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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 inhibits NF-κB transcriptional activity in primary rat hepatocytes in a dose-dependent manner.Cells were isolated and plated as described in Materials and Methods and treated with 25 to 200 μM DCA or no addition (control) for 24 h. (A) Immunoblotting of total NF-κB (top) and IκB (middle); NF-κB/IκB ratio (bottom; n = 5). Representative blots are shown. Blots were normalized to endogenous β-actin. (B) Immunoblotting of nuclear NF-κB (left) and cytoplasmatic NF-κB (right; n = 7). HDAC1 and GAPDH were used as nuclear and cytoplasmatic loading controls, respectively, as well as indicators for cross-contamination of protein fractions. Representative blots are shown. (C) NF-κB transcriptional activity (n = 5). 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. Results are expressed as mean ± SEM fold change.
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f4: DCA inhibits NF-κB transcriptional activity in primary rat hepatocytes in a dose-dependent manner.Cells were isolated and plated as described in Materials and Methods and treated with 25 to 200 μM DCA or no addition (control) for 24 h. (A) Immunoblotting of total NF-κB (top) and IκB (middle); NF-κB/IκB ratio (bottom; n = 5). Representative blots are shown. Blots were normalized to endogenous β-actin. (B) Immunoblotting of nuclear NF-κB (left) and cytoplasmatic NF-κB (right; n = 7). HDAC1 and GAPDH were used as nuclear and cytoplasmatic loading controls, respectively, as well as indicators for cross-contamination of protein fractions. Representative blots are shown. (C) NF-κB transcriptional activity (n = 5). 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. Results are expressed as mean ± SEM fold change.

Mentions: NF-κB-mediated survival signalling plays an important role in the liver, and its inhibition has been shown to result in massive hepatocellular apoptosis33. Indeed, NF-κB activation protects hepatocytes against glycochenodeoxycholic acid-induced apoptosis34. Further, miR-21 was recently described as a direct transcriptional target of NF-κB26, in response to genotoxic stress27. Consequently, we next investigated whether activation of the miR-21/PDCD4 pro-apoptotic pathway by DCA was dependent on NF-κB expression and/or activity. Primary hepatocytes were incubated with 25 to 200 μM DCA for 24 h. Total NF-κB protein levels were not significantly modulated by DCA (Fig. 4A top). NF-κB is usually located in the cytoplasm, bound to its inhibitor, IκB. Upon activating stimuli, IκB is phosphorylated, ubiquitinated and, consequently, degraded, thus allowing NF-κB nuclear translocation and activation35. Our results show that IκB levels were induced by DCA in a dose-dependent manner, for concentrations >25 μM, up to ~2-fold (at least p < 0.05) (Fig. 4A middle). As a result, the NF-κB/IκB ratio progressively decreased in cells incubated with increasing concentrations of DCA (Fig. 4A bottom). Our results further show that DCA inhibits NF-κB nuclear translocation, also in a dose-dependent manner (Fig. 4B). To unequivocally establish that DCA inhibits NF-κB activation in primary rat hepatocytes, we next transfected cells with a luciferase construct containing the NF-κB responsive element, in the presence or absence of DCA. DCA decreased NF-κB transcriptional activity in a dose-dependent manner up to ~78% for the 200 μM concentration (p < 0.01) (Fig. 4C). Because DCA hampers NF-κB transcriptional activity in a similar pattern to miR-21, these results suggest that DCA-mediated inhibition of miR-21 may constitute an NF-κB downstream event.


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 NF-κB transcriptional activity in primary rat hepatocytes in a dose-dependent manner.Cells were isolated and plated as described in Materials and Methods and treated with 25 to 200 μM DCA or no addition (control) for 24 h. (A) Immunoblotting of total NF-κB (top) and IκB (middle); NF-κB/IκB ratio (bottom; n = 5). Representative blots are shown. Blots were normalized to endogenous β-actin. (B) Immunoblotting of nuclear NF-κB (left) and cytoplasmatic NF-κB (right; n = 7). HDAC1 and GAPDH were used as nuclear and cytoplasmatic loading controls, respectively, as well as indicators for cross-contamination of protein fractions. Representative blots are shown. (C) NF-κB transcriptional activity (n = 5). 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. Results are expressed as mean ± SEM fold change.
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f4: DCA inhibits NF-κB transcriptional activity in primary rat hepatocytes in a dose-dependent manner.Cells were isolated and plated as described in Materials and Methods and treated with 25 to 200 μM DCA or no addition (control) for 24 h. (A) Immunoblotting of total NF-κB (top) and IκB (middle); NF-κB/IκB ratio (bottom; n = 5). Representative blots are shown. Blots were normalized to endogenous β-actin. (B) Immunoblotting of nuclear NF-κB (left) and cytoplasmatic NF-κB (right; n = 7). HDAC1 and GAPDH were used as nuclear and cytoplasmatic loading controls, respectively, as well as indicators for cross-contamination of protein fractions. Representative blots are shown. (C) NF-κB transcriptional activity (n = 5). 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. Results are expressed as mean ± SEM fold change.
Mentions: NF-κB-mediated survival signalling plays an important role in the liver, and its inhibition has been shown to result in massive hepatocellular apoptosis33. Indeed, NF-κB activation protects hepatocytes against glycochenodeoxycholic acid-induced apoptosis34. Further, miR-21 was recently described as a direct transcriptional target of NF-κB26, in response to genotoxic stress27. Consequently, we next investigated whether activation of the miR-21/PDCD4 pro-apoptotic pathway by DCA was dependent on NF-κB expression and/or activity. Primary hepatocytes were incubated with 25 to 200 μM DCA for 24 h. Total NF-κB protein levels were not significantly modulated by DCA (Fig. 4A top). NF-κB is usually located in the cytoplasm, bound to its inhibitor, IκB. Upon activating stimuli, IκB is phosphorylated, ubiquitinated and, consequently, degraded, thus allowing NF-κB nuclear translocation and activation35. Our results show that IκB levels were induced by DCA in a dose-dependent manner, for concentrations >25 μM, up to ~2-fold (at least p < 0.05) (Fig. 4A middle). As a result, the NF-κB/IκB ratio progressively decreased in cells incubated with increasing concentrations of DCA (Fig. 4A bottom). Our results further show that DCA inhibits NF-κB nuclear translocation, also in a dose-dependent manner (Fig. 4B). To unequivocally establish that DCA inhibits NF-κB activation in primary rat hepatocytes, we next transfected cells with a luciferase construct containing the NF-κB responsive element, in the presence or absence of DCA. DCA decreased NF-κB transcriptional activity in a dose-dependent manner up to ~78% for the 200 μM concentration (p < 0.01) (Fig. 4C). Because DCA hampers NF-κB transcriptional activity in a similar pattern to miR-21, these results suggest that DCA-mediated inhibition of miR-21 may constitute an NF-κB downstream event.

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