SPECIAL TOPIC
Pre- and Postnatal Lung Development, Maturation, and Plasticity
Insulin-like growth factor I receptor is downregulated after alveolarization in an apoptotic fibroblast subset

Suseela Srinivasan1, Jennifer Strange2, Feyisola Awonusonu1, and Margaret C. Bruce1

Departments of 1 Pediatrics and 2 Immunology, University of Kentucky Medical School, Lexington, Kentucky 40536


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

After alveolar formation, >20% of interstitial lung fibroblasts undergo apoptosis, a process that is of critical importance for normal lung maturation. The immature lung contains two morphologically distinct fibroblast populations, lipid-filled interstitial fibroblasts (LIF) and non-LIF (NLIF), which differ with respect to contractile protein content, proliferative capacity, and expression of mRNAs for fibronectin and types I and III collagen, but not tropoelastin. After alveolarization, apoptosis occurs in only one fibroblast population, the LIF. Using flow cytometry to analyze fibroblasts stained with a lipophilic, fluorescent dye, we identified a subset, designated LIF(-), that contained fewer lipid droplets. Unlike LIF that retain lipid, LIF(+), the LIF(-) do not undergo apoptosis after alveolarization. In LIF(+), apoptosis was correlated with downregulation of insulin-like growth factor I receptor (IGF-IR) mRNA and cell surface protein expression. Treatment with anti-IGF-IR decreased total lung fibroblast survival (P = 0.05) as did treatment with the phosphatidylinositol 3-kinase inhibitor LY-294002 and the ras-raf-mitogen-activated protein kinase inhibitor PD-98059 (P < 0.002), which block IGF-I/insulin receptor survival pathways. These observations implicate downregulation of IGF-IR expression in fibroblast apoptosis after alveolar formation.

lung development; PI 3-kinase; ras-raf-MAP kinase; flow cytometry; lipid interstitial fibroblasts


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

DURING DEVELOPMENT, CELLS that were once critical for the growth of an organ are often removed by apoptosis when their continued presence might adversely impact the function of the mature organ. In the immature lung, >20% of interstitial fibroblasts undergo apoptosis after the period of bulk alveolarization, resulting in a substantial reduction in interstitial volume (1, 6, 15, 25). The thinning of alveolar walls decreases the diffusion distance for inspired oxygen, thus enhancing gas exchange. In addition, the removal of a substantial fraction of interstitial fibroblasts facilitates the formation of intercellular contacts between endothelial cells of adjacent capillary layers, a process thought to promote the subsequent maturation of the lung vasculature, an integral part of which is the transformation from a double to a single capillary network in alveolar walls (25).

Apoptosis was first implicated as a contributing factor in lung development only recently. In the fetal rat lung, apoptotic mesenchymal cells were evident at days 14-18 of gestation, whereas apoptotic epithelial cells appeared somewhat later at 18 days of gestation (17, 24). The apoptotic index was observed to increase from <1% in the fetal lung to 9% at birth, followed by a decrease to 1.5% on postnatal day 2 (17). We and others have shown that after alveolar formation, the incidence of apoptotic lung fibroblasts increases sharply, reaching peak levels that exceed 20% on postnatal days 17-19 (6, 25). The onset of apoptosis was reported by Schittny and colleagues (25) to be delayed by several days when litter size exceeded 14 pups, however, suggesting the possible involvement of multiple factors in the regulatory control of this developmental apoptotic process.

After alveolar formation, apoptosis occurs in only one of two morphologically distinct rat lung fibroblast populations, the lipid-filled interstitial fibroblast (LIF). This developmental apoptotic event results in the shift from a predominantly LIF population before septation to one that consists primarily of nonlipid interstitial fibroblasts (NLIF) in the mature lung (1, 20). LIF and NLIF both express tropoelastin mRNA (1, 21), a connective tissue protein that is of critical importance for both normal fetal airway (29) and postnatal alveolar (2) development. These two subsets appear to have little else in common, however (1, 7). In addition to differences with respect to intracellular lipid content (21), LIF and NLIF differ substantially with respect to size (20), contractile protein content (20), expression of mRNAs for fibronectin (1) and types I and III collagen (3), and proliferative capacity (1), suggesting the potential for unique roles for each of these subsets during development and in response to injury.

Although developmental apoptosis has been investigated extensively in many other organ systems, the cell death pathways activated in lung fibroblasts after septation have not yet been identified. To address this issue, the possible involvement of the insulin-like growth factor I receptor (IGF-IR) in the marked increase in the incidence of fibroblast apoptosis that occurs after alveolarization was considered in view of the well-documented role of this receptor as a survival factor in both fibroblasts (18) and adipocytes (22), which are similar in many respects to the LIF. Consistent with the hypothesis that decreased signaling via the IGF-IR contributes to lung fibroblast apoptosis, IGF-IR mRNA expression in whole lung homogenate decreases substantially after alveolar formation, coinciding with the onset of apoptosis in rat lung fibroblasts (30).

Both the phosphatidylinositol (PI) 3-kinase and the ras/raf/mitogen-activated protein (MAP) kinase (MAPK) pathways have been implicated in IGF-IR-mediated survival signaling (23). The IGF-IR survival pathway is initiated by ligand binding, receptor autophosphorylation, and activation of tyrosine kinase activity. Activation of the PI 3-kinase pathway involves the tyrosine phosphorylation of insulin receptor substrates (IRS-1, IRS-2), which then interact with SH2 domains of the p85 subunit of PI 3-kinase, which in turn activates Akt/protein kinase B (PKB). Activated Akt/PKB can phosphorylate Bad, which suppresses apoptosis. IGF-IR and the insulin receptor (IR) can activate the MAPK pathway either by direct interaction with Shc proteins that bind growth factor receptor-bound protein 2 (Grb2) or indirectly through IRS-1 and IRS-2, which contain binding sites for Grb2 (31). The binding of Shc and Grb2 to the activated receptor leads to the activation of the ras-MAPK signaling pathway.

In some systems, IGF-IR activation of PI 3-kinase and its downstream effector PKB/Akt is necessary and sufficient to mediate survival (18), whereas the MAPK pathway influences survival in other systems (23). Downstream of the MAPK and PI 3-kinase pathways, Bad is thought to be a target for at least three antiapoptotic protein kinases: MAPK, protein kinase A (PKA), and PKB. Each of these protein kinases appears to phosphorylate Bad at a different site, however (10). Although a proapoptotic member of the bcl-2 family, once phosphorylated, Bad can no longer bind and inactivate bcl-2 (11, 18).

