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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

DCA-induced oxidative stress activates PIDD processing and caspase-2 in a dose-dependent manner.Primary rat hepatocytes were isolated as described in Materials and Methods and treated with 25 to 200 μM DCA or no addition (control) for 24 h. (A) Immunoblotting of PIDD-CC (top; n = 5) and active caspase-2 (bottom; n = 5). (B) Immunoblotting of PIDD-CC (left; n = 5) and active caspase-2 (right; n = 5). (C) Immunoblotting of IκB (n = 5). Cells were pre-treated with 5 mM NAC for 1 h and, when indicated, incubated with 100 μM DCA for 24 h. Representative blots are shown. Blots were normalized to endogenous β-actin. Results are expressed as mean ± SEM fold change.
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f7: DCA-induced oxidative stress activates PIDD processing and caspase-2 in a dose-dependent manner.Primary rat hepatocytes were isolated as described in Materials and Methods and treated with 25 to 200 μM DCA or no addition (control) for 24 h. (A) Immunoblotting of PIDD-CC (top; n = 5) and active caspase-2 (bottom; n = 5). (B) Immunoblotting of PIDD-CC (left; n = 5) and active caspase-2 (right; n = 5). (C) Immunoblotting of IκB (n = 5). Cells were pre-treated with 5 mM NAC for 1 h and, when indicated, incubated with 100 μM DCA for 24 h. Representative blots are shown. Blots were normalized to endogenous β-actin. Results are expressed as mean ± SEM fold change.

Mentions: To investigate this hypothesis, we measured PIDD processing and caspase-2 activation in primary rat hepatocytes incubated with 25–200 μM DCA for 24 h. In agreement with the increase in oxidative stress, levels of PIDD-CC and active caspase-2 were augmented by DCA in a dose-dependent manner up to ~2.9- and 8-fold, respectively (at least p < 0.05) (Fig. 7A). Of note, caspase-2 knockdown, using a specific siRNA, partially abrogated DCA-induced apoptosis (p < 0.05) (Figure S3), suggesting that caspase-2 activation is an important pro-apoptotic event during DCA-induced cell death. Finally, under antioxidant conditions, DCA-dependent PIDD processing was impaired (p < 0.05) and activation of caspase-2 significantly reduced (p < 0.05) (Fig. 7B). Importantly, DCA-induced IκB levels were also strongly reduced in the presence of NAC, comparing with DCA alone (Fig. 7C). Altogether, these results indicate that DCA-induced oxidative stress is at least partially responsible for inhibition of NF-κB in a PIDD-dependent manner, thus establishing a novel link between genotoxic stress, miR-21 inhibition and cellular toxicity.


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)

DCA-induced oxidative stress activates PIDD processing and caspase-2 in a dose-dependent manner.Primary rat hepatocytes were isolated as described in Materials and Methods and treated with 25 to 200 μM DCA or no addition (control) for 24 h. (A) Immunoblotting of PIDD-CC (top; n = 5) and active caspase-2 (bottom; n = 5). (B) Immunoblotting of PIDD-CC (left; n = 5) and active caspase-2 (right; n = 5). (C) Immunoblotting of IκB (n = 5). Cells were pre-treated with 5 mM NAC for 1 h and, when indicated, incubated with 100 μM DCA for 24 h. Representative blots are shown. Blots were normalized to endogenous β-actin. Results are expressed as mean ± SEM fold change.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4664913&req=5

f7: DCA-induced oxidative stress activates PIDD processing and caspase-2 in a dose-dependent manner.Primary rat hepatocytes were isolated as described in Materials and Methods and treated with 25 to 200 μM DCA or no addition (control) for 24 h. (A) Immunoblotting of PIDD-CC (top; n = 5) and active caspase-2 (bottom; n = 5). (B) Immunoblotting of PIDD-CC (left; n = 5) and active caspase-2 (right; n = 5). (C) Immunoblotting of IκB (n = 5). Cells were pre-treated with 5 mM NAC for 1 h and, when indicated, incubated with 100 μM DCA for 24 h. Representative blots are shown. Blots were normalized to endogenous β-actin. Results are expressed as mean ± SEM fold change.
Mentions: To investigate this hypothesis, we measured PIDD processing and caspase-2 activation in primary rat hepatocytes incubated with 25–200 μM DCA for 24 h. In agreement with the increase in oxidative stress, levels of PIDD-CC and active caspase-2 were augmented by DCA in a dose-dependent manner up to ~2.9- and 8-fold, respectively (at least p < 0.05) (Fig. 7A). Of note, caspase-2 knockdown, using a specific siRNA, partially abrogated DCA-induced apoptosis (p < 0.05) (Figure S3), suggesting that caspase-2 activation is an important pro-apoptotic event during DCA-induced cell death. Finally, under antioxidant conditions, DCA-dependent PIDD processing was impaired (p < 0.05) and activation of caspase-2 significantly reduced (p < 0.05) (Fig. 7B). Importantly, DCA-induced IκB levels were also strongly reduced in the presence of NAC, comparing with DCA alone (Fig. 7C). Altogether, these results indicate that DCA-induced oxidative stress is at least partially responsible for inhibition of NF-κB in a PIDD-dependent manner, thus establishing a novel link between genotoxic stress, miR-21 inhibition and cellular toxicity.

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