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Am J Physiol Lung Cell Mol Physiol 292: L1313-L1326, 2007. First published January 19, 2007; doi:10.1152/ajplung.00146.2006
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Rapid onset of gene expression in lung, supportive of formation of alveolar septa, induced by refeeding mice after calorie restriction

Donald Massaro,1 Emma Alexander,1 Kristin Reiland,1 Eric P. Hoffman,2 Gloria DeCarlo Massaro,3 and Linda Biadasz Clerch3

Lung Regeneration Laboratory, Departments of 1Medicine and 3Pediatrics, Georgetown University School of Medicine, and 2Center for Genetic Medicine, Children's National Medical Center, Washington, District of Columbia

Submitted 17 April 2006 ; accepted in final form 17 January 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Alveolar regenerative gene expression is unidentified partly because its onset, after a regenerative stimulus, is unknown. Toward addressing this void, we used a mouse model in which calorie restriction produces alveolar loss, and ad libitum access to food after calorie restriction induces alveolar regeneration. We selected four processes (cell replication, angiogenesis, extracellular matrix remodeling, and guided cell motion) that would be required to convert a flat segment of alveolar wall into a septum that increases gas-exchange surface area. Global gene expression supportive of processes required to form a septum was present within 3 h of allowing calorie-restricted mice food ad libitum. One hour after providing calorie-restricted mice food ad libitum, RNA-level expression supportive of cell replication was present with little evidence of expression supportive of angiogenesis, extracellular matrix remodeling, or guided cell motion. Cell replication was more directly assayed by measuring DNA synthesis in lung. This measurement was made 3 h after allowing calorie-restricted mice food ad libitum because translation may be delayed. Ad libitum food intake, following calorie restriction, elevated DNA synthesis. Thus RNA expression 1 h after allowing calorie-restricted mice food ad libitum supported increased cell replication; measurements at 3 h revealed increased DNA synthesis and RNA expression, supportive of the three other processes required to form a septum. These findings identify the first hour after providing calorie-restricted mice ad libitum access to food as the onset of gene expression in this model that supports processes needed for alveolar regeneration.

microarray; cell replication; angiogenesis; extracellular matrix; regeneration; guided cell motion

IDENTIFICATION OF GENE EXPRESSION in lung that initiates regeneration of pulmonary gas-exchange units (alveoli) is of biological interest and potential clinical value. It is also important to know if alveolar regeneration, following different causes of alveolar loss and induced by different experimental manipulations (16, 18, 30–33, 40, 42, 51), is brought about by the same pattern of gene expression. The use of global gene profiling, which should be a powerful tool to identify the initial gene expression that specifies alveolar regeneration, has been hampered by the lack of knowledge of the time when, following a regenerative stimulus, the very upstream gene expression determinative of alveolar regeneration begins. Having this information would narrow the period that must be studied to identify, understand, and manipulate gene expression that initiates alveolar regeneration.

Our goal is to identify the time, following a regenerative maneuver or stimulus, during which very upstream changes in gene expression occur that initiate processes required for alveolar regeneration. Therefore, as a first step toward our goal, we enumerated processes that biological knowledge (24, 26) indicates are required for alveolar regeneration, i.e., transforming a flat segment of alveolar wall into an elongating fold (septum) that increases gas-exchange surface area. These processes are lung cell replication, angiogenesis, remodeling of the extracellular matrix, and guided cell motion. Although individually these process occur during many conditions in the lung, their occurrence together in a regenerative model strongly suggest that they are the result of signaling that results in alveolar regeneration. The model of alveolar regeneration used for the present work was allowing mice ad libitum access to food following calorie restriction-induced loss of alveoli (14, 19, 20, 30, 33, 49). Microarray gene profiling was performed on RNA from lung tissue of mice after 14 days of calorie restriction and 3 h after the onset of ad libitum refeeding following 14 days of calorie restriction. Gene expression in lung supportive of cell replication, angiogenesis, extracellular matrix remodeling, and guided cell motion was present within 3 h of the onset of ad libitum access to food. Then, real-time polymerase chain reaction (RT-PCR) was used to assess the expression of a subset of genes after 14 days of calorie restriction and after allowing mice 1 h of ad libitum access to food immediately following 14 days of calorie restriction. This demonstrated RNA-level gene expression supportive of cell replication was present within 1 h of providing mice ad libitum access to food after calorie restriction for the immediately preceding 14 days. We then more directly tested for evidence of cell replication by measuring DNA synthesis in lung. Because translation of proteins required for the increased DNA synthesis associated with cell replication may be delayed, DNA synthesis was measured 3 h after calorie-restricted mice were allowed ad libitum access to food; it was depressed in calorie-restricted mice and rose in calorie-restricted mice that were allowed ad libitum access to food for 3 h.

