). In addition, both CA-IKK and NF-κB overexpression induced miR-21 expression by 40 and 80%, respectively, when compared with controls (p < 0.05). Both these effects were almost completely reverted to control levels by DCA (p < 0.05) (Fig. 5B top). Additionally, PDCD4 expression profiles displayed an opposite pattern to miR-21 expression levels, further confirming the relevance of this pro-apoptotic target during DCA-dependent modulation of miR-21 via NF-κB (Fig. 5B bottom). Finally, activation of the NF-κB pathway by CA-IKK and NF-κB overexpression plasmids resulted in increased cellular viability (at least p < 0.05), significantly hampered upon DCA co-incubation (p < 0.05) (Fig. 5C).


Inhibition of NF-κB by deoxycholic acid induces miR-21/PDCD4-dependent hepatocelular apoptosis.

M Rodrigues P, B Afonso M, L Simão A, M Borralho P, M P Rodrigues C, E Castro R - Sci Rep (2015)

Modulation of the NF-κB pathway halts DCA-modulation of miR-21/PDCD4 and cell viability.Cells were transfected with a plasmid encoding a constitutively active form of IKK (CA-IKK), NF-κB or an empty vector (pcDNA 3.1) or incubated with 10 μM BAY 11-7085 and treated with 100 μM DCA or no addition as described in Materials and Methods. (A) NF-κB transcriptional activity (n = 6). Cells were transfected with a mixture of an inducible NF-κB responsive construct, encoding the firefly luciferase reporter gene, and a constitutively expressing Renilla luciferase construct, as an internal standard control. (B) Real-Time RT-PCR analysis of miR-21 (top; n = 5) and immunoblotting of PDCD4 (bottom; n = 5). Representative blots are shown. Blots were normalized to endogenous β-actin. (C) Cell viability, measured by the ApoTox-GloTM Triplex assay (n = 5). (D) NF-κB transcriptional activity (n = 4). (E) Real-time RT-PCR analysis of miR-21 (top; n = 6) and immunoblotting of PDCD4 (bottom; n = 8). Representative blots are shown. Blots were normalized to endogenous β-actin. (F) Cell death, measured by LDH assay (top; n = 8) and caspase-3/7 activity measured by the ApoTox-GloTM Triplex assay (bottom; n = 6). Results are expressed as mean ± SEM fold change.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f5: Modulation of the NF-κB pathway halts DCA-modulation of miR-21/PDCD4 and cell viability.Cells were transfected with a plasmid encoding a constitutively active form of IKK (CA-IKK), NF-κB or an empty vector (pcDNA 3.1) or incubated with 10 μM BAY 11-7085 and treated with 100 μM DCA or no addition as described in Materials and Methods. (A) NF-κB transcriptional activity (n = 6). Cells were transfected with a mixture of an inducible NF-κB responsive construct, encoding the firefly luciferase reporter gene, and a constitutively expressing Renilla luciferase construct, as an internal standard control. (B) Real-Time RT-PCR analysis of miR-21 (top; n = 5) and immunoblotting of PDCD4 (bottom; n = 5). Representative blots are shown. Blots were normalized to endogenous β-actin. (C) Cell viability, measured by the ApoTox-GloTM Triplex assay (n = 5). (D) NF-κB transcriptional activity (n = 4). (E) Real-time RT-PCR analysis of miR-21 (top; n = 6) and immunoblotting of PDCD4 (bottom; n = 8). Representative blots are shown. Blots were normalized to endogenous β-actin. (F) Cell death, measured by LDH assay (top; n = 8) and caspase-3/7 activity measured by the ApoTox-GloTM Triplex assay (bottom; n = 6). Results are expressed as mean ± SEM fold change.
Mentions: To elucidate the role of NF-κB in our model, we next differently modulated the NF-κB pathway by using a constitutively active form of IKK (CA-IKK), that overactivates the NF-κB pathway by inducing IκB degradation36, as well as an NF-κB overexpression construct37. Transfection of cells with the CA-IKK and NF-κB plasmids increased NF-κB transcriptional activity by ~2- and 9-fold, respectively (p < 0.01), while co-incubation of cells with DCA significantly hampered NF-κB activation (p < 0.05) (Fig. 5A). In addition, both CA-IKK and NF-κB overexpression induced miR-21 expression by 40 and 80%, respectively, when compared with controls (p < 0.05). Both these effects were almost completely reverted to control levels by DCA (p < 0.05) (Fig. 5B top). Additionally, PDCD4 expression profiles displayed an opposite pattern to miR-21 expression levels, further confirming the relevance of this pro-apoptotic target during DCA-dependent modulation of miR-21 via NF-κB (Fig. 5B bottom). Finally, activation of the NF-κB pathway by CA-IKK and NF-κB overexpression plasmids resulted in increased cellular viability (at least p < 0.05), significantly hampered upon DCA co-incubation (p < 0.05) (Fig. 5C).

Bottom Line: In fact, NF-κB overexpression or constitutive activation halted miR-21-dependent apoptosis by DCA while opposite results were observed upon NF-κB inhibition.In turn, DCA-induced oxidative stress resulted in caspase-2 activation and NF-κB/miR-21 inhibition, in a PIDD-dependent manner.These signalling circuits may constitute appealing targets for bile acid-associated liver pathologies.

View Article: PubMed Central - PubMed

Affiliation: Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, 1649-003 Lisboa, Portugal.

ABSTRACT
MicroRNAs (miRNAs/miRs) are key regulators of liver metabolism, while toxic bile acids participate in the development of several liver diseases. We previously demonstrated that deoxycholic acid (DCA), a cytotoxic bile acid implicated in the pathogenesis of non-alcoholic fatty liver disease, inhibits miR-21 expression in hepatocytes. Here, we investigated the mechanisms by which DCA modulates miR-21 and whether miR-21 contributes for DCA-induced cytotoxicity. DCA inhibited miR-21 expression in primary rat hepatocytes in a dose-dependent manner, and increased miR-21 pro-apoptotic target programmed cell death 4 (PDCD4) and apoptosis. Both miR-21 overexpression and PDCD4 silencing hampered DCA-induced cell death. Further, DCA decreased NF-κB activity, shown to represent an upstream mechanism leading to modulation of the miR-21/PDCD4 pathway. In fact, NF-κB overexpression or constitutive activation halted miR-21-dependent apoptosis by DCA while opposite results were observed upon NF-κB inhibition. In turn, DCA-induced oxidative stress resulted in caspase-2 activation and NF-κB/miR-21 inhibition, in a PIDD-dependent manner. Finally, modulation of the NF-κB/miR-21/PDCD4 pro-apoptotic pathway by DCA was also shown to occur in the rat liver in vivo. These signalling circuits may constitute appealing targets for bile acid-associated liver pathologies.

No MeSH data available.


Related in: MedlinePlus