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Physiol. Genomics 31: 429-440, 2007. First published August 28, 2007; doi:10.1152/physiolgenomics.00060.2007
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Received 12 March 2007; accepted in final form 18 August 2007.
Physiological Genomics 31:429-440 (2007)
1094-8341/06 $8.00 © 2007 American Physiological Society

Global expression profiles from C57BL/6J and DBA/2J mouse lungs to determine aging-related genes

Vikas Misra 1, Hannah Lee 1, Anju Singh 1, Kewu Huang 1, Rajesh K. Thimmulappa 1, Wayne Mitzner 1, Shyam Biswal 1,2,3 and Clarke G. Tankersley 1

1 Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland
2 Division of Pulmonary and Critical Care Medicine, Johns Hopkins University, Baltimore, Maryland
3 Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, School of Medicine, Johns Hopkins University, Baltimore, Maryland


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study identified gene expression profiles that provided evidence for genomic mechanisms underlying the pathophysiology of aging lung. Aging lungs from C57BL/6 (B6) and DBA/2 (D2) mouse strains differ in physiology and morphometry. Lungs were harvested from B6 mice at 2, 18, and 26 mo and from D2 mice at 2 and 18 mo of age. Purified RNA was subjected to oligonucleotide microarray analyses, and differential expression analyses were performed for comparison of various data sets. A significant majority of differentially expressed genes were upregulated with aging in both strains. Aging D2 lungs uniquely exhibited upregulation in stress-response genes including xenobiotic detoxification cascades. In contrast, aging B6 lungs showed downregulation of heat shock-response genes. Age-dependent downregulation of genes common to both B6 and D2 strains included several collagen genes (e.g., Col1a1 and Col3a1). There was a greater elastin gene (Eln) expression in D2 mice at 2 mo, and Eln was uniquely downregulated with age in this strain. The matrix metalloproteinase 14 gene (Mmp14), critical to alveolar structural integrity, was also downregulated with aging in D2 mice only. Several polymorphisms in the regulatory and untranslated regions of Mmp14 were identified between strains, suggesting that variation in Mmp14 gene regulation contributes to accelerated aging of lungs in D2 mice. In summary, lungs of B6 and D2 mice age with variable rates at the gene expression level, and these quantifiable genomic differences provide a template for understanding the variability in age-dependent changes in lung structure and function.

Mmp14; lung elasticity; lung senescence; microarray; mouse single nucleotide polymorphisms


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
GLOBAL GENE EXPRESSION PROFILING is a powerful tool to generate hypotheses for less understood processes and has been used to study aging-associated gene expression changes in a variety of mouse tissues (8, 9, 25, 27, 35, 36). However, differential gene expression analyses have not been applied to the aging lung, a critical primary organ in the defense against environmental insults such as airborne pollutants. To date, only one study has documented global expression in healthy human lungs (18) as a function of age. Although this study did not actually achieve statistical significance in gene expression variation with age, modest global differences in gene expression between young and old subjects were observed.

The present study offers certain alternatives relative to studies using clinical samples by employing inbred mouse strains. The use of inbred mice achieves several advantages such as isogenicity and genomewide homozygosity among individuals within a strain, which significantly reduces gene expression variability between individuals. This variability was confounding in the aging human study (18). Aging mouse models have been used, for example, to evaluate global gene expression changes in skeletal muscle (35). One of the primary findings with aged skeletal muscle suggested that stress-response genes, including heat shock-response and oxidative stress-inducible genes, were upregulated. A similar study focusing on retinal tissue also found an upregulation in stress-response genes with age (25). While both of these previous studies used C57BL/6J (B6) mice, there was a common age-dependent upregulation of stress-response genes across different tissues.

In the present study, B6 mice were compared with DBA/2J (D2) mice to take advantage of the strain differences in natural longevity (14, 34), that is, D2 mice have a significantly shorter life span compared with B6 mice (average life span 21 vs. 29 mo). The B6 and D2 strains have been contrasted in other aging studies, primarily involving differences between their hematopoietic stem cell cycling characteristics (28, 58, 66). Other studies have focused on the variation in longevity and the role of caloric restriction in extending life span (16, 17). In the context of the lung, these two strains exhibit differential susceptibilities to a wide variety of stresses, including pulmonary infection (45, 50), hypersensitivity pneumonitis (19), ovalbumin-induced pulmonary eosinophilia, and cigarette smoke-induced emphysema (4). In the latter study (4), D2 mice exhibited a greater susceptibility to severe emphysematous changes following cigarette smoke exposure, which was characterized by more uniform and earlier onset of air space enlargement compared with B6 mice. With respect to basal differences in lung structure and function, B6 and D2 strains display distinguishable phenotypes in lung morphometry, airway resistance, parenchymal elasticity, and ventilatory behavior (23, 31, 63).