Thus RNA expression 1 h after calorie-restricted mice were provided food ad libitum supported increased cell replication, and measurements 3 h after allowing calorie-restricted mice food ad libitum revealed the presence of increased cell replication in the lungs of these mice as well as RNA-level gene expression supportive of the other three processes needed to form a septum, angiogenesis, extracellular matrix remodeling, and guided cell motion. Together, these findings identify the first hour after allowing calorie-restricted mice food ad libitum as the time gene expression supportive of processes needed to form a septum begins in this model. When equivalent data from other experimental models of alveolar regeneration (18, 30, 32, 40, 42, 51) is available, it will allow a comparison, among several models of alveolar regeneration, at a similar period of gene expression supportive of processes needed to form a septum. This will help identify the upstream alveolar regenerative gene expression and help determine whether it is context-dependent or independent.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Animals and experimental manipulations. Adult male C57BL/6J mice were purchased from Jackson Laboratory and, on arrival at Georgetown University School of Medicine, were housed four or five per cage in the Department of Comparative Medicine. They were on a 12:12-h light-dark cycle and were allowed ad libitum access to Ralston Purina Rodent Chow 5001. After 1 wk or more at Georgetown, the mice were housed one per cage, and the amount of chow eaten each day was measured. Using the average daily weight of chow eaten over the prior 4–5 days by each mouse of the group to be calorie restricted, we diminished the daily amount of chow for each mouse by two-thirds (30, 33); these mice will be referred to as restricted. Other mice, also housed individually, were always allowed ad libitum access to chow; these mice will be referred to as fed. All mice were always allowed ad libitum access to tap water. After 14 days of calorie restriction, we killed a group of restricted and a group of fed mice. Other mice, after 14 days of calorie restriction, were allowed ad libitum access to food for 1 or 3 h; these mice will be referred to as refed. Refed mice, together with fed mice, were killed 1 or 3 h later. Animals were killed by exsanguination, produced by cutting large abdominal blood vessels, after achieving a surgical level of anesthesia (failure to withdraw from a toe-pinch) with xylazine (~10 mg/kg) plus ketamine (~75 mg/kg). All procedures were approved by the Georgetown University Animal Care and Use Committee and comply with the American Physiological Society Guiding Principles on the Care and Use of Animals.

Expression profiling. Lungs were excised, snap-frozen in liquid N2, and stored at –80°C. Total RNA was extracted using TRIzol (Invitrogen) and further purified using RNeasy (Qiagen). RNA was converted into double-stranded DNA using SuperScript Choice System (Invitrogen) with an oligo(dT) primer containing T7 RNA polymerase promoter (Genset). Phenol-chloroform extraction was used to purify double-stranded cDNA, which was then used for in vitro transcription with an Enzo Bioarray RNA transcript-labeled kit. RNeasy (Qiagen) was used to purify biotin-labeled cRNA, which was randomly fragmented before hybridization to an Affymetrix Mouse Genome 430 2.0 Array GeneChip, which contains 39,000 transcripts, using an Affymetrix Fluidics Station 400 and a Hewlett Packard G2500/7 Gene Array scanner. All gene expression data is available at the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO).

Analysis of gene expression data. As before (30), we used an experimental design that attempts to account for potential diurnal variation in gene expression. We killed fed and 14-day restricted mice at 9 am. Other fed mice were killed at 10 am or at noon, as were 14-day, calorie-restricted, 1-h (10 am) and 3-h (noon) refed mice. To test the statistical significance of intergroup means for each group, a two-tailed unpaired t-test was performed (StatMost 32). We worked only with genes with an intergroup P value of ≤0.05. We further reduced the genes analyzed by selecting genes for which expression would be expected to support four cellular processes that biological knowledge indicates would be required to form an alveolus, i.e., cell replication, angiogenesis, extracellular matrix remodeling, and guided cell motion.

RT-PCR. The relative amount of mRNA was assessed using RT-PCR. Total RNA, isolated from mouse lung tissue using TRIzol reagent (Invitrogen), followed by cleanup with the RNeasy mini kit (Qiagen), was used to perform two-step RT-PCR. In the first step, cDNA was reverse-transcribed (RT) from total RNA using random primers from the High Capacity cDNA Archive Kit (Applied Biosystems; AB). In the second step, PCR products were synthesized using the PCR master mix from AB with AmpErase UNG and AmpliTaq Gold polymerase to cleave the reporter from the quencher dye of the TaqMan probe during PCR. The exponential increase of PCR products was monitored by following the increase in the fluorescence of the reporter dye. The sequence-specific primers (unlabeled) and the TaqMan minor groove binder 6-FAM dye-labeled probes were obtained from AB. The AB Assay ID for each mRNA measured is listed in Supplemental Table S1 (the online version of this article contains Supplemental Tables S1–S7). TaqMan 18S rRNA, VIC-labeled, primer-limited, endogenous control (AB) was used to normalize the amount of cDNA added to the reaction; all reactions were performed in the AB 7300 RT-PCR instrument. For quantitation, standard curves for 18S rRNA and the gene of interest were run for each gene expression assay.