In the present study we compared IGF-IR expression in LIF, which undergo apoptosis after alveolar formation, with expression in NLIF, which do not, to determine whether there was a correlation between IGF-IR expression and survival. We observed that IGF-IR message and cell surface protein expression were both downregulated in LIF after alveolar formation on postnatal days 16-18, strongly suggesting a role for the IGF-IR in lung fibroblast survival. In further support of this concept, lung fibroblast apoptosis could be induced in vitro by treatment with alpha IR-3, an antibody that binds the ligand binding site on the IGF-IR. Using specific inhibitors of the PI 3-kinase and the ras-raf-MAPK pathways, we observed a significant increase in cell death in response to treatment with each of these inhibitors, suggesting the involvement of both the PI 3-kinase and MAPK pathways in postnatal lung fibroblast survival.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Fibroblast isolation. Fibroblasts were isolated from the lungs of fetal and postnatal Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) on postnatal days 3-30 according to a procedure previously described in detail (5). Cells were cultured in either complete medium or serum-free (S-F) medium as indicated. Complete medium contained 1:1 (vol/vol) Dulbecco's modified Eagle's medium (DMEM)-Ham's F-12, 10% fetal bovine serum, penicillin (100 U/ml), streptomycin (10 mg/ml), and glutamine (292 µg/ml). S-F medium contained 1:1 (vol/vol) DMEM-Ham's F-12 medium, 1 mg/ml fatty acid-free bovine serum albumin (Sigma, St. Louis, MO), 10 µg/ml iron-saturated transferrin (Sigma), 2 × 10-8 M selenium (Sigma), penicillin (100 U/ml), streptomycin (10 mg/ml), and glutamine (292 µg/ml). Unless otherwise indicated, enzymes and tissue culture reagents were purchased from GIBCO BRL (Life Technologies, Grand Island, NY).

Quantitation and sorting of LIF by flow cytometry. Adherent fibroblasts were detached from flasks using 0.05% trypsin-0.02% EDTA and then either fixed in paraformaldehyde (0.5% final concentration) or resuspended in PBS. Both fixed and unfixed cells were maintained at 4°C during centrifugation at 1,200 rpm for 10 min, filtration through nylon mesh (70 µm), and treatment with Nile red (9-diethylamino-5H-benzo[alpha ]phenoxazine-5-one), a fluorescent, lipophilic dye (Molecular Probes, Eugene, OR) to stain intracellular lipid droplets, as previously described (1). The Nile red stock solution (1.0 mg/ml DMSO) was diluted in saline and then added immediately to the fibroblast suspension to achieve a final concentration of 0.1 µg Nile red/ml.

Fibroblasts were analyzed on a FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA) equipped with a 15-mW argon-ion laser operated at 488 nm. A short-pass dichroic beam splitter transmitted emission <560 nm. Nile red fluorescence was collected through a 530/30-nm band-pass filter. Forward-angle light scatter (FSC) and side-angle light scatter (SSC) measurements, estimates of cell size and of the structural complexity of the cell membrane and internal cell structures, respectively, were expressed on a linear scale. LIF were distinguished from NLIF on the basis of intracellular lipid content, relative size (FSC) and relative structural complexity (SSC). Changes in the relative percentages of fibroblast subsets were quantitated as a function of age. At least 10,000 cells were analyzed in each sample. Data were analyzed using CellQuest software from Becton Dickinson. The term LIF when used throughout the text refers to all lipid-containing fibroblasts. The terms LIF(+) and LIF(-) are used when the relative intracellular lipid content of fibroblast subsets was determined by flow cytometric analysis.

In separate experiments, fibroblasts subsets were sorted by flow cytometry on the basis of size, structural complexity, and intracellular lipid content. NLIF, LIF, LIF(+), and LIF(-) subsets were first identified on the basis of Nile red fluorescence intensity, which was analyzed with CyCLOPS Summit software (Cytomation, Fort Collins, CO). Cells that appeared in regions of overlap between two peaks were excluded. Next, each population minus the cells contained in region(s) of overlap was further characterized by plotting Nile red fluorescence intensity vs. SSC, again eliminating any cells that appeared in regions of overlap between two populations. Fibroblasts were then sorted under sterile conditions at a rate of 1-2,000 cells/s into tubes maintained at 4°C using a MoFlo flow cytometer (Cytomation) equipped with a Coherent I-70 laser tuned to 488 nm and regulated to 100 mW. After sorting, the fibroblast subsets were centrifuged at 4°C and the cell pellets were stored at -80°C.

Analysis of cell surface IGF-IR expression by flow cytometry. Lung cell homogenate was plated for 1 h in DMEM:Ham's F-12 containing 10% autologous rat serum and then detached from flasks by treatment with nonenzymatic Cell Dissociation Solution (Sigma) on a rocking platform for 10 min at 37°C. The cells were then fixed briefly at 4°C with an equal volume of 0.2% paraformaldehyde to preserve cell integrity during subsequent centrifugation steps. The fixed cells were rinsed in RPMI containing 1.0% BSA, resuspended in anti-IGF-IR (clone alpha IR-3) (8.0 µg/ml in RPMI-1.0% BSA), incubated on ice for 2.5 h, rinsed in RPMI-1.0% BSA, and stained with an R-phycoerythrin-conjugated F(ab') fragment of goat anti-mouse IgG (100 µg/ml) (Dako, Carpinteria, CA) for 45 min on ice, rinsed with RPMI-1.0% BSA, and stored in 1.0% paraformaldehyde at 4°C until they were assayed by flow cytometry. The monoclonal antibody alpha IR-3, which binds reversibly to the ligand binding site on the IGF-IR (18), was obtained from Oncogene Science (Uniondale, NY). An unstained sample and a sample stained only with the secondary antibody were analyzed in each experiment. Emission spectra for Nile red overlap with the emission spectra for phycoerythrin; thus it was necessary to stain an aliquot of each cell suspension with Nile red alone to identify the appropriate regions for each fibroblast subset. Cell surface-bound fluorescence was analyzed on a Becton Dickinson FACS Calibur flow cytometer using a 15-mW argon-ion laser operated at 488 nm. Phycoerythrin fluorescence was collected through a 585-nm band pass filter. Data were analyzed using CyCLOPS Summit software obtained from Cytomation.