The primary goal of the present study was to identify gene expression profile differences between B6 and D2 strains, which serve as genomic indicators of differential aging of the mouse lung (23). A parallel goal of this study was to propose genetic mechanisms regarding the notable age-dependent loss of lung elasticity in D2 mice, which is not seen in B6 mice. The results show that a greater number of genes were upregulated with age in the lungs of D2 mice compared with B6 mice, which we propose is a genomic indicator of accelerated aging in the lungs of D2 mice. In particular, specific genes associated with degradation of lung elasticity were regulated differently in the lungs of D2 and B6 mice. The present study also focused on strain-dependent differences with age in the gene expression of Mmp14, a critical gene essential to the maintenance of normal alveolar structural integrity (2, 26, 53). Furthermore, the disparity in Mmp2 gene expression between strains serves as a potential mechanism leading to accelerated loss of elasticity with age in the lungs of D2 mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals.
Male C57BL/6J (18 and 26 mo) and DBA/2J (18 mo) mice were procured from the National Institute on Aging (Bethesda, MD). Two-month-old mice of each strain were purchased from Jackson Laboratories (Bar Harbor, ME). Mice were housed in an antigen- and virus-free room and were provided with water and a pelleted stock diet ad libitum. All procedures were approved by the Johns Hopkins University Animal Care and Use Committee and complied with American Physiological Society guidelines.

Oligonucleotide microarray.
Lungs were isolated, and total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA). Extracted RNA was purified with the RNeasy mini kit (Qiagen, Valencia, CA). The quality of the RNA was assessed with RNA 6000 nano assay kits (Agilent Technologies, Palo Alto, CA). The isolated RNA was applied to Mouse Genome 430 2.0 GeneChip arrays (Affymetrix, Santa Clara, CA) (n = 3/group) according to procedures described previously (54). This array contains probes for detecting ~14,500 well-characterized genes and 4,371 expressed sequence tags. Scanned output files were analyzed with Affymetrix GeneChip Operating Software (GCOS) version 1.3, and were independently normalized to an average intensity of 500. To identify the differentially expressed transcripts, pairwise comparison analyses were performed with the DMT 3.0 program (Affymetrix). Only genes that changed in at least six of nine comparisons and that showed a P value ≤0.05 by Mann-Whitney test were considered statistically significant with respect to differential gene expression. An absolute fold change (FC) of ≥1.4 was also used as a criterion to select notable genes. These criteria were consistent with the bioinformatics techniques used in previous mouse studies (9, 10, 64). In addition, the following group comparisons were considered essential to test the hypothesis of the present study. These group comparisons were 1) 18 mo vs. 2 mo for B6 mice, 2) 26 mo vs. 2 mo for B6 mice, 3) 18 mo vs. 2 mo for D2 mice, and 4) B6 vs. D2 mice at 2 mo. The strain differences at 18 mo were inferred by the collective evaluation of these four primary group comparisons. For further stringency, all genes with absolute (FC – FC SE) ≤1.4 were eliminated from further analyses. NetAffx (39) was used to extract gene ontology information for the profiles. For those genes with multiple probes, the single probe with the highest magnitude FC (positive or negative) was retained as representing that gene. Microarray data sets were deposited to the Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/projects/geo/) with accession no. GSE6591.

Quantitative real-time RT-PCR (Taqman).
Quantitative real-time RT-PCR (qPCR) was performed to validate the accuracy of microarray results. The reverse transcription reaction was performed with the SuperScript First-Strand Synthesis System (Invitrogen) as described previously (60). In Table 1, the specific genes analyzed with qPCR are reported. These analyses were performed with assay-on-demand primers and probe sets in the ABI 7000 Taqman system (Applied Biosystems, Foster City, CA). GAPDH was used for normalization, all PCR values were assayed in triplicate, and the average Taqman FC values are reported.