DNA synthesis by lung. We measured DNA synthesis by lung slices as previously described in detail (15, 22). Briefly, lung slices were incubated in Krebs-Ringer bicarbonate (KRB) solution (56), previously gassed for 1 h with 95% O2-5% CO2, to which we had added 5.5 mM glucose and serum concentrations of amino acids (37) and subsequently [3H]thymidine plus sufficient nonradioactive thymidine to achieve a final medium concentration of 15 µM thymidine (15, 22). This concentration of thymidine was used to eliminate possible differences in the pool size of endogenous thymidine (15, 22). Excised lungs were sliced on a McIlwain tissue slicer at a setting of 1.0 mm (27), placed in KRB with glucose, amino acids, and [3H]thymidine, and incubated at 37°C for 1 h, shaking at 120 oscillations/min, while being gassed with 95% O2-5% CO2. The slices were then removed from the incubation mixture, homogenized with a Polytron homogenizer set at full speed, and the homogenate and medium were handled as previously described (15, 22). Briefly, radioactivity in cold 10% trichloroacetic acid (TCA) insoluble material, made soluble in hot 5% TCA, and radioactivity in medium 10% TCA soluble material was counted, and the rate of incorporation of thymidine into DNA, i.e., the rate of DNA synthesis, by lung slices was calculated.

Western blot and ELISA measurements. Lung tissue was homogenized using a Polytron set at full speed at 4°C in lysis buffer containing 24 mM Tris (pH 7.3), 0.5 mM EDTA, 40 mM KCl, Triton X-100, 1.0 mM DTT, and protease inhibitor cocktail (Calbiochem). Homogenates were centrifuged at 10,000 g at 4°C for 1 h. The protein concentration of the supernatant fluid was measured spectrophotometrically with Coomassie Plus protein assay reagent (Pierce) using bovine serum albumin as the standard. Proteins in the 10,000 g supernatant fluid were separated by electrophoresis in 4–20% gradient SDS-PAGE gels (Bio-Rad) and then transferred onto nitrocellulose Hybond ECL membranes (Amersham, Piscataway, NJ), which were blocked for 2 h at room temperature using 1% nonfat milk in Tris-buffered saline-Tween (TBS-T); 0.1% Tween 20, 20 mM Tris (pH 7.6), and 137 mM NaCl followed by an incubation overnight at 4°C with the following primary antibodies: anti-cyclin D3 monoclonal (Cell Signaling Technology, Beverly, MA) at a dilution of 1:3,000; anti-matrix metalloproteinase-3 (anti-MMP-3) polyclonal (AnaSpec, San Jose, CA) at a dilution of 1:1,000; or anti-Plexin A2 polyclonal (Santa Cruz Biotech, Santa Cruz, CA) at a dilution of 1:2,000. The membranes were then washed once for 15 min and then three times for 5 min with TBS-T and incubated for 1 h at room temperature with goat anti-mouse (1:3,000), goat anti-rabbit (1:3,000), or rabbit anti-goat (1:1,000) from Bio-Rad in 1% nonfat milk in TBS-T. Membranes were washed in TBS-T followed by detection of the immunoreactive protein with an ECL kit (Amersham). The membranes were then probed with rabbit anti-galectin-1 antibody, which acted as an internal standard. Galectin protein bands were visualized by ECL with goat anti-rabbit secondary antibody (Bio-Rad). All Western blots were quantitated by laser densitometry (Molecular Dynamics) using ImageQuant software. Cyclin D3, MMP-3, and Plexin A2 densities were expressed as relative densitometry units per galectin-1. ELISA for vascular endothelial growth factor (VEGF) protein was performed using Quantikine mouse VEGF immunoassay kit. Lung homogenates were diluted 20-fold with calibrator diluent RD5T provided in the kit. The protocol followed was as described in the manufacturer's instructions. VEGF concentrations were expressed as VEGF protein per total lung protein.

Statistical analysis of DNA synthesis and protein expression. The mean and standard deviation were calculated, and an unpaired two-tailed t-test (StatMost) was used to calculate the statistical significance of intergroup differences.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Gene expression measured by DNA microarray compared with expression of the same genes measured by RT-PCR. To test the reliability and repeatability of our microarray results on lungs of calorie-manipulated mice and mice always allowed food ad libitum, we repeated the experimental manipulations on a different group of mice and measured by RT-PCR the expression of 31 genes (Tables 1 and 2) chosen because of the function of their cognate protein, e.g., cell replication, angiogenesis, extracellular matrix remodeling, and guided cell motion. Of the 31 genes selected, 25 (81%) confirmed the results of the microarray experiments (Tables 1 and 2). When we compared array and RT-PCR expression only among genes for which fold differences by microarray were 1.5 or greater, as done by others (61), our confirmation of the microarray results by RT-PCR was 91%. Because we used a different group of mice, killed at different times of the year, for microarray and RT-PCR measurements, our high percentage of confirmation is especially reassuring as it encompasses technical, biological, and temporal variability.


