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Comparative Study
. 2013 Aug;35(4):1045-60.
doi: 10.1007/s11357-012-9426-6. Epub 2012 Jul 25.

Identification of the dichotomous role of age-related LCK in calorie restriction revealed by integrative analysis of cDNA microarray and interactome

Daeui Park  1 , Eun Kyeong Lee, Eun Jee Jang, Hyoung Oh Jeong, Byoung-Chul Kim, Young Mi Ha, Seong Eui Hong, Byung Pal Yu, Hae Young Chung
Affiliations
Comparative Study

Identification of the dichotomous role of age-related LCK in calorie restriction revealed by integrative analysis of cDNA microarray and interactome

Daeui Park et al. Age (Dordr). 2013 Aug.

Abstract

Among the many experimental paradigms used for the investigation of aging, the calorie restriction (CR) model has been proven to be the most useful in gerontological research. Exploration of the mechanisms underlying CR has produced a wealth of data. To identify key molecules controlled by aging and CR, we integrated data from 84 mouse and rat cDNA microarrays with a protein-protein interaction network. On the basis of this integrative analysis, we selected three genes that are upregulated in aging but downregulated by CR and two genes that are downregulated in aging but upregulated by CR. One of these key molecules is lymphocyte-specific protein tyrosine kinase (LCK). To further confirm this result on LCK, we performed a series of experiments in vitro and in vivo using kidneys obtained from aged ad libitum-fed and CR rats. Our major significant findings are as follows: (1) identification of LCK as a key molecule using integrative analysis; (2) confirmation that the age-related increase in LCK was modulated by CR and that protein tyrosine kinase activity was decreased using a LCK-specific inhibitor; and (3) upregulation of LCK leads to NF-κB activation in a ONOO(-) generation-dependent manner, which is modulated by CR. These results indicate that LCK could be considered a target attenuated by the anti-aging effects of CR. Integrative analysis of cDNA microarray and interactome data are powerful tools for identifying target molecules that are involved in the aging process and modulated by CR.