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  		Table 1. Genes assayed by qPCR

 
Canonical pathways identification.
The Ingenuity Pathways Analysis library of canonical pathways identified those that were most significant to the data set. Genes that were considered differentially expressed and were associated with a canonical pathway in the Ingenuity Pathways Knowledge Base are discussed in the present report. The significance of the association between a data set and a specific canonical pathway was estimated in the following two ways: 1) the proportion of genes in the data set included in the canonical pathway and 2) Fischer's exact test was used to calculate a P value determining the probability of the association between the data set and the canonical pathway. In this analysis, genes or gene products are represented as nodes, and the biological relationship between two nodes is represented as a line (i.e., an edge). All edges are supported by at least one reference from the literature. The Ingenuity Pathways Knowledge Base contains data from human, mouse, and rat orthologs. The intensity of the node color indicates the degree of up (green)- or down (red)-regulation. Nodes are displayed with various shapes that represent the functional class of a specific gene product.

Sequencing of Mmp14.
Primers for PCR amplification and sequencing of Mmp14 were designed with Primer3 software (http://fokker.wi.mit.edu) and synthesized by Integrated DNA Technologies (Coralville, IA). In tail clips of both strains, DNA was extracted by the DNeasy mini kit (Qiagen) and PCR amplification was carried out with ExTaq Premix (Takara Mirus Biosciences, Madison, WI) and 1.25 µmol of each primer. For the coding region, cDNA was sequenced with purified RNA from lungs of each strain. SuperScript One-Step RT-PCR for long templates (Invitrogen) was used to convert RNA to cDNA. The PCR products were resolved on 1% agarose gels, stained with ethidium bromide, and visualized under UV light to assess purity. PCR products were directly sequenced after purification with the QIAquick PCR purification kit (Qiagen). Sequencing was carried out at the DNA sequencing core facility at Johns Hopkins University. Sequence data were downloaded, assembled, and aligned (with ClustalX) to identify potential polymorphisms between the two strains. All polymorphisms were confirmed by sequencing both strands. Chromatograms were analyzed by Finch TV software and verified by manual review. Sequences of primers used in this study are given in Supplemental Table S1.1

Experimental design and general data analyses.
The choice of the last aging time point for each strain in this study was ~3 mo short of the strain-specific average life span (14, 34). Basal differences between strains (B6 vs. D2 at 2 mo) were provided to evaluate gene expression differences between young adults in the absence of a senescence effect. Previous reports (1, 43) suggest that alveolar septation in the mouse lung is generally complete between postnatal day (P)14 and P40. During this period, alveolar wall thinning occurs, followed by general maturation. Likewise, Amy and colleagues (1) showed very little change in lung structure after 4 wk of age. A heat map was constructed with GeneCluster and Treeview software (M. Eisen; http://rana.lbl.gov/eisensoftware.htm) (12). Values are reported as means ± SE.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Figure 1 shows three Venn diagrams that were scaled so that the areas of the individual circles were proportional to the number of genes in each data set. There were 366 genes that were differentially expressed with aging in D2 mice and not in the B6 mice (Fig. 1A, Table 2). Of the 366 genes unique to D2 mice at 18 mo, 242 genes were upregulated and 124 genes were downregulated. In contrast, 158 differentially expressed genes were unique to B6 mice at 18 mo of age; 108 genes were upregulated and 50 were downregulated. It is noteworthy that 42 genes were commonly upregulated and 51 genes were downregulated in lungs of both B6 and D2 mice at 18 mo (Fig. 1, B and C).


Figure 1
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  		Fig. 1. Venn diagrams depicting the distribution of genes in the microarray data sets into various subsets. A: distributions of total differentially expressed (both up- and downregulated) genes into different subsets. One-hundred fifty-eight (62 + 96 genes) differentially expressed genes were unique to C57BL/6 (B6) mice at 18 mo. B: upregulated genes only. Forty-two genes (10 + 32 genes) were commonly upregulated in both B6 and DBA/2 (D2) mice. C: downregulated genes only. Fifty-one genes (12 + 39 genes) were commonly downregulated in both B6 and D2 mice. Each individual subset is compared with 2-mo-old mice from the same strain. Although the area of each subset (i.e., represented by a circle) is proportional to the number of genes in that subset, the overlapping regions are not proportional. The numbers of genes common to any 2 data sets are displayed in red at the ends of dashed black arrows. The numbers of genes common to all 3 data sets are in black at the end of a solid black arrow.