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  		Table 1. Calorie restriction for 14 days compared with ad libitum access to food for 14 days: gene expression assessed by microarray vs. RT-PCR

 

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  		Table 2. Calorie restriction for 14 days followed by ad libitum fed for 3 h, compared with always ad libitum food: gene expression assessed by microarray vs. RT-PCR

 
Microarray-assessed expression of cell replication-related genes after 14 days of calorie restriction and after 14 days of calorie restriction followed by 3 h of ad libitum access to food. Expression of three key inhibitors of cell cycle-dependent kinases, p27, p57KIP2, and p16INK4 (17, 36, 64), was higher in lungs of 14-day restricted mice compared with lungs of 14-day fed mice (Table 3A). E1A, an adenoviral protein, alters gene expression in a manner that promotes cell cycle proliferation; its repressor, cellular repressor of E1A-stimulated genes (59, 60), was higher in 14-day restricted than in fed mice (Table 3A). The RNA of retinoblastoma-like 2/bp130 (44), for which protein is highly homologous to the tumor suppressor protein pRb (62), and Wee-1 kinase, an inhibitor of cell division cycle control protein 2 (63), was higher in restricted than in fed mice. Abundant in neuroepithelial area (ANA), for which expression was higher in restricted than in fed mice, impairs serum-induced cell cycle progression from G0/G1 to S phase in serum-starved cells (Table 3A; Ref. 2). The higher expression of TPA-inducible sequence 21 (TIS21), a member of the antiproliferative gene family (35), in lungs of restricted mice than in lungs of fed mice supports a conclusion that there is less replication in lungs of restricted mice than in lungs of fed mice. Most broadly important to progression from G0 to G1, Smad4, which activates negative regulators of cyclin-dependent kinases 2, 4, and 6, and of the D and E cyclins (52), was higher in lungs of restricted than in lungs of fed mice (Table 3A). Consistent with the higher expression of Smad4, the mRNA of cyclin D1 and D2 was lower in restricted compared with fed mice (Table 3B).


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  		Table 3. Expression of cell replication-related genes in lungs of mice calorie restricted for 14 days, compared with always ad libitum fed: microarray

 
Cyclin C RNA was higher in restricted than in fed mice (Table 3C). However, although cyclin C acts with c-Myc to induce proliferation of hematopoietic cells (29), it functions mainly, in cooperation with cyclin-dependent kinase-8, to regulate RNA polymerases (2, 25, 46) or to regulate cell adhesion (28). The fold elevation of mCDC 47, p130PITSL, cdk/Cdc-28-like protein kinase-3 (Clk-3), and Cdc-6-related protein (Table 3C), which favors cell replication, act via the cell cyclins, the expression of most of which was lower in lungs of restricted compared with lungs of fed mice (Table 3B; Ref. 5).

Expression of five genes in lungs of restricted mice, for which downregulation would increase cell replication, was lower than in lungs of fed mice (Table 3D). However, lower expression of D cyclins in restricted compared with fed mice (Table 3B), which are downstream of cyclin-dependent kinases 4 and 6 inhibitor-18 protein (5), and the lower expression of cyclin B1 in restricted mice (Table 3B), which is downstream of p53, would abrogate the enhancing effect of p53 (Table 3D) on cell replication. From these data we conclude that, after 14 days of calorie restriction, gene expression at the RNA level indicates cell replication is suppressed in the lung.

Comparison of gene expression in lungs of mice refed for 3 h after 14 days of calorie restriction (Table 4) to gene expression in lungs of mice restricted for 14 days (Table 3) showed the expression of only one gene, cyclin D1, was different between groups at both times. The expression of the other 27 genes, which were different between mice restricted for 14 days and fed for 14 days, was not different within 3 h of refeeding. The absence in Table 4 of Smad4 and the other suppressive genes present in Table 3 means their expression at the RNA level, supporting suppression of cell replication, was abrogated by 3 h of refeeding. This conclusion is confirmed by our studies of lung DNA synthesis (Fig. 1) (vide infra).


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  		Table 4. Expression of cell replication-related genes in lungs of mice calorie restricted for 14 days and then ad libitum fed for 3 h, compared with always ad libitum fed: microarray

 

Figure 1
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  		Fig. 1. Calorie-related changes in DNA synthesis by lung. Means ± SD are given. Numbers within the bars indicate the number of mice. CR, calorie restricted 14 days; Fed 1, ad libitum fed 14 days; CR-3h RF, calorie restricted 14 days then allowed food ad libitum for 3 h; Fed 2, always allowed food ad libitum and killed at the same time as CR-3h RF.