Extraction of total RNA and RT-PCR. Total RNA extracted from freshly isolated fibroblasts sorted under sterile conditions was purified and reverse transcribed as described previously (5). The number of rats used in each assay varied with postnatal age. In each of the three experiments conducted on 4-day rat pups, fibroblasts were obtained from 6-8 rats, and at 17 days each of four experiments was conducted on fibroblasts obtained from 10-12 rats. The PCR products were resolved by electrophoresis on 12.5% polyacrylamide gels and stained with SYBR-Gold (Molecular Probes). Fluorescent PCR products were quantitated on a Storm 840 using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). The identities of amplified cDNAs were confirmed on an automatic sequencer (Perkin-Elmer) located in the Macromolecular Structure Analysis Facility at the University of Kentucky.

PCR primers. To amplify the IGF-IR 364-bp product, the 5'-forward and 3'-reverse complement primer sequences used were 5-ATGAGTACAACTACCGCTGC-3' and 5'TACATGCTCTGGGTGCTGTT-3, respectively. The primer pair used to amplify cyclophilin has been published previously (5).

Survival assay. Freshly isolated fibroblasts were allowed to adhere for 1 h and then were thoroughly rinsed to remove nonadherent cells. To assess the influence of IGF-IR expression on fibroblast survival, cells were cultured for 24 and 48 h either in complete or S-F medium in the presence of monoclonal antibodies (2.0 µg/ml) directed against the IGF-IR (clone alpha IR-3) or against the beta -subunit of the IR (clone CT-3) (Oncogene Science) (18). To assess the influence of specific kinase pathways on lung fibroblast survival, cells were treated with specific inhibitors of PI 3-kinase (20 µM LY-294002, Cayman Chemical, Ann Arbor, MI) and MAPK kinase (MEK; 20 µM PD-98059, CalBiochem, San Diego, CA) for 24 h in complete or S-F media. Viability was assessed after staining cells with 1.0 µg/ml propidium iodide and 1.0 µg/ml calcein AM (Molecular Probes) to identify dead and live cells, respectively. Cells were viewed on a Nikon Diaphot 300 microscope equipped with filter sets for fluorescence.

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. Chromatin fragmentation was assessed using a fluorescent terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay (In Situ Cell Death Detection Kit, Boehringer Mannheim, Indianapolis, IN). In brief, adherent treated and control cells were fixed in freshly prepared 4% paraformaldehyde (pH 7.4) for 1 h at room temperature, rinsed in PBS, permeabilized for 2 min on ice, and rinsed twice with PBS. Next, the TUNEL reaction mix was applied to each slide, covered with Parafilm, incubated for 1 h in the dark at 37°C, and rinsed 3× with PBS. To assess chromatin condensation, slides were counterstained for 10 min with 1.0 µg/ml Hoechst 33258 (Molecular Probes), rinsed with PBS, mounted in glycerol-PBS (1:2), and viewed under a Nikon Diaphot 300 microscope equipped with filter sets for fluorescence [Chroma fluorescein isothiocyanate filter cube for TUNEL; Nikon 4'-6-diamidino-2-phenylindole filter cube for Hoechst].

Statistical analysis of data. Values are presented as means ± SD. The significance of decreases in the relative percentage of LIF and in cell surface expression of IGF-IR with postnatal age was evaluated by Student's t-test. To assess the effects on fibroblast survival of treatment with specific kinase inhibitors in the presence and absence of serum, we used a two-factor ANOVA with Fisher's least-significant difference correction for multiple comparisons (Systat version 9.0, SPSS, Chicago, IL). Values were considered to be significantly different when P <=  0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Intracellular lipid content of a subpopulation of LIF decreases during postnatal lung development. In the late fetal and early postnatal rat lung, the LIF is the predominant fibroblast subset, accounting for 80% of lung fibroblasts on postnatal day 4 (1). At the end of the period of bulk alveolar formation, LIF undergo apoptosis (1), resulting in a fibroblast population in the mature lung that consists primarily of NLIF. In addition to the decrease in the relative percentage of LIF during postnatal development, there is also a substantial loss of lipid droplets from many of the LIF that remain in the lung (1, 9, 20). One objective of this investigation was to further characterize the population of LIF that loses intracellular lipid droplets as a function of postnatal age.

Using flow cytometry to quantitate the intracellular lipid content in freshly isolated rat lung fibroblasts stained with the lipophilic fluorescent dye Nile red, we identified, at the beginning of the second postnatal week, a separate population consisting of fibroblasts that stained less intensely with Nile red than did LIF (Fig. 1A). Light microscopic analysis of fibroblasts sorted by flow cytometry on the basis of Nile red fluorescence intensity and then stained with oil red O, indicated that cells in this subpopulation were similar in appearance to LIF but contained substantially fewer lipid droplets than LIF. Accordingly, these cells were designated LIF(-).


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Fig. 1.   Flow cytometric analysis of intracellular lipid content in freshly isolated rat lung fibroblasts stained with Nile red, a lipophilic fluorescent dye, as described in METHODS. A and C: representative histograms are presented for cells obtained on postnatal days 9 and 17. Fluorescence intensity height (FL1) is plotted on a log scale on the x-axis. Cells in region R3 were more fluorescent due to their intracellular lipid content than were cells in either regions R2 or R1. B and D: dot plots of forward-angle light scatter (FSC), indicative of relative size, vs. side-angle light scatter (SSC), indicative of relative structural complexity, are shown for each of 3 populations from 9- and 17-day rats. FSC and SSC characteristics of nonlipid interstitial fibroblasts (NLIF), shown in yellow, are essentially the same in cells from 9- and 17-day rats. In 17-day rats, lipid-filled interstitial fibroblasts [LIF(-)], shown in blue, have FSC and SSC characteristics similar to those in 9-day LIF(-). In contrast, when compared with FSC and SSC characteristics on postnatal day 9, 17-day LIF(+), shown in red, shift to the left along the x-axis, indicative of a decrease in apparent size, consistent with apoptosis.