 

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  		Table 2. Summary of numbers of genes in different subsets of microarray data

 
In comparison to D2 mice at 18 mo, there were 242 genes uniquely expressed in the aging B6 mice at 26 mo, with 167 genes being upregulated and 75 genes being downregulated (Fig. 1). There were 134 differentially expressed genes common to B6 mice at 26 mo and D2 mice at 18 mo (Fig. 1A). Of these genes, 66 were upregulated and 68 were downregulated. As summarized in Table 2, separating upregulated and downregulated data sets reveals a trend toward a larger fraction (i.e., 66–69%) of genes being upregulated with aging in both B6 and D2 strains (Fig. 1, B and C, and Table 2).

Age-dependent gene networks uniquely expressed in lungs of D2 mice.
Several components of xenobiotic detoxification cascades were upregulated uniquely in the D2 strain with aging (Table 3). Changes in expression of these genes were not observed in the aging lung of B6 mice. Specifically, these genes encompassed sulfur metabolism (e.g., Sult1a1 and Sult1e1), glutathione conjugating enzymes (e.g., Gsta3), and cytochrome P-450 genes. Generally, several of these genes related to xenobiotic metabolism were differentially expressed between B6 and D2 mice at 2 mo of age, including the cytochrome P-450 genes (Cyp3a13, and Cyp1a1) upregulated in B6 mice, for example. The expression of Cyp1a1 showed dichotomous age-dependent changes between strains, with upregulation in D2 mice and downregulation in B6 mice.


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  		Table 3. Selected networks of stress response on aging

 
Age-dependent gene networks uniquely expressed in lungs of B6 mice.
The heat shock response, a classical defense mechanism against various stresses in mammalian organisms (6, 38, 69), was markedly downregulated with aging in the B6 strain (Table 3). For example, the heat shock-response genes (Hspa1a and Hspa1b) were repressed at a magnitude of four- to fivefold in the aging lung of B6 mice. Two other heat shock genes (Hspb7 and Slc8a1) were shown to be expressed at a greater magnitude in D2 compared with B6 mice at 2 mo of age.

Variation in gene networks between B6 and D2 mice at 2 mo.
To assess genes differentially expressed with aging in this two-strain model, it was important to identify genes that showed baseline differences between the two strains. One classification of genes expressed differently between the two strains was related to B cell receptor signaling (Supplemental Table S2). Other classifications of genes expressed differently between strains were related to the antigen presentation pathway, PTEN signaling, and IGF-I signaling (Supplemental Table S3). Excluding the upregulation of the IGF-I binding protein (Igfbp2) and Ywhaz genes, 13 of 15 IGF-I signaling genes were downregulated in B6 compared with D2 mice. For example, Igfbp4, Pik3r1, Ptk2, Rras2, and Ywhag were downregulated in B6 mice, suggesting that the lungs of the two strains may have different activity states for the IGF-I pathway leading to variation in the rates of aging (Fig. 2).


Figure 2
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  		Fig. 2. Strain differences in basal IGF-I signaling. Network depicts the differential expression of IGF-I signaling pathway between B6 vs. D2 mice at 2 mo of age. Intensity of color reflects the magnitude of fold change. Green, upregulated; red, downregulated. Mosaic of colors indicates >1 isoform having magnitudes in the opposite direction.

 
Age-dependent gene expression changes related to lung structural integrity.
As shown by the profile in Fig. 3, age-dependent gene expression changes common to both B6 and D2 mice are related to lung structural integrity. From this profile, a subgroup of genes differed between strains at 2 mo of age (Supplemental Table S4). For example, a majority of collagen-encoding genes associated with the lung (e.g., Col1a1, Col3a1, and Col5a2) were downregulated with age in both strains, but these genes showed a greater expression in D2 mice at 2 mo. Other genes (e.g., Adamts2 and Fbn1) associated with the extracellular matrix (ECM) were also downregulated with age in both strains (Supplemental Table S5). Mmp9 was upregulated with age in both strains; however, the increase in expression was 13.6-fold in D2 mice compared with 2.9-fold in B6 mice. This increase in B6 mice occurred between 18 and 26 mo of age. Several other genes, including Gzma, Igfbp3, Pdgfrb, and Agtrl1, showed similar age-dependent downregulation in both strains (Supplemental Table S4). There was also a differential loss of WNT1-inducible signaling pathway protein 1 gene (Wisp1) expression with age, which was greater in D2 than in B6 mice.