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Figures

Fig. 1
Fig. 1
Comparison of differentially expressed genes in the aging and CR transcriptomes. The red circle (aging) represents genes differentially expressed between young and old. The blue circle (CR) represents genes differentially expressed between old and CR. The microarrays used were 84 cDNA microarrays from GEO (GDS355, GDS356, GDS2612, and GDS3102). The dataset was tested on kidney, skeletal muscle, heart, and white adipose tissues in mouse and rat, and the all microarray experiments were designed as models of young versus old and old versus CR. The DEGs were identified by the criteria of a >2-fold change and a false discovery rate <5. As a result, we obtained 1,075 DEGs, which we collectively termed the aging and CR transcriptome; 740 genes were expressed differentially in aging, whereas 565 genes were expressed differentially in CR. A total of 230 genes were changed significantly in both aging and the CR condition; 103 genes were upregulated in aging but downregulated by CR, and 121 genes were downregulated in aging but were upregulated by CR. The differentially expressed genes in aging and CR overlapped (31 and 41 % of the age- and CR-related DEGs, respectively)
Fig. 2
Fig. 2
Gene sets enriched in the aging and CR transcriptomes. Classification of the aging and CR transcriptomes in the context of KEGG terminologies. The mapping terminologies (pathways) were selected by Fisher’s exact t test and false discovery rate (filtering options: p < 10−4 and FDR < 5). Red bars indicate upregulated gene sets and green bars represent downregulated gene sets in each condition. Pval means the modified significant value that is calculated by the following equation: ± [−log(p value)]. + means upregulated gene set, and − means downregulated gene set. a Distribution of the aging transcriptome with respect to biological process. b Distribution of the CR transcriptome with respect to biological process. c Distribution of genes shared in both the aging and CR transcriptomes
Fig. 3
Fig. 3
Protein–protein interaction network composed of common differentially expressed genes in both aging and CR. a The PPI network, in which the nodes represent proteins, and the edges represent the interactions between the proteins inferred from the HPRD database. The network consisted of 119 proteins and 1,156 interactions. A total of 59 proteins that were upregulated in aging but downregulated by CR are represented in red, and 60 proteins that were downregulated in aging but upregulated by CR are represented in green. The node sizes correlate with the number of interacting partners (degrees). Gray colors are the proteins that were obtained from the HPRD database and interacted with the DEGs. b The distribution and hierarchical clustering of degree and betweenness centrality for proteins upregulated by aging but downregulated by CR. c The distribution and hierarchical clustering of degree and betweenness centrality for proteins downregulated by aging but upregulated by CR. We used clustering analysis, with the aim of dividing the group that has high degree and betweenness centrality and extra groups. From this clustering result, we chose LCK, PLSCR1, and NDRG1 from the DEGs that are upregulated in aging but downregulated by CR, and MBP and SLC2A4 (GLUT4) from the genes that are downregulated in aging but upregulated by CR
Fig. 4
Fig. 4
Changes in the mRNA levels, protein levels, and activity of LCK, and the effect of SFKs and LCK on PTK activity upregulation during aging. a RT-PCR analysis of BLK, cSrc, LCK, FYN, HCK, and LYN was performed using RNA isolated from young (Y) and old (O) AL and CR rats. ß-Actin primers were used in separate PCR reactions to control for the efficiency of cDNA synthesis in each sample. b and c Western blotting was performed to detect total LCK protein levels and the tyrosine phosphorylation status (p394 and p505) of LCK from AL and CR rats. ß-Actin was used as a loading control for the cytosolic and membrane fraction samples. One representative blot is shown from three independent experiments that yielded similar results. d PTK activity was measured in kidney homogenates from Y and O rats. PTK activity was measured in the presence or absence of tyrosine kinase inhibitor, Src kinase inhibitor (PP2, 5 μM), or LCK inhibitor [damnacanthal (Dam), 40 nM]. Each value is the mean ± SE of five rats. The results of one-factor ANOVA: *p < 0.05 vs. young rats; ###p < 0.001 vs. age-matched old rats
Fig. 5
Fig. 5
NF-κB activation by LCK induction in Raw 264.7 cells stimulated with LPS. a LPS (100 ng/mL)-induced accumulation of intracellular ONOO− was monitored by DHR123. The time course of changes in fluorescence was monitored with a GENios Plus fluorescent microplate reader. b Raw 264.7 cells were exposed to LPS (100 ng/mL) for 12 h. The levels of anti-LCK and anti-p LCK antibody were detected by Western blotting. ß-Actin was used as a loading control. c Raw 264.7 cells were pre-incubated for 2 h with l-NIO (100 μM), penicillamine (100 μM), or damnacanthal (170 nM) before treatment with LPS (100 ng/mL) for 12 h. The level of p-p65 NF-κB was detected in the nucleus by Western blotting. TFIIB was used as a loading control. d (left) Cell lysates were immunoprecipitated with anti-IκBα antibody followed by Western blotting with anti-p-tyrosine antibody. d (right) Cell lysates were analyzed by Western blotting with anti-p-TAK1 or anti-p-IκBα (serine 32) antibody. ß-Actin was used as a loading control. One representative blot is shown from three independent experiments that yielded similar results
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References

    1. Abraham KM, Levin SD, Marth JD, Forbush KA, Perlmutter RM. Thymic tumorigenesis induced by overexpression of p56lck. Proc Natl Acad Sci U S A. 1991;88(9):3977–3981. doi: 10.1073/pnas.88.9.3977. - DOI - PMC - PubMed
    1. Anderson DH, Radeke MJ, Gallo NB, Chapin EA, Johnson PT, Curletti CR, Hancox LS, Hu J, Ebright JN, Malek G, Hauser MA, Rickman CB, Bok D, Hageman GS, Johnson LV. The pivotal role of the complement system in aging and age-related macular degeneration: hypothesis re-visited. Prog Retin Eye Res. 2010;29(2):95–112. doi: 10.1016/j.preteyeres.2009.11.003. - DOI - PMC - PubMed
    1. Artyomov MN, Lis M, Devadas S, Davis MM, Chakraborty AK. CD4 and CD8 binding to MHC molecules primarily acts to enhance Lck delivery. Proc Natl Acad Sci U S A. 2010;107(39):16916–16921. doi: 10.1073/pnas.1010568107. - DOI - PMC - PubMed
    1. Barabasi AL, Gulbahce N, Loscalzo J. Network medicine: a network-based approach to human disease. Nat Rev Genet. 2011;12(1):56–68. doi: 10.1038/nrg2918. - DOI - PMC - PubMed
    1. Chung HY, Kim HJ, Kim KW, Choi JS, Yu BP. Molecular inflammation hypothesis of aging based on the anti-aging mechanism of calorie restriction. Microsc Res Tech. 2002;59(4):264–272. doi: 10.1002/jemt.10203. - DOI - PubMed

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