Further characterization of LIF(-) by flow cytometry confirmed light microscopic observations that cells with decreased Nile red fluorescence were much less structurally complex than the remainder of the LIF, consistent with a loss of intracellular lipid droplets in LIF(-). Lung fibroblasts from 9-day rats are shown in Fig. 1A to segregate into three populations based on Nile red fluorescence intensity. Nile red fluorescence was the least intense in NLIF (shown in yellow); the minimal fluorescence seen in these cells is likely to be due to the uptake of Nile red by cell membrane lipids (14). Nile red fluorescence was intermediate in intensity in LIF(-) (shown in blue) and most intense in LIF(+) (shown in red), indicating that LIF(-) contained fewer lipid droplets than LIF(+).

FSC, indicative of relative cell size, and SSC, indicative of the structural complexity of the cell surface and internal structures, were also analyzed for each of these three populations. Three distinct populations of fibroblasts are apparent in Fig. 1, B and D, in which dot plots of FSC vs. SSC for the 9- and 17-day fibroblasts, respectively, are shown. The data presented are representative of cell-distribution patterns seen in three similar experiments. The least fluorescent cells, the NLIF (yellow, Fig. 1, A and C), are shown in Fig. 1, B and D, to be the least structurally complex of the fibroblast subtypes. The most fluorescent cells, the LIF(+) (red, Fig. 1, A and C), had the greatest structural complexity, as indicated by the y-axis coordinates, and were intermediate in size, relative to the NLIF and the LIF(-), as indicated by the x-axis coordinates. The LIF(-), shown in blue in Fig. 1, A and C, are seen in Fig. 1, B and D, to be the largest of the three subsets, as indicated by their position on the x-axis, and intermediate with respect to structural complexity.

On day 9, the geometric mean values for SSC were 611 for LIF(+) compared with 343 for LIF(-), indicative of decreased structural complexity in LIF(-) and consistent with a diminished lipid content in LIF(-) (Fig. 1B). Geometric mean values for FSC were 563 for LIF(-), 445 for LIF(+), and 423 for NLIF, indicating that NLIF were the smallest cells, whereas LIF(-) were the largest.

A comparison of the Nile red fluorescence for 9- and 17-day cells indicated that the same three populations were present at both postnatal ages, although the relative percentage of LIF(+) was decreased and that of NLIF was increased at day 17 (Fig. 1C). Because we had previously shown that LIF, but not NLIF, undergo apoptosis after alveolar formation, we compared FCS and SSC characteristics for LIF(-) and LIF(+) to determine whether there was any evidence of flow cytometric changes typical of apoptosis in one or both LIF subpopulations (Fig. 1D). A decrease in FSC due to changes in membrane refraction and decreased cell size has been demonstrated in various cell types to be indicative of apoptosis (8, 28). We found that relative cell size was, in fact, decreased in 17-day LIF(+) when compared with 9-day LIF(+), as indicated by the shift of the population shown in red to the left along the x-axis. At 9 days, the FSC geometric mean of LIF(+) was 105% of the geometric mean of NLIF, whereas at 17 days the FSC geometric mean of LIF(+) was only 84% of the geometric mean of NLIF. These findings indicate that LIF(+) were larger than NLIF before the onset of apoptosis, whereas during apoptosis at 17 days, LIF(+) were smaller than NLIF. An increase in SSC due to chromatin condensation occurring during the early stages of apoptosis is also characteristic of apoptosis; however, we saw no evidence of an increase in SSC in NLIF, LIF(+), or LIF(-). It is likely, however, that an increase in SSC of LIF(+) due to chromatin condensation could be offset by the loss of a fraction of lipid droplets in LIF(+) due to increased permeability of apoptotic cell membranes.

Having identified three distinct fibroblast populations based on differences in relative size, structural complexity, and lipid content, we then quantitated changes in the relative percentages of NLIF, LIF(-), and LIF(+) as a function of postnatal age. As shown in Fig. 2A, the relative percentage of LIF(+), shown in region R3, decreased with advancing postnatal age. Initially the least abundant subset, NLIF increased from 27% on days 9-12 to a relative percentage of 71% at 1 mo (Fig. 2B). The relative percentage of LIF(-) was consistently 20-24% from the time of the initial appearance of this subset on postnatal day 9 until 1 mo of age. In contrast, we observed a significant decrease in the relative percentage of LIF(+) as a function of postnatal age from a mean value of 45 ± 7.5% on days 9-12 to 6.1 ± 2.0% on postnatal days 23-30 (P = 0.009). Consistent with our earlier findings that TUNEL-positive 17-day lung fibroblasts were LIF as opposed to NLIF (1), these observations imply that LIF shown previously to be apoptotic were primarily LIF(+). The decrease in the relative percentage of LIF(+) from day 9 to day 30, during which time the relative percentage of LIF(-) remained constant, provides compelling evidence that, in the postnatal rat lung, developmental apoptosis occurs primarily in the LIF(+) subpopulation.


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Fig. 2.   LIF lose lipid droplets with advancing postnatal age. A: representative histograms are presented for fibroblasts obtained on postnatal days 4, 10, 17, and 23. FL1 is plotted on a log scale. Cells in region R3 were more fluorescent due to their greater intracellular lipid content than cells in regions R2 or R1. B: relative percentages of NLIF, LIF(+), and LIF(-) at each of 3 postnatal age ranges. Values are means ± SD: 9-12 days, n = 3 experiments, 4-6 rats/experiment; 17-18 days, n = 4 experiments, 8-10 rats/experiment; and 23-30 days, n = 4 experiments, 8-10 rats/experiment. *Significantly less than days 9-12, P < 0.009.

Temporal correlation of developmental apoptosis with a decrease in steady-state expression of IGF-IR mRNA in LIF vs. NLIF. To explore the possibility that downregulation of IGF-IR mRNA expression might be a factor in the developmental apoptosis seen in LIF, we compared IGF-IR mRNA expression in NLIF vs. LIF obtained both before (postnatal day 4) and during developmental apoptosis (day 17). Freshly isolated fibroblasts were stained with Nile red and sorted by flow cytometry, based on their intracellular lipid content and on FCS and SSC characteristics. Cells in the region of overlap between the NLIF, LIF(+), and LIF(-) peaks were excluded to ensure the collection of highly enriched populations of each subset. IGF-IR mRNA expression levels in the sorted subsets were then assessed by RT-PCR. As shown in Fig. 3, IGF-IR mRNA expression in NLIF was essentially the same on postnatal days 4 and 17. In LIF, however, IGF-IR mRNA expression was markedly lower on day 17, during apoptosis, than on day 4, before the onset of apoptosis associated with the postseptation period. Expressed as a ratio of LIF to NLIF, mean mRNA values for IGF-IR normalized to cyclophilin were 2.37 ± 0.84 at 4 days (n = 3) vs. 0.46 ± 0.26 at 17 days (n = 4), indicating a significant decrease in the relative expression of IGF-IR mRNA that was temporally correlated with the onset of apoptosis after alveolar formation (P = 0.007).


