Protection against inflammatory β-cell damage by lysine deacetylase inhibition and microRNA expression?

TY - CONF

T1 - Protection against inflammatory β-cell damage by lysine deacetylase inhibition and microRNA expression?

AU - Vestergaard, Anna Lindeløv

AU - Pallesen, Emil Marek Heymans

AU - Novotny, Guy Wayne

AU - Noesgaard, Daniel

AU - Lundh, Morten

AU - Regazzi, Romano

AU - Mandrup-Poulsen, Thomas

PY - 2015/9/15

Y1 - 2015/9/15

N2 - Background and aims: Pro-inflammatory cytokines contribute to pancreatic β-cell apoptosis in type 1 and 2 diabetes mellitus. The detrimental effects resulting from cytokine-induced signaling in the β cell can be reduced by inhibition of class I classical lysine deacetylases (KDACi), especially HDAC1 or HDAC3, and is associated with down-regulation of inflammatory gene expression, only in part through hyperacetylation of NFB. We therefore hypothesize that HDACi-mediated hyperacetylation of histones and/or other proteins upregulate expression of microRNAs (miR), which repress translation of oxidative stress proteins responsible for β-cell death. The aim of the study is to identify novel and specific therapeutic targets for β-cell protection by mapping the miR profile of β cells rescued from inflammatory assault by inhibition of lysine deacetylation, thereby identifying miR that repress cytokine-induced mitochondrial and oxidative β-cell stress. Materials and methods: Stable INS-1 knockdown (KD) clones of HDAC1, -2, and -3 (class 1 KDACs) or empty vector were generated by Lentiviral shRNA transduction. The cells were incubated 6h with or without cytokines (150 pg/mL IL-1β + 5 ng/mL IFNγ), and RT-qPCR-based miR array was performed. Regulation of several miR was verified by TaqMan RT-qPCR, and medium nitrite was determined with Griess’ reagent. Results: Following systematic analysis using NormFinder, miR-103 was chosen for normalization of the qPCR array data. Constitutive expression of 103 miR, and cytokine-induced expression of 84 miR were up- or down-regulated more than 3-fold by KD of HDAC1, -2, and/or -3 (see figure). MiR-146a, -146b, -21 and -34a were chosen for further analysis, and their expression was assessed by RT-qPCR normalized to U6. Cytokine exposure induced a 15-fold expression of miRNA-146a, which was reduced by 65% in HDAC1- but not in HDAC2- or HDAC3-deficient INS-1 cells (p< 0.05, n=5). Expression of miRNA-146b, miR-21 and miR-34a were unaffected by HDAC1-3 deficiency, while cytokine-induced NO production was reduced by 27% and 44% in the HDAC1 (n=8) and HDAC2 (n=5) but not HDAC3 deficient clones respectively. The qPCR array analysis revealed several additional novel miR potentially important for understanding the beneficial effects of KDAC inhibition, which are currently being functionally validated. Conclusion: The interesting association between HDAC1 KD-mediated protection against cytokine-induced INS-1 cell destruction, and the inhibitory effect of HDAC1 knockdown on the β-cell pro-apoptotic miR-146a and other miR under investigation, warrants further investigations aiming to show a causal role of miR in HDAC inhibitor-mediated protection of pancreatic β cells. The perspective of this study is to develop novel anti-diabetic drugs targeting HDAC1 and/or associated miR.

AB - Background and aims: Pro-inflammatory cytokines contribute to pancreatic β-cell apoptosis in type 1 and 2 diabetes mellitus. The detrimental effects resulting from cytokine-induced signaling in the β cell can be reduced by inhibition of class I classical lysine deacetylases (KDACi), especially HDAC1 or HDAC3, and is associated with down-regulation of inflammatory gene expression, only in part through hyperacetylation of NFB. We therefore hypothesize that HDACi-mediated hyperacetylation of histones and/or other proteins upregulate expression of microRNAs (miR), which repress translation of oxidative stress proteins responsible for β-cell death. The aim of the study is to identify novel and specific therapeutic targets for β-cell protection by mapping the miR profile of β cells rescued from inflammatory assault by inhibition of lysine deacetylation, thereby identifying miR that repress cytokine-induced mitochondrial and oxidative β-cell stress. Materials and methods: Stable INS-1 knockdown (KD) clones of HDAC1, -2, and -3 (class 1 KDACs) or empty vector were generated by Lentiviral shRNA transduction. The cells were incubated 6h with or without cytokines (150 pg/mL IL-1β + 5 ng/mL IFNγ), and RT-qPCR-based miR array was performed. Regulation of several miR was verified by TaqMan RT-qPCR, and medium nitrite was determined with Griess’ reagent. Results: Following systematic analysis using NormFinder, miR-103 was chosen for normalization of the qPCR array data. Constitutive expression of 103 miR, and cytokine-induced expression of 84 miR were up- or down-regulated more than 3-fold by KD of HDAC1, -2, and/or -3 (see figure). MiR-146a, -146b, -21 and -34a were chosen for further analysis, and their expression was assessed by RT-qPCR normalized to U6. Cytokine exposure induced a 15-fold expression of miRNA-146a, which was reduced by 65% in HDAC1- but not in HDAC2- or HDAC3-deficient INS-1 cells (p< 0.05, n=5). Expression of miRNA-146b, miR-21 and miR-34a were unaffected by HDAC1-3 deficiency, while cytokine-induced NO production was reduced by 27% and 44% in the HDAC1 (n=8) and HDAC2 (n=5) but not HDAC3 deficient clones respectively. The qPCR array analysis revealed several additional novel miR potentially important for understanding the beneficial effects of KDAC inhibition, which are currently being functionally validated. Conclusion: The interesting association between HDAC1 KD-mediated protection against cytokine-induced INS-1 cell destruction, and the inhibitory effect of HDAC1 knockdown on the β-cell pro-apoptotic miR-146a and other miR under investigation, warrants further investigations aiming to show a causal role of miR in HDAC inhibitor-mediated protection of pancreatic β cells. The perspective of this study is to develop novel anti-diabetic drugs targeting HDAC1 and/or associated miR.

UR - http://www.easdvirtualmeeting.org/resources/protection-against-inflammatory-beta-cell-damage-by-lysine-deacetylase-inhibition-and-microrna-expression--3

UR - http://www.easdvirtualmeeting.org/resources/protection-against-inflammatory-beta-cell-damage-by-lysine-deacetylase-inhibition-and-microrna-expression--2

M3 - Poster

T2 - 51st EASD Annual Meeting

Y2 - 14 September 2015 through 18 September 2015

ER -