Figure 3
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  		Fig. 3. Representative heat map of aging genes. Heat map depicts the magnitude of differential expression for profiles of aging genes (with or without baseline strain difference at 2 mo) in mouse lung. Green, upregulated; red, downregulated; black, no significant differential expression. A comprehensive list is reported in Supplemental Table S4.

 
Several age-dependent genes related to the lung ECM were expressed differently between the strains (Supplemental Table S5). The expression levels of Eln were downregulated with age in both strains but to a greater extent in D2 compared with B6 mice (5.1- vs. 1.7-fold) at 18 mo of age. Eln was also expressed at a greater magnitude at 2 mo in D2 compared with B6 mice. Although the expression level of Mmp2 was downregulated with age in both strains, the D2 mice showed markedly elevated levels at both 2 and 18 mo (qPCR FC values = 55- and 37-fold, respectively; Table 4) compared with B6 mice. Finally, the expression level of Mmp12 was upregulated with age uniquely in B6 mice. This differential expression occurred between 2 and 18 mo and remained unchanged up to 26 mo of age.


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  		Table 4. FC values for Figure 2 for different comparison data sets

 
Validation of microarray data.
Oligonucleotide microarray data were validated by qPCR to evaluate FC of selected genes reported in the microarray comparisons. In Fig. 4, a correlation between FC results comparing microarray with qPCR is shown. The data points depicted were derived from the four group comparisons detailed in MATERIALS AND METHODS. A Pearson's correlation coefficient of 0.91 was observed between microarray and qPCR data. This result suggested a high degree of consistency between the microarray and qPCR data and raised confidence regarding the other gene expression data obtained only by oligonucleotide microarray analyses. The values included in the correlation are also reported in Table 4.


Figure 4
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  		Fig. 4. Validation of microarray data by quantitative real-time RT-PCR (qPCR; Taqman) expressed as fold change (FC). A highly positive correlation (r value = 0.91) was observed between microarray and qPCR values. Data points arise from the 4 main comparisons depicted in Table 4. The gene expression results for Mmp2 comparing B6 and D2 mice at 2 mo are not shown (see Table 4).

 
Sequence variation of Mmp14 gene.
We observed strain- and age-dependent changes in the expression of Mmp14 that were consistent with other reports demonstrating its importance for maintenance of lung architecture (2, 26, 53). The microarray results show the expression levels of Mmp14 to uniquely fall with age in D2 mice by a FC of 2.2. A similar trend was demonstrated with qPCR. In this case, downregulation was observed in both strains (Table 4), but the magnitude of repression was greater in D2 mice by a FC of 3.1. We sequenced the gene region, including its promoter, coding region, and untranslated regions (UTRs; 5' and 3'), and the sequencing results are reported in Table 5. There were 14 single nucleotide polymorphisms (SNPs) of Mmp14 between the B6 and D2 strains, consisting of 3 in the promoter, 7 in the coding region, and 4 in the 3' UTR. Among these 14 SNPs, 8 were previously shown in the Mouse Genome Informatics resequencing project (http://www.informatics.jax.org), which was reported in October 2006. This is based on NCBI mouse build 36.1 (reference assembly). Entire B6 regions that were sequenced in this study were verified to have 100% identity with the B6 sequence stored in public databases.


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  		Table 5. Summary of SNPs found in Mmp14 in B6 and D2 strains