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Fig. 3.   Expression of insulin-like growth factor I receptor (IGF-IR) mRNA varies with fibroblast type and postnatal age. Lung fibroblasts were isolated from 4-day and 17-day rat pups, stained with Nile red, and separated by fluorescence-activated cell sorting (FACS) into subsets that were Nile red negative (NLIF) or Nile red positive (LIF). Total RNA was then reverse transcribed and amplified by PCR using primer pairs specific for IGF-IR and cyclophilin. PCR products were separated by PAGE, gels were stained with SYBR-Gold, and the fluorescent PCR products were quantitated on a Storm 840 using ImageQuant software. In 4-day (4d) cells, IGF-IR mRNA expression was greater in LIF than in NLIF. By day 17, in each of two experiments, IGF-IR mRNA expression in NLIF was essentially the same as in 4-day cells. In 17-day (17d) LIF, however, IGF-IR mRNA levels were decreased relative to day 4. IGF-IR ImageQuant values normalized to cyclophilin were: 4d NLIF = 2.10, 4d LIF = 4.49, 17d LIF = 1.05, 17d NLIF = 2.50, 17d LIF = 0.53, 17d NLIF = 3.26.

Cell surface IFG-IR expression decreases in LIF(+) after alveolarization. We then assessed cell surface IGF-IR expression on lung fibroblasts obtained during the initial phase of alveolar formation (postnatal days 3, 7, and 8) and after alveolar formation (days 16-18) to determine whether there was a correlation between the onset of apoptosis and downregulation of cell surface receptor expression. Freshly isolated adherent fibroblasts were removed from tissue culture flasks using a nonenzymatic cell dissociation solution and then stained with alpha IR-3. Cell surface IGF-IR protein expression was evaluated by flow cytometry, and expression levels in NLIF, LIF(+), and LIF(-) were compared before and after the onset of apoptosis. Because the broad emission spectra of Nile red precluded dual labeling with phycoerythrin, the regions containing NLIF, LIF(+), and LIF(-) were identified in an aliquot of cells stained with Nile red alone by flow cytometric analysis of fluorescence plotted on a log scale, and SSC plotted on linear scale. In lung fibroblasts obtained from 3- to 8-day rats, IGF-IR expression was greater in LIF than in NLIF (Fig. 4). Surface IGF-IR expression remained essentially unchanged in NLIF from days 3 through 18 and in LIF(-) obtained from rats at 8-18 days of age. In contrast, there was a significant decrease in the mean value for cell surface IGF-IR expression for LIF(+) from 20.5 ± 4.1 on days 3-8 to 1.2 ± 2.1 on days 16-18 (P = 0.005).


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Fig. 4.   Flow cytometric analysis of cell surface IGF-IR expression. Freshly isolated lung fibroblasts were fixed in 0.1% paraformaldehyde and then stained with alpha IR-3, followed by an R-phycoerythrin-conjugated F(ab') fragment of goat anti-mouse IgG. Unstained cells and cells stained only with the secondary antibody served as controls. Values for positively stained cells were corrected for nonspecific staining seen in each subset. Regions containing NLIF, LIF(-), and LIF(+) were identified by analyzing FL1 on a log scale and SSC on a linear scale in separate aliquots of the cell suspensions stained with Nile red. Cell surface IGF-IR expression was decreased significantly in LIF(+) from postnatal days 3-8 to postnatal days 16-18 (P = 0.005).

IGF-IR is a survival factor for rat lung fibroblasts. To determine the extent to which rat lung fibroblasts are dependent on survival signaling via the IGF-IR, cells obtained from the lungs of 7- to 9-day rats were treated with alpha IR-3, a specific blocking antibody for this receptor. Fibroblast survival was assessed by quantitating the percentage of calcein AM-positive (live) vs. propidium iodide-positive (dead) cells in response to treatment. After treatment with alpha IR-3 for 24 h in the absence of serum, the mean value for the survival rate in three separate experiments was 52 ± 17.8%, which was significantly lower than that in untreated S-F control cultures, 80 ± 3.6% (P = 0.05). When cells were treated with both alpha IR-3 and the blocking antibody to the IR in combination, the additive effect was negligible. In a separate experiment, we documented the presence of apoptotic cells seen in response to treatment of 8-day rat lung fibroblasts with alpha IR-3 and with both alpha IR-3 and anti-IR antibodies in combination (Fig. 5). Chromatin fragmentation was assessed using a fluorescent DNA end-labeling TUNEL technique, and chromatin condensation was detected using a Hoechst counterstain.


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Fig. 5.   IGF-IR mediates survival in postnatal rat lung fibroblasts. Freshly isolated 8-day lung fibroblasts were plated on 8-well slides and treated for 48 h with serum-free (S-F) medium alone (A, B); alpha IR-3 (2.0 µg/ml; C, D); or both alpha IR-3 (2.0 µg/ml) and anti-IR (2.0 µg/ml) (E, F). The cells were then fixed in 4% paraformaldehyde and labeled by fluorescent terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) to detect DNA fragmentation (B, D, F) and counterstained with Hoechst 33258 to detect chromatin condensation (A, C, E). A field viewed sequentially through FITC (TUNEL) and 4'-6-diamidino-2-phenylindole (DAPI, Hoechst) filter cubes is shown for the control and for each of the treatment groups.