 
Microarray-assessed angiogenesis-related gene expression after 14 days of calorie restriction and after 14 days of calorie restriction followed by ad libitum access to food for 3 h. Angiogenesis is a multiphase process that includes remodeling of extracellular matrix, disruption of the vessel wall, endothelial cell migration and proliferation, and formation of a capillary tube. Among the genes for which expression was higher in lungs of mice restricted for 14 days than in lungs of fed mice, fms-like kinase-1 (Flt-1) is a potent inhibitor of VEGF and of placental growth factor, two powerful angiogenic agents (21) (Table 5A). Expression of mRNA of important angiogenic molecules (10, 48, 54), VEGF-A, tyrosine kinase receptors 1 and 2, which are receptors for angiopoietin-2 (50), a protector for stressed endothelial cells (8), was low in lungs of restricted mice, as were Flt-3, angiogenin, and thrombomodulin, potent inducers of angiogenesis (3, 10). Other mediators of angiogenesis platelet-derived growth factor (PDGF)-beta, its receptor, fms-like kinase-3, and transforming growth factor (TGF)-beta2, Mac 25, coagulation factor VII, and Flt-3 (13) were lower in lungs of restricted than in lungs of fed mice (Table 5A). It is interesting that expression of caveolin-1, which is required for liver regeneration (9) following partial hepatectomy, was depressed in lungs of restricted mice. Expression of Dutt-1 (Robo1), a receptor for Slit (4), and neuropilin-1, a receptor for VEGF (54), which are cellular movement guidance molecules (53), was lower in lungs of restricted mice than in lungs of fed mice (Table 5A). PDGF-beta and TGF-beta2, both important proangiogenic mediators (10, 41), exhibited lower expression in lungs of restricted than in lungs of fed mice (Table 5A).


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  		Table 5. Gene expression in lungs of mice calorie restricted by two-thirds for 14 days, compared with always ad libitum fed: microarray

 
MMP are involved in angiogenesis; they remodel extracellular matrix and, in doing so, release growth factors, e.g., VEGF sequestered within the matrix (34, 57). The mRNA of MMP-3, -9, -11, and -15 was diminished in lungs of restricted mice compared with lungs of fed mice (Table 5B). Inhibition of MMP-9 diminishes endothelial cell migration (34); increase of MMP-11 is thought to be angiogenic (58), and degradation products of MMP 3 are angiogenic (45). Tissue inhibitor of metalloproteinase (TIMP)-3 mRNA was elevated at 14 days in restricted compared with fed mice (Table 5B); its protein inhibits angiogenesis by blocking binding of VEGF by its receptor (45). The difference in expression of MMP-3, -9, -11, and -15 and TIMP-3 between lungs of mice restricted for 14 days compared with lungs of fed mice (Table 5B) is consistent with an absence of angiogenesis during calorie restriction. The data in Table 5, A and B, led us to conclude that calorie restriction impairs gene expression supportive of angiogenesis.

Within 3 h of refeeding, the expression of genes important to angiogenesis, for which expression was low after 14 days of calorie restriction (Table 5A), did not remain differently expressed between refed mice and fed mice (Table 6A), i.e., none of the genes that exhibit an intergroup difference in Table 5A appear in Table 6A. Those angiogenesis-related genes are absent from Table 6A because, 3 h after refeeding, these genes did not exhibit a statistically significant intergroup difference of expression. Put differently, they went from being lower to a statistically significant degree between groups at 14 days to not being statistically different between groups at 14 days plus 3 h, i.e., their expression increased during 3 h of refeeding. This indicates that within 3 h of refeeding, gene expression in lung, at the RNA level, is supportive of angiogenesis.


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  		Table 6. Expression of genes in lungs of mice calorie restricted for 14 days and then ad libitum fed for 3 h, compared with always ad libitum fed: microarray

 
Microarray-assessed expression of extracellular matrix-related genes. Among extracellular matrix-related genes for which expression was significantly different (P < 0.05) between lungs of mice restricted for 14 days and lungs of fed mice, 80% were lower in the former than in the latter (Table 5B). Within 3 h of refeeding, these differences were eliminated except for the intergroup difference of procollagen type VI {alpha} I (compare Tables 5B and 6B). These findings demonstrate that the lung is capable of changing gene expression at the RNA level that supports loss and, as shown by Kuhn et al. (23), regeneration of lung elastin and collagen.

Microarray-assessed expression of guided cell motion-related genes. Of 30 cell motion and guidance-related genes, expression of 21 (70%) was lower in lungs of mice restricted for 14 days compared with fed mice (Table 5C). Within 3 h of refeeding, only one mRNA, chemokine (C-X-C motif) receptor-3, that exhibited an intergroup difference at 14 days of calorie restriction maintained an intergroup difference 3 h after refeeding (compare Tables 5C and 6C). Thus, of 30 mRNAs that exhibited an intergroup difference at 14 days, 29 failed to exhibit an intergroup difference of their mRNA within 3 h after refeeding.

