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. 2016 Sep 29;8(9):2232-2252.
doi: 10.18632/aging.101056.

Global Gene Profiling of Aging Lungs in Atp8b1 Mutant Mice

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Free PMC article

Global Gene Profiling of Aging Lungs in Atp8b1 Mutant Mice

Ramani Soundararajan et al. Aging (Albany NY). .
Free PMC article

Abstract

Objective: Recent studies implicate cardiolipin oxidation in several age-related diseases. Atp8b1 encoding Type 4 P-type ATPases is a cardiolipin transporter. Mutation in Atp8b1 gene or inflammation of the lungs impairs the capacity of Atp8b1 to clear cardiolipin from lung fluid. However, the link between Atp8b1 mutation and age-related gene alteration is unknown. Therefore, we investigated how Atp8b1 mutation alters age-related genes.

Methods: We performed Affymetrix gene profiling of lungs isolated from young (7-9 wks, n=6) and aged (14 months, 14 M, n=6) C57BL/6 and Atp8b1 mutant mice. In addition, Ingenuity Pathway Analysis (IPA) was performed. Differentially expressed genes were validated by quantitative real-time PCR (qRT-PCR).

Results: Global transcriptome analysis revealed 532 differentially expressed genes in Atp8b1 lungs, 157 differentially expressed genes in C57BL/6 lungs, and 37 overlapping genes. IPA of age-related genes in Atp8b1 lungs showed enrichment of Xenobiotic metabolism and Nrf2-mediated signaling pathways. The increase in Adamts2 and Mmp13 transcripts in aged Atp8b1 lungs was validated by qRT-PCR. Similarly, the decrease in Col1a1 and increase in Cxcr6 transcripts was confirmed in both Atp8b1 mutant and C57BL/6 lungs.

Conclusion: Based on transcriptome profiling, our study indicates that Atp8b1 mutant mice may be susceptible to age-related lung diseases.

Keywords: Atp8b1 mutant; aging; gene profiling; lungs; transcriptome.

Conflict of interest statement

The authors have no conflict of interests to declare.

Figures

Figure 1
Figure 1. Differentially expressed genes in C57BL/6 and Atp8b1 mutant lungs
(A) Differentially expressed genes in the young (7-9 wk) (N=6) vs. aged (14 M) (N=6) C57BL/6 and Atp8b1 mutant lungs from a total of 34,000 genes that were analyzed by Affymetrix microarray, respectively. p < 0.05. (B) Venn diagram depicting overlapping and unique genes in aged C57BL/6 and Atp8b1 mutant lungs. Comparison of the differentially expressed genes (7-9 wks vs 14 M) in C57BL/6 and Atp8b1 mutant lungs revealed 37 overlapping genes between the two datasets, 350 unique genes in Atp8b1 mutant lungs and 85 unique genes in C57BL/6 lungs.
Figure 2
Figure 2. Ingenuity Pathway Analysis (IPA) identified perturbation of cellular assembly, organization, function, and maintenance in aged network to be perturbed in aged C57BL/6 lungs
Some of the key molecules in this network are shown in the figure. The genes Col1a, Col3a1, Zbtb16 and Rgcc were downregulated, whereas Mkx and Lair were upregulated in aged C57BL/6 lungs.
Figure 3
Figure 3. Ingenuity Pathway Analysis revealed genes in Xenobiotic metabolism to be affected in aged Atp8b1 mutant mice
One of the most important transcript in Xenobiotic metabolism namely NQO1 was decreased in aged Atp8b1 mutant lungs. Members that were decreased in Xenobiotic metabolism included CREBBP and p300. The transcript encoding members in PI3K complex (PIKCD) and PKC α,β (PRKCB) were increased in aged Atp8b1 mutant lungs.
Figure 4
Figure 4. Ingenuity Pathway Analysis shows perturbation in Nrf2 signaling in aged Atp8b1 mutant mice
The transcripts encoding NQO1, CREBBP, p300 were decreased in aged Atp8b1 mutant mice. Members in PI3K complex (PIK3D), PKC α,β (PRKCB, PKc(s) and DNAJC3 were increased in aged Atp8b1 mutant lung.
Figure 5
Figure 5. Ingenuity Pathway Analysis reveals dys-regulated Calcium signaling in aged Atp8b1 mutant mice
The transcripts Atp2b1 and Atp2b2 encoding ATP2B1 and ATP2B2 that play an important role in calcium signaling were decreased in Atp8b1 mutant lung in age-dependent manner.
Figure 6
Figure 6. Ingenuity Pathway Analysis of RhoGD1 signaling in aged Atp8b1 mutant mice
The transcript encoding key molecules in this pathway, namely, RHOA, RHOH and Ras homologs were increased, whereas CREBBP, MYL3, Mlcp, and Ppp1r12 were decreased in aged Atp8b1 mutant mice. Symbols and Color Keys. In the Figures (2-6), upregulated genes are depicted in red and downregulated genes in green. The solid lines depict direct interaction and the dashed lines depict indirect interaction between genes. The arrow represents interaction between genes. The symbols represent the following. A= Activation, B= Binding, E= Expression, I= Inhibition, PP = Protein-Protein binding, P = Phosphorylation/Dephosphorylation, RB= regulation of binding, MB = Group/Complex membership.
Figure 7
Figure 7. Quantitative Real-time RT-PCR analysis confirmed several transcripts in Atp8b1 mutant and C57BL/6 lungs at 7-9 wks vs. 14 M
(A) In C57BL/6 mice, CxCr6 transcript increased significantly at 14M relative to 7-9 wks.*** p < 0.001 relative to 7-9 wks time point. (B) In C57BL/6 mice, Col1a1 transcript significantly decreased at 14M compared to 7-9 wks. * p < 0.05 relative to 7-9 wks time point. (C) In Atp8b1 mutant, Adamts2 was significantly decreased at 14M when compared to 7-9 wks. * p < 0.05 relative to 7-9 wks time point. (D) CxCr6 transcript was significantly increased in 14M Atp8b1 mutant lungs versus 7-9 wks Atp8b1 mutant lungs. * p < 0.05 relative to 7-9 wks time point. (E) Col1a1 transcript levels decreased significantly at 14M relative to 7-9 wks in Atp8b1 mutant lungs. * p < 0.05 relative to 7-9 wks time point. (F) Mmp13 was significantly increased in aged Atp8b1 mutant lungs versus young adult lungs (7-9 wks). * p < 0.05 relative to 7-9 wks time point. For the qRT-PCR, N=6 mice were tested per group. Student's T test was used to calculate statistical significance between the two groups.
Figure 8
Figure 8. Western blot analysis of Nrf2
Equal amounts of protein from C57BL/6 and Atp8b1 mutant lung homogenates (7-9 wks and 14 M timepoints) were separated on 10% SDS-PAGE and probed with anti-Nrf2 antibody. There was no change in Nrf2 protein levels at 7-9 wks in both the groups. There was a significant decrease in Nrf2 protein in Atp8b1 mutant lungs at 14 M compared to C57BL/6 control. This is a representative blot. The experiment was repeated three times. β–Actin used as a loading control shows equal loading in all the lanes.

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