Inhibitors of the PI 3-kinase and MAPK pathways decrease fibroblast survival. Both IGF-IR and the IR can signal via either the PI 3-kinase pathway or the MAPK pathway to promote fibroblast survival. To assess the contributions of each of these downstream pathways in 7- to 9-day rat lung fibroblasts, we assessed survival in cells treated with 20 µM LY-294002, a specific inhibitor of the PI 3-kinase pathway and with 20 µM PD-98059, a specific inhibitor of MEK in the MAPK pathway (Fig. 6). Treatment with LY-294002 and PD-98059, alone and in combination, resulted in a significant increase in cell death, both in the presence of serum (P = 0.002) and in the absence of serum (P < 0.0001). The presence of serum conferred protection against cell death induced by treatment with PI 3-kinase and MAPK inhibitors, but only in the case of treatment with PD-98059 did the degree of protection achieve statistical significance (P = 0.007). Treatment with both inhibitors in combination resulted in a small increase in cell death relative to the effect seen after treatment with either agent alone. This increase was not significant, however. Lung fibroblasts in which apoptosis was induced by treatment with inhibitors of PI 3-kinase and MAPK are shown in Fig. 7. Chromatin fragmentation was assessed using a fluorescent DNA end-labeling TUNEL technique. Chromatin condensation was detected in the same cells counterstained with Hoechst.


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Fig. 6.   Primary 7- to 9-day fibroblasts cultured in 24-well plates in S-F or complete media were treated for 24 h with 20 µM LY-294002, 20 µM PD-98059, or with both kinase inhibitors in combination. Viability was assessed on a Nikon Diaphot 300 microscope equipped with filter sets for fluorescence. Viable cells were identified as those that incorporated the vital dye calcein AM (1 µg/ml), whereas cells that stained positive for propidium iodide (1 µg/ml) were counted as dead. In each of 3 separate experiments, a minimum of 100 cells was counted per well in each of 6 wells per treatment group. *Significantly different from S-F controls (P < 0.0001); **significantly different from controls cultured in complete media (P = 0.002); ***significantly less than S-F PD-98059 treatment (P = 0.007).



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Fig. 7.   Primary 7- to 9-day fibroblasts plated on 8-well slides were treated for 24 h with 20 µM LY-294002 or 20 µM PD-98059 in S-F media, then fixed in 4% paraformaldehyde, and labeled by fluorescent TUNEL to detect DNA fragmentation (B, D, F) and counterstained with Hoechst 33258 to detect chromatin condensation (A, C, E). A field viewed sequentially through FITC (TUNEL) and DAPI (Hoechst) filter cubes is shown for the control and for each of the treatment groups: control (A, B); 20 µM LY-294002 (C, D); and 20 µM PD-98059 (E, F).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Within several days after the completion of the period of bulk alveolarization in the postnatal rat lung, >20% of interstitial fibroblasts undergo apoptosis (1, 6, 17, 25). This substantial decline in fibroblast number serves to decrease alveolar wall thickness, which, in turn, enhances gas exchange and may also facilitate the maturation of the microvasculature from a double to a single capillary layer (25). Our initial observations that the relative percentage of LIF decreased during the first month of life suggested that apoptosis occurred primarily, if not exclusively, in LIF (1). The occurrence of apoptosis in LIF, but not NLIF, was verified using the TUNEL assay to identify DNA strand breaks in fibroblasts shown by flow cytometric analysis of intracellular lipid content and relative size and structural complexity to be LIF (1).

In the present study, we extended these observations by further characterizing the LIF removed by apoptosis. Flow cytometric analysis of the intracellular lipid content of lung fibroblasts obtained during the second postnatal week demonstrated the presence of a population of LIF, designated LIF(-), that had lost a substantial fraction of their lipid droplets. LIF(-) could be distinguished from NLIF and from LIF that retained lipid droplets, LIF(+), by flow cytometric quantitation of intracellular lipid content and by the analysis of FCS and SSC characteristics, indicators of relative cell size and structural complexity, respectively. On day 9, LIF(-) constituted ~20% of total lung fibroblasts, a relative percentage that did not change substantially throughout the remainder of the first month of life. In contrast, the relative percentage of LIF(+) decreased from 45% of total fibroblasts on days 9-12 to 6% at 1 mo. Taken together with flow cytometric evidence of changes consistent with apoptosis only in LIF(+), these data indicate that the apoptosis previously shown to occur in LIF (1) occurs primarily in those cells that retain lipid droplets, LIF(+).

A similar decrease in intracellular lipid content of lung fibroblasts beyond postnatal day 8 was also noted by Kaplan and colleagues (4, 14). Lipid droplet content, determined at the electron microscopy (EM) level, was constant from postnatal days 4-8 (4), followed by an 88% decrease from day 8 to the adult lung (14). Although these authors concluded that there was only a modest decrease in the relative percentage of LIF in the mature lung, this apparent discrepancy with our results could be due to the fact that relatively few cells (200/lung) were evaluated at the EM level, whereas we were able to quantitate intracellular lipid in at least 10,000 cells per experiment, a number not feasible with light or EM techniques.

Although the regulatory control of intracellular lipid content in LIF is poorly understood, lipid accumulation in a similar cell, the adipocyte, has been well characterized. Both cell types acquire neutral lipid droplets rich in triglycerides during development. These droplets appear late in gestation and then decrease in number during the perinatal period. Chen and colleagues (9) have shown in rat LIF that expression of the peroxisome proliferator-activated receptor-gamma (PPAR-gamma ), an orphan nuclear receptor implicated in lipid homeostasis and adipogenesis in the adipocyte, peaks shortly before birth. This increase in PPAR-gamma precedes the increased expression at birth of genes containing response elements for PPAR-gamma that control lipid hydrolysis and transport lipoprotein lipase and adipocyte lipid binding protein (12, 16). A role for insulin/IGF signaling in the activation of adipogenesis has also been suggested by the observation that differentiation of 3T3-L1 preadipocytes into adipocytes requires either insulin or IGF-I (26). However, there is, as yet, no evidence to support a causal relationship between a decrease in ligand binding and the loss of lipid from adipocytes.

On the basis of our results and those of other investigators (14), it appears likely that LIF(-) are derived from LIF, since both contain lipid droplets, in contrast to NLIF. In addition, both LIF(+) and LIF(-) are larger and more structurally complex than NLIF. Our results suggest that loss of intracellular lipid in LIF(-) at the beginning of the second postnatal week confers protection against the marked increase in fibroblast apoptosis that occurs after the period of bulk alveolar formation. This observation would seem to imply that interference with the loss of intracellular lipid may render a larger fraction of lung fibroblasts susceptible to apoptosis, an event that could result in excessive thinning of alveolar walls and thus compromise mature lung function. Although beyond the scope of the current investigation, it will be of interest to determine in future studies the mechanisms responsible for the loss of intracellular lipid in rat lung fibroblasts. Furthermore, comparison of adipogenic gene expression in LIF(+) vs. LIF(-) could clarify the roles played by these genes during what appears to be a dedifferentiation process.

