Limits...
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 inhibits the NF-κB/miR-21/PDCD4 axis in vivo.Wistar male rats (n = 6) were treated with 125 mg/kg/day of DCA by oral gavage for 1 and 3 days before sacrifice. Rats in the control group (n = 6) were administered with water as described in Material and Methods. (A) Real-Time RT-PCR analysis of miR-21. Results are expressed as mean ± SEM fold change. (B) Representative blots and quantitation, in fold-change from controls, of PDCD4, IκB/NF-κB ratio, PIDD-CC and active caspase-2 from liver of rats treated with DCA for 1 day (left) and 3 days (right). Blots were normalized to endogenous β-actin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4664913&req=5

f8: DCA inhibits the NF-κB/miR-21/PDCD4 axis in vivo.Wistar male rats (n = 6) were treated with 125 mg/kg/day of DCA by oral gavage for 1 and 3 days before sacrifice. Rats in the control group (n = 6) were administered with water as described in Material and Methods. (A) Real-Time RT-PCR analysis of miR-21. Results are expressed as mean ± SEM fold change. (B) Representative blots and quantitation, in fold-change from controls, of PDCD4, IκB/NF-κB ratio, PIDD-CC and active caspase-2 from liver of rats treated with DCA for 1 day (left) and 3 days (right). Blots were normalized to endogenous β-actin.

Mentions: To validate our in vitro results and further investigate the physiological relevance of the modulation of the miR-21/PDCD4 pathway by DCA, rats were administered with DCA by oral gavage for 1 to 3 days. As expected, total plasma bile acids, as well as ALT and AST levels increased with time in DCA-treated animals, compared with controls (data not shown). Hepatic miR-21 expression was reduced by  >25% (p < 0.05) and ~10% in animals administered with DCA for 1 and 3 days, respectively (Fig. 8A). Concomitantly, liver PDCD4 protein levels were increased by DCA at both 1 and 3 days of treatment (Fig. 8B). In parallel, hepatic total NF-κB protein levels were reduced in DCA-treated animals, while IκB levels increased, leading to a progressive and significant decrease in the NF-κB/IκB ratio throughout time, further reinforcing the role of this transcription factor in modulating the miR-21/PDCD4 pathway. Finally, both PIDD processing and caspase-2 activation were also increased in DCA-treated animals. Curiously, rats administered with DCA for 5 days displayed slightly increased miR-21 expression levels, although positive modulation of PDCD4 was still observed (data not shown), suggesting that sustained DCA administration engages activation of additional pathways of cell death. Overall, our results suggest that PIDDosome activation and consequent inhibition of the NF-κB/miR-21/PDCD4 pathway by DCA occurs both in vitro and in vivo, providing new mechanistic insights into its role as a putative pathogenic factor for hepatic disorders.


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 inhibits the NF-κB/miR-21/PDCD4 axis in vivo.Wistar male rats (n = 6) were treated with 125 mg/kg/day of DCA by oral gavage for 1 and 3 days before sacrifice. Rats in the control group (n = 6) were administered with water as described in Material and Methods. (A) Real-Time RT-PCR analysis of miR-21. Results are expressed as mean ± SEM fold change. (B) Representative blots and quantitation, in fold-change from controls, of PDCD4, IκB/NF-κB ratio, PIDD-CC and active caspase-2 from liver of rats treated with DCA for 1 day (left) and 3 days (right). Blots were normalized to endogenous β-actin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f8: DCA inhibits the NF-κB/miR-21/PDCD4 axis in vivo.Wistar male rats (n = 6) were treated with 125 mg/kg/day of DCA by oral gavage for 1 and 3 days before sacrifice. Rats in the control group (n = 6) were administered with water as described in Material and Methods. (A) Real-Time RT-PCR analysis of miR-21. Results are expressed as mean ± SEM fold change. (B) Representative blots and quantitation, in fold-change from controls, of PDCD4, IκB/NF-κB ratio, PIDD-CC and active caspase-2 from liver of rats treated with DCA for 1 day (left) and 3 days (right). Blots were normalized to endogenous β-actin.
Mentions: To validate our in vitro results and further investigate the physiological relevance of the modulation of the miR-21/PDCD4 pathway by DCA, rats were administered with DCA by oral gavage for 1 to 3 days. As expected, total plasma bile acids, as well as ALT and AST levels increased with time in DCA-treated animals, compared with controls (data not shown). Hepatic miR-21 expression was reduced by  >25% (p < 0.05) and ~10% in animals administered with DCA for 1 and 3 days, respectively (Fig. 8A). Concomitantly, liver PDCD4 protein levels were increased by DCA at both 1 and 3 days of treatment (Fig. 8B). In parallel, hepatic total NF-κB protein levels were reduced in DCA-treated animals, while IκB levels increased, leading to a progressive and significant decrease in the NF-κB/IκB ratio throughout time, further reinforcing the role of this transcription factor in modulating the miR-21/PDCD4 pathway. Finally, both PIDD processing and caspase-2 activation were also increased in DCA-treated animals. Curiously, rats administered with DCA for 5 days displayed slightly increased miR-21 expression levels, although positive modulation of PDCD4 was still observed (data not shown), suggesting that sustained DCA administration engages activation of additional pathways of cell death. Overall, our results suggest that PIDDosome activation and consequent inhibition of the NF-κB/miR-21/PDCD4 pathway by DCA occurs both in vitro and in vivo, providing new mechanistic insights into its role as a putative pathogenic factor for hepatic disorders.

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