RT-PCR assessment of gene expression in lung after 14 days of calorie restriction and 1 h after allowing calorie-restricted mice ad libitum access to food. Next, we sought to determine if gene expression at the RNA level, supportive of alveolar regeneration, was in place in restricted mice less than 3 h after refeeding. In this case, the approach was less global than the microarray work; we assessed expression of genes for which proteins would support processes required to form a septum. Among 13 cell cycle-related genes, all except cyclin D3 were lower in restricted mice compared with fed mice (Table 7). One hour after refeeding (Table 8), expression of three of these genes (cyclin-dependent kinase-2, cyclin E2, and cyclin-dependent kinase-1b inhibitor) was greater in refed mice compared with always-fed mice (compare Table 8 with Table 7). Expression of two genes (cyclin-dependent kinase-4 and cell division cycle 37 homolog) was lower in 14-day restricted mice than in fed mice; this difference was not present between lungs of mice refed for 1 h compared with lungs of fed mice (compare Tables 8 and 7). Therefore, within 1 h of refeeding, expression of 5 of 12 previously downregulated cell replication-related genes was increased. Among genes for which expression was elevated within 1 h of refeeding were key genes needed for entry into G1, i.e., cyclin-dependent kinases 2 and 4. From these data we conclude that within 1 h of refeeding, gene expression at the RNA level supportive of cell replication was in place.


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  		Table 7. Expression of cell replication-related genes in lungs of mice calorie restricted for 14 days, compared with always ad libitum fed: RT-PCR

 

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  		Table 8. Expression of cell replication-related genes in lungs of mice calorie restricted for 14 days and then ad libitum fed for 1 h, compared with always ad libitum fed: RT-PCR

 
Among angiogenesis-related genes for which expression was assessed by RT-PCR, there was little evidence of a change in gene expression supportive of angiogenesis within the first hour of refeeding compared with expression after 14 days of calorie restriction (Tables 9 and 10). A similar lack of change was found at 1 h for genes supportive of extracellular matrix remodeling (Tables 11 and 12) and among guided cell motion compared with gene expression before refeeding (Tables 13 and 14).


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  		Table 9. Expression of angiogenesis-related genes in lungs of mice calorie restricted for 14 days, compared with ad libitum fed: RT-PCR

 

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  		Table 10. Expression of angiogenesis-related genes in lungs of mice calorie restricted for 14 days and then ad libitum fed for 1 h, compared with always ad libitum fed: RT-PCR

 

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  		Table 11. Expression of extracellular matrix-related genes in lungs of mice calorie restricted for 14 days, compared with always ad libitum fed: RT-PCR

 

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  		Table 12. Expression of extracellular matrix-related genes in lungs of mice calorie restricted for 14 days and then ad libitum fed for 1 h, compared with always ad libitum fed: RT-PCR

 

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  		Table 13. Expression of guided cell motion-related genes in lungs of mice calorie restricted for 14 days, compared with always ad libitum fed: RT-PCR

 

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  		Table 14. Expression of guided cell motion-related genes by lungs of mice calorie restricted for 14 days and then ad libitum fed for 1 h, compared with always ad libitum fed

 
DNA synthesis and protein expression in lung. Cell replication was assayed more directly than by RNA-level gene expression by measuring DNA synthesis in lung. DNA synthesis by slices of lung from mice restricted for 14 days and then refed for 3 h was 17% higher than DNA synthesis by lung slices from mice restricted for 14 days (Fig. 1). Viewed differently, DNA synthesis by slices of lungs from restricted mice was 4.1-fold lower than DNA synthesis by slices of lung from fed mice (Fig. 1), whereas DNA synthesis by slices of lungs from mice refed for 3 h was 3.0-fold lower than DNA synthesis by slices of lung from fed mice (P < 0.004 between the fold differences; Fig. 1).

To assess translation of select RNAs into protein concentration, we used Western blot analysis and ELISA. The relative concentration of VEGF-A protein and MMP-3 protein was lower and that of Plexin A2 was higher in lungs of mice refed for 1 h than in lungs of fed mice (Table 15).


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  		Table 15. Relative protein concentration in lungs of mice calorie restricted for 14 days and then ad libitum fed, compared with always ad libitum fed

 
These results of DNA synthesis and relative protein concentration (Fig. 1 and Table 15) support a conclusion that DNA synthesis, reflecting cell replication, was elevated within 1 h of refeeding but do not support a conclusion that the processes of angiogenesis, extracellular matrix remodeling, or guided cell motion had been initiated.