Whereas the NLIF is the predominant fibroblast population in the mature rodent lung, the LIF is predominant during the late saccular and early alveolar stages of lung development, suggesting a critical role for the LIF during the late fetal and early postnatal stages. One such developmental role proposed by McGowan and Torday (21) is that of providing a source of lipid that could be used in the synthesis of surfactant phospholipid by the alveolar type II epithelial cell. Also of potential relevance to postnatal lung development is the fact that expression of mRNA for fibronectin, a major constituent of the pulmonary extracellular matrix, is ninefold higher in LIF than in NLIF during the first week of life (1). The ability of this cell-adhesive protein to influence migration, proliferation, and differentiation of specific lung cell types suggests that fibronectin production by LIF is of critical importance during the early stages of lung development.

There is, perhaps, a greater degree of uncertainty regarding the specific roles of LIF and NLIF in the mature lung, however. It has been assumed, although never rigorously tested, that the LIF is a precursor of the myofibroblast, a cell identified by the presence of alpha -smooth muscle actin (alpha -SMA) and vimentin and the absence of desmin (21). Myofibroblasts play a critical role in repair because of their ability to proliferate and secrete abundant extracellular matrix in response to injury. In sorted 16-day fibroblasts, we observed that NLIF cultures were essentially devoid of alpha -SMA, whereas the majority of LIF(-) were alpha -SMA positive (M. C. Bruce, unpublished results). alpha -SMA-positive filaments were absent in the majority of LIF(+), suggesting that the LIF(-) may be the predominant source of myofibroblast precursors. In the absence of a specific marker for each subset, however, the rapid loss of lipid droplets in vitro and the differential rates of proliferation of these subsets precluded the definitive identification of the fibroblast subset(s) that contain alpha -SMA-positive cells.

In addition to the unique contributions of each fibroblast subset, several lines of evidence suggest that lung fibroblast behavior may also be a function of the interactions between subsets. Sorted populations of NLIF tend to adhere more readily and to proliferate more rapidly when plated on fibronectin-coated surfaces than when plated on plastic (M. C. Bruce, unpublished observations). We have also observed that after sorting by flow cytometry, NLIF proliferate more rapidly when cocultured with LIF than when cultured alone (1). Although the increased adherence and proliferation seen in NLIF cocultured with LIF may well be the result of a complex series of interactions, the increased fibronectin production by LIF relative to that by NLIF appears likely to be a contributing factor.

The identification of LIF(+) as the fibroblast subtype that undergoes apoptosis in the postnatal lung enabled us to compare levels of expression of IGF-IR among fibroblast subsets to assess the role of this receptor as a survival factor. The IGF-IR was considered a logical survival factor candidate both because of its documented role in this capacity in fibroblasts and adipocytes and because the decreased expression of this receptor in the developing rat lung coincides with the onset of apoptosis after alveolar formation (6, 30). In the present study, a significant decrease in lung fibroblast expression of IGF-IR, at both the cell surface protein and mRNA levels, was seen only in LIF(+), implying that downregulation of this growth factor receptor is responsible, at least in part, for the developmental apoptosis that occurs in this lung fibroblast subset.

The role of the IGF-IR as a survival factor in rat lung fibroblasts was further established in a series of experiments in which the ligand binding region was blocked using the alpha IR-3 antibody, a technique used successfully by others to induce apoptosis in a variety of cell types. Treatment with this blocking antibody substantially decreased survival of 7- to 9-day fibroblasts. The decreased survival observed in cells treated with blocking antibodies in S-F medium, which lacks IGF-I, suggests that lung fibroblasts are capable of producing this growth factor in vitro, consistent with earlier reports of IGF-I production by primary cultures of rat lung fibroblasts (27).

The PI 3-kinase and MAPK pathways have each been implicated in IGF-IR and IR survival signaling, although the degree to which these pathways are activated by these receptors varies considerably with cell type (18). In the present studies, blocking IGF-IR and IR survival pathways by treatment with inhibitors of the PI 3-kinase and the MAPK pathways also resulted in a significant decrease in fibroblast survival. Survival was further decreased in cells treated with both inhibitors in combination, consistent with the possibility that signaling via each of these pathways can promote fibroblast survival.

Although the concept of lung fibroblast heterogeneity has been accepted for more than two decades, the findings presented herein suggest a greater degree of complexity than was previously appreciated. Significant progress has been made in recent years in the identification of specific roles for lung fibroblast subsets during development. However, in light of our observations that there are, in fact, two distinct subpopulations of lipid-containing fibroblasts, LIF(+) and LIF(-), further comparisons of the developmental changes in gene and protein expression in these subsets appear warranted. The observed differences with respect to intracellular lipid content, IGF-IR expression, and the incidence of apoptosis in these two LIF subtypes suggest that these cells are likely to have distinct roles during alveolar formation and injury repair in the mature lung.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-31172 (to M. C. Bruce).


    FOOTNOTES

Address for reprint requests and other correspondence: M. C. Bruce, Dept. of Pediatrics, Div. of Neonatology, Univ. of Kentucky Medical School, Lexington, KY 40536 (E-mail: mbruce{at}uky.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.

10.1152/ajplung.00050.2001

Received 7 February 2001; accepted in final form 9 May 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Awonusonu, F, Srinivasan S, Strange J, Al-Jumaily W, and Bruce MC. Developmental shift in the relative percentages of lung fibroblast subsets: role of apoptosis postseptation. Am J Physiol Lung Cell Mol Physiol 277: L848-L859, 1999[Abstract/Free Full Text].

2.   Bostrom, H, Willets K, Pekny M, Leveen P, Lindahl P, Hedstrand H, Pekna M, Hellstrom M, Gebre-Medhin S, Schalling M, Nilsson M, Kurland S, Tornell J, Heath JK, and Betsholtz C. PDFG-A signaling is a critical event in lung alveolar myofibroblast development and alveogenesis. Cell 85: 863-873, 1996[ISI][Medline].