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The objective of this study was to use global gene expression to examine the effects of differential aging in lungs of B6 and D2 mice. These strains are known to have different average life spans, with D2 mice showing a significantly shorter life span (~21 mo vs. 29 mo) than B6 mice (14, 34). The final time point selected in the present study was intended to be ~3 mo earlier than the strain-specific average life span. An intermediate time point was chosen for the B6 strain, to chronologically coincide with the 18-mo-old D2 mice. In addition, our laboratory has shown (23) that the lung ages differently between these strains at these time points, as demonstrated by greater losses of parenchymal elasticity and tissue density. The accelerated aging in D2 lungs was also characterized by substantially greater collagen fiber content. Numerous other studies have compared these same inbred strains to explore the effects of aging in other tissues (13, 22, 56). In addition, these strains have been studied with a variety of toxin exposures on the lungs (4, 33). With respect to genomic variation in the progression of aging between strains, the present results suggest that there was a notably greater quantity of genes differentially expressed with age in lungs of D2 compared with B6 mice (Fig. 1A). The disproportionate number of upregulated relative to downregulated genes was also notable in the aging lungs of both strains. There was distinct strain variation in the aging profiles in the lungs, with D2 mice showing a propensity toward upregulation of stress response genes while B6 showed downregulation in a comparable class of genes (i.e., heat shock-response genes). There was also evidence to hypothesize that the greater downregulation of Mmp14 expression and the sequence variation in D2 relative to B6 mice played a role in the accelerated aging of the lung in this strain.

Our observation that global gene expression differences with age favor a greater proportion of upregulated genes is consistent with another study in retinal tissue (25). In contrast, however, aging skeletal muscle from B6 mice shows an equal number of up- and downregulated genes (35). In the same study, the number of genes differentially expressed with aging was significantly reduced after caloric restriction, an intervention known to extend life span. If caloric restriction facilitates a slower rate of aging (i.e., extends life span), a lower number of differentially expressed genes appears to be associated with a delayed progression of aging, at least in skeletal tissue. Our results in lung tissue were consistent with this previous study by showing a progressively increased number of differentially expressed genes with age of B6 mice (Fig. 1A). Moreover, the aging of the lung from D2 mice was advanced relative to B6 mice at a similar age (23), and this accelerated aging of the lung was associated with an amplification of the number of differentially expressed genes. In each case, however, these quantities represent a relatively small number compared with the total number of genes in the mouse genome. The present study is also consistent with previous studies showing a greater expression of stress-response genes with age, suggesting that this may be a genomic compensatory response to aging (11, 51, 65).

Mariani and colleagues (42) conducted a related genomic study on the developing lung from embyronic day (E)12 to P21 in the B6 strain. In their study, genes expressing proteins important to the lung ECM were also emphasized. For example, the elastin gene (Eln) was coexpressed with the platelet-derived growth factor receptor (Pdgfrb) and fibulin (Fbln) genes. Also, expression levels of several members of the collagen gene family (Col6a1 and Col6a2) were coexpressed together with the fibrillin 1 gene (Fbn1) in the developing lung. In the present study, similar gene clusters appeared to be coexpressed in the aging lung of both strains. The coexpression between Eln and Pdgfrb, for example, was downregulated in D2 mice, suggesting that the age-dependent downregulation of Eln expression may have been modulated by Pdgfrb repression (41). On the other hand, Eln expression was relatively maintained with age in B6 mice, which likely occurred through alternative regulatory mechanisms rather than changes in Pdgfrb expression. In contrast to Mariani's work, the present results showed coexpression of different collagen genes (Col1a1, Col3a1, and Col5a2) and Fbn1, and this cluster was downregulated in both strains. It was also noteworthy that TGF-ß regulatory mechanisms did not appear to be involved in the aging lung of either strain. The TGF-ß pathway is frequently associated with lung pathological changes, such as carcinogenesis and fibrosis (5). With respect to the present study, there were no overt signs of lung pathological changes in either strain at any age.

The aging lung of D2 mice increased several stress-response cascades, including the upregulation in detoxification networks, while preserving the heat shock response network. The upregulation of oxidative stress-response genes, such as the superoxide dismutase gene SOD1, has been shown to extend life span by protecting tissues against reactive oxygen species (55). Stress response induction has also been reported in gene expression profiles from aging skeletal muscle (35). This profile included Ckmt2, Aldh2, Aldh3, DnaJ homolog, Hsp105, Cyp1b1, Cyp3a, and Cbr. Furthermore, caloric restriction tempered the age-associated upregulation of these stress-response genes. Specific genes upregulated in lungs of D2 mice were associated with xenobiotic metabolism (Table 3). In contrast, Hspa1a and Hspa1b were downregulated uniquely in B6 mice. The mechanisms by which differential expression of detoxification genes in D2 mice or heat shock-response genes in B6 mice contribute to variable rates of aging in the lung require future study.