Changes in food intake and gene expression in liver and kidney. During calorie restriction, lung (33), but not liver (12) or kidney (1), exhibits calorie-related changes in the amount of DNA present. Therefore, as a comparison between lung, which exhibits calorie-related changes in its amount of DNA, and liver and kidney, which do not, we used RT-PCR and assessed gene expression related to cell replication and to angiogenesis in liver and to cell replication in kidney. Expression of cyclins D1, D2, and E2 was lower, and expression of cyclin-dependent kinase inhibitor-1b was higher, in liver of restricted mice than in liver of fed mice (Supplemental Table S2); elevation of cyclin-dependent kinase inhibitor-1b is expected to prevent the action of the elevated cyclin-dependent kinase-2. There were no other intergroup differences among the genes tested. Overall, these findings indicate that cell replication is decreased in the liver of mice after 14 days of calorie restriction. At 1 h after refeeding, the higher expression of the cyclin-dependent kinase-6, cyclin D3, and cell cycle division cycle 37 homolog provided some evidence of increased cell replication in liver (Supplemental Table S3). However, the majority of cell cycle-related genes tested were either not changed (cyclin-dependent kinases 2 and 4, cyclin-dependent kinase inhibitor-1b, and cell cycle division cycle 2A homolog) or were lower in liver of mice refed for 1 h (cyclins A2, B2, D1, D2, E2, and Mcm2). Thus the preponderance of evidence failed to support robust cell replication in liver 1 h after refeeding. Liver did not exhibit calorie restriction-induced changes in expression of angiogenesis-related genes (Supplemental Table S4), but, upon refeeding, did demonstrate some change in gene expression consistent with the presence of angiogenesis, i.e., a higher expression of Flt-1, a receptor for VEGF-A (Supplemental Table S5). These findings are not consistent with the reported failure of changes of calorie intake to alter the DNA content of liver (12). However, as noted below for the kidney, a subset of cells in the liver may experience calorie-related changes in cell replication, but the magnitude of the change in cell replication may be too small to be identified by measuring total organ DNA.

Calorie restriction did not alter expression of cell cycle-related genes in kidney (Supplemental Table S6). One hour after refeeding, gene expression at the RNA level was also not strongly supportive of cell replication in kidney (Supplemental Table S7), e.g., the greatly higher expression of cyclin-dependent kinase inhibitor-1b, which would prevent entry into the cell cycle by the less elevated expression of cyclin-dependent kinases 2 and 4. Of the cyclins tested, only B2 and E2 were elevated after 1 h of refeeding. These findings are consistent with the failure of calorie restriction to alter the DNA content of kidney (1), despite recent evidence that indicates renal tubular epithelium does regenerate (38). If the regenerating cells represent a small portion of renal cells, their replication might be insufficient to measurably alter the total amount of DNA in the kidney.


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Aim and modus operandi. The absence of remedial therapy for chronic obstructive pulmonary disease (COPD) led to studies to identify pharmacological reagents to induce alveolar regeneration in animal models of emphysema. Four reagents, namely all-trans-retinoic acid (18, 32, 42), adrenomedulin (40), granulocyte monocyte colony-stimulating factor (18), and hepatic growth factor (51), selected on the basis of their biological actions, induce alveolar regeneration in lungs of rodents with experimentally produced emphysema. Furthermore, a recent feasibility trial of the use of retinoids in individuals with COPD has provided evidence of a delayed improvement of the health-related quality of life and an isolated, statistically significant decrease of pulmonary hyperinflation, but it was concluded that retinoids could not yet be recommended for treatment of COPD (47). It seems reasonable to conclude from these observations that a fundamental understanding of gene expression determinative of alveolar regeneration, which could be manipulated for therapeutic purposes, would accelerate achieving the goal of alveolar regeneration in humans. Toward achieving such an understanding, we have assumed that initial signal(s) induce(s) a complex repertoire of downstream changes of gene expression that result in processes needed to turn a flat area of alveolus into a fold (septum) capable of gas exchange. These processes, among others, include cell replication, angiogenesis extracellular matrix remodeling, and guided cell motion. To keep our experimental approach manageable, we have focused on these gene expression related to these four biological processes.

We chose not to make assumptions about candidate genes that initiate alveolar regeneration; instead we used microarray gene profiling to obtain a global view of gene expression induced by our experimental maneuvers. Fourteen days of calorie restriction was chosen as a time to obtain lungs for microarray analysis because, at that time, the rate of calorie restriction-related alveolar loss has become asymptotic, and hence the lungs are close to an architectural steady state (30, 33). Three hours of ad libitum access to food, following 14 days of calorie restriction, was chosen on the assumption that the organism's survival in the wild requires a rapid response to full feeding, including regeneration of alveoli to meet the organism's calorie-related higher O2 consumption (39). Based on the results from the microarray experiments, which revealed gene expression at the RNA-level supportive of cellular processes needed to form a septum, i.e., cell replication, angiogenesis, extracellular matrix remodeling, and guided cell motion, we assayed gene expression less globally, but more informed, using RT-PCR. Finally, we used more functional indicators of the presence or absence of these processes, i.e., DNA synthesis and expression of selected proteins in lung.

Interpretation of microarray and RT-PCR findings. We interpret the results from cell replication-related gene expression (Table 3) to mean cell replication in the lung is depressed after 14 days of calorie restriction. More specifically, very upstream regulators of the cell cycle, i.e., cyclin-dependent kinase inhibitors, are elevated, two D cyclins, which initiate entry into G1, are low, as is B1, a controller of passage through M phase of the cell cycle. The role of cyclin G is less well-characterized than that of several other members of cyclin family. However, cyclin G can be a negative regulator of the p53 activation pathway (43). Within 3 h of refeeding, expression of several key inhibitors of cell replication (cyclin-dependent kinase inhibitors), which were elevated at 14 days of calorie restriction (Table 3), were no longer elevated (compare Tables 3 and 4).