3.   Breen Falco, EVM, Absher M, and Cutroneo KR. Subpopulations of rat lung fibroblasts with different amounts off type I and type III collagen mRNAs. J Biol Chem 265: 6286-6290, 1990[Abstract/Free Full Text].

4.   Brody, JS, and Kaplan NB. Proliferation of alveolar interstitial cells during postnatal lung growth. Am Rev Respir Dis 127: 763-770, 1983[ISI][Medline].

5.   Bruce, MC, and Honaker CE. Transcriptional regulation of tropoelastin expression in rat lung fibroblasts: changes with age and hyperoxia. Am J Physiol Lung Cell Mol Physiol 274: L940-L950, 1998[Abstract/Free Full Text].

6.   Bruce, MC, Honaker CE, and Cross RJ. Lung fibroblasts undergo apoptosis following alveolarization. Am J Respir Cell Mol Biol 20: 228-236, 1999[Abstract/Free Full Text].

7.   Cannigia, I, Tseu I, Han RRN, Smith BT, Tanswell K, and Post M. Spatial and temporal differences in fibroblast behavior in fetal rat lung. Am J Physiol Lung Cell Mol Physiol 261: L424-L433, 1991[Abstract/Free Full Text].

8.   Carbonari, M, Cibati M, and Fiorilli M. Measurement of apoptotic cells in peripheral blood. Cytometry 22: 161-167, 1995[ISI][Medline].

9.   Chen, S, Jackson S, Doro M, and McGowan S. Perinatal expression of genes that may participate in lipid metabolism by lipid-laden lung fibroblasts. J Lipid Res 39: 2483-2492, 1998[Abstract/Free Full Text].

10.   Cross, TG, Scheel-Toellner D, Henriquez NV, Deacon E, Salmon M, and Lord JM. Serine/threonine protein kinases and apoptosis. Exp Cell Res 256: 34-41, 2000[ISI][Medline].

11.   Datta, SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, and Greenberg ME. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 91: 231-241, 1997[ISI][Medline].

12.   Forman, BM, Tontonoz P, Chen J, Brun RP, Spiegelman BM, and Evans RM. 15-Deoxy-delta-12,14-protstaglandin J2 is a ligand for the adipocyte determination factor PPAR-gamma . Cell 83: 803-812, 1995[ISI][Medline].

13.   Greenspan, P, and Fowler SD. Spectrofluorometric studies of the lipid probe, nile red. J Lipid Res 26: 781-789, 1985[Abstract].

14.   Kaplan, NB, Grant MM, and Brody JS. The lipid interstitial cell of the pulmonary alveolus. Am Rev Respir Dis 132: 1307-1312, 1985[ISI][Medline].

15.   Kauffman, SL, Burri PH, and Weibel ER. The postnatal growth of the rat lung. II. Autoradiography. Anat Rec 180: 63-76, 1974[ISI][Medline].

16.   Kliewer, SA, Lenhard JM, Willison TM, Patel I, Morris DC, and Lehman JM. A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor-gamma and promotes adipocyte differentiation. Cell 83: 813-819, 1995[ISI][Medline].

17.   Kresch, MJ, Christian C, Wu F, and Hussain N. Ontogeny of apoptosis during lung development. Pediatr Res 43: 426-431, 1998[Abstract].

18.   Kulik, G, Klippel A, and Weber MJ. Antiapoptotic signalling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt. Mol Cell Biol 17: 1595-1606, 1997[Abstract].

20.   Maksvytis, HJ, Vaccaro C, and Brody JS. Isolation and characterization of the lipid-containing interstitial cell from the developing rat lung. Lab Invest 45: 248-259, 1981[ISI][Medline].

21.   McGowan, SE, and Torday JS. The pulmonary lipofibroblast (lipid interstitial cell) and its contributions to alveolar development. Annu Rev Physiol 59: 43-62, 1997[ISI][Medline].

22.   Navarro, P, Valverde AM, Benito M, and Lorenzo M. Insulin/IGF-I rescues immortalized brown adipocytes from apoptosis down-regulating Bcl-xS expression, in a PI 3-kinase- and MAP kinase-dependent manner. Exp Cell Res 243: 213-21, 1998[ISI][Medline].

23.   Parrizas, M, Saltiel AR, and LeRoith D. Insulin-like growth factor 1 inhibits apoptosis using the phosphatidylinositol 3'-kinase and mitogen-activated protein kinase pathways. J Biol Chem 272: 154-161, 1997[Abstract/Free Full Text].

24.   Scavo, LM, Ertsey R, Chapin CJ, Allen L, and Kitterman JA. Apoptosis in the development of rat and human fetal lungs. Am J Respir Cell Mol Biol 18: 21-31, 1998[Abstract/Free Full Text].

25.   Schittny, JC, Djonov V, Fine A, and Burri PH. Programmed cell death contributes to postnatal lung development. Am J Respir Cell Mol Biol 18: 786-793, 1998[Abstract/Free Full Text].

26.   Smith, PJ, Wise LS, Berkowitz R, and Rubin CS. Insulin-like growth factor-1 is an essential regulator of the differentiation of 3T3-L1 adipocytes. J Biol Chem 263: 9402-9408, 1988[Abstract/Free Full Text].

27.   Stiles, AD, and Moats-Staats BM. Production and action of insulin-like growth factor I/somatomedin C in primary cultures of fetal lung fibroblasts. Am J Respir Cell Mol Biol 1: 21-26, 1989[ISI][Medline].

28.   Swat, W, Ignatowicz L, and Kisielow P. Detection of apoptosis of immature CD4+8+ thymocytes by flow cytometry. J Immunol Methods 137: 79-87, 1991[ISI][Medline].

29.   Wendel, DP, Taylor DG, Albertine KH, Keating MT, and Li DY. Impaired distal airway development in mice lacking elastin. Am J Respir Cell Mol Biol 23: 320-326, 2000[Abstract/Free Full Text].

30.   Werner, H, Woloschak M, Adamo M, Shen-Orr Z, and Roberts CT, Jr. Developmental regulation of the rat insulin-like growth factor I receptor gene. Proc Natl Acad Sci USA 86: 7451-7455, 1989[Abstract].

31.   White, MF. The IRS-signaling system: a network of docking proteins to IRS-1 in insulin signaling. Mol Cell Biochem 182: 3-11, 1998[ISI][Medline].


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