Another important signaling cascade that differed between B6 and D2 strains was the IGF-I pathway. It is noteworthy that IGF-I signaling is a well-established determinant of longevity (29, 57). While IGF-I is known to regulate antioxidant defense and cellular stress response systems, it is also known to stimulate Eln expression in neonatal pulmonary fibroblast cells (52). In the present study, differences in IGF-I signaling between B6 and D2 strains at 2 mo of age were observed with respect to the expression of Igfbp2, Igfbp4, and Pik3r1 (Supplemental Table S3). Specifically, Igfbp2 was downregulated and Igfbp4 and Pik3r1 were upregulated in D2 relative to B6 mice. Igfbp2 and Igfbp4 are known to affect IGF-I signaling by essentially inhibiting the interaction between IGF and its receptors (15, 48). Generally, this inhibition leads to a suppression of cell growth and proliferation and, therefore, may vary the physiological rates of aging in lung tissue between B6 and D2 mice. These results suggested that the activation status of IGF-I signaling may differ between strains, and this signaling variation appears to be maintained throughout life. It is also possible that this variation in activation status of IGF-I signaling is involved in the Eln expression differences (52) at 2 mo of age.

The concept that specific genetic determinants modulate the rate of aging in the lung has been explored by other investigative groups. Mori and coworkers (49) proposed that the regucalcin gene (Rgn) was essential for the integrity of the lung ECM, and its deficiency led to alveolar enlargement, a characteristic of lung senescence (67). In a similar fashion, the klotho gene (Kl) has been proposed as a gene crucial to lung architecture (62). Our lab has previously investigated the age-dependent changes in lung mechanics and morphometry in aging B6 and D2 mice. The D2 strain demonstrated a loss of lung elasticity and tissue density at 20 mo, which was not observed in B6 mice at a similar age. In the present study, the qPCR results demonstrate a 55-fold greater expression of Mmp2 at 2 mo of age in D2 compared with B6 mice (Table 4). While both strains showed an age-dependent decline in the expression levels of Mmp2 at 18 mo of age, the approximate 55-fold greater expression level in D2 mice was largely maintained throughout life (~37-fold greater in D2 mice at 18 mo). It is known that Mmp2 activity plays a role in the fragmentation of elastic fibers with aging (44, 68).

The primary objective of the present study was to identify pivotal genetic mechanisms that determine the variable rates of aging in the lung between B6 and D2 mice. A parallel objective was to better understand the molecular bases for age-dependent loss of lung elasticity in the D2 strain as a primary indicator of accelerated aging in the lung. One proposed mechanism of age-dependent elastic fiber fragmentation involves the interaction between Mmp2 and Mmp14. The regulatory pathway suggesting that Mmp14 activates Mmp2 on a biochemical level involves Timp2 (37). It has also been suggested that Mmp2, Mmp14, and Timp2 are coregulated at the transcriptional level, having similar promoter sequences (32, 40). One hypothesis to explain the loss of elastic recoil of the lung of D2 mice implicates the markedly greater expression of Mmp2, which may lead to a greater degradation of elastin with age in the lung tissue of this strain. As the expression level of Mmp2 falls with age in D2 mice, there is a parallel drop in Mmp14 and Eln expression. If these associations represent a mechanism by which elastin fragmentation occurs in the lung, then Mmp14 expression is a critical focal point for regulation. Therefore, the sequence variation between B6 and D2 strains in Mmp14 reported in the present study serves as a potential site for variation in transcriptional regulation. Mmp14 is a tethered membrane collagenase, which belongs to the family of membrane-type matrix metalloproteinases (MT-MMPs), found in lung epithelial and fibroblast cells (2). Mmp14 is known to be critical for normal postnatal alveolar development (2, 26, 53), and unlike other Mmp-null mice, the knockout phenotype of Mmp14 causes severe connective tissue abnormalities and death after 50 days of age (21). The SNPs found in the coding region were synonymous, suggesting that these two strains likely have the same Mmp14 primary protein structure. Furthermore, the SNPs located in the regulatory region do not overlap the SP1 or EGR1 sites as previously described (20).