The evidence that gene expression in the lung after 14 days of calorie restriction and 3 h after refeeding indicates downregulation at 14 days and upregulation at 3 h led us to test if gene expression supportive of cell replication was evident 1 h after refeeding. The data in Table 7, which show 12 of 13 cell cycle-related genes tested, were lower in calorie-restricted compared with fed mice, indicating cell replication is suppressed in lungs of mice restricted for 14 days. By contrast, expression of key genes, for which protein products result in leaving the arrest stage of the cell cycle, e.g., cyclin-dependent kinase-2 and cyclin E2, is higher in mice refed for 1 h compared with fed mice (Table 8). These conclusions are supported by the lower rate of DNA synthesis in the lung of 14-day restricted mice than in the lung of always-fed mice and by the higher rate of DNA synthesis in lung of refed mice compared with 14-day restricted mice (Fig. 1).

Our interpretation of microarray and RT-PCR experiments on cell replication-related gene expression is that calorie restriction results in suppression of cell replication in the lung, and refeeding after calorie restriction induces gene expression supportive of cell replication in the lung within 1 h. The studies of DNA synthesis in lung support this interpretation.

Comparison of calorie-related changes of gene expression among lung, liver, and kidney. Unlike the lung (30, 33), the DNA content of the liver (12) and kidney (1) does not diminish during calorie restriction. Therefore, for comparison with the lung, we tested the effect of calorie restriction and refeeding on cell replication-related gene expression in liver and kidney. In liver at 14 days of calorie restriction, the RNA cyclins D1, D2, and E2 were lower in restricted mice than in fed mice. After 1 h of refeeding, cyclin-dependent kinase-6 RNA was higher in liver of refed mice than in always-fed mice. However, except for cyclin D3, the other D cyclins and cyclin E2, which act as partners with cyclin-dependent kinase-6 to initiate the cycle, are lower in liver of restricted mice than in mice refed for 1 h. Based on these observations, we conclude there is evidence to support the notion that calorie restriction depresses cell replication in liver but less evidence to support the notion that refeeding after calorie restriction alters cell replication in the liver. These changes in RNA-level gene expression are not consistent with the measurements of DNA that demonstrate neither calorie restriction nor refeeding after calorie restriction alters the total number of cells in the liver (12). However, assuming that the RNA-level changes in gene expression are translated into changes in protein concentration, it seems very possible that increased replication by a small number of cells would not be detected by measuring total liver DNA. The liver is the exemplar of organ regeneration (55). Therefore, to further our comparison with lung, we tested for the presence of an angiogenic response at the RNA level to altered calorie intake. Calorie restriction did not result in angiogenesis-related changes in gene expression, but upon refeeding, the liver did have changes in gene expression supportive of angiogenesis. Unlike lung, in kidney, expression at the RNA level of genes supportive of cell replication was not changed by calorie restriction. However, as in lung, gene expression in kidney is consistent with a conclusion that refeeding results in new cell formation. It is possible that calorie-related regeneration of a small number of cells in the kidney is not detected by measurement of total kidney DNA (38) or that the RNA is not translated. Irrespective, calorie-related gene expression differs among lung, liver, and kidney, demonstrating a selective response of these organs to calorie restriction and refeeding.

Conservation of gene expression determinative of alveolar regeneration. Because a major goal of our animal work is translation into patient care, the relevance, or lack of relevance, of the animal models used to humans is very important. Calorie restriction in rats (14, 20, 49), hamsters (19), and mice (30, 33) induces alveolar loss; ad libitum refeeding after calorie restriction, where tested (rats and mice; Refs. 30, 33, 49), results in alveolar regeneration. People starved to death (11) and individuals with severe weight loss due to anorexia nervosa (6, 7) lose alveoli as determined by microscopic examination (11) and computed tomography (7). This demonstrates that calorie-related loss of alveoli has been conserved from rodents to humans. Whether alveolar regeneration in humans with alveolar loss due to starvation or calorie restriction occurs following an increased consumption of food has not, to our knowledge, been reported. However, in view of the conservation of alveolar loss due to calorie restriction from rodents to humans, it seems likely that alveolar regeneration following increased calorie intake, which occurs in rodents (30, 33, 49), has to also be conserved in humans as a response to periodic episodes of food scarcity and plenty during evolution.


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This study was supported by National Heart, Lung, and Blood Institute Grants HL-020366, HL-73558, and HL-37666.


   DISCLOSURES
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REFERENCES
 
D. J. Massaro and G. D. Massaro hold a patent for the use of retinoids in pulmonary diseases.


   ACKNOWLEDGMENTS
 
We thank Andrea DeBiase at the Center for Genetic Medicine, Children's National Medical Center, Washington, DC, for technical expertise in Affymetrix microarray analysis.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. J. Massaro, Lung Biology Laboratory, Box 571481, 3900 Reservoir Rd. NW, Washington, DC 20057-1481 (e-mail: massarod{at}georgetown.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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