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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

PDCD4 silencing hampers hepatocyte cell death induced by DCA.Primary rat hepatocytes were transfected with a specific siRNA against PDCD4 (siRNA PDCD4) or a control (siRNA C) and treated with 100 μM DCA or no addition for 48 h. (A) Immunoblotting of PDCD4 (n = 4). Representative blots are shown. Blots were normalized to endogenous β-actin. (B) Cell viability, measured by the ApoTox-GloTM Triplex assay (n = 4). (C) General cell death measured by LDH assay (n = 11). (D) Apoptotic cells detected by Hoechst staining. Representative images of control, DCA, PDCD4 silencing and PDCD4 silencing + DCA are shown. Bar, 30 μM. Arrows indicate apoptotic nuclei. Results are expressed as mean ± SEM fold change.
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f3: PDCD4 silencing hampers hepatocyte cell death induced by DCA.Primary rat hepatocytes were transfected with a specific siRNA against PDCD4 (siRNA PDCD4) or a control (siRNA C) and treated with 100 μM DCA or no addition for 48 h. (A) Immunoblotting of PDCD4 (n = 4). Representative blots are shown. Blots were normalized to endogenous β-actin. (B) Cell viability, measured by the ApoTox-GloTM Triplex assay (n = 4). (C) General cell death measured by LDH assay (n = 11). (D) Apoptotic cells detected by Hoechst staining. Representative images of control, DCA, PDCD4 silencing and PDCD4 silencing + DCA are shown. Bar, 30 μM. Arrows indicate apoptotic nuclei. Results are expressed as mean ± SEM fold change.

Mentions: To further evaluate the functional role of PDCD4 during DCA-induced apoptosis of primary rat hepatocytes, we next transfected cells with a specific siRNA against PDCD4, or a control siRNA, in the presence or absence of DCA. Upon silencing, PDCD4 protein levels were significantly inhibited (p < 0.01), comparing with control cells (Fig. 3A). PDCD4 inhibition led to a modest but significant increase in cellular viability (Fig. 3B), and decrease in general cell death (Fig. 3C). These results suggest that basal PDCD4 protein levels may not actively contribute to hepatocellular death. However, its activation by DCA is necessary for induction of apoptosis, as PDCD4 silencing significantly impaired the ability of DCA to reduce cellular viability or to increase cell death and apoptosis (Fig. 3,B–D).


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)

PDCD4 silencing hampers hepatocyte cell death induced by DCA.Primary rat hepatocytes were transfected with a specific siRNA against PDCD4 (siRNA PDCD4) or a control (siRNA C) and treated with 100 μM DCA or no addition for 48 h. (A) Immunoblotting of PDCD4 (n = 4). Representative blots are shown. Blots were normalized to endogenous β-actin. (B) Cell viability, measured by the ApoTox-GloTM Triplex assay (n = 4). (C) General cell death measured by LDH assay (n = 11). (D) Apoptotic cells detected by Hoechst staining. Representative images of control, DCA, PDCD4 silencing and PDCD4 silencing + DCA are shown. Bar, 30 μM. Arrows indicate apoptotic nuclei. Results are expressed as mean ± SEM fold change.
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Related In: Results  -  Collection

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f3: PDCD4 silencing hampers hepatocyte cell death induced by DCA.Primary rat hepatocytes were transfected with a specific siRNA against PDCD4 (siRNA PDCD4) or a control (siRNA C) and treated with 100 μM DCA or no addition for 48 h. (A) Immunoblotting of PDCD4 (n = 4). Representative blots are shown. Blots were normalized to endogenous β-actin. (B) Cell viability, measured by the ApoTox-GloTM Triplex assay (n = 4). (C) General cell death measured by LDH assay (n = 11). (D) Apoptotic cells detected by Hoechst staining. Representative images of control, DCA, PDCD4 silencing and PDCD4 silencing + DCA are shown. Bar, 30 μM. Arrows indicate apoptotic nuclei. Results are expressed as mean ± SEM fold change.
Mentions: To further evaluate the functional role of PDCD4 during DCA-induced apoptosis of primary rat hepatocytes, we next transfected cells with a specific siRNA against PDCD4, or a control siRNA, in the presence or absence of DCA. Upon silencing, PDCD4 protein levels were significantly inhibited (p < 0.01), comparing with control cells (Fig. 3A). PDCD4 inhibition led to a modest but significant increase in cellular viability (Fig. 3B), and decrease in general cell death (Fig. 3C). These results suggest that basal PDCD4 protein levels may not actively contribute to hepatocellular death. However, its activation by DCA is necessary for induction of apoptosis, as PDCD4 silencing significantly impaired the ability of DCA to reduce cellular viability or to increase cell death and apoptosis (Fig. 3,B–D).

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