Generally, the results of the present study suggest three genetic mechanisms that potentially contribute to variable rates of aging in the lungs of B6 and D2 mice. First, there was an age-dependent change in gene expression in Mmp14 that occurs uniquely in D2 mice. While the specific age at which this occurs is unclear, the depression in Mmp14 may lead to a loss of alveolar structural integrity, as shown previously (2, 26, 53). Second, time-dependent changes in lung elasticity are subject to gene expression differences occurring early in life and persisting with age. In this regard, the strain differences in Mmp2 observed at 2 mo of age, which continue through 18 mo, may have a greater adverse effect chronically on lung elasticity in D2 relative to B6 mice. A third potential difference between B6 and D2 mice is the age-dependent changes in specific Mmps. In the context of the D2 mice, upregulation of Mmp9 occurs, while in a different setting the upregulation of Mmp12 occurs in B6 mice. Likewise, the gene expression of Mmp9 in D2 mice is upregulated to a notably greater magnitude at 18 mo than observed in B6 mice at 26 mo of age. The effects of Mmp9 on the degradation of elastin are well established (30, 61). In light of these findings, we hypothesize that the aging lung of D2 mice is uniquely susceptible to accelerated loss of elasticity attributable to several of these potential molecular mechanisms.

The potential genetic mechanisms proposed in the present study must be considered within certain limitations. In general, the choice of age categories for the present study corresponds to similar age groups used in two previous studies (23, 24) from our laboratory outlining the age-dependent changes in the lung and the variation between strains. However, there may be some concern over the use of 2-mo-old mice as young controls. In gerontological studies, the mouse at this age is considered the biological equivalent of a postpubertal organism in the latter stages of maturational development (46). The primary factor leading to our use of 2-mo-old mice as young controls was the belief that the lung structure and architecture is generally complete by 40 days (1, 7, 43, 47, 59). While there may be ongoing maturation of other systems that impact the further development of the lung, such as immunologic function, alveolar septation (1) and lung elastic properties (7, 59) are essentially complete by 2 mo of age in mice. Moreover, mouse lung volumes do not change from 2 to 3 mo of age (47). On the other hand, we cannot rule out the indirect effects that these latter development stages might have on the global gene expression levels in the lung. Furthermore, our experimental design would have been improved had it included one or more intermediate age groups, especially for the D2 strain (46). With regard to lung development, maturation, and aging, there is a dearth of knowledge at these intermediate ages. Future studies on the genomic mechanisms of the aging lung would be considerably strengthened by understanding the time course of changes in expression levels of specific genes at closer spaced time intervals.

In conclusion, our findings suggest that the genomic bases for variation in rates of aging with respect to the mouse lung involve a specific differential gene expression profile. First, there was an increasing number of differentially expressed genes in the aging mouse lung, and 18-mo-old D2 mice showed a notably greater number of genes compared with B6 mice at either 18 or 26 mo of age. This might suggest greater stochasticity across gene expression networks, tending toward a loss of homeostasis with age. Future studies are required to focus on cell-specific stochasticity in aging lung similar to that seen in aging cardiomyocytes (3). Second, a higher fraction of differentially expressed genes were upregulated in the lungs of both strains with age. Finally, strain-dependent variation in gene expression levels of Mmp2 and Mmp14 are potentially important genetic mechanisms, and their downregulation is associated with accelerated loss of elasticity in the lung of aging D2 mice.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Institute on Aging Grant AG-21057 (to C. G. Tankersley), National Heart, Lung, and Blood Institute Grants HL-010342 (to C. G. Tankersley) and HL-081205 (to S. Biswal), the Flight Attendant Medical Research Institute's Young Clinical Scientist Research Award (to S. Biswal), Maryland Cigarette Restitution Fund (to S. Biswal), and National Institute of Environmental Health Sciences Center Grant P30-ES-038819.


   ACKNOWLEDGMENTS
 
We thank Catherine Scollick and Alexander Balbir for assistance with generation and uploading of raw microarray data sets to NCBI GEO.

Present address of K. Huang: Department of Respiratory Medicine, Beijing Chaoyang Hospital, Beijing 100020, China.


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: C. G. Tankersley, Div. of Physiology, Bloomberg School of Public Health, Johns Hopkins Univ., 615 N. Wolfe St., E7612, Baltimore, MD 21205 (e-mail: ctankers{at}jhsph.edu).

1 The online version of this article contains supplemental material. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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